CHARACTERIZATION OF THE HUMAN FACTOR XII (HAGEMAN ...

CHARACTERIZATION OF T H E HUMAN FACTOR XII (HAGEMAN FACTOR)

CDNA AND THE GENE

DEBORAH E. COOL

A THESIS SUBMITTED IN PARTIAL FULFILMENT OF

THE REQUIREMENTS FOR THE DEGREE OF

DOCTOR OF PHILOSOPHY

- T H E FACULTY OF GRADUATE STUDIES

Department of Biochemistry

We accept this thesis as conforming

to the required standard

THE UNIVERSITY OF BRITISH COLUMBIA

October, 1987

In presenting this thesis in partial fulfilment of the requirements for an advanced

degree at the University of British Columbia, I agree that the Library shall make it

freely available for reference and study. I further agree that permission for extensive

copying of this thesis for scholarly purposes may be granted by the head of my

department or by his or her representatives. It is understood that copying or

publication of this thesis for financial gain shall not be allowed without my written

permission.

Department

The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3

DE-6(3/81)

ABSTRACT

A human liver cDNA library was screened by colony hybridization with two mixtures of

synthetic oligodeoxyribonucleotides as probes. These oligonucleotides encoded regions of

0-factor X l l a as predicted from the amino acid sequence. Four positive clones were isolated

that contained D N A coding for most of factor XI I mRNA. A second human liver cDNA

library was screened by colony hybridization with 3 zP-labeled cDNA clones obtained from

the first screen and two identical clones were isolated.

D N A sequence analysis of these overlapping clones showed that they contained D N A coding

for the signal peptide sequence, the complete amino acid sequence of plasma factor XII , a

T G A stop codon, a 3' untranslated region of 150 nucleotides, and a poly A + tail. The cDNA

sequence predicts that plasma factor XII consists of 596 amino acid residues. Within the

predicted amino acid sequence of factor XII , were identified three peptide bonds that are

cleaved by kallikrein during the formation of j3-factor X l l a .

Comparison of the structure of factor XII with other proteins revealed extensive sequence

identity with regions of tissue-type plasminogen activator (the epidermal growth factor-like

region and the kringle region) and fibronectin (type I and type II homologies). As the type

II region of fibronectin contains a collagen-binding site, the homologous region in factor XII

may be responsible for the binding of factor XII to collagen. The carboxyl-terminal region of

factor XII shares considerable amino acid sequence homology with other serine proteases

including trypsin and many clotting factors.

A human genomic phage library was screened by using a human factor XII cDNA as a

hybridization probe. Two overlapping phage clones were isolated which contain the entire

human factor XII gene. D N A sequence and restriction enzyme analysis of the clones

indicate that the gene is approximately 12 kbp in size and is comprised of 13 introns and 14

exons. Exons 3 through 14 are contained in a genomic region of only 4.2 kbp with introns

ranging in size from 80 to 554 bp.

The multiple regions found in the coding sequence of FXII that are homologous to putative

domains in fibronectin and tissue-type plasminogen activator are contained on separate

exons in the factor XII gene. The intron/exon gene organization is similar to the serine

protease gene family of plasminogen activators and not to the clotting factor family.

Analysis of the 5' flanking region of the gene shows that it does not contain the typical

T A T A and C A A T sequences found in other genes. This is consistent with the finding that

transcription of the gene is initiated at multiple start sites.

LIST OF ABBREVIATIONS

A Adenosine

ATP Adenosinetriphosphate

bp Base Pair(s)

BSA Bovine Serum Albumin

C Cytosine

Ca Calcium ions

dNTP Deoxynucleotidetriphosphate

DNA Deoxyribonucleic Acid

DNase Deoxyribonuclease

ddNTP Dideoxynucleotidetriphosphate

DTT Dithiothreitol

EDTA Ethylenediaminetetraacetic Acid

EtBr Ethidium Bromide

G Guanosine

Gla 7-Carboxyglutamic Acid

GuHCl Guanidine Hydrochloride

hnRNA Heterogenous Nuclear RNA

IPTG Isopropyl-beta-D-Thiogalactopyranoside

Kbp Kilobase Pair(s)

Krpm Thousand Revolutions Per Minute

LB Luria Broth

mA Milliamps

min Minute (s) mRNA Messenger RNA

N Any Nucleotide (G, A, T or C)

OD Optical Density

pfu Plaque Forming Unit

PA Plasminogen Activator(s)

R Purine (A or G)

RNA Ribonucleic Acid

RNase Ribonuclease

rRNA Ribosomal RNA

TEMED N,N,N',N'-Tetramethylethylenediamine

tPA Tissue-Type Plasminogen Activator

Tris ' Tris(hydroxymethyl)aminomethane

tRNA Transer RNA

TJ Uridine

UV Ultra Violet

V Volts

T Thimidine

W Watts

X-Gal 5-Bromo-4-Chloro-3-Indoyl-|3-D

-Galactopyroside

Y Pyrimidine (T or C)

T A B L E O F C O N T E N T S

Abstract ii

List of Abbreviations iv

Table of Contents vi

List of Tables ix

List of Figures x

Acknowledgements xii

I. Introduction 1 A . T H E C O N T A C T S Y S T E M OF P L A S M A 1

1. The Biochemistry of the Surface Activation Components 2 a. The Catalytic Mechanism of Serine Proteases 5 b. Factor XII 6 c. Prekallikrein and Factor X I 12 d. High Molecular Weight Kininogen 14

2. A Model of the Surface Activation Mechanism 15 B. P H Y S I O L O G I C A L F U N C T I O N S OF T H E C O N T A C T S Y S T E M 18

1. Blood Coagulation 18 2. Fibrinolysis 21

a. Plasminogen and Its Activators 22 3. The Inflammatory Response 24 4. Conversion of Prorenin to Renin 26

C. T H E S T R U C T U R E OF E U K A R Y O T I C S T R U C T U R A L G E N E S 26 1. Promoters 26 2. Introns and Exons 28 3. Transcription and Processing 28

D. M E C H A N I S M S OF G E N E E V O L U T I O N 29 1. Gene Duplication 29 2. Gene Fusions 30 3. Exon Shuffling 30

E . E V O L U T I O N OF A M I N O ACID A N D D N A S E Q U E N C E 30 1. Molecular Clock 30 2. Intron Sliding 31

F . P R O T E I N DOMAINS OF T H E C O A G U L A T I O N A N D P L A S M I N O G E N A C T I V A T O R G E N E S 32

1. Trypsin-Like Family of Serine Proteases 32 G. E V O L U T I O N OF T H E SERINE P R O T E A S E S 36 H . O B J E C T I V E S 37

II. Materials and Methods 39 A . M A T E R I A L S 39 B. STRAINS, VECTORS A N D M E D I A 40

1. Bacterial Strains 40 2. Vectors 41

3. Media . 41 C. G E L ELECTROPHORESIS 42

1. Neutral Agarose Gels 42 2. Formaldehyde Agarose Gels 43 3. Neutral Polyacrylamide Gels 43 4. Denaturing Polyacrylamide Gels 44

D. ISOLATION OF D N A 45 1. Small-scale Isolation of Plasmid D N A 45 2. Large-scale Isolation of Plasmid D N A 46 3. Small-scale Isolation of X D N A 47 4. Large-scale Isolation of X D N A 48 5. Isolation of Single-stranded Plasmid D N A 49

E . ISOLATION O F P O L Y A+ RNA 50 F . O L I G O N U C L E O T I D E SYNTHESIS A N D PURIFICATION 51 G. L A B E L I N G O F D N A 53

1. Labeling With 3 2P by Nick Translation 53 2. Labeling with T4 Polynucleotide Kinase 53

H . SCREENING A PLASMID LIBRARY 54 I. SCREENING XPHAGE LIBRARIES 56 J . D N A SUBCLONING 57

1. Production of D N A Fragments for Ligation 57 2. Ligation of D N A into p U C l 3 or M13 Vectors 58 3. Transformation of D N A into Bacteria 59

K. B L O T HYBRIDIZATIONS 60 1. Southern Blot Analysis of Genomic and Cloned D N A 60 2. Northern Blot Analysis 61

L . D N A S E Q U E N C E A N A L Y S I S 62 1. Screening M13 Clones 62 2. D N A Sequence Analysis 63 3. Computer Analysis of D N A Sequence Data 65

M . MAPPING T H E E N D OF A mRNA TRANSCRIPT 65 1. Primer Extension Analysis 65 2. Nuclease S1 Mapping 67

III. Results 69 A. C H A R A C T E R I Z A T I O N O F T H E H U M A N FXII cDNA 69

1. Isolation of Factor XII cDNA Clones 69 2. Sequence Analysis of Human Factor XII cDNA Clones 76 3. Predicted Amino Acid Sequence of Human Factor XII 79 4. 5'End of the Factor X n c D N A 80 5. A 2.40 kb mRNA From Liver Codes For Human Factor XII 83

B. T H E H U M A N F A C T O R XII G E N E 86 1. Isolation and Characterization of Factor XII Genomic Clones 86 2. Southern Blot Analysis of the Human FXII Gene 91 3. Localization of Intron/Exon Junctions 94 4. Nucleotide Sequence of the Human Factor XII Gene 97 5. Transciption Initiation Site of the Factor XII Gene 101

IV. Discussion 109 A. CHARACTERIZATION O F T H E H U M A N F A C T O R XII cDNA 109

1. Characterization of Factor XII cDNA Clones 109 2. Factor XII Is Synthesized as a Prescursor 110

B. HOMOLOGIES WITH O T H E R PROTEINS I l l 1. Amino-Terminal End of the Zymogen Form of Factor XII 113 2. Fibronectin Homology 114 3. Epidermal Growth Factor Homology. 118 4. Kringle Homology 118 5. Proline-Rich Domain 119 6. Serine Protease Domain 119 7. Plasminogen Activator Homology 124 8. Physiological Significance of the Homologous Protein Domains 125

C. A PERSPECTIVE O N T H E R O L E O F T H E C O N T A C T S Y S T E M IN HOMEOSTASIS 126

D. D N A S E Q U E N C E A N A L Y S I S OF T H E H U M A N F A C T O R XII G E N E .... 128 E . A N A LY SIS OF T H E PROMOTER REGION IN T H E H U M A N FXII G E N E ....

129 1. The C A A T Element 130 2. The T A T A Box 131

F. P U T A T I V E PROTEIN DOMAINS A R E F O U N D O N S E P A R A T E E X O N S 133

G. E V O L U T I O N O F T H E F A C T O R XII G E N E 136 1. Evolution of the Amino Terminal Half of Factor XII 137 2. Evolution of the Serine Protease Domain of Factor XII 142 3. Intron/Exon Sliding 146

H . A M O D E L FOR T H E E V O L U T I O N O F T H E H U M A N F A C T O R XII G E N E ... 147

Concluding Remarks 150

V. Literature Cited 151

LIST OF TABLES

I. D N A Sequencing Mixes 64

II. Nucleotide Sequence of Intron/Exon Junctions in the FXII Gene 95

III. Frequencies of Nucleotides at Intron/Exon Junctions 96

IV. Size and Position of Exons and Introns in the FXII Gene 97

V. Per-cent Homologies Between the Amino Acid Sequence in the/3-factor X l l a and Pancreatic Serine Proteases Catalytic Domains 123

VI. Summary of TATA-Like Elements Found in Liver Specific and Coagulation Genes . 130

LIST OF FIGURES

1. The Blood Coagulation Cascade 3

2. Schematic Diagram of 0-factor X l l a 8

3. Schematic Diagram of the Surface-Dependent Contact Activation Mechanism 16

4. Amino Acid Sequence Homologies in Coagulation Factor Zymogens 33

5. Predicted Oligonucleotide Sequences for 0-factor X l l a 70

6. Double Oligonucleotide Screen For Factor XII cDNA 72

7. Restriction Enzyme Map and Sequencing Strategy for Factor XII cDNA 74

8. Nucleotide Sequence of Factor XII cDNA 77

9. Nucleotide Sequence of a cDNA for the FXII Signal Peptide 82

10. Size Determination of the Factor XII mRNA Using rRNA Markers 84

11. Southern Blot Analysis of FXII Genomic Clones 87

12. Intron/Exon Organization of the FXII Gene 90

13. Genomic D N A Southern Blot Analysis of the FXII Gene 92

14. Partial D N A Sequence of the FXII Gene 98

15. Nuclease S i Mapping of the FXII Gene 102

16. Primer Extension Analysis of the FXII Gene 104

17. Northern Blot Analysis of the 5' End of the FXII Gene 107

18. Proposed Model for Factor XII 112

19. Amino Acid Sequence Homologies of FXII With Other Proteins 115

20. Amino Acid Sequence Alignment of the Catalytic Domain of FXII With Other Serine Proteases 121

21. Schematic Diagram of FXII and Positions of Introns 134

22. N-Terminal Exon Organization in the Plasminogen Activators and FXII 138

23. Comparison of Exon Organization of the Serine Protease Domain in Some Serine Proteases 143

A Model for the Evolution of the Plasminogen Activators and FXII .

ACKNOWLEDGEMENTS

I would like to thank the members of my supervisory committee, Drs. Jim Richards and

Peter Candido for their kind support and advice throughout this project. I would also like to

thank the members of the MacGillivray lab, those past and present, for their good natured

humour, for tolerating Molly and for the much appreciated technical advice and for their

tremendous support during my work in the lab. I would especially like to acknowledge my

great supervisor, Dr. Ross MacGillivray (or "Ross") who has taught me always to try to

achieve a high standard of excellence in science and to take pride and pleasure in the work.

Thank you Ross, I am grateful for all the assistance you have given me. I would like to

thank Dr. M . J . Smith who was my Masters of Science Degree supervisor and who became

a good friend and encouraged me throughout my Ph.D. degree. Finally, I would especially

like to thank my husband, for his unending encouragement during my graduate degrees and

for all his help in preparation of my manuscripts and this thesis.

I. INTRODUCTION

A. THE CONTACT SYSTEM OF PLASMA

Organisms can respond to invasion by foreign substances, to trauma and to internal

disorders by the activation of four plasma systems: they are the complement systems, the

contact (Hageman) activated clotting system, the extrinsic clotting system and the

fibrinolytic system (Ratnoff and Saito, 1979; Cochrane and Griffin, 1982; Pesce and

Dosekun, 1983; Sundsmo and Fair, 1983; Colman, 1984). Maintenance of a homeostatic

environment in vivo may be a result of the synergism that exists between these plasma

systems (Cochrane, 1980; Bennett and Ogston, 1981). At least the coagulation and

fibrinolytic systems can be activated both in vivo and in vitro by a mechanism described as

"surface-mediated" or "contact phase" activation which requires a negatively charged

surface as well as the appropriate plasma proteins (reviewed in Ratnoff and Saito, 1979).

In vitro studies have also demonstrated that the contact activation mechanism plays a role

in the inflammatory response, in the production of kinins and in the conversion of prorenin

to renin (Bennett and Ogston, 1981; Cochrane and Griffin, 1982). The proteins which are

primarily responsible for the surface activation reactions are factor XII (Hageman factor),

prekallikrein (Fletcher factor), high molecular weight (HMW) kininogen (Fitzerald factor)

and factor XI (PTA, plasma thromboplastin antecedent) (Davie et al., 1979). Factor XII

and H M W kininogen have surface binding properties and therefore are essential in the

surface activation mechanism. The following review will describe the physical and

biochemical properties of the components involved in contact activation; this will include

mainly the role of factor XII in surface activation of the plasma systems listed above and

the subsequent relevance of factor XII to the maintenance of homeostasis in living

Introduction / 2

organisms.

1. The Biochemistry of the Surface Activation Components

The characteristic enzymatic reaction in the plasma systems described above is the action of

serine proteases. Similar to the pancreatic serine proteases, trypsin (Marquart et al.,

1983), chymotrypsin (Cohen et al., 1981) and elastase (Sawyer et al., 1978), the proteases

involved are synthesized in precursor forms and are activated by limited proteolysis (Davie

et al., 1979; Jackson and Nemerson, 1980). The active enzymes have a trypsin-like

specificity with a preference for argininyl peptide bonds and contain the conserved

activation and catalytic site residues described for the pancreatic enzymes (Jackson and

Nemerson, 1980). Unlike the digestive enzymes, the proteases required in the complex

plasma systems have long amino terminal polypeptide domains that appear to be important

in functions such as metal-binding, surface binding or fibrin binding and in their regulation

(Jackson and Nemerson,11980). The zymogens in the contact system are activated by

limited proteolysis (discussed below) resulting in a cascade of enzymatic reactions in which

inactive zymogens are converted to active serine proteases that act upon each other in a

controlled and step-wise manner (Jackson and Nemerson, 1980). The physiological

significance of the cascade mechanism is the amplification of reactions by a few initiator

molecules resulting in a massive amount of end-product such as fibrin from the blood

coagulation cascade (Fig. 1) (Davie et al., 1979; Jackson and Nemerson, 1980) or plasmin

from the fibrinolytic cascade (Collen, 1979).

Figure 1: The Blood Coagulation Cascade

Outline of the mammalian blood coagulation cascade with the intrinsic pathway (left) and

extrinsic pathway (right) converging at the activation of factor X to factor Xa, and ending

with the formation of the insoluble fibrin clot. Bars represent the polypeptide chains

(proportional to polypeptide chain length) with molecular weights indicated below. Intra

molecular disulfide bridges are indicated by lines between the two chains (from Neurath,

1984).

S u r f a c e H M W K i n i n o g e n K a l l i k r c i r i

: 60K 27K' • S O K 27K<

• 2 5 K J 2 5 K

K a l l i k r e l n X I L . X L

va C o 2 * , P - l i p i d

5K 32K 70 K P r o t h r o m b i n T h r o m b i n

F i b r i n o g e n F i b r i n

XCI„

F i b r i n ( X - l l n k e d )

Introduction / 5

a. The Catalytic Mechanism of Serine Proteases

The catalytic mechanism of peptide bond cleavage is the same for all serine proteases and

has been intensively studied. The classical model of serine protease catalysis is the

"charge-transfer relay" mechanism (see Creighton, 1984) involving the catalytic triad of

amino acid residues which consists of His 5 7 , Asp l t 2 and the active site Ser l i S in

chymotrypsin. These amino acids are conserved in all serine proteases and are essential

residues in the hydrolysis of amide and ester bonds. In the charge-transfer relay

mechanism the polypeptide binds to the S i binding site pocket, and the oxygen of the

carbonyl group in the amide linkage undergoes nucleophilic attack by the hydroxyl group of

S e r 1 9 5 , which has been polarized by H i s 5 7 ; a tetrahedral intermediate is formed and is

further stabilized by the transfer of a hydrogen ion to A s p x 0 2 . The hydrogen ion from

S e r x , 5 is then transferred to the leaving group, and an acyl-enzyme intermediate is formed.

This intermediate undergoes nucleophilic attack by an incoming water molecule, and the

tetrahedral intermediate is formed again resulting in the transfer of the polypeptide chain to

water and restoration of the serine hydroxyl group in the protease active site.

Aspects of the charge-transfer relay theory have been disputed, however, based on high

resolution X-ray diffraction studies on trypsin-substrate complexes and on neutron

diffraction studies which can readily position hydrogen atoms (Creighton, 1984). One

fundamental change is that interactions between the enzyme and substrate take place

which make the active site more energetically suited to the tetrahedral intermediate

conformation than to the substrate, products or the acyl-enzyme intermediate

conformations. The carbonyl group in the scissile bond becomes distorted when entering the

active site and begins assuming the tetrahedral conformation. This is stabilized by three

Introduction / 6

hydrogen bonds. The first hydrogen bond is between the backbone carbonyl group at

residue 214 and the backbone -NH- group of the P x residue (the amino terminal residue to

the scissile bond). The other two hydrogen bonds are between the carbonyl oxygen of P i

and the two backbone -NH- groups of residues 213 and 215 of the enzyme, which form the

oxyanion binding site. Polarization between the negative charge on the oxygen atom and

the positive charge on the carbon atom of the carbonyl group is large enough that the carbon

atom will be susceptible to nucleophilic attack by the oxygen in the hydroxyl group of

Ser x , s . When this occurs the hydrogen atom of the serine residue is transferred to His 5 7 ,

and the acyl-enzyme intermediate is formed. An important feature of this reaction is that

the Asp x 0 2 residue is not needed for the extra positive charge assumed transiently by

His 5 7 but the residue appears to be essential in orienting the substrate in the active site

through hydrogen bonding and in maintaining the His 5 7 conformation during catalysis so

that the hydrogen atom of the imadazole ring is not on the N e (which accepts the Serx , s

hydrogen atom) but on the N ° atom of the ring (see Creighton, 1984).

b. Factor XII

Factor XII, or Hageman factor, was so named after the individual in which the deficiency

was first discovered (Ratnoff and Colopy, 1955). Although factor XII circulates in plasma

at very low concentrations (approximately 30 Mg/ml), it has been highly purified from

human, bovine, rabbit and guinea pig plasmas (Cochrane and Wuepper, 1971; Movat and

Ozge-Anway, 1974; Revak et al., 1974; Griffin and Cochrane, 1976; Fujikawa et al.,

1977a,b; Claeys and Collen, 1977; Fujikawa and Davie, 1981; Yamamoto and Cochrane,

1981). The complete amino acid sequence of 596 amino acid residues and the identification

of glycosylation sites of human factor XII have only recently been determined for the human

Introduction / 7

protein (Fujikawa and McMullen, 1983; McMullen and Fujikawa, 1985) because of the

difficulty in isolating large quantities of the protein from plasma for amino acid sequence

analysis. Prior to the commencement of the work described in this thesis, the only known

amino acid sequence for bovine factor XII was at the amino-terminus of the zymogen and

around the active site serine residue in the serine protease domain. Soon after this project

was initiated, however, the entire amino acid sequence of the serine protease domain of the

human protein, 0-factor X l l a (Fig. 2) was reported (Fujikawa and McMullen, 1983) which

aided in the identification of cloned cDNAs of human factor XII.

Human factor XII is a glycoprotein containing 16.8% carbohydrate including 4.2% hexose,

4.7% hexosamine and 7.9% N-acetylneuraminic acid. The protein is synthesized in the liver

and secreted into blood as a single polypeptide chain with N-glycosylation on Asn and

O-glycosylation on Thr and Ser residues (Fujikawa and McMullen, 1983;McMullen and

Fujikawa, 1985). Glycosylation is intimately involved in protein secretion (Blobel et al.,

1979). Carbohydrate attachment occurs during protein synthesis (N-glycosylation) and in

the Golgi apparatus (O-glycosylation). Proteins which are to be glycosylated are

synthesized on the rough endoplasmic reticulum and contain an amino-terminal extension

called a "signal sequence" which is 16 to 30 residues in length and has a hydrophobic core

(Blobel et al., 1979). During protein synthesis, the signal peptide sequence is bound by a

"signal recognition protein", and the complex is subsequently directed to the endoplasmic

reticulum membrane. The nascent strand is transported through the membrane and is

cleaved on the lumen side by a resident signal peptidase. The specificity of the enzyme

recognition site has been described (Watson, 1984). The protein is then shuttled to the

Golgi apparatus by an unknown mechanism where it is glycosylated. Secreted proteins are

transported from the Golgi apparatus to the plasma membrane in secretory vesicles and

Figure 2: Schematic Diagram of 0-factor Xlla

The open bars represent the human factor XII molecule with "Site 1" and "Site 2" arrows

indicating the kallikrein cleavage sites of the zymogen generating the three polypeptide

chains of M r 40,000 (amino terminal fragment), 12,000 and 30,000 (carboxy terminal

fragment with serine protease activity) (from Fujikawa and McMullen, 1983). The amino

acid sequence shows the amino terminal nanopeptide (L-chain of (3-factor Xlla) generated by

kallikrein cleavage at unknown sites in either the M r 40,000 or the 12,000 fragments;

partial amino acid sequence of the serine protease domain is given in the H-chain of 0-factor

Xl la .

Site2 Site I

1 1 lleProProTrp 40,000 H 12,000 |—fvdiValGlyGly 30,000

AsnGlyProLeuSerCysGlyGlnArg Vol ValGlyQy Cys GlyAsp(g)GlyGly-

L-Chain of£ -Factor Xlla H-Chain of£-Factor Xlla

Introduction / 10

enter plasma upon membrane fusion. The form of factor XII found in plasma is a

single-chain polypeptide ( M r 80,000) with an isoleucine residue at the amino terminus. In

the prefactor XII, this He residue is presumedly preceded by the bond cleaved by signal

peptidase.

The zymogen form of factor XII is activated in plasma by partial proteolysis by kallikrein

generating an active serine protease (Davie et al., 1979; Jackson and Nemerson, 1980).

There are multiple sites in factor XII that are susceptible to cleavage by a number of

proteases including kallikrein, plasmin and trypsin (Margolis, 1958; Kaplan and Austen,

1971; Meier et al., 1977; Dunn and Kaplan, 1982). Limited proteolysis by kallikrein

activates surface-bound factor XII and produces two active enzyme forms, a-factor X l l a and

/3-factor X l l a . A single cleavage of the zymogen generates a-factor X l l a while two more

subsequent cleavages by kallikrein yield 0-factor X l l a (Revak et al., 1974; Dunn et al.,

1982; Fujikawa and McMullen, 1983). Both active enzymes consist of two polypeptide

chains held together by a disulfide bond (Revak et al., 1974, 1977). A/p/io-factor X l l a is

comprised of two polypeptide chains of M r 52,000 and 28,000 while j3-factor X l l a consists of

two polypeptide chains of M r 2,000 and 28,000. Amino-terminal sequence analysis has

shown that the M r 52,000 fragment in a-factor X l l a is derived from the amino-terminal

region of factor XII (Fujikawa et al., 1980b) and contains the surface-binding site (Revak

and Cochrane, 1976; Revak et al., 1977). However, the M r 2,000 fragment of /3-factor X l l a

does not bind to surfaces. The M r 28,000 fragments of both a- and j3-factor X l l a are

identical and contain the catalytic domain derived from the carboxy-terminal region of factor

XII (Revak et al, 1974, 1977). The catalytic domain of a- and B-factor X l l a shares

extensive amino acid sequence homology with other serine proteases, including many blood

clotting factors (see Jackson and Nemerson, 1980; Fujikawa and McMullen, 1983). The

Introduction / 11

sequence homology is highest at the activation regions and near the active sites of these

proteases.

Factor XII and a-factor X l l a readily bind to anionic surfaces such as silicates, dextran

sulfate and sulfatides in vitro (Ratnoff and Rosenblum, 1958; Fujikawa et al., 1977, 1980a).

In vivo, these binding properties may be responsible for the activation of factor XII when it

comes in contact with collagen or platelet membranes (Wilner et al, 1968; Harpel, 1972).

Surface-bound factor XII has enzyme activity towards its protein substrates, prekallikrein

and factor XI (McMillin et al., 1974; Saito, 1977; Heimark et al., 1980) (see below).

Surface-bound a-factor X l l a is further cleaved by kallikrein resulting in 0-factor X l l a which

is free to enter plasma since it no longer has the fragment of M r 52,000 which contains the

surface binding property of the molecule.

Various in vivo and in vitro substrates for activated factor XII have been described in the

literature and include prekallikrein, factor XI and factor VII (reviewed in Cochrane and

Griffin, 1982). The serine protease recognizes the sequence Thr-Arg-Ile (in prekallikrein)

and Pro-Arg-Ue (in FXI) and cleaves the polypeptide bond carboxy-terminal to the Arg

residues.

The serine proteases including the digestion enzymes show little amino acid sequence

identity (less than 40%) except in a few regions which are important in the catalytic

reaction. However this amount of homology may transfer to an 85% tertiary structural

homology (James et al., 1978). This observation has allowed the construction of computer

generated models of the serine protease domains of a number of clotting factors including

thrombin, FIXa, F X a (Furie et al., 1982) and FXII (Cool et al., 1985). The means of

Introduction / 12

substrate specificity of the enzymes remains unclear (Furie et al., 1982) but probably

results from the surface topology which would be unique for each protein. This kind of

specificity is essential in the ordered amplification of the coagulation and fibrinolytic

cascades (see Davie et al., 1979; Jackson and Nemerson, 1980).

Activated factor XII is regulated in plasma by inhibitors. The major plasma inhibitor for

0-factor X l l a is the Cl-inhibitor (de Agostini et al., 1984) which is the major proteolytic

inhibitor of the proteolytic enzymes derived from the first component of complement

(Ziccardi, 1981). The Cl-inhibitor is also the primary inactivator of plasma kallikrein

(Schapira et al., 1982; Van der Graaf et al., 1983). Kinetic studies (de Agostini et al., 1984)

with human plasma and purified Cl-inhibitor, a2-antiplasmin and antithrombin III showed

that 74% of ^-factor X l l a was inhibited by Cl-inhibitor and the remaining activated protein

inhibited equally by a2-antiplasmin antithrombin III. A complex of 145,000 daltons was

detected by SDS-polyacrylamide gel electrophoresis which represents a 1:1 stoichiometric

relationship between 0-factor X l l a and Cl-inhibitor (de Agostini et al., 1984).

c. Prekallikrein and Factor XI

Prekallikrein and factor XI circulate in plasma as zymogen forms of serine proteases and

are involved in the surface-dependent initiation of the coagulation cascade (Davie et al.,

1979; Jackson and Nemerson, 1980). Prekallikrein is also involved in the contact activation

of fibrinolysis (Bouma et al., 1980; Mandle and Kaplan, 1977; Colman, 1969), kinin

generation (Kerbiriou and Griffin, 1979; Nakayasu and Nagasawa, 1979; Mori and

Nagasawa, 1981) and the inflammatory response (Kaplan, 1978; Collen, 1979; Cochrane,

1980; Bouma and Griffin, 1981; Colman, 1984).

Introduction / 13

Prekallikrein and factor XI circulate in plasma as inactive zymogens in complexes with

H M W kininogen (Mandel et al, 1976; Thompson et al, 1977). The proteinases, purified

from rabbit, bovine and human plasmas (reviewed in Cochrane and Griffin, 1982), are also

glycoproteins synthesized by the liver and secreted (Davie et al., 1979; Jackson and

Nemerson, 1980). Prekallikrein enters plasma as a single chain polypeptide with a

molecular weight of 88,000 (Mandle and Kaplan, 1977; Bouma et al., 1980; Heimark and

Davie, 1981) whereas factor XI (Bouma and Griffin, 1977; Kurachi and Davie, 1977, 1981)

is the only serine protease secreted as a dimer with a molecular weight of 55,000 to 63,000

for the monomer. The proteins are activated by a single proteolytic cleavage by factor X l l a

(Revak et al, 1974; Fujikawa et al, 1980c; Bock et al, 1981; Tans and Griffin, 1982;

Griffin and Cochrane, 1976; Bouman and Griffin, 1977; Kurachi et al, 1980; Ohkubo et al,

1982) producing two chain molecules with amino-terminal heavy chains containing the

substrate-HMW kininogen-binding sites and carboxy-terminal light chains which bear the

serine protease domains (Mandle and Kaplan, 1977; van der Graaf et al, 1983b).

Recently the complete amino acid sequence of both human prekallikrein (Chung et al, 1986)

and factor XI (Fujikawa et al, 1986) have been determined by partial amino acid sequence

of the purified proteins and by prediction of amino acid sequences from isolated cDNA

clones. The two proteins were found to share 86% amino acid sequence identity.

Alignment of half-cysteine residues shows that these two proteins share a structural domain

of 91 amino acid residues which is repeated four times in both proteins. This repeat is

present in the heavy chains of the prekallikrein and factor XI molecules and does not exhibit

sequence homology with any other known protein. It is possible that this repeat is

important in the H M W kininogen interaction in the molecule.

Introduction / 14

d. High Molecular Weight Kininogen

High molecular weight kininogen is a nonenzymatic cofactor which greatly enhances the

surface activation of the various plasma systems described below (Griffin and Cochrane,

1976; Meier et al., 1977; Revak et al, 1977; Griffin, 1978; van der Graaf et al, 1982). The

protein is synthesized in the liver and is secreted into plasma as a glycoprotein ( M r

110,000) containing 13% carbohydrate (Jackson and Nemerson, 1980; Burgess and

Esmouf, 1985). Human H M W kininogen is cleaved twice by kallikrein giving a two chain

molecule held together by a disulfide bond and releasing a small peptide (approximately

1200 daltons) called bradykinin (Saito, 1977; Jackson and Nemerson, 1980). This cleavage

reaction occurs only when the H M W kininogen/ prekallikrein complex has bound to a

negatively charged surface and prekallikrein is activated by factor Xl la . The sites of

interaction between prekallikrein (or factor XI) and H M W kininogen are on the light chain in

H M W kininogen (the carboxy-terminal region) and on the heavy chain of prekallikrein (the

amino-terminal half of the molecule) (or FXI) (Bock et al., 1985). The interaction is

noncovalent, and a 1:1 complex is formed between these proteins. The light chain of H M W

kininogen has a histidine rich region that is adjacent and carboxy-terminal to the bradykinin

domain in the molecule and is probably responsible for binding to surfaces since deletion of

this region removes coagulation enhancement ability of the molecule (Cochrane and Griffin,

1979; Kerbiriou etal., 1980; Silverberg et al., 1980).

An important product of the kallikrein-HMW kininogen reaction is the nonapeptide,

bradykinin. The physiological functions of kinins have been studied intensely; their roles in

the organism are varied and include participation in the inflammatory response, in renal

function and in the regulation of the cardiovascular system (Bennett and Ogston, 1981; Diz,

Introduction / 15

1985).

2. A Model of the Surface Activation Mechanism

The enzymatic mechanism for surface activation (Cochrane and Griffin, 1982) is still

speculative but a schematic diagram (Fig. 3) from Griffin and Cochrane (1976) illustrating

coagulation initiation can be used to describe the essential steps (Bouma and Griffin, 1981).

The complexes, prekallikrein/HMW kininogen and factor XI /HMW kininogen, and factor XII

bind to negatively charged surfaces, presumably surfaces that are produced during injury

and involve the vascular basement membrane. However, highly purified forms of collagen

added to plasma did not activate factor XII as might be expected as collagen is an important

interstitial molecule (Griffin et aZ.,1975; Fujikawa et al., 1980a). Surfaces play an essential

role in the contact system during the coagulation and fibrinolytic cascade reactions as they

allow the molecules involved to assemble and to act upon one another. For example, the

result of the factors binding to the surface during contact activation is a burst of enzymatic

activity to produce fibrin or plasmin in a matter of seconds.

Factor XII is the first zymogen to be activated in the contact activation mechanism (Griffin,

1981) although it is still unknown how the first molecule is activated. The protein becomes

more susceptible to cleavage when bound to a surface (Griffin, 1978), and it is possible that

small amounts of a-factor X l l a in plasma could be responsible for the initial reaction

(Heimark et al., 1980; Rosing et al., 1985). Therefore, according to the model, an active

form of factor XII cleaves surface-bound factor XII molecules in a slow enzymatic reaction

which is followed by the activation of surface-bound prekallikrein generating kallikrein

(Griffin, 1981). The initiation reactions are now greatly enhanced since prekallikrein and

Figure 3: Schematic Diagram of the Surface-Dependent Contact Activation

Mechanism

Schematic diagram of the surface-dependent reactions involved in contact phase activation

of various plasma systems including the coagulation and fibrinolysis cascades (from Griffin

and Cochrane, 1979). PK: Prekallikrein; Kal: Kallikrein; HMWKgn: H M W kininogen; H F :

Hageman Factor (factor XII).

Introduction / 18

factor XII can undergo reciprocal proteolysis (Figure 3). Kallikrein, binding weakly to

H M W kininogen, (Thompson et al., 1983) dissociates from the cofactor and activates many

other factor XII molecules (Tans et al., 1986) as well as generating bradykinin from H M W

kininogen. AZp/ia-factor X l l a displays greater activity towards surface-bound factor XI

than to prekallikrein (Griffin, 1981) and so FXI is preferentially hydrolyzed, resulting in a

cascade of enzymatic reactions. Factor XIa does not dissociate readily from H M W

kininogen (Bouma et al., 1983) and is probably inhibited by its plasma inhibitor (a x -protease

inhibitor) (Scott et al., 1982) in its complex form. Kallikrein cleaves factor X l l a a second

time and generates 0-factor X l l a which is no longer surface bound and enters plasma. The

lifetime of both activated factor XII and prekallikrein in plasma is very short since they are

inhibited rapidly by the Cl-inhibitor (Schreiber et al., 1973).

B. PHYSIOLOGICAL FUNCTIONS OF THE CONTACT SYSTEM

1. Blood Coagulation

Haemostasis is the spontaneous arrest of blood flow during injury to a blood vessel (Guyton,

1977). Survival of the organism with a closed circulatory system such as in the

vertebrates, depends on rapid initiation at the site of injury resulting in blood coagulation

(Davie et al., 1979; Jackson and Nemerson, 1980). Platelets circulate in plasma and adhere

to the subendothelial walls of torn vessels forming a platelet plug. Circulating molecules,

the coagulation proteases, respond to platelet aggregation with the subsequent deposition of

fibrin at the site of injury (Guyton, 1977). Many of the proteins involved are inactive

zymogen forms of serine proteases and were discovered in patients with abnormal

thromboplastin times resulting in a tendency to bleed (Bloom, 1981). At least 14 proteins

Introduction / 19

found in plasma and tissue are required in blood coagulation as well as phospholipid and

C a 2 + (Jackson and Nemerson, 1980; Burgess and Esnouf, 1985). The participating

molecules interact with one another in a cascade effect (Fig. 1) whereby one molecule

activates many others resulting in the conversion of a large number of fibrinogen molecules

to fibrin (Davie and Ratnoff, 1964; MacFarlane, 1964). Fibrin is cross-linked and forms an

insoluble fibrin clot which reinforces the platelet plug and prevents loss of fluid.

The order in which the coagulation factors are activated depends on which factors are

involved when the coagulation cascade is initiated (Davie et al., 1979; Jackson and

Nemerson, 1980). There are two enzymatic pathways, the intrinsic and the extrinsic

pathway, which converge at a common step involving the activation of factor X. Figure 1

depicts the reactions in the coagulation cascade described in mammals. The intrinsic

pathway is initiated by factor XII activation of prekallikrein and factor XI in the presence of

a negatively charged surface and high molecular weight (HMW) kininogen (Griffin, 1981),

as described previously. Initiation of the extrinsic pathway requires tissue factor released

from tissue during injury. Tissue factor is a lipoprotein and acts as a cofactor which

accelerates the activation of factor X by factor V i l a (Davie et al., 1979; Zur and Nemerson,

1981). The intrinsic pathway (Davie and Ratnoff, 1964) derives its activation molecules

strictly from plasma whereas the extrinsic pathway (Zur and Nemerson, 1981) utilizes

proteins from tissue extract; however, both pathways are usually activated upon injury to a

blood vessel. Figure 1 also shows that the extrinsic pathway can be initiated by the

kallikrein/factor X l l a systems by activating factor VII (Kisiel et al., 1977; Radcliffe et al.,

1977; Seligsohn et al., 1979). Coagulation initiation via the extrinsic pathway has also been

demonstrated in vitro by cold dependent activation of factor VII (Seligsohn et al. 1978).

Introduction / 20

The blood coagulation cascade is an elegant illustration of regulation of a complicated

biological process through the action of a single type of enzymatic reaction- polypeptide

chain cleavage by a serine protease (Davie et al., 1979; Jackson and Nemerson, 1980). The

proteins involved are inactive zymogen forms of serine proteases and upon activation will

recognize their specific zymogen substrate in the cascade and activate it by limited

proteolysis at a specific site(s) and always after an arginine residue (Davie et al., 1979;

Jackson and Nemerson, 1980). For example, coagulation factors XII, XI, X, IX, VII and

prothrombin are partially cleaved to produce active serine proteases (Xlla, XIa, Xa, IXa

and thrombin) shown in Figure 1. Cofactors such as V, VIII, H M W kininogen or tissue

factor are required in some of these proteolytic reactions in addition to phospholipid and

C a 2 + for the vitamin K-dependent coagulation proteins (factors VII, IX, X and prothrombin)

(Suttie, 1985). Specific glutamic acid residues found at the amino-terminal regions of these

coagulation proteins are converted to 7-carboxyglutamic acid (Gla) by a vitamin

K-dependent carboxylase. The Gla residues are required in the C a 2 + bridging of the

Gla-containing proteins to phospholipid membranes prior to their activation (Suttie, 1985).

The culmination of all these reactions in the cascade is the polymerization of fibrin molecules

(Doolittle, 1984) to reinforce the platelet plug. The fibrin network is strengthened by the

formation of covalent cross links between monomers by the transglutaminase factor XIHa

which effectively stops the loss of blood (Curtis, 1981). The activated serine proteases are

rapidly inhibited by plasma inhibitors and by feedback inhibition loops (Davie et al., 1979;

Jackson and Nemerson, 1980) which will prevent the proteases from acting at any other

Although factor XII and kallikrein are central components of the surface activation

mechanism in blood coagulation, individuals with either plasma deficiency are

Introduction / 21

assymptomatic. However, plasma coagulation times are greatly reduced and during

extreme trauma or surgical procedures, the bleeding disorder may become life-threatening

(Ratnoff and Saito, 1979; Griffin, 1981). It is reasonable that the coagulation system has

evolved in such a way that it is activated by more than one mechanism since it is crucial

that upon injury, the flow of blood must be arrested.

2. Fibrinolysis

In mammals the dissolution of fibrin clots requires the activation of the fibrinolytic

enzymatic system (Collen, 1979). Plasminogen is the major precursor which is activated

upon cleavage by a serine protease called plasminogen activator (Wiman, 1978). The

product, plasmin, is a trypsin-like serine protease that cleaves 50-60 bonds in fibrin

(Doolittle, 1981). Plasmin exhibits broad substrate specificity and therefore is inhibited

rapidly in plasma by a 2-antiplasmin in a 1:1 stoichiometric complex (Wiman and Collen,

1977). Within the complex the active site serine residue of plasmin interacts with the

carbonyl group of a leucine residue of the inhibitor to form a covalent bond (Wiman and

Collen, 1977). The rate of the inhibition reaction is one of the fastest described for

protein-protein interactions and is one order of magnitude greater than the inhibition rate of

reaction for trypsin by its inhibitors (Wiman and Collen, 1978). The action of the

fibrinolytic enzymes is not limited to the dissolution of blood clots but also function in other

biological phenomena such as tissue repair (Astrup, 1978), malignant transformation

(Reich, 1975), macrophage function (Reich, 1975), ovulation (Strickland, 1978) and embryo

implantation (Strickland, 1978).

Introduction / 22

a. Plasminogen and Its Activators

Human plasminogen is a single chain glycoprotein (M 90,000) of which the entire amino

acid sequence has been determined (Sottrup-Jensen et al., 1978a,b). Two forms of the

molecule are found in plasma, Glu-plasminogen and Lys-plasminogen. Lys-plasminogen is

a proteolytic degradation form of Glu-plasminogen. Lys-plasminogen is the most common

form isolated from plasma and is activated by plasminogen activators by limited proteolysis

between A r g 5 6 0 - V a l 5 t l . The resulting plasmin molecule consists of a heavy chain and a

light chain held together by a disulfide bond. The heavy chain has five characteristic

kringle domains which are responsible for the binding of plasmin to fibrin. The light chain

has the active site which has the His, Asp and Ser of the catalytic triad found in all serine

proteases. Activation of Glu-plasminogen is much slower than Lys-plasminogen but the

final product is the same, Lys-plasmin. Activation of plasminogen by the plasminogen

activators is greatly enhanced in the presence of fibrin.

Plasminogen activation is a complicated process which is not yet clearly understood. The

protein may be activated along three different pathways: an intrinsic pathway in which all

the components are found in plasma; an extrinsic pathway in which the enzymes required

originate from tissues and, an exogenous pathway in which therapeutic substances such as

urokinase and streptokinase are involved (Collen, 1979; Gaffney, 1981;Mullertz, 1984).

Intrinsic fibrinolysis is activated in the presence of factor XII, prekallikrein and H M W

kininogen and a negatively charged surface (Collen, 1979). The mechanism of activation is

not known, and its physiological relevance is unclear. However, plasma deficient in factor

XII shows a greatly reduced plasmin generation time (Ratnoff, 1969; Meier et al., 1977).

Introduction / 23

Physiologicially, a few factor XII deficient individuals have shown a tendency towards deep

venous thrombosis and pulmonary embolism (see Ratnoff and Saito, 1979; Cochrane and

Griffin, 1982).

Plasminogen activators are serine proteases which are present in many different tissues,

organs and secretions and have various functions (Collen, 1980; Bachmann and Kruithof,

1984). Tissue-type plasminogen activator (tPA) (M 70,000) (Pennica et al, 1983) is a

single-chain glycoprotein of 530 amino acids. It is not clear whether tissue-type

plasminogen activator is synthesized as a zymogen form of the serine protease since both

single-chain and double-chain forms are active and are found in plasma (Bachmann and

Kruithof, 1984). The double-chain molecule is produced by limited proteolysis of the bond

between A r g 2 7 „-I le 2 7 b y plasmin, plasma kallikrein or by factor Xa (Wallen et al., 1983;

Ichinose et al., 1984). In the activation of plasminogen, the enzyme is active only when

bound to fibrin and not when bound to fibrinogen. Because of its high affinity for fibrin

(dissociation constant, 150 nM) and considering the high concentration of plasma fibrinogen

(5 to 10 MM), tPA is almost quantitatively bound to the clot during blood coagulation. In the

presence of fibrin, the catalytic activity of t-PA towards Glu-plasminogen increases more

than 500-fold, making the activator a very specific and potent fibrinolytic effector molecule.

Urokinase plasminogen activator ( M r 53,000) has been isolated from urine and kidney

tissue and is a single-chain glycoprotein consisting of 411 amino acid residues (Salerno et al.,

1984; Riccio et al., 1985). The physiological importance of this plasminogen activator has

been associated with the role of plasminogen activators in tissue degradation and

remodeling although it is not known how urokinase functions (see Nagamine et al., 1985;

Riccio et al., 1985). The protein is synthesized as a zymogen form of a serine protease and

Introduction / 24

is activated by partial proteolysis generating a two-chain molecule (Wun et al., 1982).

3. The Inflammatory Response

The inflammatory response to vascular injury is the process whereby the blood flow is

increased by vasodilatation, vascular permeability is increased, leucocytes are summoned

by chemical attractants (chemotaxis) and fibrin is deposited and removed (Bennett and

Ogston, 1981; Wright, 1982). Closer examination of these reactions reveals that the

mulitiple plasma systems, coagulation, fibrinolysis, complement and kinin generation,

become intimately linked (Pesce and Dosekun, 1983; Sundsmo and Fair, 1983; Colman,

1984). Vasodilatation is caused by a number of agents including bradykinin which is a

plasma product from the contact activation mechanism and results from cleavage of H M W

kininogen by kallikrein. Other vasodilators are found in plasma and platelets and interact

with collagen, thrombin or components of the complement system. Permeability of vascular

tissue has been associated with activation of factor XII, with kinins generated from H M W

kininogen and with small peptides derived from the complement systems (C3a and C5a)

that stimulate the release of vasoactive amines from mast cells. Neutrophils

(polymorphonuclear leukocytes) are elicited from plasma to the site of injury through the

action of chemotaxis (Wright, 1982). Kallikrein and tissue plasminogen activator are

chemotactic for neutrophils as well as the complement components, C3, C5 and C5b67

(Bennett and Ogston, 1981). As part of the host defense mechanism, neutrophils penetrate

the vascular site, undergo degranulation and remove foreign material by phagocytosis

(opsonized microorganisms, cellular debris or immunoglubulin and complement fixed to a

substrate which is not susceptible to phagocytosis) (Wright, 1982). Extracellular release of

chemical substances such as proteases and lysosomal constituents by neutrophils causes

tissue damage and inflammation (Wright, 1982).

Introduction / 25

The role of kallikrein and factor X l l a in the neutrophil aggregation and degranulation

reactions has been described (Schapira et al., 1982; Wachtfogel et al., 1983; Wachtfogel et

al., 1986). The involvement of factor X l l a was measured by elastase and lactoferrin

released from neutrophil granules in vitro (Wachtfogel et al., 1986). This release was

stimulated by increasing amounts of FXIIa. Furthermore, Plow (1982) showed that

neutrophil release of elastase was associated with blood coagulation. Pertinent to these

events is that elastase is a major fibrinolytic protease that dissolves clots during the

inflammatory process (Plow, 1980). The possible role of contact activation in the

inflammatory response mechanism is clearly demonstrated in the neutrophil reactions and

the release of elastase. Components of the complement system may also be acted upon by

the contact activation proteases since it has been demonstrated that rabbit kallikrein

generated chemotactic activity for neutrophils from rabbit C5 (Wiggins et al., 1981).

Dissolution of the fibrin deposited at sites of inflamed tissue is required in the resolution

phase of the inflammatory process (Bennett and Ogston, 1981). As described above,

elastase released from neutrophils is one mechanism which is independent of the fibrinolytic

system that results in the removal of fibrin. However, plasmin is also often available at

these sites of injuries (Bennett and Ogston, 1981). Plasminogen is probably activated by

plasminogen activators released from endothelial cells. The plasminogen activators can be

activated via the contact activation system. Factor X l l a and kallikrein would be available

since coagulation would have been initiated earlier on at the damaged tissue site to produce

the fibrin clot.

Introduction / 26

4. Conversion of Prorenin to Renin

Angiotensin II is a hypertensive agent required in the regulation of blood pressure.

Angiotensinogen is synthesized as an inactive precursor which is partially hydrolyzed by

renin to produce Angiotensin I, a less active form of Angiotensin II (Derkx et al., 1976). A

dipeptidase-converting enzyme, found in pulmonary endothelial cells, removes a dipeptide

from Angiotensin I producing Angiotensin II (Erdos, 1979). The enzyme also destroys

bradykinin in a similar reaction by cleaving the carboxy-terminal dipeptide, Phe-Arg (Erdos,

1979). Renin also circulates in plasma as an inactive zymogen. Prorenin can be activated

in vitro by partial proteolysis by plasmin, trypsin, urinary kallikrein or plasma kallikrein

(Cochrane and Griffin, 1982). It has been suggested that factor Xlla does not cleave

prorenin directly but becomes necessary in its activation of plasma prekallikrein (Bennett

and Ogston, 1981; Cochrane and Griffin, 1982). The importance of the contact system

activation mechanism in vivo in the prorenin conversion reaction is questionable although

plasma deficient in factor Xlla or kallikrein exhibit abnormal surface dependent activation

of prorenin (Derkx et al., 1979; Sealy et al, 1979).

C. THE STRUCTURE OF EUKARYOTIC STRUCTURAL GENES

1. Promoters

Expression of genes transcribed by RNA polymerase II is regulated by regions in the

genome which are usually 5' to the coding sequence and are not transcribed (reviewed in

Hansen and Sharp, 1984; Dynan and Tjian, 1985). These regions are important in

regulating transcription levels, in determining specific sites of transcript initiation and in

Introduction / 27

determining rates of gene transcription. These various functions of the 5' region are

accomplished by recognition of sequence motifs by RNA polymerase II and by important

regulatory proteins, most of which are unknown (Hansen and Sharp, 1984). The composite

promoter consists of three regions. The first is the initiation region, spanning from

nucleotide -50 to +10 where nucleotide +1 is the point of transcription initiation, and

including the " T A T A " and cap sites. The second region is the immediate upstream

sequence extending from nucleotides -50 to -110. The third region consists of the enhancer

(activator or potentiator) sequences whose positions vary widely (Hansen and Sharp, 1984).

The best described and most commonly found features of 5' regions in genes are the

" T A T A " and "CAAT" sequences that are located approximately 30 bp and 80 bp

respectively, from the start of the coding sequence in the gene (Breathnach and Chambon,

1981; Hansen and Sharp, 1984). The role of the " T A T A " sequence is to allow transcription

to proceed at approximately 25 bp downstream at a single site and in some cases to enhance

transcription rates (Hansen and Sharp, 1984). The "CAAT" sequences are upstream

regulatory elements initially found in globin genes where they act by increasing

transcription rates (Benoist et al., 1980; Hansen and Sharp, 1984). However, sequence

elements that enhance transcription can vary depending on the gene and not all genes have

C A A T elements (Hansen and Sharp, 1984). For example, the presence of inducible

enhancer sequences have been described for a number of genes (Kessel and Khoury, 1983)

including the heat shock genes (Lindquist, 1986), mouse metallothioniein I gene (Brinster et

al., 1982), the human interferon-^ gene (Kessel and Khoury, 1983) and the human

interferon-a1 gene (Ragg and Weissmann, 1983). Inducible genes are responsive to heat,

high metal concentrations, hormones and other agents (Kessel and Khoury, 1983).

Introduction / 28

2. Introns and Exons

R N A Polymerase II transcribed genes and some t R N A genes are found i n the genome as

mosaics of coding (exon) and noncoding (intron) regions. The discovery of heterogeneous

nuclear R N A lead to the realization that transcripts entering the cytoplasm that were

polyadenylated were in reduced forms of this nuclear R N A (Breathnach and Chambon,

1981). Introns were described (Breathnach et al., 1977) and the splicing apparatus

revealed (Abelson, 1979) which elucidated the heterogeneous nuclear R N A processing

mechanism in the nucleus (see L e w i n , 1983) . This splicing mechanism for R N A Polymerase

II depends on recognition of conserved sequences at the donor and recipient sites of the

introns by smal l nuclear R N A s and the subsequent cleavage by splicing proteins (Lerner

and Steitz, 1979). The sizes of introns are not conserved between members of gene families

and va ry greatly. The m i n i m u m size of a eukaryotic intron is about 80 bp which m a y be

due to the constraints caused by the splicing mechanism (Wieringa et al., 1984).

3. Transcription and Processing

N e w l y transcribed h n R N A s are processed in the nucleus in order to direct their export from

the nucleus to the cytoplasm and to allow correct protein translat ion to occur at the

appropriate start site in the m R N A . Sites of init iation (Baker and Ziff, 1981; Man ley ,

1983) and termination (Birnstiel et al., 1985) of transcription in genes are poorly defined

and are subject to variat ion amongst eukaryotic genes. Transcr ipt ion of most genes begins

wi th a purine but cytosine or uridine have also been found (Baker and Ziff, 1981; Man ley ,

1983). Nontransla ted 5' ends of m R N A s are usual ly 20 to 80 bp in length in 70% of m R N A

5' sequences compiled by Kozak (1984). The processing reactions include (i) 5' capping, (ii)

Introduction / 29

removal of part of the 3' untranslated region and subsequent addition of a poly A + tract

(approximately 200 bases in length) and (iii) removal of the the introns. Capping at the 5'

end is important for efficient splicing of introns (Grabowski et al., 1985) and in translation

(Shatkin, 1985). Polyadenylation occurs after part of the 3' untranslated region has been

removed by a nuclease (Breathnach and Chambon, 1981) approximately 20 bp downstream

of a conserved AAUAAA site (Proudfoot and Brownlee, 1976). This sequence is required

for nuclease activity but is not required in the polyadenylation reaction (Montell et al.,

1983). After the introns have been excised the mature transcript can pass through the

nuclear matrix and enter the cytoplasm where it is translated on the ribosome to produce

the polypeptide chain (Nevins, 1983).

D. MECHANISMS OF GENE EVOLUTION

1. Gene Duplication

Gene families have arisen by the mechanism of gene duplication (Li, 1983) resulting in

proteins with common structure and function (Edgell et al., 1983). If the time of divergence

between two related genes is large, then the genes begin to evolve differently and may

assume new functions such in the lysozyme-a lactalbumin family (Hall et al., 1982) and in

the immunoglobulin superfamily (Hood et al., 1985). The serine protease family of proteins

provides a good example of gene duplication events with subsequent mutations during

divergence. The resulting proteins maintain some amino acid sequence identity such that

they retain the basic structure for their enzymatic reactions as serine proteases

(Hewett-Emmett et al.', 1981; Patthy, 1985).

Introduction / 30

2. Gene Fusions

Variety in the genes can result from the fusion of one gene, or parts thereof, with another

unrelated gene to give a new kind of protein (Doolittle, 1985). Polymorphism in the clotting

genes has been attributed partially to this mechanism (Doolittle, 1985; Neurath, 1985)

during gene duplication events (Calos and Miller, 1980).

3. Exon Shuffling

Complex proteins are usually encoded by genes made up of introns and exons (see above).

It has been suggested (Gilbert, 1978; Blake, 1978; 1983; Rogers, 1985) that the proteins

consist of functional and structural domains that can be identified in related proteins. In

addition, introns demarcate these coding regions as separate exons in eukaryotic genes.

The importance of this concept in evolution is that these exons can be inserted by an exon

shuffling mechanism into other genes within the genome (Gilbert, 1978; Blake, 1978; 1983;

Rogers, 1985). In this model, protein domains on separate exons can be exchanged between

genes and may confer new structure and possibly new function to the recipient molecule.

E. EVOLUTION OF AMINO ACID AND DNA SEQUENCE

1. Molecular Clock

A phylogeny of species can be established by determining the rate of evolution of a

conserved protein family within different species (Zuckerkandl and Pauling, 1965; Wilson et

al., 1977; L i et al., 1985). The validity of this argument is dependent on random mutation

Introduction / 31

of D N A occuring at a constant rate which manifests itself as a constant rate of amino acid

substitution (Zuckerkandl and Pauling, 1965). This constant rate of change of protein

sequence is called the "molecular clock" and when established for a particular protein,

divergence times between species can be assessed. Sometimes, protein evolution is not

constant due to selective pressures on organisms (Wilson et al., 1977). Also, it has been

shown (Wilson et al., 1977; Li et al., 1985) that regions within a protein diverge at different

rates. This allows the detection of protein domains that may be more functionally

important to the organism than other regions of the molecule.

2. Intron Sliding

Members of protein families exhibit regions of variation in the protein that would have

arisen not from amino acid point mutations nor from insertion of domains from other

proteins but from intron sequence within the gene (Craik et al., 1983). The mechanism by

which small amino acid regions have been inserted into proteins is called intron/exon sliding,

and if the junction has shifted in such a way as to maintain the correct translational reading

frame, then it is possible that the new protein sequence could be retained through natural

selection. These small inserts are often found on the surface of molecules where tertiary

structural modifications are most easily tolerated, and they are usually not greater than 13

amino acids in length (Craik et al., 1983). Examples of intron sliding have been found in the

dihydrofolate reductase and serine protease gene families (Craik et al., 1983).

Introduction / 32

F. PROTEIN DOMAINS OF THE COAGULATION AND PLASMINOGEN

ACTIVATOR GENES

1. Trypsin-Like Family of Serine Proteases

The trypsin-like family of serine proteases has been conserved since the time of divergence

of prokaryotes and eukaryotes (Neurath, 1984). These enzymes exhibit diverse functions

such as digestion, coagulation, fibrinolysis, complement activation, neuropeptide processing

and fertilization of germ cells (Neurath and Walsh, 1976). The serine protease domains of

the trypsin-like enzymes share approximately 40% amino acid sequence identity and have

been assumed to share a common mechanism of proteolysis (Young et al, 1978;

Hewett-Emmett et aZ.,1981).

The trypsinogen, chymotrypsinogen and proelastase proteins differ from the coagulation

and fibrinolysis enzymes in their amino-terminal domains. The digestive enzymes all have

short amino-terminal extensions which are released upon activation by proteolysis.

However, the coagulation and fibrinolysis proteins have very large amino-terminal

fragments that consist of multiple putative domains within these proteases (Figure 4)

(Jackson and Nemerson, 1980; Zur and Nemerson, 1981; Doolittle, 1985). Protein

homologies were detected by alignment of common amino acid residues, especially the

cysteine residues which suggest conserved tertiary structure in disulfide bridging patterns.

These regions may have important roles in regulation and selective activation on and by

other serine proteases of the cascade (Jackson and Nemerson, 1980).

The vitamin K-dependent proteins (see above) have a common "Gla-rich" domain which is

Figure 4: Amino Acid Sequence Homologies in Coagulation Factor Zymogens

Comparison of the structures of coagulation and fibrinolytic zyymogens to trypsinogen. The

solid bar represents the catalytic region in the proteases, the cross hatched region

represents the Gla region, K represents the kringles, E represents regions homologous to

epidermal growth factor precursor, 1 represent regions homologous to the type I homology

of fibronectin, and A represents the homologous regions found in factor XI and prekallikrein.

the lengths of the bars are approximately proportional to the lengths ofthe polypeptide

chains. Arrows represent the locations of peptide bonds that are cleaved during the

activation of the zymogens. Solid lines below the proteins represent disulfide bridges and do

not necessarily represent their true locations.

PROTHROMBIN V/A K I K

FACTOR VII

FACTOR IX

FACTOR X

PROTEIN C

FACTOR XI

PREKALLIKREIN

FACTOR XII

A 1 A I A 1 A

PLASMINOGEN K I K I K 1 K I K T

TISSUE T Y P E PLASMINOGEN ACTIVATOR

1 I E I K .1 K. I

UROKINASE E K

TRYPSINOGEN

Introduction / 35

contiguous with a prepro leader sequence (see Fung et al., 1985). These proteins include

prothrombin (MacGillivray and Davie, 1984; Degen et al., 1983), factor VII (Hagen et al.,

1986), factor IX (Kurachi and Davie, 1982; Jaye et al., 1983), factor X (Fung et al., 1984,

1985; Leytus et al., 1986) and protein C (Long et al., 1984; Foster and Davie, 1984;

Beckmann et al., 1985) and protein S (Dahlback etal., 1986; Lundwall et al., 1987; Hoskins

et al., 1987). The amino acid sequence of a seventh vitamin K-dependent protein called

protein Z has recently been determined (Hojrup et al., 1985) though the role of this protein is

not known.

A "kringle" structure that consists of 80 amino acid residues with six characteristic cysteine

residues (Magnusson et al., 1975) is found in plasminogen (Sottrup-Jensen et ah, 1978),

tissue-type plasminogen activator (Pennica et al., 1983), urokinase plasminogen activator

(Verde et al., 1984) as well as in prothrombin (Magnusson et al., 1975). The function of the

kringle is not known but plasminogen kringle 4 may be associated with binding to fibrin

molecules.

Another region found in both blood coagulation and fibrinolysis cascade zymogens is the

epidermal growth factor like domain (Blomquist et al, 1984; Doolittle et al., 1984). This

region has been identified in factor VII (Hagen et al., 1986), factor IX (Kurachi and Davie,

1982; Jaye et al, 1983) factor X (Fung et al, 1984, 1985; Leytus et al, 1984) protein C

(Long et al, 1984; Foster and Davie, 1984; Beckmann et al, 1985), protein Z (Hojrup et al,

1985) and the plasminogen activators (Pennica et al, 1983; Verde et al, 1984). The

epidermal growth factor domain is present in the 19 kdal fragment of vaccinia virus and

several other proteins (Blomquist et al, 1984; Doolittle et al, 1984). In many of the

EGF-like regions, a specific aspartic acid or asparagine residue is converted to

Introduction / 36

j3-hydroxyaspartic acid or 0-hydroxyasparagine, respectively.

Tissue-type plasminogen activator has a region that is homologous to the type I homology

found in fibronectin (Ny et al., 1983; Freizner-Degen et al., 1985). This region has fibrin

binding activity in fibronectin (Petersen et al., 1983; Yamada, 1983) and bears

approximately 40% amino acid sequence identity with tissue-type plasminogen activator

(Ny et al., 1984; Friezner-Degen et al, 1985).

G. EVOLUTION OF THE SERINE PROTEASES

Analysis of the blood coagulation and fibrinotysis families of serine proteases aids in

determining the evolutionary process of the genes and how they diversified. Furthermore,

by comparing these genes between species it is possible to determine a time scale within

which these evolutionary events had occured. Patthy (1985) provides a model utilizing the

exon shuffling mechanism to describe how these families of proteins arrived at their present

day form and from this type of speculation we may be able to determine the relationships of

newly characterized blood coagulation and fibrinolysis proteins. With the help of

biotechnology it has become possible to predict the amino acid sequence of many of these low

abundance plasma and tissue proteins. For example cDNA sequences of blood coagulation

proteins prothrombin (MacGillivray et al, 1980; Degen et al, 1983; MacGillivray and

Davie, 1984; MacGillivray et al, 1986; Jorgensen et al, 1987), factor VII (Hagen et al,

1986), factor IX (Kurachi and Davie, 1982; Jaye et al, 1983; McGraw et al, 1986) factor X

(Fung et al, 1984, 1985; Leytus et al, 1984; 1986) protein C (Long et al, 1984; Foster and

Davie, 1984; Beckmann et al, 1985) and protein S (Dahlback et al, 1986; Lundwall et al,

1987; Hoskins et al, 1987) have been determined. Furthermore, the cDNA sequences of

Introduction / 37

the fibrinolytic proteins plasminogen (Malinowski et al., 1984), tissue-type plasminogen

activator (Pennica et al., 1983) and urokinase (Verde et al, 1984) have been determined.

From the cDNA sequences it is possible to predict the amino acid sequences of the zymogens

as well as to predict any regions which are cleaved by the cell prior to secretion such as the

prepro peptides and the signal peptides. Therefore, this kind of technology has been

extremely beneficial in the complete characterization of proteins. However, these

techniques do not provide secondary structure data such as disulfide bridging patterns nor

the identification of glycosylated residues.

fl. OBJECTIVES

The contact system of activation of the various plasma systems described above may be an

important physiological phenomenon required for maintaining homeostasis in the organism.

Central to this mechanism is the glycoprotein, factor XII, which possesses the

surface-binding property that is critical in the initiation reaction. The physical and

biochemical means by which factor XII plays a role in the contact activation may be inferred

from the protein sequence. Recombinant D N A technology will be used to obtain cDNA

sequences from which the amino acid sequence can be predicted to circumvent the expensive

and time consuming processes of protein purification and sequencing using the standard

Edman degradation protein sequencing method. The cDNA can also be used as a probe for

obtaining the factor XII gene.

The evolutionary history of factor XII will be determined from the gene structure since the

family of serine proteases is well characterized and it may be possible to gain a better

Introduction / 38

understanding of the evolution of this old and complex family of genes. Many proteins with

common function belong to the same family and it is possible that FXII is more closely

related to the surface activating proteases, prekallikrein or factor XI, or it may even have

regions of protein homology related to the binding domain of HMW kininogen that had been

acquired through an exon shuffling mechanism in the genome. Clearly, from the

determination of both the amino acid sequence and the gene structure for human factor XII,

greater insight will be gained into the function and evolution of this protein.

II. MATERIALS AND METHODS

A. MATERIALS

Agarose, acrylamide, bisacrylamide, urea, ammonium persulphate, Sephadex G-25 and

T E M E D were from Bio-Rad Laboratories. Oligo (dT) cellulose (type 7) was from P .L .

Biochemicals. Yeast extract, casamino acids, bacto-tryptone and bacto-agar were Difco

grade from the Grand Island Biological Company. NZamine type A was from Sheffield

Products. Nitrocellulose sheets and circles (82 and 132 mm) (BA-85) of 0.45 ym pore size

were from Millipore or Schleicher and Schuell. 3 2 P-labeled deoxyribonucleotide

triphosphates (3000 Ci/mmole; 1 Ci = 3.7 x I O 1 0 Bq) were from Amersham. Phenol

(British Drug Houses Ltd.) was redistilled before use (boiling point 180°C) and frozen in

portions at -20°C. Deoxy- and dideoxy-ribonucleotide triphosphates and the M13

sequencing primer (heptadecanucleotide) were from PL-Pharmacia.

Isopropyl-0D-thiogalactopyranoside (IPTG),

5-bromo-4-chloro-3-indolyl/3-D-galactopyranoside (X-Gal), ethidium bromide, yeast transfer

R N A (tRNA), ampicillin, tetracycline, chloramphenicol, ribonuclease A and

deoxyribonuclease I were from Sigma. Cesium chloride was from Cabot Berylco Ltd.

Ultragel AcA54 was from L K B . Most of the oligodeoxyribonucleotides including some of the

M13 sequencing primer used, were synthesized on an Applied Biosystems 890A D N A

Synthesizer (by Tom Atkinson, Dept. of Biochemistry) and purified by denaturing

polyacrylamide gel electrophoresis prior to use (Atkinson and Smith, 1984). One

oligonucleotide pool was synthesized and purified by Dr. M . Zoller at Cold Spring Harbor

Laboratories, New York. A l l other chemicals were of reagent grade or better and were

purchased from either Sigma Chemical Co., Fisher Scientific, or British Drug Houses Ltd.

Materials and Methods / 40

Restriction endonucleases, T4 D N A ligase, T4 D N A Polymerase, T4 polynucleotide kinase

and bovine serum albumin (nuclease free) were from New England Biolabs, Bethesda

Research Laboratories or PL-Pharmacia. Nuclease S i was from Boehringer-Mannheim

and avian myoblastosis virus reverse transcriptase was from Promega Biotec. D N A

polymerase I and D N A polymerase I Klenow fragment were from Boehringer-Mannheim or

PL-Pharmacia. Adult human liver was obtained from kidney donor patients. Liver

samples were cut into 0.5 cm slices, rinsed in sterile PBS (10 mM sodium phosphate pH7.5 ,

150 m M NaCl), frozen in liquid nitrogen and stored at - 7 0 ° C . Human genomic D N A was

isolated by Dr. Colin Hay (Dept. of Biochemistry) and Escherichia coli rRNA was provided

by Dr. P. Dennis (Dept. of Biochemistry).

B. STRAINS, VECTORS AND MEDIA

1. Bacterial Strains

E. coli MC1061 (araD 139, A(ara, leu) 7697, AlacX74, galU-, gal K - , hsr - , hsm +, str A)

(Casadaban and Cohen, 1980) was host for the liver cDNA library cloned into the plasmid

pKT218 containing cloned liver cDNA (Prochownik et al, 1983). E. coli C600 (F-, thi-1,

thr-1, leuB6, l acYl , tonA21, supE44,\-) (Maniatis et al., 1982) was host for human

genomic D N A cloned into the XCharon28 vector (Blattner, et al., 1977). E. coli JM83 (ara,

Alacpro, strA, thi-, 080, lacZAMl5) (Vieira and Messing, 1982) was host for the

transformation of pUC13 vector (Vieira and Messing ,1982) and was obtained from Dr.

Jim Stone, Dept. of Microbiology, U B C . E. coli JM103 (Alacpro, supE, thi-, straA, sbcB15,

endA, hsdR-, F \ traD36, proAB, lacIQ, lacZAM15 (Messing, 1983) was host for

transformation of M13 mp 8, 9, 18 and 19 vectors (Messing, 1983). E. coli RY1088

[AlacU169, supE, supF, hsdR-, hsdM + , metB, trpR, tonA21, proC::Tn5(pMC9), pMC9 is

pBR322-lacIQ] (Young and Davis, 1983a,b) was host for screening and isolation of D N A

cloned into the Xgt l l vector (Young and Davis, 1983a,b).

2. Vectors

The M13 mp 8, 9, 18 and 19 vectors (Messing, 1983) were used for D N A sequence analysis.

Small fragments of cloned D N A were subcloned into the vector p U C l 3 (Vieira and Messing,

1982), obtained from Dr. Mark Zoller, (Dept. of Biochemistry, University of British

Columbia) for restriction endonuclease mapping and hybridization probes.

3. Media

The medium for growth of E. coli hosts for X clones was N Z C Y M (Maniatis et al, 1982),

which is lOg NZamine type A, 2g M g C l 2 , 5g NaCl, 5g yeast extract, lg casamino acids per

litre and the pH was adjusted to 7.5 with NaOH. Phage X libraries were plated on

NZCYM-agar plates (1.5% w/v) plates with an overlay of NZCYM-agarose (0.7% w/v) when

screening or NZCYM-agar (0.7% w/v) when titering phage libraries or stocks. Phage were

diluted or stored in S M buffer (5.8g NaCl, 2g M g S 0 4 - 7 H 2 0 , 50 ml IM Tris-HCl pH7.5, 5

ml 2% gelatin per liter). Luria broth (5g yeast extract, lOg bacto-tryptone and lOg NaCl

per liter) (Maniatis et al., 1982) was used as medium for growth of E. coli JM83 containing

the p U C l 3 plasmid derivative. For selection of E. coli JM83 colonies transformed with pUC

derivatives containing cloned DNA, cultures were plated on LB-agar (1.5% w/v) plates

supplemented with 50 ug/ml ampicillin. A liver cDNA library in E. coli MCl061/pKT218

vector screened on LB-agar (1.5% w/v) plates supplemented with tetracycline (12.5 ug/ml).

All D N A cloned into M13 vectors and transformed into JM103 was amplified in Y T medium

(Maniatis et al., 1982), which is 5g yeast extract, 8g bacto-tryptone and 5g NaCl per liter.

Phage M13 transformants were plated on YT-agar (1.5% w/v) plates overlayed with

YT-agar (0.7% w/v). E. coli JM103 was maintained on minimal medium plates (Messing,

1983), which were made up as follows: 3g of agar in 160 ml H 2 0 (sterilized and cooled to

5 5 ° C ) was mixed with 40 ml 5X salts (2.1g K 2 H P 0 4 , 0.9g K H 2 P 0 4 , 0.2g ( N H 4 ) 2 S 0 4 ,

0. 1g NaCitrate.7H 2 0 per 40 ml), 2 ml 20% glucose, 0.2 ml 20% M g S 0 4 - 7 H 2 0 and 0.1 ml

10 mg/ml thiamine. Each of these solutions was sterilized separately by autoclaving except

for thiamine which was filter sterilized. Large-scale cultures (more than 500 ml) of E. coli

JM83 or MC1061 containing pUC or pKT218 plasmid vectors, respectively, were grown in

Luria broth in the presence of the appropriate antibiotic. These cultures were further

treated with chloramphenicol (250 ug/ml) in the mid-log phase of growth.

C. GEL ELECTROPHORESIS

1. Neutral Agarose Gels

Electrophoresis grade agarose was boiled in d H 2 0 (added according to the percentage w/v

agarose gel required) for 10 minutes, cooled to 5 0 ° C and the appropriate volume of 5OX

T A E buffer (2M Tris-OH, I M glacial acetic acid, 0.1M E D T A , pH7.2) was added to a final

dilution of IX T A E (Maniatis et al., 1982). To stain nucleic acids in the gel, a volume of

ethidium bromide solution (10 mg/ml) was added to the cooled gel before pouring so that the

final concentration of ethidium bromide was 0.5 ug/ml. The gel was poured into a mold and

was allowed to set for 1 hour. D N A samples were adjusted to IX loading buffer with a 10

times concentrated stock (in 30% ficoll, 0.2% xylene cyanol, 0.2% bromophenol blue) before

loading. Electrophoresis was carried out in IX TAE buffer at 1-3 volts/cm after which the

DNA was visualized by irradiation of UV light (260 nm or 360 nm).

2. Formaldehyde Agarose Gels

RNA fragments were separated in agarose gels prepared with a denaturing agent such as

formaldehyde (Maniatis et al., 1982) as follows. An agarose solution (usually 0.6% w/v

final concentration) was boiled and cooled to 60°C. The gel solution was adjusted to 2.2M

formaldehyde (pH less than 4.0) and IX running buffer from a 5X running buffer stock

(0.2M MOPS pH7.0, 50mM sodium acetate, 5mM EDTA) and poured into a mini-gel mold.

RNA samples were prepared in a 20M1 loading volume consisting of 4.5yg RNA (up to 20/ig),

2.0/ul 5X running buffer, 3.5M1 formaldehyde (pH4.0) and lO.Oyl formamide and heated for

20 minutes at 50°C. The samples were electrophoresed for 1.5 hours at 15V/cm, and lanes

containing ribosomal markers were excised and prepared for staining while the region of the

gel containing mRNA was treated for a Northern blot transfer (below). The gel with the

marker lanes was washed with sterile d H 20 for 2 hours with 5 changes of water. The gel

was soaked in 0.1M ammonium acetate for 1 hour with one change of buffer followed by a

final solution of 0.1M ammonium acetate, 0.1M 0-mercaptoethanol and 0.5yg/ml ethidium

bromide for 1 to 3 hours. Prior to visualization with UV light, the gel was destained for 2

hours in the same buffer but without ethidium bromide.

3. Neutral Polyacrylamide Gels

A 30% (w/v) stock solution of acrylamide (29:1 acrylamide:bisacrylamide) in H 2 0 was

prepared and diluted according to the percent gel required. A 10X TBE buffer stock

solution (0.89M Tris-OH, 0.89M boric acid, 25 m M E D T A , pH 8.3) was added so that the

gel solution was IX T B E buffer and the solution was degassed using a water aspirator.

Ammonium persulphate (0.066% w/v) and T E M E D (0.04% w/v) were added to initiate

polymerization, the gel was poured and was allowed to polymerize for 1 hour. A 10X stock

of loading buffer was added to the D N A samples which were then adjusted to 0.3% ficol,

0.002% bromophenol blue, 0.002% xylene cyanol. The gel was subject to electrophoresis

6V/cm in IX T B E buffer. DNA was visualized by staining the gel with a solution of

ethidium bromide (lug/ml) in IX T B E buffer followed by irradiation under U V light

(260nm).

4. Denaturing Polyacrylamide Gels

A 40% (w/v) stock solution of acrylamide (38:2 acrylamide:bisacrylamide) was prepared,

and an appropriate volume was added to d H 2 0 , 10XTBE buffer (for a final concentration of

1XTBE buffer) and urea (for a final concentration of 8.3M) to attain the correct percentage

of gel required. The urea was dissolved in the solution in a 3 7 ° C water bath, and then the

solution was degassed. Polymerization was initiated with the addition of ammonium

persulphate and T E M E D to final concentrations of 0.066% (w/v) and 0.024% (w/v),

respectively. The gel was poured and allowed to polymerize for 2 hours. The D N A samples

were applied after heating at 9 0 ° C in loading buffer (98% deionized formamide, lOmM

E D T A , 0.02% xylene cyanol, 0.02% bromophenol blue) for 3 minutes. D N A fragments

separated in denaturing gels were visualized by autoradiography after drying under

vacuum with a Bio-Rad gel drier at 8 0 ° C for 20-30 minutes, and exposing to Kodak XK-1

film, with or without intensifying screens (Dupont Lightning Plus).

D. ISOLATION OF DNA

1. Small-scale Isolation of Plasmid DNA

Overnight cultures of E. coli containing recombinant plasmids were grown in Luria broth or

Y T medium in the presence of the appropriate antibiotic at 3 7 ° C . Plasmid DNA was

isolated from 1.5 ml of the culture according to the method of Birnboim and Doly (1979) and

described in Maniatis et al. (1982). The bacterial culture was centrifuged for 30 seconds in

an Eppendorf microfuge and the supernatant was discarded. The pellet was resuspended in

I O O M I of a solution (at 4°C) containing 50 mM glucose, lOmM E D T A , 25 m M Tris-HCl, pH

8.0, and 4 mg/ml lysozyme and incubated at room temperature for 5 minutes. A lysis

solution (200/il of 0.2% NaOH, 1% SDS) was added and incubated on ice for 5 minutes. The

SDS was precipitated by the addition of 150M1 of a potassium acetate solution (60 ml 5M

potassium acetate, 11.5 ml glacial acetic acid, 28.5 ml d H 2 0 , pH 4.8) followed by incubation

at 4 ° C for 5 minutes. Cellular debris and potassium dodecylsulfate were removed by

centrifugation in an Eppendorf centrifuge for 10 minutes at 4 ° C . The supernatant (350^1)

was removed carefully and extracted with an equal volume of phenohchloroform (1:1, v:v).

The phases were separated by centrifugation for 2 minutes in an Eppendorf centrifuge, and

two volumes of 95% ethanol were added to the supernatant. The mixture was incubated at

room temperature for 5 minutes. The D N A was recovered by centrifugation in an

Eppendorf centrifuge for 5 minutes. The pellet was washed with 1 ml of 70% ethanol,

recentrifuged and air dried. The plasmid D N A pellet was resuspended in 2bu\ T E buffer

(lOmM Tris-HCl, pH 8.0, 1 m M EDTA) .

2. Large-scale Isolation of Plasmid DNA

Large volumes (500 ml) of Luria broth were innoculated with 3 ml of a fresh overnight

culture of E. coli containing plasmid DNA (antibiotics were added depending on the host

resistance) and grown at 37°C with vigorous shaking until the OD600nm of the culture was

0.5. At this time 250 mg chloramphenicol was added, and the culture was shaken for 12-16

hours at 37°C. Cells were collected by centrifugation at 5 krpm for 10 minutes in a GS-3

rotor (Sorvall) at 4°C. The supernatant was discarded, and the cells were frozen at -20°C.

The cells were resuspended on ice in a solution of 25% (w/v) sucrose, 50 mM Tris-HCl, pH

8.0 and 1.5 mg/ml lysozyme so that the final volume of the cell suspension was 5.0 ml. The

suspension was transferred to a 25 ml Oakridge tube (Beckman) and kept on ice. The cells

were lysed by the addition of 10ml of a 0.2N NaOH, 1%SDS solution and incubated on ice

for 10 minutes. The SDS and cell debris were precipitated with 7.5ml of a 3M potassium

acetate solution pH4.8 and incubated on ice for 10 minutes. (The potassium acetate

solution was prepared from 60ml of 5M potassium acetate to which were added 11.5 ml

glacial acetic acid and 28.5 ml dH20.) The lysis mixture was centrifuged at 35 krpm, 4°C

for 30 minutes in a Ti60 rotor (Beckman). The supernatant was decanted into a 50 ml

centrifuge tube, 0.6 volumes of isopropanol was added, and the DNA was precipitated at

room temperature for 15 minutes. The plasmid DNA was collected by centrifugation at 9

krpm for 30 minutes at room temperature in an HB-4 rotor (Sorvall). The pellet was

resuspended in T E buffer, and solid CsCl was added for a final concentration of l g CsCl/ml

plasmid DNA solution. The solution was adjusted to a final volume of 13.5ml to which was

added 300 M1 of a solution of ethidium bromide (10 mg/ml). The solution was placed into a

Beckman heat sealable tube and was centrifuged in a Ti70.1 rotor (Beckman) for 20 hours

at 20°C at 60 krpm. Plasmid DNA was observed with UV light irradiation (360 nm) as a

band midway in the tube. The band was isolated by puncturing the tube with an 18 gauge

syringe, and the ethidium bromide was removed from the DNA solution by multiple

extractions with water saturated isopropanol. When the DNA solution was no longer pink

in color, the extraction was repeated two more times, and the solution was diluted 4 times

with dH 20. The DNA was precipitated by the addition of 3M sodium acetate (to a final

concentration of 0.2M) and two volumes of 95% ethanol. The suspension was left at -20°C

overnight. The precipitated DNA was recovered by centrifugation at 9 krpm and 4°C for

30 minutes in an HB-4 rotor.

3. Small-scale Isolation of X DNA

A fresh culture of host cells was grown overnight in NZCYM medium containing 0.02%

maltose at 37°C with shaking. A plug of agar containing a single phage plaque was added

to I O O M I of the culture, and the phage were preabsorbed to the host cells for 15 minutes at

37°C. The culture was transferred to 20 ml of prewarmed NZCYM and shaken vigorously

at 37°C until total cell lysis was observed. Chloroform (lOOyl) was added, and the culture

was shaken for a further 5 minutes. Cell debris was removed by centrifugation at 5 krpm

and 4°C for 5 minutes in an HB-4 rotor. The supernatant was transferred to a 50ml

centrifuge tube which contained 6ml of 50% PEG (polyethylene glycol 6000, Carbowax

8000) and 3ml of 5M NaCl, and the phage were precipitated at 4°C overnight. The phage

particles were collected by centrifugation at 5 krpm and 4°C for 15 minutes in an HB-4

rotor, and the supernatant was completely removed. The phage pellet was resuspended in

500M1 of DNAsel buffer (50mM Tris-HCl pH7.5, 5mM MgCl 2, 0.5mM CaCl 2) which

contained 5*ig DNAsel and 50/ig RNAseA. The reaction mixture was incubated at 37°C for

30 minutes, and then the cellular debris removed by centrifugation in an Eppendorf

centrifuge at 4°C for 5 minutes. The supernatant was adjusted to 1% SDS, 5mM EDTA,

and then 100/Lig of proteinase K (predigested for 1 hour at 65°C) was added and incubated

at 65°C for 1 hour. The DNA solution was extracted twice with phenohchloroform (1:1),

and the DNA was precipitated by the addition of sodium acetate to 0.3M and 2 volumes of

95% ethanol followed by incubation at -20°C overnight. The precipitated DNA was

collected by centrifugation at 9 krpm and 4°C for 15 minutes in an HB-4 rotor. The pellet

was washed with 1 ml 70% ethanol, air dried and resuspended in 50/ul TE buffer.

4. Large-scale Isolation of X DNA

The large-scale isolation of XDNA has been described in Maniatis et al. (1982). A fresh

overnight culture of host bacteria was grown in NZCYM containing 0.02% maltose, and the

OD600nm was taken. A volume of culture was centrifuged (at 3 krpm for 5 minutes in an

HB-4 rotor at 4°C) which corresponded to 2 x l O 1 0 bacteria (1 OD600nm = 8 x 10 s

cells/ml). The bacteria were resuspended in 1 ml SM medium, and 2 x 10' phage were

added. The phage were preabsorbed to the bacteria at 37°C for 15 minutes after which

they were transferred to 450 ml of pre warmed NZCYM medium and shaken vigorously

until total cell lysis was observed. The culture was adjusted to a final concentration of 1%

chloroform and 1M NaCl and shaken for a further 10 minutes. The debris was removed by

centrifugation in a GS-3 rotor at 5 krpm and 4°C for 10 minutes. DNAsel and RNaseA

(50Mg each) were added to the phage supernatant and incubated at 4°C for 1 hour. The

phage particles were precipitated overnight at 4°C by the addition of 33g PEG. Phage were

collected by centrifugation in a GS-3 rotor at 5 krpm and 4°C for 10 minutes. The pellet

was resuspended in SM for a final volume of 5 ml after which the suspension was extracted

with chloroform. The aqueous supernatant was adjusted to a final volume of 13.5 ml with

S M buffer containing 0.75 g CsCl/ml S M buffer and transferred to a Beckman heat sealable

tube. The phage were subjected to isopycnic centrifugation in a Ti70.1 rotor for 16-18

hours at room temperature and 60 krpm. A blue band midway in the gradient was

observed by shining a light on the gradient. The band was extracted with an 18 gauge

needle, and the CsCl solution containing Xphage was adjusted to 0.02% gelatin and then

transferred to a dialysis bag. The CsCl solution was exchanged with a solution of lOmM

Tris-HCl pH8.0, 25mM NaCl, lOmM M g C l 2 by dialyzing two times against a 1000-fold

volume excess of buffer. Extraction of D N A from the CsCl purified phage particles was

performed as described for the small-scale preparation of Xphage DNA.

5. Isolation of Single-stranded Plasmid DNA

D N A sequence was obtained from purified (Messing, 1983) single-stranded M13 DNA.

Agar plugs containing M13 phage plaques representing D N A cloned into the M13mp8,9,18

or 19 vectors were removed from the agar plate, transferred to 2ml Y T and grown

overnight with shaking at 3 7 ° C . Bacterial cells from 1.5 ml of the overnight culture were

removed by centrifugation in an Eppendorf centrifuge, and 650M1 of the supernatant was

transferred to a tube containing 300yl 40% P E G and 300M1 5M NaCl. The phage were

allowed to precipitate for 15 minutes at room temperature and were collected by

centrifugation in an Eppendorf centrifuge for 5 minutes. The pellet was resuspended in

200M1 of low salt buffer (20mM Tris-HCl pH7.5, 20mM NaCl, ImM EDTA). The phage

were extracted with 200^1 of phenol (redistilled and saturated in T E buffer) followed by two

extractions with an equal volume of phenol/chloroform (1:1, v:v). The D N A in the aqueous

phase was precipitated overnight at - 2 0 ° C in a final concentration of 0.3M sodium acetate

with 2 volumes of 95% ethanol. The D N A was collected by centrifugation, and the pellet

was resuspended in 50u\ 0.3M sodium acetate and reprecipitated with 2 volumes of 95%

ethanol overnight at - 2 0 ° C . The final D N A pellet was resuspended in 40yl low salt buffer.

E. ISOLATION OF POLY A* RNA

All glassware was baked at 1 8 0 ° C overnight, and pipet tips, centrifuge and Eppendorf

tubes and solutions were autoclaved. Human liver R N A was isolated by the method of

Chirgwin et al. (1979). Frozen liver (-70°C) was homogenized using a Polytron

homogenizer in an appropriate volume of buffer [7.5M guanidine hydrochloride (GuHCl),

25mM sodium citrate pH7.0 and 0.1M DTT] so that lOml/g tissue was obtained. A

10%(w/v) N-lauryl sarcosine solution was added to a final concentration 0.5% (w/v), and the

insoluble matter was removed by centrifugation at 5 krpm for 30 minutes in an HB-4 rotor

at 4 ° C . The R N A solution was adjusted to 33% ethanol, and R N A was precipitated

overnight at - 2 0 ° C . RNA was collected by centrifugation at 5 krpm in an SS-34 rotor at

0 ° C for 30 minutes, and the pellet resuspended in half of the starting volume of GuHCl

buffer. The removal of insoluble material by low speed centrifugation, precipitation of RNA

and resuspension in a half volume of GuHCl buffer was repeated two more times. The final

RNA pellet was resuspended in a small volume of d H z O , and the concentration of RNA was

determined from absorption at 260nm [lOD260nm = 40 mg/ml (Davis et al., 1980)]. RNA

was stored in d H 2 0 at - 7 0 ° C .

PolyA + mRNA was isolated by chromatography on a column of oligo-dT cellulose (Edmonds

et al., 1971; Aviv and Leder, 1972). The solution of total R N A was adjusted to a final

concentration of 0.4M sodium acetate pH7.5, 0.1% SDS and applied to the column

equilibrated in column buffer (0.4M sodium acetate pH7.5, 0.1% SDS and 1 m M EDTA).

The unbound R N A fraction was reapplied three times. The column was washed until the

OD260nm of the eluate was below 0.05. PolyA + RNA was eluted from the column with 1

m M E D T A , 0.1% SDS. The OD260nm of each fraction was determined and those

containing R N A were pooled. The solution of RNA was adjusted to 0.3M sodium acetate

pH4.8 to which two volumes of 95% ethanol were added, and the RNA was precipitated

overnight at - 2 0 ° C . RNA was resuspended in d H 2 0 at a high concentration and stored at

- 7 0 ° C .

F. OLIGONUCLEOTIDE SYNTHESIS AND PURIFICATION

Oligonucleotides were synthesized (by Tom Atkinson, Dept. of Biochemistry) with an

Applied Biosystems 380A D N A Synthesizer (Atkinson and Smith, 1984). Two pools of

mixed heptadecadeoxyribonucleotides were used as hybridization probes in the screening of

the human cDNA liver library. Pool I (5 ' -dTCRAAYTGRTGNCCCCA-3' , where R

represents both dG and dA, Y represents dT and dC, and N represents dG, dA, dT and dC)

was complementary to the mRNA sequence coding for amino acid residues 133-138 of

(3-factor X l l a (Fujikawa and McMullen, 1983) (Fig. 5). Pool II

(5'-dCCYTGRCANGCYTCNGT-3') was complementary to the mRNA coding for residues

182-188 of ^-factor Xl la . Two oligonucleotides were synthesized which correspond to parts

of the cDNA sequence (5 ' -CGAAAGTGTTGACTCCA-3 ' and

5 ' -GGCCAAAGGTCTTGGAAA-3 ' ) and used to probe for specific regions in the gene and for

nuclease S i mapping and Northern blot analysis.

The synthetic products required purification by polyacrylamide gel electrophoresis and

passage through Sep-Pac columns (Atkinson and Smith, 1984). Dried pellets of the newly

synthesized oligonucleotides were resuspended in 90u\ d H 2 0 . A I O M I volume was

transferred to 20yl deionized formamide and heated at 90 °C for 3 minutes. The

oligonucleotide sample and a sample of dye-formamide [xylene cyanol (mobility equivalent

to 45 bases) and bromophenol blue (mobility equivalent to 12 bases) (Maniatis et al., 1982)]

were applied to a 20% polyacrylamide-8M urea gel (42 cm) and electrophoresed at 1500V

until the bromophenol blue had migrated two-thirds of the gel length. The gel was

transferred to Saran Wrap and placed over a thin layer chromatography (TLC) plate, and

the single-stranded D N A was visualized with U V irradiation (260nm). The band

corresponding to the correct length of oligonucleotide was excised and eluted in 1.5 ml of

buffer containing 0.5M ammonium acetate, lOmM magnesium acetate by incubation at

3 7 ° C overnight. The solution was vortexed vigorously and centrifuged for 5 minutes in an

Eppendorf centrifuge and the supernatant removed. The debris was resuspended in 0.5ml

of the elution buffer, vortexed, centrifuged and the supernatant pooled with the 1.5ml

obtained previously. A Sep-Pac was attached to a 5ml syringe and was washed with 10ml

H P L C grade acetonitrile followed by 10ml d H 2 0 . A new syringe was used for each

solution. The sample was loaded onto the small column and pushed through while collecting

the eluate in Eppendorf tubes. The column, loaded with DNA, was washed with 5ml d H 2 0

and 1ml fractions of the wash solution were collected. To elute the oligonucleotide from the

SepPac column, 6ml of 60% methanol (or 20% acetonitrile) were pushed through the

column, and 1 ml fractions collected. The D N A eluted in the first three fractions was

detected by absorbance at 260nm. The concentration was determined from the OD260nm

(lOD = 33yg). Fractions containing significant amounts of D N A were dried in a Speed vac

at 80 °C for 3 hours. The pellet was resuspended in the appropriate volume of d H 2 0 and

was stored at - 2 0 ° C .

G. LABELING OF DNA

1. Labe l ing With 3 2 P by Nick Translation

D N A for use as hybridization probes was labeled with 3 2 P by nick translation (Maniatis et

al, 1975). D N A (100-500 ng) was labeled in 50^1 of 50 m M Tris-HCl pH7.5, 5 m M M g C l 2 ,

0.05 mg/ml BSA, 10 m M (3-mercaptoethanol, 20 tfM dGTP, 20 «M TTP, 1.4 iM dATP, 1.4

uM dCTP, 70 yCi a - 3 2 P dATP, 70 M C i 3 2 P dCTP, 0.2mM C a C l 2 , 50 pg DNasel and 20

units E. coli D N A polymerase I (Romberg). The reaction mixture was incubated at room

temperature for 30 minutes and then terminated by the addition of three volumes of 1%

SDS-lOmM E D T A , 25 Mg tRNA and by heating at 68°C for 10 minutes. The unicorporated

isotope was separated from the labeled D N A by chromatography on a column of Ultrogel

AcA54 (5 ml) equilibrated in lOmM Tris-HCl pH7.5, 200mM NaCl and 0.25mM E D T A .

Labeled D N A was eluted in the same buffer and collected in 200M1 fractions from the

column. Single-stranded labeled D N A used as hybridization probes was obtained by

treating the labeled duplex D N A with a tenth of a volume I M N a O H at 68°C for 10

minutes followed by the addition of a tenth of a volume 1.5M N a H 2 P 0 4 (untitrated) just

prior to use.

2. Labe l ing wi th T4 Polynucleotide Kinase

Oligonucleotides were 5'-end labeled with T4 polynucleotide kinase (Chaconas and van de

Sande, 1980) in a 20u\ reaction volume containing 33 pmoles oligonucleotide, 2.5MCi/ul

7 . 3 2 p A T P , 0.1M Tris-OH pH8.0, 0.005M dithieothrieotol, 0.01M M g C l 2 and 4 units T4

polynucleotide kinase. The reaction was incubated at 37°C for 45 minutes and stopped

with 0.005M E D T A (pH8.0) and heated at 6 8 ° C for 10 minutes. Unincorporated y-32P

A T P was removed by chromatography on a column of Sephadex G-25 (superfine)

•equilibrated and eluted with 5 m M E D T A (pH 8.0). The excluded fraction was added

directly to the hybridization mixture. The specific activities obtained were usually

approximately 10 6 cpm/pmole.

H. SCREENING A PLASMID LIBRARY

A n adult human liver cDNA library (Prochownik et al., 1983) was generously provided by

Dr. S. H . Orkin (Children's Hospital Medical Center, Boston). This library consists of

double-stranded cDNA (>500 bp in length) cloned into the PstI site of pKT218 by

homopolymeric dG:dC tailing. Initially, 2 x 10 s colonies of the cDNA library were plated

directly onto ten cellulose nitrate filters (132mm diameter) placed on Luria broth plates

containing tetracycline (12.5 mg/ml). The colonies were grown at 3 7 ° C until they reached a

diameter of l-2mm from which two sets of replica filters were prepared (Hanahan and

Meselson, 1980; Fung et al., 1984). The lawn of bacteria was oriented by stabbing with an

18 gauge needle through the filter and the agar. The filters containing colonies were

removed and another filter disc was placed on top of the first filter, and the orientation

marks were placed on the new filter. The plaque lift procedure was repeated with a new

filter disc. The replica filters were transferred to fresh LB-tetracycline plates, and the

colonies were grown at 3 7 ° C until they were 3-4mm in diameter after which the filters were

transferred to LB-chloramphenicol (250Mg/ml) plates and incubated overnight at 3 7 ° C . The

master filters were stored on LB-tetracyline plates at 4 ° C . Colonies were lysed on the

filters by soaking the filters twice for 20 minutes on Whatman 3 M M paper wetted in a

solution of 0.5M NaoH, 1.5M NaCl. The replica discs were neutralized in a similar manner

in a solution of IM Tris-HCl pH7.5 for 20 minutes followed by treatment with 0.5M

Tris-HCl pH7.5, 1.5M NaCl for 20 minutes. The nitrocellulose filters discs were air dried

and baked at 6 8 ° C for 16-18 hours.

The filters were hybridized and washed according to conditions described in Fung et al.

(1984). Prior to hybridization, the filter discs were washed three times in 6XSSC (1XSSC

is 0.15M NaCl, 0.015M sodium citrate pH7.0) to remove cell debris and prehybridized in

6XSSC, 2X Denhardt's solution (IX Denhardt's is 0.02% BSA, 0.02% ficol, 0.02%

polyvinylpyrrolidone) at 6 8 ° C for at least 6 hours. One set of replica filters was hybridized

with the 3 2P-labeled Pool I oligonucleotides, and the other set of filters was hybridized to the

3 2P-labeled Pool II oligonucleotides. Each hybridization solution contained 6XSSC, 2X

Dendhardt's solution, 0.2% SDS and 50-100 x 10« cpm 3 2P-labeled oligonucleotide. The

filters were hybridized for 18 hours at 3 7 ° C and then washed in 6XSSC at room

temperature for 5 minutes, 3 7 ° C for 5 minutes and then two times at 4 2 ° C for 5 minutes.

Positive clones were detected by autoradiograhy at - 7 0 ° C with an intensifier screen.

Colonies that hybridized to both pools of oligonucleotides were rescreened at lower colony

density until all the colonies plated were positive.

The cDNA library was also screened using a 3 2 P-nick translated hybridization probe

specific for FXII. Preparation of replica nitrocellulose filters and their prehybridization

conditions were the same as that for oligonucleotides but the hybridization conditions were

changed. Replica filters were hybridized in a solution of 6XSSC, 2X Dehardt's solution,

0.5% SDS and 50 x 10« cpm at 6 8 ° C for 16-18 hours. The washing conditions for the

specific probe were 4XSSC, 0.2%SDS at room temperature for 10 minutes followed by a

wash in the same buffer at 6 8 ° C . More stringent conditions for washing can be used with

specific hybridization probes and so the filters were further washed at 1XSSC, 0.2%SDS for

1 hour at 6 8 ° C with three changes of buffer.

/. SCREENING XPHAGE LIBRARIES

Two Xphage libraries were screened for FXII containing sequences according to the methods

described in Benton and Davis (1977). The first was a human genomic phage library kindly

provided by Dr. P. Leder (Harvard University). This library consists of a partial Sau3A

digest of human placental D N A ligated into the BamHl site of Charon 28. One million

recombinant phage were screened by in situ hybridization. Phage were plated on E. coli

strain C600 at a density of 20,000 pfu per 15 cm diameter petri dish and grown at 3 7 ° C .

Duplicate plaque lifts were obtained using nitrocellulose filters. The filters were placed on

the plates when the phage were pin-point size and were not touching one another. The

nitrocellulose disc was oriented by stabbing the filter and the agar using a syringe needle.

The master plates were placed at 4 ° C . The Filters were transferred to a fresh plate of

medium (NZYCM) and grown overnight at 3 7 ° C . The phage were lysed on the filters in

0.5M NaOH, 1.5M NaCl for 5 minutes and neutralized in 1M Tris-HCl, pH 7.5 for 5

minutes followed by 0.5M Tris-HCl, pH7.5, 1.5M NaCl for 5 minutes. The filters were then

washed with gentle rubbing to remove debris in 3XSSC and allowed to air dry. The filters

were baked at 6 8 ° C overnight and were prepared for hybridization with specific cDNA

probes for FXII. The nitrocellulose discs were prehybridized in 6XSSC, 2X Denhardt's

solution and 0.2%SDS at 68 °C for 6 hours. A hybridization solution, consisting of the same

buffer and 50-100 x 10' cpm of a FXII hybridization probe of FXII cDNA that was labeled

by 3 2 P by nick translation, was prepared and the filters incubated at 6 8 ° C overnight. The

filters were washed at room temperature in 4XSSC, 0.5%SDS followed by two washes for

30 minutes at 6 8 ° C in 2XSSC, 0.5%SDS. Positive phage were identified by

autoradiography at - 7 0 ° C with intensifier screens. Positive phage found on both replica

filters were replated and rescreened until all the phage hybrized with the probe.

A second library (Koschinsky et al., 1986) was screened and consisted of human liver cDNA

fragments (less than 500 bp in size) cloned into the EcoRl site of Xgt l l (Huynh et al, 1984).

A total of 0.5 x 10 6 phage were plated on E. coli strain RY1088 at 20,000 phage per plate

and incubated at 3 7 ° C . The screening procedure was the same as that described above for

the Xphage human genomic D N A library. The hybridization probe used was a 3 2 P-nick

translated 280 bp fragment of cDNA representing sequence encoding the N-terminal region

of FXII.

J. DNA SUBCLONING

1. Production of DNA Fragments for Ligation

D N A fragments to be subcloned were generated either by restriction endonuclease digestion

or by randomly shearing large pieces of D N A by sonication (Deininger, 1983). Restriction

endonuclease digestions were performed by using the conditions recommended by the

suppliers. Restriction fragments were electrophoresed through agarose or acrylamide gels,

and those fragments that were to be cloned were excised and recovered from the gel by

electroelution (Maniatis era/., 1982).

Random fragments of plasmid D N A were produced by sonication using a Heat Systems

Sonifier at output level 2. The D N A (10-lOOug) was sheared in 500/zl of 0.5M NaCl, 0.1M

Tris-HCl, pH7.4, lOmM EDTA with 5 pulses of 5 seconds. The solution was cooled on ice

and vortexed between pulses. The resulting DNA fragments were fractionated on a 5%

polyacrylamide gel, and fragments (300-500 bp in size) were recovered by electroelution.

The DNA solution (450MD was removed from the dialysis bag and adjusted to 0.3M sodium

acetate. Two volumes of 95% ethanol were added, and the DNA was precipitated by

incubation at -20°C overnight. The fragments were collected by centrifugation in an

Eppendorf centrifuge, and the pellet was resuspended in 25yl dH 20. The ends were made

blunt-ended in a 50yl reaction of 33mM Tris-acetate, pH7.8, 66mM potassium acetate,

lOmM magnesium acetate, lOOmg/ml BSA, 0.2mM each deoxynucleotide triphosphate and

6 units T4 DNA polymerase. The reaction conditions varied depending on the conditions

recommended by the supplier. The repaired fragments were extracted with phenol and

precipitated in 0.3M sodium acetate by the addition of two volumes of 95% ethanol and

incubation at -20°C overnight. The DNA was recovered by centrifugation in an Eppendorf

centrifuge for 5 minutes, and the pellet was resuspended in lO/il TE buffer.

2. Ligation of DNA into pUCl3 or M13 Vectors

Fragments of DNA were ligated into the vectors, pUC13 or M13, in a reaction volume of

15wl consisting of DNA, 66mM Tris-HCl, pH7.5, 5mM MgCl 2, 5mM dithiothreiotol, 0.4mM

ATP and T4 DNA ligase [1 unit for blunt-ended ligations and 0.1 unit for sticky-ended

ligations (Maniatis et al., 1982)]. The amounts of insert and vector DNA used in the

ligation reactions depended on the vector: for pUCl3, 200ng insert DNA and lOOng vector

DNA were used while for M13, lOng sticky-ended insert DNA and 10 ng vector DNA were

used, and 50ng blunt-ended insert DNA and lOng vector DNA were used. Ligation of

sticky-ended DNA was for 4 hours at 16°C whereas ligation reactions of blunt-ended DNA

were incubated at 1 6 ° C overnight. The ligation reactions were stored at - 2 0 ° C .

3. Transformation of DNA into Bacteria

Host bacterial cells were made competent for D N A transformation using calcium chloride

(Messing, 1983). E. coli JM83 or JM103 were grown in 50 ml L B or Y T , respectively, at

3 7 ° C with vigorous shaking until the culture reached an OD600hm of 0.2-0.4. The cells

were collected by centrifugation for 5 minutes in an HB-4 rotor at 2.5 krpm at 4 ° C , and the

pellet was resuspended in ice-cold 50mM C a C l 2 at half the original volume of culture. The

cells were incubated on ice for 40 minutes and then recovered by centrifugation as before.

The calcium chloride treated cells were resuspended in one-tenth the original volume of ice

cold C a C l 2 solution and incubated at 4 ° C for 2-18 hours. Aliquots (0.3ml) of competent

cells were transferred to glass tubes (13x100mm) placed on ice. Volumes of 2-3 u\ of the

ligation mixture containing either M13 or pUC13 vectors were used to transform JM103

and JM83 competent cells, respectively. Transformation proceeded for 40 minutes on ice

and then for 2 minutes at 4 2 ° C . JM103 transformations were plated immediately with the

addition of 10/d 100 m M IPTG, 35-50yl X-Gal (2% w/v in dimethylformamide), 0.2ml host

cells and 3ml soft Y T agar and spread on Y T plates. JM83 transformed cells were rescued

with the addition of 0.7ml L B followed by shaking at 3 7 ° C for one hour. The cells were

spread in 100/ul aliquots with 50<d X-Gal (2% w/v) onto LB-ampicillin (50yg/ml) plates. The

JM103 and JM83 plated transformants were incubated overnight at 3 7 ° C . Phage plaques

or colonies which were clear or white, respectively, contained cloned D N A whereas plaques

or colonies transformed with religated vector without insert D N A appeared blue on the

plates.

K. BLOT HYBRIDIZATIONS

1. Southern Blot Analysis of Genomic and Cloned DNA

D N A was transferred to nitrocellulose filter sheets from agarose gels essentially as

described by Southern (1975). D N A samples (1 to 10 Mg) were digested by restriction

endonucleases and were separated by electrophoresis through agarose gels.

Single-stranded nicks were introduced into the D N A by irradiation using 260nm ultraviolet

light for 30 seconds. The gel was soaked in 0.5N NaOH, 0.6M NaCl for 45 minutes to

denature the D N A and then the gel was neutralized by soaking twice in a solution of 1M

Tris-HCl pH7.5, 0.6M NaCl for 30 minutes. The gel was placed on Whatman 3 M M paper

wicks which extended into the transfer buffer (10XSSC). A piece of nitrocellulose filter was

cut to the size of the gel and soaked for 10 minutes in d H 2 0 and then 10XSSC. The filter

was placed on the gel followed by two sheets of Whatman 3 M M paper and stacks of paper

towels which were cut smaller than the nitrocellulose sheet. The D N A was transferred to

the filter paper for 16 hours for plasmid or lambda D N A but 36-48 hours for genomic DNA.

After transfer, the paper towels and 3 M M paper were removed and the lane origins were

marked on the nitrocellulose filter. The filter was removed and washed in 3XSSC, air dried

and baked at 68 °C for 6 hours.

Hybridization of 3 2 P-labeled D N A to genomic DNA Southern blots was performed according

to Kan and Dozy (1978) whereas Southern blots of plasmid or lambda D N A were hybridized

as described in Maniatis et al. (1982). The nitrocellose filter sheet containing genomic

D N A was wetted in 3XSSC and prehybridized for 16 hours at 3 7 ° C in 10 ml of a solution

containing 50% formamide, 6XSSC, ImM E D T A , 0.1% SDS, lOmM Tris-HCl pH7.5, 10X

Denhardt's solution, 0.05% sodium pyrophosphate, 100Mg/ml denatured herring sperm

D N A , 25/ng polyA + and 50 Mg/ml tRNA. The hybridization solution was identical to that for

prehybridization except for the addition of 20-50 x 10 6 cpm of denatured 3 2P-labeled DNA.

Hybridization was allowed to proceed for 48 hours at 3 7 ° C , and then the blot was washed

for 1 hour at room temperature in 2XSSC, IX Denhardt's solution followed by two washes

at 5 0 ° C for 45 minutes in 0.1XSSC, 0.1% SDS. The blot was rinsed twice at room

temperature in 0.1XSSC, 0.1% SDS followed by four rinses at room temperature in

0.1XSSC. After air drying the hybridized blot was exposed to Kodak XK-1 film with an

intensifier screen for 1 to 7 days at - 7 0 ° C .

The hybridization conditions used for plasmid and lambda D N A containing inserted

fragments of D N A were similar to those described in the screening of the Xphage libraries.

The baked filter was wetted in 3XSSC and prehybridized for 4 hours in 6XSSC, 2X

Denhardt's solution, 0.2% SDS at 6 8 ° C . A n identical solution was prepared which also

contained 1 to 10 x 10 6 cpm of denatured 3 2P-labeled D N A was exchanged for the

prehybridization solution. The blot was hybridized for 16 hours at 6 8 ° C followed by

washing in 4XSSC, 0.5% SDS for 10 minutes at room temperature. Subsequent washes

were in 1XSSC, 0.5% SDS at 6 8 ° C . The hybridized blots were air dried and exposed to

Kodak film 2-16 hours at room temperature.

2. Northern Blot Analysis

RNA was separated by electrophoresis in formaldehyde agarose gels and then transferred

to nitrocellulose filter sheets (Maniatis et al., 1982). The RNA was denatured in the

presence of formaldehyde (Lehrach et al., 1977) which was present in the gel and in the

loading solution. After electrophoresis, the gel was treated with a solution of 50mM N a O H

for 45 minutes and then neutralized in 0.1M Tris-HCl pH7.5 for 45 minutes. The gel was

soaked in a final solution of 20XSSC for 45 minutes and then placed on Whatman 3 M M

filter paper wicks extending into a solution of transfer buffer (10XSSC). The nitrocellulose

filter paper was wetted in d H 2 0 and 10XSSC, and the RNA was transferred to the paper

for 16 hours as described above for Southern blotting. Specific mRNA species were detected

by hybridization and washing as described for Southern blots of genomic D N A (see above).

L. DNA SEQUENCE ANALYSIS

1. Screening M13 Clones

D N A fragments to be sequenced by the chain termination method (Sanger et al., 1977) were

ligated into the M13 sequencing vectors (Messing et al., 1981; Messing, 1983) as described

above. To identify the M13 phage which contain the desired insert, the M13 plaques were

screened by in situ hybridization (Benton and Davis, 1977). The M13 phage were plated on

1.5%(w/v) Y T agar plates at a density of approximately 500 plaques/plate. Nitrocellulose

filter discs (85mm) were placed over the plaques, the filter oriented with an 18 gauge needle

and then the filter was transferred to a solution of 0.5N NaOH, 1.5M NaCl for 5 minutes to

lyse the phage particles and denature the D N A on the filter. The filters were neutralized in

I M Tris-HCl pH7.5 for 5 minutes followed by a solution of 0.5M Tris-HCl pH7.5, 1.5M

NaCl for 5 minutes. The filters were air dried and baked for 6 hours at 6 8 ° C . The

nitrocellulose filter discs were washed with 3XSSC for 10 minutes at room temperature and

then prehybridized in 6XSSC, 2X Denhardt's solution and 0.2% SDS for 4 hours at 6 8 ° C .

The hybridization solution was the same as the prehybridization solution but also contained

1 to 50 x 10 6 cpm of denatured 3 2P-labeled D N A to identify phage containing the D N A to

be sequenced. The filters were incubated at 6 8 ° C for 16 hours and then washed in 4XSSC,

0.5% SDS at room temperature for 10 minutes. The excess probe was removed by further

washes at 6 8 ° C in 1XSSC, 0.5% SDS with multiple exchanges of the washing buffer. The

filters were dried and exposed to Kodak-XKl film at - 7 0 ° C for 16 hours with an intensifier

screen. Selected phage plaques were transferred to Y T media, diluted approximately 10*

times and replated. M13 phage containing insert D N A from these plates were grown and

their single-stranded D N A isolated.

2. DNA Sequence Analysis

The nucleotide sequence of single stranded D N A isolated from M13 clones was determined

by the chain termination method (Sanger et al., 1977) as modified for phage M13 templates

(Messing et al., 1981). Table I shows the concentrations of the deoxy- and

dideoxy-nucleotides used in the sequencing reactions. An M13 primer ( I M I o f a solution with

0.03 OD260nm/ml) having the nucleotide sequence of 5 ' - G T A A A A C G A C G G C C A G - 3 ' was

hybridized to single stranded template D N A (in 4 ul) in a total reaction volume of 8jul that

also contained I M I d H z O and 2M1 10X Hin buffer (600 m M NaCl, lOOmM Tris-HCl pH7.5,

70mM MgCl 2 ) . The hybridization mixture was incubated at 6 8 ° C for 10 minutes and then

was allowed to cool slowly to room temperature followed by the addition of I M I of 15juM

dATP and 1.5M1 3 2 P-a-dATP (10 uCi/ul). Aliquots consisting of 2.0/xl of the hybridization

mixture were dispensed to four tubes containing 1.5/d of the appropriate

deoxy/dideoxy-nucleotide mixture (Table I). The reactions were started with the addition of

0.4 units D N A polymerase I Klenow fragment and incubated at room temperature for 15

minutes. A chase solution was added (lul of 0.5mM dATP) and the reaction mixtures were

SEQUENCING MIXES

N u c 1 e o t 1 d e d/ddG d /ddA d / d d T d /ddC

dG 7.9 1 0 9 . 4 1 5 8 . 7 1 5 7 . 9

dT 157 .6 1 0 9 . 4 7.9 1 5 7 . 9

dC 157 .4 109 .4 1 5 8 . 7 10 .5

ddG 157 .4 - - -ddA - 1 1 6 . 7 - -

ddT - - 550 .3 -

ddC _ 191 .6

T a b l e I : DNA S e q u e n c i n g M i x e s .

The numbers In the t a b l e r e f e r to the yuM c o n c e n t r a t i o n s of the

d i d e o x y and d e o x y - r I b o n u c I e o t I de t r i p h o s p h a t e s used in the s e q u e n c i n

m i x e s f o r M13 DNA s e q u e n c i n g . The v a l u e s used were t h o s e d e t e r m i n e d

D r . Joan M c P h e r s o n , D e p t . of B o t a n y , UBC.

incubated for 20 minutes at room temperature followed by the addition of 5yl of a stop-dye

mixture (98% formamide, 10 m M E D T A pH8.0, 0.02% xylene cyanol, 0.02% bromophenol

blue). The extended products were denatured by heating to 9 2 ° C for three minutes, and

l-2yl of each reaction mixture was analyzed on 6% and 8% polyacrylamide-8M urea gels in

1XTBE buffer at lW/cm. After electrophoresis, the gels were dried and exposed to Kodak

XK-1 film overnight at room temperature.

3. Computer Analysis of DNA Sequence Data

Analysis of the D N A sequence data obtained from the sequencing gels was assisted by the

computer programs of Staden (1982) and Delaney (1982).

M. MAPPING THE END OF A MRNA TRANSCRIPT

1. Primer Extension Analysis

A hybridization primer was prepared as follows. Single stranded D N A of a fragment

representing the 5' end of the FXII mRNA was subcloned M13mpl9 and was made double

stranded and radioactive with D N A polymerase I-Klenow fragment in the following

reaction. The M13 primer was hybridized to the single stranded D N A template at 6 8 ° C for

10 minutes in a mixture consisting of 2.5yl M13 primer, 2.5yl D N A , 2.5yl 10X Hindi

buffer (1M NaCl, 0.1M Tris-HCl pH7.4, 0.07M M g C l 2 , 0.01M dithiothreiotol) and 7.5yl

d H 2 0 . The reaction was cooled slowly to room temperature and then adjusted with 2yl

15yM dATP, 3M1 ^P-a-dATP ( I O M C I / M D , 2yl 0.5mM dGTP, dCTP and T T P and 1.25 units

of Klenow fragment (2.5yl). The extension reaction was incubated for 5 minutes at room

temperature and transferred to 6 8 ° C for 10 minutes. The reaction was cooled to 3 7 ° C , and

20 units of the restriction endonuclease Hinc II was added and the D N A digested for 20

minutes. A n equal volume of stop-dye mixture (98% deionized formamide, 10 m M E D T A ,

pH 7.5, 0.02% bromophenol blue, 0.02% xylene cyanol) was added and the D N A was

denatured at 9 0 ° C for 5 minutes. The D N A fragments were subjected to denaturing gel

electrophoresis through a 6% polyacrylamide-8 M urea gel. After autoradiography for 5

minutes, the gel slice containing the primer D N A (180 bp) was excised and the D N A

recovered by electroelution. The D N A was precipitated with ethanol and resuspended in

6.5M1 of a buffer containing 40 m M Pipes pH 6.7, 1 m M E D T A and 0.4 M NaCl (Kingston et

al., 1984). The mixture was added to 3yl polyA + mRNA from human liver (18yg) and 30M1

deionized formamide. Another reaction was prepared in the same manner but without

mRNA. The reaction mixtures were heated at 8 0 ° C for 15 minutes and then transferred to

a 3 0 ° C water bath for 16 hours (Kingston et al., 1984). The nucleic acids were precipitated

with 140/il of 0.3M sodium acetate (pH 5.0) and 400/il of 95% ethanol. The pellets,

containing approximately 60,000 cpm, were resuspended in 50M1 of A M V reverse

transcriptase buffer (50 m M Tris-OH pH 8.3, 20 m M KC1, 10 m M M g C l 2 , 5mM

dithiothreiotol)(Kingston et al., 1984) to which was added 30 units of A M V reverse

transcriptase. The reactions proceeded at 3 7 ° C for 1 hour and then the nucleic acids were

precipitated with 5yl of 3M sodium acetate and 125 ul of 95% ethanol. The pellets were

resuspended in 5M1 of load-dye mix and denatured at 9 0 ° C for 5 minutes. The

primer-extended product was analyzed on a 6% polyacrylamide-8 M urea gel. D N A

sequencing reactions of the template from which the original primer was obtained were used

as size markers. Following electrophoresis, the gel was dried and exposed to Kodak XK-1

film overnight at room temperature and then 3 to 14 days at - 7 0 ° C with an intensifier

screen.

2. Nuclease S i Mapping

A hybridization probe was prepared in the same manner as that described above. The

single stranded primer, however, was obtained from an M13 subclone of the 450 bp Hpall

fragment that represents the 5' end of the mRNA transcript and 5' most flanking

sequences. An oligonucleotide primer (5 ' -CGAAAGTGTTGACTCCA-3' ) was synthesized on

an Applied Biosystems 380A D N A Synthesizer (Atkinson and Smith, 1984) and is

complementary to a region within the 450 bp Hpall fragment. The preparation of the long

3 2P-labeled primer was the same as that described for the primer extension reaction above.

To generate the correct hybridization probe for nuclease SI mapping, the Klenow

3 2 P-labeled D N A was digested with EcoRl and the single stranded primer was isolated from

a 6% polyacrylamide-8M urea gel as described above. Hybridization of this primer (210 bp

in length) to poly A + mRNA was as described for the primer extension reaction. Another

identical reaction was prepared except that d H 2 0 was added instead of poly A + mRNA.

After hybridization, the nucleic acids were precipitated by the addition of 140jul of 0.3M

sodium acetate and 400/ul of 95% ethanol followed by incubation at - 7 0 ° C for 20 minutes.

The nucleic acids were collected by centrifugation at 4 ° C for 10 minutes in an Eppendorf

centrifuge. To the pellets were added 270M1 nuclease S i buffer (270ul of 0.28 M NaCl,

50mM sodium acetate, 4.5mM ZnS0 4 ) , lOug denatured herring D N A and 10 to 30 units of

nuclease S i . The reaction mixture was incubated at 3 7 ° C for 15 minutes to 1 hour. The

digested D N A was precipitated with 10/ug tRNA and 600^1 95% ethanol and incubated at

- 7 0 ° C for 15 minutes. The D N A was recovered by centrifugation and the pellet

resuspended in 2.5M1 of ImM Tris-HCl pH 7.5, 0.6mM NaCl, O. lmM M g C l 2 and 2.5yl of

stop-dye mix. The sample was heated for 3 minutes at 90 °C and analyzed on a 6%

polyacrylamide-8M urea gel together with the D N A sequencing reactions of the template

from which the original probe was prepared. The nuclease S1 reaction on the same

hybridization mixture was repeated with the addition of 10 ug tRNA instead of 10ug of

denatured herring DNA and 200 units of enzyme. The gel was dried and exposed to film as

described for the primer extension reaction above.

III. RESULTS

A. CHARACTERIZATION OF THE HUMAN FXII CDNA

1. Isolation of Factor XII cDNA Clones

A human liver cDNA library (provided by Dr. Stuart Orkin at Children's Hospital,

Harvard University) was screened to isolate the human FXII cDNA. Two hundred

thousand bacterial colonies of the cDNA library were screened by in situ hybridization with

two mixtures of synthetic oligonucleotides that encoded two regions of 0-factor X l l a as

predicted from the known amino acid sequence (Fig. 5). Although each of the oligonucleotide

mixtures hybridized to about 60 different colonies, only two colonies hybridized to both

(Fig. 6). After rescreening at lower colony density, plasmid D N A was isolated from the two

positives that were designated pcHXII-11 and pcHXII-14 (Fig. 7). Subsequent sequence

analysis revealed that these plasmids contained D N A coding for the known amino acid

sequence of /3-factor Xl la . Because pcHXII-11 and pcHXII-14 contained only short cDNA

inserts, 2 x 10 5 colonies of the cDNA library were rescreened with the [3 2P]-labeled Psil

insert of pcHXn-11 as a hybridization probe. Thirty-six positive clones of various lengths

were obtained; plasmids pcHXII-17 and pcHXII-501 contained the longest cDNA inserts

and were studied further. Restriction endonuclease mapping of the four positive plasmids

showed that they contained overlapping D N A (Fig. 7) except that pcHXII-17 appeared to

contain a deletion (Fig. 7, open dashed bar). This was confirmed by subsequent D N A

sequence analysis (see next section).

Figure 5. Predicted Oligonucleotide Sequences for 0-factor Xlla

Flow Chart indicating the regions of the 0-factor X l l a amino acid sequence (Fujikawa and

McMullen, 1983) from which the oligonucleotide sequences were predicted; mixtures of

synthetic heptodecadeoxyribonucleotides were used as hybridization probes in screening the

liver cDNA library. The underlined numbers represent the two pools of oligonucleotides

used in the double screening technique (Figure 6). Oligo 1 and Oligo 2 sequences represent

amino acids 537 to 542 (Thr to Gly) and 486 to 491 (Trp to Glu), respectively, in the

0-factor X l l a amino acid sequence (Fig. 6).

g-factor Xlla

H,N - AsnGlyProLeuSerCys — GlyGlnArg I S I S 2

ValValGly - Cys -TrpGlyHisGlnPheGlu.

mRNA Sequence

Complementary Oligo Sequence

5 lTC^AASG\GTCCCCA 3' G T G C

32 Possible Sequences

ThrAspAlaCysGlnGly—(Ser)—ThrSerVal - COOH

J mRNA Sequence

Complementary Oligo Sequence

5'-CC^TG^CATCC^TCTGT- 31

C G C G C

128 Possible Sequences

Figure 6: Double Oligonucleotide Screen For Factor XII cDNA

A liver cDNA library was plated at a high density (100,000 colonies/filter) and duplicate

filters were hybridized with either 3 2P-labeled oligo-1 or oligo-2 pools of synthetic

heptodecadeoxyribonucleotides (upper discs). The overlapping positive colony (arrows) was

rescreened at a lower density (200 colonies/filter) in duplicate and hybridized with the same

oligonucleotide mixtures (lower discs). Positive colonies which overlapped (arrows) were

selected and their D N A sequence determined.

Figure 7: Restriction Enzyme Map and Sequencing Strategy for Factor XII cDNA

The bars below the restriction map represent cDNA clones; pcHXII-11, pcHXII-14,

pcHXII-17 and pcHXII-501 and include regions coding for the signal peptide (dotted bar),

the heavy chain of a-factor X l l a (solid bars) and the 3' untranslated region (slashed bar).

The open dashed line in pcHXII-17 represents a deletion (see text for details). Arrows

below the cDNA clones represent the sequencing strategy used, where each arrow

represents the M13 clone. The extent of sequencing is indicated by the length of arrows.

D N A sequence determined on the coding strand is shown by an arrow pointing left;

sequence determined on the non-coding strand is shown by an arrow pointing right. Thick

arrows represent D N A sequences obtained from randomly sheared cDNA fragments and

thin arrows represent sequence obtained from restriction enzyme fragments. The scale at

the bottom represents nucleotides in kilobase pairs.

p c H X I I - 1 1

p c H X I I - 1 4

p c H X I I - 1 7

p c H X I I - 5 0 1 HE

Is\SSS\SN •SWWNSM

2. Sequence Analysis of Human Factor XII cDNA Clones

Results / 76

The strategy used to determine the nucleotide sequence of the four clones, pcHXII-11, 14,

17 and 501 is shown in Figure 7. The thick arrows represent the sequence determined from

M13 phage containing randomly sheared DNA. Although more than 90% of the nucleotide

sequence was determined using this strategy, few random clones were isolated that

overlapped the Aval site or the 5' end of factor XII cDNA (Fig. 7). To complete the

nucleotide sequence analysis, plasmid pcHXII-501 (Fig. C) was digested with PstI and the

1200 bp and 280 bp fragments were isolated by polyacrylamide gel electrophoresis. The

280 bp fragment was cloned directly into the PstI site of M13mp8. The 1200 bp fragment

was digested with both Aval and Smal, and the ends were made blunt-ended using the

Klenow fragment of E. coli D N A polymerase I (Smith et al., 1979). The resulting Pstl/Aval

and AvallSmal fragments were isolated by polyacrylamide gel electrophoresis and

subsequently ligated into the Smal site of M13mpl8. The D N A sequence analysis of these

clones is shown in Fig. 7 (thin arrows).

The nucleotide sequences of the four factor XII cDNA clones were compiled into a consensus

sequence (Fig. 8) using the computer program designed by Staden (1982). Each nucleotide

in the sequence was determined an average of 6.6 times and 84% of the sequence was

determined on both strands. In the regions of overlap, the four factor XII cDNA clones

contained identical nucleotide sequences with one exception. In agreement with the

restriction map of pcHXII-17 (Fig. 7), the D N A sequence analysis showed that this plasmid

contains a deletion of 53 bp between nucleotides 1330 and 1383 (Fig. 8). The deleted region

is flanked by an inverted repeat having the sequence: CTCGC(G/C)CGTC (nucleotides

1320-1329 and nucleotides 1383-1392, Fig. 8). This deletion alters the reading frame of

Figure 8: Nucleotide Sequence of Factor XII cDNA

The sequence was determined by analysis of the overlapping clones shown in Figure 7. The

predicted amino acid sequence of human factor XII is shown above the D N A sequence, the

oligonucleotide sequences used for screening the cDNA library are represented by boxes at

nucleotides 1,474-1,490 and 1,627-1,643. The signal peptide is numbered backwards from

the putative signal peptidase cleavage site (open arrow). The proteolytic cleavage sites that

give rise to (3-factor X l l a are shown by the heavy arrows (at amino acid residues 334, 343

and 353). The residues that make up the catalytic triad (His 393, Asp 442 and Ser 544)

are underlined. The T G A stop codon is located at nucleotides 1,807-1,809 and followed by

the 3' untranslated region (nucleotides 1,810-1,959) and a poly A + tail. The poly A +

recognition site A A T A A A is encoded by nucleotides 1,937-1,942. A carbohydrate

attachment site (Fujikawa and McMullen, 1983) is indicated by the solid diamond.

10 20 Leu Glu Ser Thr Leu Ser He Fro Pro Trp Clu Ala Pro Lys Clu His Lys Tyr Lys Ala Glu Glu His Thr Val Val Leu Thr Val Thr Gly Glu Pro Cys His TTG GAG TCA ACA CTT TOG ATT CCA CCT TGG GAA GCC CCC AAG GAG CAT AAG TAC AAA GCT GAA GAG CAC ACA GTC GTT CTC ACT CTC ACC GGG GAG CCC TGC CAC

15 30 45 60 75 90 105

30 40 . 5 0 60 Phe Pro Phe Gin Tyr His Arg Gin Leu Tyr His Lys Cys Thr His Lys Gly Arg Pro Gly Pro Gin Pro Trp Cys Ala Thr Thr Pro Asn Phe Asp Gin Asp Gin TTC CCC TTC CAG TAC CAC CCG CAG CTG TAC CAC AAA TGT ACC CAC AAG GGC CGG CCA GGC CCT CAG CCC TGG TGT GCT ACC ACC CCC AAC TTT GAT CAG CAC CAC

120 135 150 165 180 195 210

70 80 90 Arg Trp Gly Tyr Cys Leu Glu Pro Lys Lys Val Lys Asp His Cys Ser Lys His Ser Pro Cys Gin Lys Gly Gly Thr Cys Val Asn Het Pro Ser Gly Pro His CCA TGG GGA TAC TGT TTG GAG CCC AAG AAA GTG AAA GAC CAC TGC AGC AAA CAC AGC CCC TGC CAG AAA GGA GGG ACC TGT GTG AAC ATG CCA AGC GGC CCC CAC

225 240 255 270 285 300 315

100 110 120 130 Cys Leu Cys Pro Gin His Leu Thr Gly Asn His Cys Gin Lys Glu Lys Cys Phe Glu Pro Gin Leu Leu Arg Phe Phe His Lys Asn Glu l i e Trp Tyr Arg Thr TGT CTC TGT CCA CAA CAC CTC ACT GGA AAC CAC TGC CAG AAA GAG AAG TGC TTT GAG CCT CAG CTT CTC CGG TTT TTC CAC AAG AAT GAG ATA TGG TAT AGA ACT

330 345 360 375 390 405 420

140 150 160 Glu Gin Ala Ala Val Ala Arg Cya Gin Cys Lys Gly Pro Asp Ala His Cys Gin Arg Leu Ala Ser Gin Ala Cys Arg Thr Asn Pro Cys Leu His Gly Gly Arg GAG CAA GCA GCT GTG GCC AGA TGC CAG TGC AAG GGT CCT GAT GCC CAC TGC CAG CGG CTG GCC AGC CAG GCC TGC CCC ACC AAC CCG TGC CTC CAT GGC GCT CGC

435 450 465 480 495 510 525

170 180 190 200 Cys Leu Glu Val Glu Gly His Arg Leu Cys His Cys Pro Val Gly Tyr Thr Gly Pro Phe Cys Asp Val Asp Thr Lys Ala Ser Cys Tyr Asp Gly Arg Gly Leu TGC CTA GAG GTG GAG GGC CAC CGC CTC TGC CAC TGC CCG GTG GGC TAC ACC GGA CCC TTC TGC GAC GTG CAC ACC AAG GCA AGC TGC TAT GAT GGC CGC GGG CTC

540 555 570 585 600 615 630

210 220 230 Ser Tyr Arg Gly Leu Ala Arg Thr Thr Leu Ser Gly Ala Pro Cya Gin Pro Trp Ala Ser Glu Ala Thr Tyr Arg Asn Val Thr Ala Glu Gin Ala Arg Asn Trp AGC TAC CGC GGC CTG GCC AGG ACC ACG CTC TCC GGT GCG CCC TGT CAG CCG TGG GCC TCC GAG GCC ACC TAC CGG AAC GTG ACT GCC GAG CAA GCC CGG AAC TGG

645 660 675 690 70S 720 735

240 250 260 270 Gly Leu Gly Gly His Ala Phe Cys Arg Asn Pro Asp Asn Asp He Arg Pro Trp Cys Phe Val Leu Asn Arg Asp Arg Leu Ser Trp Glu Tyr Cys Asp Leu Ala GGA CTG GGC GGC CAC GCC TTC TGC CGG AAC CCG GAC AAC GAC ATC CGC CCG TGG TGC TTC GTG CTG AAC CGC GAC CGG CTG AGC TGG GAG TAC TGC GAC CTC GCA

750 765 780 795 810 825 840

280 290 300 Gin Cys Gin Thr Pro Thr Gin Ala Ala Pro Pro Thr Pro Val Ser Pro Arg Leu His Val Pro Leu Met Pro Ala Gin Pro Ala Pro Pro Lys Pro Gin Pro Thr CAG TGC CAG ACC CCA ACC CAG GCG GCG CCT CCG ACC CCG GTG TCC CCT AGG CTT CAT GTC CCA CTC ATG CCC GCG CAG CCG GCA COG COG AAG CCT CAG CCC ACG

855 870 885 900 915 . 930 945

310 320 330 Thr Arg Thr Pro Ser Gin Ser Gin Thr Pro Gly Ala Leu Pro Ala Asn Gly Glu Gin Pro Pro Ser Leu Thr Arg rAsn Gly Pro Leu Ser Cys Gly Gin Arg'Leu ACC CGG ACC CCC TCT CAG TCC CAG ACC CCG GGA GCC TTG CCG GCG AAC GGG GAG CAG CCG CCT TCC CTG ACC AGG AAC GGC CCA CTG AGC TGC GGG CAG CGC CTC

960 975 990 1,005 1,020 1,035 1,050

f 340 f GGA GCC TTG CCG GCG AAC GGG GAG CAG CCG CCT TCC CTG ACC AO

990 1.005 1.02

350 \f 360 370 Arg Lys Ser Leu Ser Ser Het Thr Arg'val Val Gly Gly Leu Val Ala Leu Arg Gly Ala'His Pro Tyr He Ala Ala Leu Tyr Trp Gly His Ser Phe Cys Ala CCC AAG ACT CTG TCT TCG ATG ACC CGC GTC GTT GGC GGG CTG GTG GCG CTA CGC GGG GCG CAC CCC TAC ATC GCC GCG CTG TAC TGG GGC CAC ACT TTC TGC GCC

1,065 1,080 1,095 1,110 1,125 1,140 1,155

380 390 400 410 ^ Gly Ser Leu He Ala Pro Cys Trp Val Leu Thr Ala Ala His Cys Leu Gin Asp Arg Pro Ala Pro Glu Asp Leu Thr Val Val Leu Gly Gin Glu Arg Arg Asn GGC AGC CTC ATC GCC CCC TGC TGG GTG CTG ACG GCC GCT CAC TGC CTG CAG GAC CGG CCC GCA CCC GAG CAT CTG ACC GTG GTG CTC GGC CAC CAA CGC CCT AAC

1,170 1,185 1,200 1,215 1,230, 1,245 1,260

420 430 440 His Ser Cys Glu Pro Cys Gin Thr Leu Ala Val Arg Ser Tyr Arg Leu His Glu A La Phe Ser Pro Val Ser Tyr Gin His Asp Leu Ala Leu Leu Arg Leu Gin CAC AGC TGT GAG CCG TGC CAG ACG TTG GCC GTG CGC TCC TAC CGC TTG CAC GAG GCC TTC TCC CCC GTC AGC TAC CAG CAC GAC CTG GCT CTG TTG CGC CTT CAG

1,275 1,290 1,305 1,320 1,335 1,350 1,365

450 460 470 480 Glu Asp Ala Asp Gly Ser Cys Ala Leu Leu Ser Pro Tyr Val Gin Pro Val Cys Leu Pro Ser Gly Ala Ala Arg Pro Ser Glu Thr Thr Leu Cys Gin Val Ala GAG GAT GCG GAC GGC AGC TGC GCG CTC CTG TCG CCT TAC GTT CAG CCG GTG TGC CTG CCA AGC GGC GCC GOG CGA CCC TCC GAG ACC ACG CTC TGC CAG GTG GCC

1,380 1,395 1,410 1,425 1,440 1,455 1,470

490 500 510 Gly Trp Gly His Gin Phe Glu Gly Ala Glu Glu Tyr Ala Ser Phe Leu Gin Glu Ala Gin Val Pro Phe Leu Ser Leu Glu Arg Cys Ser Ala Pro Asp Val His GGC trGG GGC CAC CAG TTC GJfS. GGG GCG GAG GAA TAT GCC AGC TTC CTG CAG GAG GCG CAG GTA CCG TTC CTC TCC CTG GAG CGC TGC TCA GCC CCG GAC GTG CAC

1,485 1,500 1,515 1,530 1,545 1,560 1,575

520 530 540 550 Gly Ser. Ser He Leu Pro Gly Met Leu Cys Ala Gly Phe Leu Glu Gly Gly Thr Asp Ala Cys Gin Gly Aap Ser Gly Gly Pro Leu Val Cys Glii Asp Gin Ala GGA TCC TCC ATC CTC CCC GGC ATG CTC TGC GCA GGG TTC CTC GAG GGC GGC |ACC GAT GCG TGC CAG OSh1 GAT TCC GGA GGC CCG CTG GTG TGT GAG GAC CAA GCT

l',S90 1,605 1,620 1,635 1,650 1,665 1,680

560 570 580 Ala Glu Arg Arg Leu Thr Leu Gin Gly He Ha Ser Trp Gly Ser Gly Cys Gly A Bp Arg Asn LyB Pro Gly Val Tyr Thr Asp Val Ala Tyr Tyr Leu Ala Trp GCA GAG CCC CGG CTC ACC CTG CAA GGC ATC ATC AGC TGG GGA TCG GGC TGT GGT GAC CGC AAC AAG CCA GGC GTC TAC ACC GAT GTG GCC TAC TAC CTG GCC TGG

1,695 1,710 1,725 1,740 1,755 1,770 1,785

590 596 He Arg Glu His Thr Val Ser STOP ATC CGG GAG CAC ACC GTT TCC TGA TTG CTC AGG GAC TCA TCT TTC CCT CCT TGG TGA TTC CGC AGT GAG AGA GTG GCT GGG GCA TGG AAG GCA AGA TTG TCT CCC

1,800 1,815 1,830 1,845 1,860 1,875 1,890

ATT CCC CCA GTG CGG CCA GCT CCG CGC CAG GAT GGC GCA GGA ACT CAA TAA AGT GCT TTG AAA ATG CTC Poly A 1,905 1,920 1,935 1,950 1,959

Results / 79

factor XII mRNA and probably represents a cloning artifact.

3. Predicted Amino Acid Sequence of Human Factor XII

Translation of the consensus cDNA sequence using the standard genetic code results in a

single open reading frame coding for human factor XII (Fig. 8). The derived amino acid

sequence agrees well with those regions of human factor XII that have been determined by

protein chemistry techniques. Nucleotides 19-78 encode the amino-terminal region of factor

XII (Fujikawa et al., 1980b; Fujikawa and Davie, 1981). Since the amino-terminal residue

of plasma factor XII is encoded by nucleotides 19-21 (Fig. 8), the factor XII cDNA sequence

also encodes part of an amino-terminal extension (nucleotides 1-18, Fig. 8). Nucleotides

1021-1047 and 1078-1806 encode the light and heavy chains of 0-factor X l l a as determined

by Fujikawa and McMullen (1983). In the presence of an anionic surface, kallikrein cleaves

factor XII at a position carboxy-terminal to A r g 3 5 3 , resulting in the formation of a-factor

X l l a (Fujikawa and McMullen, 1983). Further proteolytic cleavages occur

carboxy-terminal to A r g 3 3 4 and A r g 3 4 3 , and give rise to 0-factor Xl la . During the

formation of ^-factor X l l a , residues 1-334 and 345-353 of factor XII are removed. The

codon for the carboxy-terminal residue of the heavy chain of /3-factor X l l a (Ser-596) is

followed by a T G A stop codon (nucleotides 1807-1809, Fig. 8), a 3' untranslated region of

150 nucleotides and a polyA + tail. Nucleotides 1937-1942 contain the sequence A A T A A A

that is involved in the polyadenylation of mRNA (Proudfoot and Brownlee, 1976; Wickens

and Stephenson, 1984). This sequence occurs 17 nucleotides upstream of the polyA + tail.

The predicted amino acid sequence of factor XII corresponds to a mature protein of 596

amino acids. The amino acid composition of plasma factor XII was determined to be as

Results / 80

follows: A l a 5 1 , A r g 3 9 , A s n 1 3 , A s p 2 2 , l / 2 - C y s 4 0 , G l n 3 7 , G l u 3 1 , G l y 4 8 , H i s 2 7 , He,,

L e u 5 3 , L y s l 9 , Met 4 , P h e 1 5 , P r o 5 6 , S e r 3 6 , T h r 3 4 , T r p 1 3 , T y r 1 9 , V a l 3 0 . From the

predicted amino acid sequence, the molecular weight of plasma factor XII is 66,915 in the

absence of carbohydrate and 80,427 with the addition of 16.8% carbohydrate (Fujikawa and

Davie, 1981). Within experimental error, both the predicted amino acid composition and

molecular weight of factor XII are in excellent agreement with those determined for the

purified protein (Griffin and Cochrane, 1976; Fujikawa and Davie, 1981).

The complete amino acid sequence for human a-factor X l l a was also determined by

McMullen and Fujikawa (1985). The amino acid sequence was determined by automated

Edman degradation using peptides produced by chemical and enzymatic cleavages of factor

XII. These results appeared in the literature after the completion of the cDNA sequence for

factor XII described above. The two amino acid sequences were compared, and a serine

residue at position 314 is found in place of a proline residue reported by McMullen.and

Fujikawa (1985).

4. 5' End of the Factor XII cDNA

The factor XII cDNA clone, pcHXII-501, did not extend to the initiator methionine residue

and so another human liver cDNA library (prepared by W. Funk in Dr. MacGillivray's

laboratory) was screened with a [3 2P]-labeled 280 bp PstI probe representing nucleotides 1

to 280 of pcHXII-501. The library was prepared by using oligonucleotides as random

primers for reverse transcription of mRNA. Fragments were separated on a column of

Agarose A 0.5M, and were cloned into the expression vector Xgt l l . A library containing

small inserts (less than 500 bp) was screened for a clone that would extend further 5' to

Results / 81

pcHXII-501. A total of 1 x 10 6 Xgt l l recombinant phage were screened and two positive

clones were isolated. The nucleotide sequences of the cDNA clones (pcHXII-WF7 and

-WF9) were determined (Fig. 9). The two clones were identical and contained 220 bp

inserts and extended a further 40 bp 5' to pcHXII-501. Translation of the sequence

revealed an identical amino acid sequence to pcHXII-501 but extended the signal sequence

by a further 13 amino acid residues. At this point, there is an A T G sequence in the same

reading frame as the putative signal peptide. Therefore, the signal peptide sequence for

factor XII probably consists of 19 amino acid residues with the characteristic hydrophobic

core (Watson, 1984). Two other cDNA libraries were screened in order to obtain a longer

cDNA clone which would contain the 5' untranslated end of the mRNA but no longer clones

were isolated.

5. A 2.40 kb mRNA From Liver Codes For Human Factor XII

To determine the size of factor XII mRNA, human liver polyA + RNA was electrophoresed

on an agarose gel under denaturing conditions and transferred to nitrocellulose. The blot

was hybridized to [ 3 2P]labeled pcHXII-501, which contains 1959 bp of coding sequence.

Autoradiography revealed a single band that was 2,400 ± 100 nucleotides in size (Fig. 10).

The total length of mRNA sequenced from the cDNA clones, pcHXII-501 and pcHXII-WF7,

is 2000 nucleotides. Since eukaryotic mRNAs usually contain polyA + tails of 180-200

nucleotides (Perry, 1976) it seems that there is approximately 200 bases of mRNA

sequence still to be determined.

Figure 9: Nucleotide Sequence of a cDNA for the, FXII Signal Peptide

A human liver cDNA library was screened with a 3 2P-labeled 5' fragment from pcHXII-501

(Figure 7) and a positive clone (pcHXII-WF7) was isolated. The nucleotide sequence of this

clone was determined and is shown. The predicted amino acid sequence is given below the

nucleotide sequence. The numbers correspond to the cDNA sequence given in Figure 8.

The open arrow indicates the signal peptidase cleavage site and the diamond shows the

initiator Methionine residue. Only 3 nucleotides of the 5' untranslated end were contained

in the pcHXII-WF7 clone.

• GCC ATG AGG GCT CTG

Met Arg Ala Leu -19

60 GAG TCA ACA CTT TCG Glu Ser Thr Leu Ser

105 AAG TAC AAA GCT GAA Lys Tyr Lys Ala Glu

150 GAG CCC TGC CAC TTC Glu Pro Cys His Phe

195 AAA TGT ACC CAC AAG Lys Cys Thr His Lys

30 CTG CTC CTG GGG TTC Leu Leu Leu Gly Phe

• * ATT CCA CCT TGG GAA l i e Pro Pro Trp Glu

120 GAG CAC ACA GTC GTT Glu His Thr Val Val

165 CCC TTC CAG TAC CAC Pro Phe Gin Tyr His

210 GGC CGG CCA GGC CCT Gly Arg Pro Gly Pro

45 CTG CTG GTG AGC TTG Leu Leu Val Ser Leu -10

90 GCC CCC AAG GAG CAT Ala Pro Lys Glu His

135 CTC ACT GTC ACC GGG Leu Thr Val Thr Gly

180 CGG CAG CTG TAC CAC Arg Gin Leu Tyr His

225 CAG CCC TGG TGT GCT Gin Pro Trp Cys Ala

228 ACC Thr

Figure 10: Size Determination of the Factor XII mRNA Using rRNA Markers

Human liver poly A + RNA (10 Mg) was electrophoresed in a denaturing

agarose-formaldehyde gel and was transferred to nitrocellulose. The filter was hybridized

with 3 2P-labeled pcHXII-501. The filter was exposed for 18 hours at - 7 0 ° C with Lightning

Plus intensifying screens (Dupont). The positions of RNA size standards are indicated,

including mammalian 28 S (4.72kb) and 18 S (1.87 kb) rRNA and E. coli 23 S (2.90 kb) and

16 S (1.54 kb) rRNA (Noller, 1984).

4.72 _ 2.90 _ 1.87 1.54 >

B. THE HUMAN FACTOR XII GENE

Results / 86

1. Isolation and Characterization of Factor XII Genomic Clones

The factor XII gene was isolated from a human genomic D N A library (provided by Dr. P.

Leder) which contains partial Sau3A fragments (10-20 kbp in length) inserted into the

BarriHI site of XCharon 28. One million phage from this library were screened with a

human factor XII cDNA (pcHXII-501) as a hybridization probe. This cDNA contains D N A

representing coding sequence for the complete zymogen of factor XII, 6 amino acids of the

signal peptide and the 3' untranslated region (150 bp). Two recombinant phage designated

XHFXII-27 and XHFXII-76 were purified to homogeneity by rescreening four times with the

same hybridization probe.

D N A was isolated from large scale lysates of each of the two phage and analyzed by

restriction enzyme digestion. Southern blot analysis indicated that the 5' and 3' ends of the

cDNA were represented in the two phage. SamHI and PstI digests of the clones were

hybridized with 3 2P-labeled 280bp or 360 bp PstI fragments, representing the 5' and 3' end

of the cDNA, respectively. Figure 11 shows that the 280bp PstI fragment which encodes

exons 2,3 and part of 4 (see Fig. 12) hybridized to the 2.5 and 0.6 kbp PstI fragments in

XHFXII-76 and only to the 0.6 kbp PstI fragment in XHFXII-27. The 360 bp PstI fragment

representing exon 14 (see Fig. 12) hybridized to XHFXII-27 and not to XHFXII-76. The 2.0

kbp and the 0.3 kbp PstI fragments and a very large BamHl fragment in XHFXII-27

hybridized to the 3' probe. Therefore, XHFXII-76 contains the 5' but not the 3' end of the

cDNA whereas XHFXII-27 contains the 3' end and not the 5' end of the cDNA.

Figure 11: 5' and 3' Southern Blot Analysis of FXII Genomic Clones

Two clones (XHFXII-27, -76) were isolated from a genomic X phage library encoding the

human factor XII gene. The D N A was cleaved by restriction endonucleases, the fragments

were electrophoresed through neutral agarose gels and Southern (1975) blotted to

nitrocellulose filter paper. Two sets of filters were prepared and one set was hybridized

with a 3 2P-labeled 280 bp PstI (nucleotides 1-280, 5' end of the cDNA, Figure 9) or with a

3 2P-labeled 360 bp PstI fragment (nucleotide 1,680-3' end of the pcHXII-501 insert, Figure

9). The blots were autoradiographed for 18 hours. Left side blot is hybridized with the 280

bp fragment: A, XHFXII-76 digested with PstI (Lane P) or Bam HI (Lane B); B, XHFXII-27

digested with PstI (Lane P) or Bam HI (Lane B). Right side blot is hybridized with the 360

bp fragment: C, XHFXII-76 digested with PstI (Lane P) or BamHl (Lane B)\ D, XHFXII-27

digested with PstI (Lane P) or Bam HI (Lane B).

Results / 89

A partial restriction enzyme map of the cloned genomic D N A for FXII was constructed from

multiple restriction endonuclease digestions and Southern blot analysis of the fragments

hybridized to various 3 2P-labeled cDNA probes. The map (Fig. 12) shows that XHFXII-27

and -76 contained D N A sequence which overlapped but were not identical. Together, the

two phage span approximately 30 kb of genomic D N A , with the factor XII gene mapping to

a 12 kbp region in the middle of this D N A (Fig. 12). A second, unamplified human genomic

phage library (prepared by V. Geddes in Dr. MacGillivray's lab) was screened with

[3 2P]-labeled 280 bp PstI fragment of pcHXII-501. However, of 0.5 x 10* phage screened,

no positive clones for factor XII were isolated.

2. Southern Blot Analysis of the Human FXII Gene

The BamHl, HindTH and PstI fragments in the restriction enzyme map correspond to those

in the Southern blot (Fig. 13) of genomic D N A that was hybridized to [3 2P]-labeled factor

XII cDNA (pcHXII-501). Autoradiography revealed several hybridizing bands in each of

the restriction enzyme digests (Fig. 13). For example, the lengths of the BamHl (greater

than 9.5 and 7.6 kbp), the flmdIII (9.5 and 5.8 kbp) and the PstI (2.5, 1,5, 1.0 and 0.6 kbp)

fragments agree closely with the fragment lengths which contain exon sequence in the

restriction enzyme map (Fig. 12). The 7.6 kbp BamHl bands observed in the genomic

Southern blot compares to the 3' end of the gene including most of exon 14. The 3' BamHl

site is not shown in the restriction enzyme map. Since no other bands were observed in the

genomic Southern blot, it appears that the human genome contains a single factor XII gene.

Figure 12: Partial Restriction Map and Intron/Exon Organization of the FXII Gene.

A partial restriction enzyme map of the gene is given. Abbreviations used are: B-BamHI;

Hp-ffpall; Bg-BglL; P-Pstl; H-HindIU; Hc-Hincll. Exons are shown in the bar above the

restriction map as black boxes and are numbered 1 through 14. The two genomic phage

clones. XHFXII-76 and XHFXII-27 are shown below the restriction enzyme map as open

bars. Breaks in the bars indicate that the phage D N A extends approximately 5.0 and 13.0

kb 5' and 3' from the gene, respectively. The scale represents kilobase pairs.

0 1.0 2.0 3.0 4.0 5.0 6.0 7.0 8.0 9.0 10.0- 11.0 12.0 13.0 14.0 15.0 Scale (kb) I I I l I I I I I I I I I I I I

Exons 1 2 3 4 5 6 7 8 9 10 11 12 13 14

Gene 5' I I I I 3'

Restriction BP P Bg Hp P P H H P H P P B P B P B H

Enzyme Map II j l ( l I ; L _ l I I M I I II 11 _ | Hp He p

X Clones ] / / . I XHFXII-76

XHFXII-27 I Ij —

Figure 13: Genomic DNA Southern Blot Analysis of the FXII Gene

Human liver D N A (lOjug) was digested by various restriction enzymes and electrophoresed

in a 1.0% agarose gel. After denaturation, the D N A was trasferred to nitrocellulose and

hybridized with 3 2P-labeled pcHXII-501. The filter was autoradiographed for 4 days at

- 7 0 ° C with intensifying screens. Lane M shows the positions of the 3 2P-labeled \-Hindlll

D N A used as molecular weight markers. The human D N A was digested with EcoRl (Lane

1), Hindlll (Lane 2), Pstl (Lane 3), and BamHl (Lane 4).

M 1 2 3 4 23.70 4

2.25 1 .96 -

0.59 -A

Results / 94

3. Localization of Intron/Exon Junctions

To identify exon-containing D N A sequences, sonicated fragments of the genomic clone,

XHXII-27, were subcloned into M13 vectors and transformed into E. coli JM103. The

phage were plated and screened with 3 2P-labeled pcHXII-501. The D N A sequences were

determined from purified single stranded D N A of the positive phage. The intron/exon

junction sequences for exons 3 to 14 were obtained on both strands and are given in Table

II. The positions of exons 1 and 2 in XHXII-76 were determined by restriction enzyme

analysis. Small fragments containing the exons were subcloned and their D N A sequences

determined on both strands (Table II). All introns follow the G T - A G rule (Breathnach and

Chambon, 1981) except for the donor sequence of intron J which has a GC dinucleotide

instead of GT. The consensus sequences surrounding splice junctions in R N A polymerase II

transcribed genes (Mount, 1982) were also found to be in agreement with the intron/exon

junctions of the FXII gene (Table III). The localization of exons by D N A sequence analysis

and restriction enzyme mapping has shown that the FXII gene consists of 13 introns (A

through M , Table IV) and 14 exons (1-14, Table IV) of which 12 are located within 4.2 kb of

genomic D N A (Fig. 12).

4. Nucleotide Sequence of the Human Factor XII Gene

The partial D N A sequence for the human factor XII gene is shown in Fig. 14. A total of

4,735 bp of D N A sequence was determined of which 92% was obtained on both strands.

Sequence data that was obtained on one strand only was determined at least twice and

includes nucleotides -120 to -357 of the 5' end of the gene. There are 357 and 195 bp of 5'

and 3' flanking regions, respectively, given in Fig. 14. As determined by D N A sequence

EXON 5 ' SPL ICE 1NTRON 3' SPL ICE CODON NUMBER DONOR ACCEPTOR PHASE

1 T T C G g t g a g t A c c t t c c c t c c t c c c t t g t a gATTC 0

2 G T C T g t a a t g B a gag t g t g t t g t c c c t g c a gTTCT

3 C C T G g t a a g a C a g c c t t g t c t c t t t c t a c a gGTGT 1 1

4 AAAGg t g c t a D c c a c c a t g t c c a t c t c t c a gACCA >

5 AAAGgtgagg E c t a t c c c t c t t t g t c c c c a gAGAA

6 CAGG g t g a g c F c a c t g c g t t c c c t c c c c a a gCCTG

7 G T G G g t g a g t G g g a g c c c c c t t t c t c c t c a gACAC

8 G C C G g t g c g c H g c c c c g t c g t g t g g c t a c a gGAAC 1 1

9 GGAGgt t a gg 1 c a g c c c c t g c t c c t c c a c a gCCTT

1 0 ACCGgcgag t J c t c c c c t a c c c t c c c c g c a gGCCC 1 1

1 1 C T G G g t g c g t K c t c c t c t c c g c c c g g g t t a gCTCT

12 GAGCg taggc L a a g g c g c t c t g t g t t c gcagGGGC

1 3 CCAGg tgagc M g c t g c g t g t t t c c g a c c c a gGGTG 0

14 3 1 END

T a b l e 1 1 . N u c 1 e o t 1 d e S e q u e n c e o f I n t r o n / E x o n J u n c t i o n s In the Fa

XI I G e n e .

Exon s e q u e n c e i s shown in upper c a s e ; i n t r o n s e q u e n c e in lower c a s e .

The codon phase r e f e r s to the p o s i t i o n o f the I n t r o n In the codon

t r i p l e t . 0 - i n t r o n o c c u r s be tween c o d o n s , l - i n t r o n o c c u r s a f t e r the

f i r s t n u c l e o t i d e and I l - i n t r o n o c c u r s a f t e r the s e c o n d n u c l e o t i d e In

t h e c o d o n .

Donor F r e q u e n c i e s

+ 4 + 3 + 2 + 1 -1 -2 - 3 - 4 -5 - 6

G 5 1 4 10 1 3 0 9 1 1 1 3 A 3 4 4 0 0 0 3 9 0 2 T 1 4 1 1 0 1 2 1 0 2 4 C 4 4 4 2 0 1 0 3 0 4 CON N A A G G T R A G T

C A c c e p t o r F r e q u e n c i e s

- 2 0 - 1 9 -1 8 - 1 7 -1 6 -1 5-1 4 - 1 3 -1 2-1 1 -1 0 - 9 -8 - 7 - 6 -5 - 4 -3 -2 -1 + 1 + 2 + 3 + 4

G 3 3 2 4 1 4 2 3 2 1 3 0 3 3 1 1 4 0 0 1 3 5 3 2 2 A 3 3 4 0 0 1 0 1 0 0 0 1 0 0 1 0 3 1 1 3 0 4 1 3 2 T 0 3 2 3 3 1 6 6 4 7 5 7 3 5 3 4 3 2 0 0 1 4 4 4 C 7 4 5 6 9 7 5 3 7 5 5 5 7 5 8 8 3 1 0 0 0 3 5 4 5 CON Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y Y N Y A G G N N N

T a b l e I I I . F r e q u e n c i e s of N u c l e o t i d e s at I n t r o n / E x o n J u n c t i o n s .

The f r e q u e n c i e s of the d i f f e r e n t n u c l e o t i d e s at the i n t r o n / e x o n

j u n c t i o n s of the human FXI I gene a re compared to the c o n s e n s u s

(CON) of Mount ( 1 9 8 2 ) . S p l i c e j u n c t i o n s a re be tween -1 and +1.

NUCLEOTIDE POSIT ION

4 7 4 1 - 4 7 9 8

7 7 9 0 - 7 8 8 9

8 0 4 6 - 8 1 1 6

8 4 1 7 - 8 5 2 7

8 6 6 6 - 8 7 9 7

8 9 3 9 - 9 0 4 3

9 1 9 1 - 9 3 5 6

9444-9661

9 7 6 7 - 9 9 9 8

1 0 2 4 2 - 1 0 3 7 8

1 0 4 6 0 - 1 0 6 0 3

1 1 1 5 9 - 1 1 3 0 7

1 1 3 9 8 - 1 1 7 1 5

LENGTH

INTRON

NUCLEOTIDE POSIT ION

1 0 6 - 4 7 4 0

4 7 9 9 - 7 7 8 9

7 8 9 0 - 8 0 4 5

8 1 1 6 - 8 4 1 6

8 5 2 8 - 8 6 6 5

8 7 9 8 - 8 9 3 8

9 0 4 4 - 9 1 9 0

9 3 5 7 - 9 4 4 3

9 6 6 2 - 9 7 6 6

9999 -10241

1 0 3 7 9 - 1 0 4 5 9

1 0 6 0 4 - 1 1 1 5 8

1 1 3 0 8 - 1 1 3 9 6

LENGTH

4 5 0 0 »

3 0 0 0 *

T a b l e IV. S i z e and P o s i t i o n s o f Exons and I n t r o n s in the F a c t o r XI I Gene .

• S i z e s of t h e s e i n t r o n s were e s t i m a t e d f r o m r e s t r i c t i o n enzyme a n a l y s i s

( F i g . 1 2 ) . A l l o t h e r s i z e s were d e t e r m i n e d f rom the n u c l e o t i d e s e q u e n c e

of the gene ( F i g . 1 4 ) .

Figure 14: Partial DNA Sequence of the FXII Gene

The sequence was determined by the D N A sequence analysis of M13 clones. The predicted

amino acid sequence of FXII is given above the nucleotide sequence. Five transcriptional

start sites are indicated by triangles and circles between +1 and +33. Solid and open

triangles indicate strong and weak primer extension products, respectively; solid and open

circles represent strong and weak nuclease S i products, respectively. A solid line indicating

a possible T A T A sequence is shown at position -26. The CAAT-like sequence at position

-103 is shown as a double line. Inverted repeats are marked by arrows under the nucleotide

sequence. An open diamond represents Hpall sites that demarcate the nuclease S i primer.

A closed diamond indicates the complemented 5' end of the 180 bp primer used in the

extension reaction. The probable initiator methionine at amino acid -19 is indicated by a

box. The sizes of the larger introns are approximated from restriction enzyme mapping

(Fig. 13). The polyadenylylation signal A A T A A A (nucleotides 11693-11698) is boxed. In

the protein coding region, the cleavage site which gives rise to plasma FXII is denoted by a

closed arrow, and the two sites of cleavage which produces (3-FXIIa by kallikrein are

denoted by an open arrow.

99 CT GGG TAT TGT TCT AAG ATG CTC AGT TTA TCG TAG TTT GTT ACA TCA CAA TAG AAA ATC AAC ACA CTT CAC AGT GGA CTC CAA GAT CCC CAT GAT CTT TCA TCT

-356 -330 -300 -270

CCT TAA CCT CCT GAT CTC CAC AGG ACC CAG AGC ATA AGA ATG TCC CTT CTT CTG CTT CCA GTC CCA CTA TCT AGA AAA GAG AGG AGG AGC CCA UCT CTT CAT TTC -240 -210 -180 -150

0 ACC CCC ACC CAC AAA CTC CCA ACT TTC CGG CCC TCA AGG GGT GAC CAA GGA AGT TGC TCC ACT TCG CTT TCC ACA AAC AGC CTC TCC CCC ACC AGG CTC AGG AGG

-120 -90 -60 • -19

Q # • O O M e t A r 9 A l a L e u t e u

GCA GCT TGA CCA ATC TCT ATT TCC AAG ACC TTT GGC CAG TCC TAT TGA TCT GGA CTC CTG GAT AGG CAG CTG GAC CAA CGG ACG GAC GCC ATC AGG GCT CTG CTC -30 • 1 A • A 30 A 6 0

1 1 - A i • -io 0 • "l

Leu Leu Gly Phe Leu Leu Val Ser Leu Glu Ser Thr Leu Ser CTC CTG GGG TTC CTG CTG GTG AGC TTG GAG TCA ACA CTT TCG GTG AGT GCT GTG GGA ACC AGG ATT GTC CCA GGA TTG TTC TGG GGG GTC GCT ATC ACA GCC ATG

90 120 150 * AGC CAT GGC CTC TGC TCA TGA CCT GTG GGT CCA GGT GAC TAG GAG GCC TAT GTG GAA AGG TGA GGC CAJ CCC 4300 bp AG GCC AGC CCG GAA GGC CCA

180 210 240 GGC AGA GGA GAC AGA CAA CCA GAC TGG GTG GAT ACA AGG GCA CAG CCT GCA TTT CTG GGG GAG ATG GGC CTT AAG AAG ACA ACG GGG GGA GGT AGA AAG CGA AAG

4530 4560 4590 4620

GGT CTT GGG AAG AAA TCT CTG CAT TTC TGG GCV GTG AGA GGA AGC TGC AGA CTA GCA ACA GAT CGG TGG CAG GCT ATG ACT TAT AGT CAG TTC CCT GCC TTC TTC 4650 4680 4710

\ i 10 l i e Pro Pro Trp Glu Ala Pro Lys Glu His Lys Tyr Lys Ala Glu Glu His Thr Val V

TCT CCC TTG TAG ATT CCA CCT TCG GAA GCC CCC AAG GAG CAT AAG TAC AAA GCT GAA GAG CAC ACA GTC GGT AAG TGG CCT GGC TCC TCC TCC CGG GAA CCC TTC 4740 4770 4800 4B30

GGT GGG GAT GTG TAT GGT GCA GTC TCT GCA GTC TCA GGG CAG TCT AGT CTA GTC CCT ACC TCG TCC TAG GTC TTA TGC CCA TCG GCA CTA GAG TCA TCG TCA GCT 4860 4890 4920

GTC TCA TCC TTG AGG GCA GGG TAT GGG CTG TGT CTA AGT GCC CAC GAG CCT GGC TCG GAG CAG GTC CTT GAG ATA TGT GCT GCT GGC GCC ATC ACA CCT GGG CTC 4950 4980 5010 5040

CTC CCA GCC TTC CTC AGT TTC CCC AGC TTC TCC CCT TCT TTT CCT TTC CCC AGT ACG TCT CAT GGG CAT CAT TCA TGC CAC ACA GAG GCC AGG GCC TTC AAT GGG 5070 5100 5130

CAA GGA AGG ATC AAG AGC TTC TCT CTG GCA TCT GAA TGC CTC TGA AGC CCA'GCT TTA TGA GTT ATG AGC TGG GTC ACT CTC GGC GAG GGA TTT GAG TTC TCC AAG 5160 5190 5220 5250

20 30 al Leu Thr Val Thr Gly Glu Pro Cys His Phe Pro Phe Gin Tyr His Arg Gin Leu Tyr

CTT CAA TTT C 2500 bp — T C AGA GAG TGT GTT GTC CCT GCA GTT CTC ACT GTC ACC GGG GAG CCC TGC CAC TTC CCC TTC CAG TAC CAC CGG CAG CTG TAC 7800 7830

40 50 His Lys Cys Thr His Lys Gly Arg Pro Gly Pro Gin Pro Tr CAC AAA TGT ACC CAC AAG GGC CGG CCA GGC CCT CAG CCC TGG TAA GAC TAC GCA GAG GAG TTG GAG CAG GGG CCT GGG AGA CAT GTA CCC TCC CTC TCC TTC TCT

7860 7890 7920 7950

p Cys Ala Thr Thr CCA AGG AAC TCT GCT TGG AGA GAG GGG ACT GTG ATA GGG CAG GGT GGG CCA GGC CCC TCG GTA GAG CAG GGA AGC CTT GTC TCT TTC TAC AGG TGT GCT ACC ACC

7980 8010 8040

60 70 Pro Asn Phe Asp Gin Asp Gin Arg Trp Gly Tyr Cys Leu Glu Pro Lys Lys Val Lys A CCC AAC TTT GAT CAG GAC CAG CGA TGG GGA TAC TGT TTG GAG CCC AAG AAA GTC AAA GGT GCT ACA CAC AGC CTC TGG GGT GGC CTC GGG CTC TCT CCT CCC GCC

8070 8100 8130 8160

TCA TTA CTC TCC TGG TAT CAC CAG ACC CCA CAC ACC TGG GAT TCT GGA CCC AGC CCC TTC TCT CCC TCC ACA ATA CCC TTT GGA AGT CCA GAG GGA GAG TTC TCG 8190 8220 8250

GAA GGA GTG GTC CCA TTT TCC AGG TCG GTA AAC CAA GCT TGG AAA CTT GGA GTA GCA AGG TCA CAA GGC AAG TAG GTT CAA GAA GGG CCT TCG CCC CAG CTG TCT 8280 8310 8340 8370

BO 90 sp His Cys Ser Lys His Ser Pro Cys Gin Lys Gly Gly Thr Cys Val Asn Het Pro Ser Gly

GAC TCA GCT CCC TGC TCT TCC TTC CAC CAT GTC CAT CTC TCA GAC CAC TCC AGC AAA CAC AGC CCC TCC CAG AAA GGA GGG ACC TGT GTG AAC ATC CCA AGC GGC 8400 8430 8460

100 110 Pro His Cys Leu Cys Pro Gin His Leu Thr Gly Asn His Cys Gin Lys G CCC CAC TCT CTC TCT CCA CAA CAC CTC ACT GGA AAC CAC TGC CAG AAA GGT GAG GAG ATC TGG AGG ACC TGG GCG GGG TGC TCG GGG ACA GGG GCA ACC CTG GGC

8490 8520 8550 8580

120 lu Lys Cys Phe Glu Pro Gin Leu

CTA CAG AAT AGG TTC CTC GAT ACT CGG AGA CTT GGC ATC GTC CTA GAC TCT CCT GAG ACC ACT ATC CCT CTT TCT CCC CAG AG AAG TCC TTT GAG CCT 'CAG CTT 8610 8640 8670

130 140 150 Leu Arg Phe Phe His Lys Asn Glu l i e Trp Tyr Arg Thr Glu Gin Ala Ala Val Ala Arg Cys Gin Cys Lys Gly Pro Asp Ala His Cys Gin Arg Leu Ala Ser CTC CGG TTT TTC CAC AAG AAT GAG ATA TCG TAT AGA ACT GAG CAA GCA GCT GTC GCC AGA TCC CAG TCC AAG GGT CCT GAT GCC CAC TCC CAG CGG CTC GCC AGC

8700 8730 8760 8790

CAG GGT GAG CAG ATG GTT GGG AAC GGG CCA GGG AGG AGC GTC AGG AAG ACA GGC TGG CAG GAG GCC GGG TCG TCT GCC GGG AAG GAG AGC TCT CTC GGG GGG TCT 8820 8850 8880

160 170 la Cys Arg Thr Asn Pro Cys Leu His Gly Gly Arg Cys Leu Glu Val Glu Gly His Arg Leu Cys

TTA GGC CCA GGG GTC GCT CAC TCC GTT CCC TCC CCA AG CC TCC CGC ACC AAC CCG TCC CTC CAT GGG GGT CGC TCC CTA GAG GTC GAG GGC CAC CGC CTC TGC 8910 8940 8970 9000

180 190 His Cys Pro Val Gly Tyr Thr Gly Pro Phe Cys Asp Val A CAC TCC CCG GTC GGC TAC ACC GGA CCC TTC TGC GAC GTC GGT GAG TCA GGG TCT GGG GCA AGC AGA AGG CCA GCC CCC AGG TGG GAC GGG CTT GCC AGG AAG GAG

9030 9060 9090

200 GAG GGA GAG TCC GGA AAG CAG ATG AGA GGG AGG CAG GAG AGC CCA GCC TTC GCT GCC CAG GGA GCC CCC TTT CTC CTC AG AC ACC AAG GCA AGC TCC TAT GAT

9120 9150 9180 , 2 1 0

210 220. 230 Gly Arg Gly Leu Ser Tyr Arg Gly Leu Ala Arg Thr Thr Leu Ser Gly Ala Pro Cys Gin.Pro Trp Ala Ser Glu Ala Thr Tyr Arg Asn Val Thr Ala Glu Gin GGC CGC GGG CTC AGC TAC CGC GGC CTC GCC AGG ACC ACG CTC TCG GGT GCG CCC TCT CAG CCG TCG GCC TCG GAG GCC ACC TAC CGG AAC GTG ACT GCC GAG CAA

9240 9270 9300

Ala Arg Asn Trp Gly Leu Gly Gly His Ala Phe Cys Ar GCG CGG AAC TGG GGA CTG GGC GGC CAC GCC TTC TGC CGG TGC GCC GCG TGG GGC TGG GTG ACC CCT CCG CCC CAG GGC TCC GGG CTC CCG GCG CTC TAA CGG CGC

9330 9360 9390 9420

250 260 270 g Asn Pro Asp Asn Asp l i e Arg Pro Trp Cys Phe Val Leu Asn Arg Asp Arg Leu Ser Trp Glu Tyr Cys Asp Leu Ala Gin Cys

CCC GTC GTG TGG CTA CAG G AAC CCG GAC AAC GAC ATC CGC CCG TGG TGC TTC GTG CTG AAC CGC GAC CGG CTG AGC TGG GAG TAC TGC GAC CTG GCA CAG TGC 9450 9480 951°

280 290 300 . 310 Gin Thr Pro Thr Gin Ala Ala Pro Pro Thr Pro Val Ser Pro Arg Leu His Val Pro Leu Met Pro Ala Gin Pro Ala Pro Pro Lys Pro Gin Pro Thr Thr Arg CAG ACC CCA ACC CAG GCG GCG CCT CCG ACC CCG GTG TCC CCT AGG CTT CAT GTC CCA CTC ATG CCC GCG CAG CCG GCA CCG CCG AAG CCT CAG CCC ACG ACC CGG

9540 9570 9600 9 6 3 0

320 Thr Pro Pro Gin Ser Gin Thr Pro Gly A * ACC CCG CCT CAG TCC CAG ACC CCG GGA GGT TAG GAA GTG GGG GGG GAA GGA GGA GCC GAG AGG GCG CCG GGC GAG CTA GAT TCC GGC CAG CCG GCC GCG GGC TCC

9690 9720

CCG TCC TCA GCC CCT GCT CCT CCA CAG 9750

330 VT 340 la Leu Pro Ala Lys Arg Glu Gin Pro Pro Ser Leu Thr Arg Asn Gly Pro Leu Ser Cys Gly Gin Arg Leu Arg Lys CC TTG CCG GCG AAG CGG GAG CAG CCG CCT TCC CTG ACC AGG AAC GGC CCA CTG AGC TGC GGG CAG CGG CTC CGC AAG

. 9780 9810 9840

360 370 380 Ser Leu Ser Ser Met Thr Arg'val Val Gly Gly Leu Val Ala Leu Arg Gly Ala His Pro Tyr He Ala Ala Leu Tyr Trp Gly His Ser Phe Cys Ala Gly Ser ACT CTG TCT TCG ATG ACC CGC GTC GTT GGC GGG CTG GTG GCG CTA CGC GGG GCG CAC CCC TAC ATC GCC GCG CTG TAC TGG GGC CAC ACT TTC TGC GCC GGC AGC

9870 9900 9930

390 Leu He Ala Pro Cys Trp Val Leu Thr Ala Ala His Cys Leu Gin Asp Ar CTC ATC GCC CCC TGC TGG GTG CTG ACG GCC GCT CAC TGC CTG CAG GAC CGG CGA GTA CCC GCC CGC CCA GAG CCG CCC CAG GGG CCG CGG CTC CTC CGT CTC CCA

9960 9990 10020 10050

GCG CAG CTT CCA CGC TGC ACC CGA ACC CGT GCC CTA CCT TCT CCC GCC CCA CCC TTC TTT CCA CGC CCC TCC GGA GCT CCC GGG GAG GAA.GCT GGA ACA CGG GAT 10080 10110 10140

TGG GGT TCG GGA GCA GGG GGC TTC CCC AGA ACG CTT GTG GCC AGG TCT GAG AGC GCT GCC TCT CCC CTA CCC TCC CCG CAG 10170 10200 10230

g Pro Ala Pro Glu Asp Leu Thr G CCC GCA CCC GAG GAT CTG AOG

410 420 430 440 Val Val Leu Gly Gin Glu Arg Arg Asn His Ser Cys Glu Pro Cys Gin Thr Leu Ala Val Arg Ser Tyr Arg Leu His Glu Ala Phe Ser Pro Val Ser Tyr Gin GTG GTG CTC GGC CAG GAA CGC CGT AAC CAC AGC TGT GAG CCG TGC CAG ACG TTG GCC GTG CGC TCC TAC CGC TTG CAC GAG GCC TTC TCG CCC GTC AGC TAC CAG

10290 10320 10350

His Asp Leu A l a Leu Leu Arg Leu CAC GAC CTG GGT GCG TGG GGG CGC CCC GCG GGG ACG GGA AGA GAG CTT GGG CAC GGC GTC CCC GCC TCA CCC TCC TCT CCG CCC GGG TTA GCT CTG TTG CGC CTT

10380 10410 10440 10470

450 460 470 480 Gin Glu Asp Ala Asp Gly Ser Cys Ala Leu Leu Ser Pro Tyr Val Gin Pro val Cys Leu Pro Ser Gly Ala Ala Arg Pro Ser Glu Thr Thr Leu Cys Gin Val CAG GAG GAT GCG GAC GGC AGC TGC GCG CTC CTG TCG CCT TAC GTT CAG CCG GTG TGC CTG CCA AGC GGC GCC CCG CGA CCC TCC GAG ACC ACG CTC TGC CAG GTG

10500 10530 10560

490 Ala Gly Trp Gly His Gin Phe Glu G GCC GGC TGG GGC CAC CAG TTC GAG GGT AGG CAC AAC TGC TAC GGG CAG GCC TAG GGG ACC AGA CCT TTG ATC ACT GGC TTA CGC GGA AGA AGC CCG CGA CTT TGG

10590 10620 10650 10680

TAT CGT TCC GGG TGC CTA CAG AAT GGG TGG CGC TGA CCT GAT GGG TTG TCA CAA TGT GTA CGT GAA TCC CAC GTA GAA TCC CAG GGC CTG CCA TTC ACT GCT GGG 10710 10740 10770

ATC CCC AAA TCT CCT GGG GAT ACA GGG AGA ATC GAA CTT GCT CTT GGT TCC CTC TGG GCC CCC CCC TCC AAA CGC CAA CTA GGA CGC TGG CCC CGC GCT CCG GGC 10800 10830 10860 10890

TAG TGT GGG AGC CAG GTT CTG CGA CTC TGC ATG GGT GGT GGG GGA GGG GTT TCT GTT TCC GCT CCC CCC ATT CAA ATC CTC GCT TTT CTC TCC ACC TCA CCC TCC 10920 10950 10980

TTG CCT ATG AAA TTG AAT TAA TGG CAC CTC CTC CCC TTC GGG CTT CCT GCG AGA CAC GAA GCG CAT CAG TGG GTT TAC AAG CGC CTG GAG CAC CTT TGT CCA TCG 11010 11040 11070 11100

500 ly Ala Glu Clu Tyr Ala Ser Phe Leu Gin Glu Ala Gin Val Pro Phe Leu

TCC GGG CGG CAA GCG TTG TCA GAT GGG GTC TGA AGA AGC CGC TCT GTG TTC GCA GGG GCG GAG CAA TAT GCC AGC TTC CTG CAG GAG GCG CAG GTA CCG TTC CTC 11130 11160 11190

510 520 530 540 Ser Leu Glu Arg Cys Ser Ala Pro Asp Val His Gly Ser Ser He Leu Pro Gly Met Leu Cys Ala Gly Phe Leu Glu Gly Gly Thr Asp Ala Cys Gin TCC CTG GAG CGC TGC TCA GCC CCG GAC GTG CAC GGA TCC TCC ATC CTC CCC GGC ATC CTC TCC CCA GGG TTC CTC GAG GGC GGC ACC GAT GCG TGC CAG GTG AGC

11220 11250 11280 11310

Gly Asp Ser Gly Gly Pro Leu TCT TAG CCC GGT TGG CGC CCT TCC CCG AGG CCG TCA GCC ACA AAT CTC AGG TCC ACA GCG CTC ACC TGC CTG TTT CCG ACC CAG GGT GAT TCC GGA GGC CCG CTG

11340 11370 U400

550 560 570 5 8 0

Val Cys Glu Asp Gin Ala Ala Glu Arg Arg Leu Thr Leu Gin Gly He l i e Ser Trp Gly Ser Gly Cys Gly Asp Arg Asn Lys Pro Gly Val Tyr Thr Asp Val GTG TGT GAG GAC CAA GCT GCA GAG CGC CGG CTC ACC CTG CAA GGC ATC ATC AGC TOO GGA TOO GGC TGT CGT GAC CGC AAC AAG CCA GGC GTC TAC ACC CAT GTG

11430 11460 11490 11520

590 596 Ala Tyr Tyr Leu Ala Trp H e Arg Glu His Thr Val Ser STOP GCC TAC TAC CTG GCC TGG ATC CGG GAG CAC ACC GTT TCC TGA TTG CTC AGG GAC TCA TCT TTC CCT CCT TGC TGA TTC CCC AGT GAG ACA GTG GCT CGG GCA TGG

11550 11580 1 1 6 1 0

AAG GCA AGA TTG TGT CCC ATT CCC CCA GTG CGG CCA GCT CCG CGC CAG GAT GGC GCA GGA ACT 0>A TAA ifcT GCT TTG AAA ATG CTG ACA AGG AAA CCT CTT TTC 11640 11670 TTTOO 11730

TTC ATG GGT CCG CCG GGA AAT GCC AAG ACA GAA AAG CGA TTC ACA GCT TCT CCA CAG CTC TCA GAG AAC AAG GTC TAT GAG ATC TTA ACG TCC AAA ATC TAG ATG 11760 11790 11820

CCA GCC CAG CTA ATG TTT ACT GAG CCT AGG ATA CTG TAT ACC AAG CCC TGT GCA AGG AGA AGC TGC ATG TTA TT 11850 11880 11910

Results / 100

analysis of the 4.2 kb region described above and by sizing of introns A and B by restriction

enzyme analysis the total length of the FXII gene is approximately 12 kb. Transcription

initiation was determined by nuclease S1 mapping and primer extension analysis (described

below) and is designated as nucleotide +1 in Figure 14. The nucleotide sequence G A C GCC

precedes the A T G codon. This sequence agrees with the consensus sequence found prior to

initiator methionine residues (Kozak, 1984). The conserved A A T A A A (Proudfoot and

Brownlee, 1976) in the 3' flanking sequence was identified at position 11693-11698. D N A

sequence analysis showed that there is an AZuI-like repetitive element (Schmid and Jelinek,

1982) at nucleotide -370 (data not shown) preceeding the 5' end of the gene for factor XII.

Furthermore, two other Alul elements were identified in intron B by D N A sequence

analysis although their positions in the gene were not determined.

The sizes of the exons and introns and their positions in the gene are given in Table IV.

Exons range in size from 57 to 320 bp, and introns range in size from 80 to 4,500 bp. Exon

14 is the largest exon (320 bp) and includes the 3' untranslated region of the mRNA. The

average length of the remaining exons is 132 bp which is similar to the average length of

eukaryotic exons of 150 bp (Naora and Deacon, 1982). The predicted size of the mRNA is

2,036 bp which is in close agreement with the size of the mRNA determined by Northern

blot analysis (2,400 +/- 100 bp, including a poly A + tail). The predicted amino acid

sequence of the gene coding sequence and the cDNA sequence were identical except for a

proline 3 1 4 (CCT) in the gene compared to a serine residue (TCT) in the cDNA sequence.

Results / 101

5. Transciption Initiation Site of the Factor XII Gene

The site of transciption initiation was determined by primer extension and nuclease SI

mapping analysis of the 5' end of the gene. The primers used in these experiments are

indicated in Fig. 14 and the reaction products are shown in Figs. 15 and 16. The nucleotide

sequences of the templates from which the primers were prepared are also given in Figs. 15

and 16. The furthest extension product is a weak band seen with a long exposure of the gel

(7 days) and corresponds to position +1 in Fig. 14. This extension product corresponds to

the longest nuclease Sl-resistant fragment. Other nuclease SI and primer extension

products were observed and could be aligned within 1 nucleotide of each other. These

positions in the 5' end of the gene are: +14 and + 22, represented by two strong bands in

the primer extension reactions, and +27 and +33, represented by weak bands in the

autoradiograms of both the primer extension and nuclease SI reactions. Higher molecular

weight bands were not observed even with prolonged exposures (two weeks) of the gels (Fig.

15). Since the nuclease S l and primer extension products correspond to one another, it

appears that there is no intron within the 5' untranslated region of the factor XII gene. The

primer extension experiment was repeated three times resulting in the identical banding

pattern seen in Fig. 15. Furthermore, longer (220 bp) and shorter (50 bp) primers were

used in the extension reaction; the longer primer folded upon itself and could not prime

RNA, and the shorter primer resulted in only a few nucleotides of extension. No extended

product was observed when oligonucleotide primers (17 to 20 bp in length) were used in the

primer extension reaction. In separate experiments, the primer extension and nuclease S l

reactions of the primer without mRNA showed no extended product and no protected

fragments, respectively (data not shown).

Figure 15: Nuclease S l Mapping of the 5' End of the FXII Gene.

A 450 bp Hpall fragment containing all of exon 1 and 5' flanking sequence was used to

obtain a 228 bp probe for the nuclease S l mapping reaction. The probe begins at the

position 90 and extends to position -120 (Fig. 15). The 3 2P-labeled fragment was

hybridized to 18 ug Poly A + mRNA and was digested with 200 units nuclease S l for 1 hour

at 3 7 ° C . The reaction was electrophoresed in a 6% polyacrylamide-8M urea gel along with

a D N A sequencing ladder of the probe D N A (Lanes GATC). The gel was autoradiographed

for 18 hours (Lanes GATC) and 3 days with an intensifier screen and - 7 0 ° C (Lane 1). Lanes

GATC are Sanger sequencing reactions of the probe DNA. Lane 1 is the nuclease S l

reaction of poly A + mRNA from liver. The wide arrows show the nuclease S l digestion

products and the narrow arrows, the exact nucleotide to which the nuclease S l band

corresponds.

G A T C

Figure 16: Primer Extension Analysis of the 5' End of the FXII Gene.

A 180 bp 3 zP-labelled fragment of sequence complementary to FXII mRNA was hybridized

to 18 Mg of poly A + mRNA from liver and extended from the Hindll site at position 96 bp

(Fig. 15). The reaction products were electrophoresed in a 6% polyacrylamide-8M urea gel

along with Sanger sequencing reactions performed on the pcHXII-WF7 used in preparation

of the primer. Lanes 1 and 2 are primer extension products of mRNA from liver reacted

with reverse transcriptase at 3 7 ° C and 4 5 ° C for 1 hour. The gel was exposed for 18 hours

[Lanes GATC) and for 7 days at - 7 0 ° C with an intensifying screen {Lanes 1 and 2). Lanes

G A T C are the sequencing reations of the primer DNA. P indicates the primer before

extension and wide arrows indicate the extended products. Met indicates the A T G initiator

methionine sequence. Hell is the Hindi restriction site used to generate the single stranded

primer used in the primer extention reaction. The numbers beside the sequencing gel

correspond to the extended product sizes counted from the Met (ATG) (as "CAT" in the gel

shown) sequence.

Results / 106

The position of the nuclease Sl-protected product was verified further by hybridizing an

oligonucleotide complementary to nucleotides -23 to -6 (Fig. 14) to a Northern blot (Fig. 17)

of human liver polyA + mRNA. This oligonucleotide did not hybridize to the mRNA although

another oligonucleotide complementary to the region +89 to +106 did hybridize.

Therefore, the sequence immediately upstream of the putative transcriptional start site does

not contain coding sequence.

The D N A sequence of the 5' flanking region to the transcription initiation site was

determined and was analyzed for the characteristic recognition sequences " T A T A " and

"CAAT". The closest sequences to these consensus sequences were a " C T A T T T C " at -26

and a " C C A A G " at -105. The SV40 enhancer core consensus TGG(A/T)(A/T)(A/T) was

found at position -330 (ATGGTAGT). A similar sequence, A T G G T A T G , was observed at

position -123 in the promoter region of the albumin gene (Gorski et al., 1986).

Figure 17: Northern Blot Analysis of the 5' End of the FXII Gene

Human liver poly A + mRNA (10 Mg) was electrophoresed in a denaturing

agarose-formaldehyde gel. The gel also contained BamtLVHindUI digestion product of a

subcloned fragment (pB/H76-5.5) of XHXII-76 representing the 5.5 BamHI/ffmaTII

fragment at its 5' end (see Fig. 14). The nucleic acids were transferred to nitrocellulose and

hybridized with two 3 2P-labeled oligonucleotides ("a" and "b") representing -23 to -6 (oligo

"a") and +89 to +106 (oligo "b")(Fig.l5) of the gene. The filter was autoradiographed for

24 hours at - 7 0 ° C with an intensifying screen. Lane A, pB/H76-5.5 digested with

BamHI/HindlU and hybridized with the "b" oligo; Lane B, poly A + mRNA hybridized with

oligo "b"; Lane C, polyA* mRNA hybridized with oligo "a"; Lane D, pB/H76-5.5 digested

with BamHI/i/mdIII and hybridized with the "a" oligo. The numbers indicate the lengths of

the XDNA-Hmdll l marker fragments.

A B C D

9.46-6-66-

4 .25- •

2.25-1 9 6 -

IV. DISCUSSION

A. CHARACTERIZATION OF THE HUMAN FACTOR XII CDNA

1. Characterization of Factor XII cDNA Clones

A factor XII'cDNA of 1959 bp was isolated from a cDNA library prepared from human liver

mRNA. This cDNA (pcHXII-501) codes for part of a leader peptide followed by the entire

coding region of plasma factor XII, a T G A stop codon, a 150 bp non-coding region at the 3'

end and a poly A + tail of at least 75 bp. A second human liver cDNA library was screened

and two identical clones (pcWFHXII-WF7,-WF9) were isolated that contained cDNA

sequence coding for the signal peptide region, the putative initiator methionine residue of

factor XII and three nucleotides of 5' untranslated end of the message.

The cDNA sequence data have enabled us to locate the kallikrein cleavage sites (solid

arrows, Fig. 8) that produce a- and 0-factor X l l a during zymogen activation (Cochrane et

al., 1973; Revak et al., 1977; Dunn et al., 1982). The three cleavage sites occur carboxy-

terminal to arginine residues in the sequences: Leu-Thr-Arg, Gly-Gln-Arg and Met-Thr-Arg.

Because kallikrein does not recognize a unique sequence, however, the locations of other

kallikrein cleavage sites in the M r 52,000 fragment of a-factor X l l a cannot be determined

from the cDNA sequence data.

Discussion / 110

2. Factor XII Is Synthesized as a Prescursor

Factor XII is a secreted protein and would be expected to contain a signal peptide which

functions in the transport of the protein across the rough endoplasmic reticulum membrane

(Blobel etal., 1979). The cDNA sequence predicts that factor XII is synthesized as a

precursor containing an amino-terminal extension of at least 19 amino acid residues

(encoded by nucleotides 1-18, Fig.D and nucleotides 3-40, Fig.9). Cleavage of a Ser-Ile bond

(encoded by nucleotides 16-21, Fig.8) in the precursor would give rise to plasma factor XII.

This bond cleavage is consistent with the specificity of signal peptidase (Watson, 1984).

Several clotting factors require vitamin K for their biosynthesis, including prothrombin,

factor X , factor IX, factor VII and protein C (Jackson and Nemerson, 1980; Suttie, 1985).

Each of these proteins undergoes a post-translational modification of 10-12 glutamic acid

residues by a vitamin K-dependent carboxylase. The resulting 7-carboxyglutamate

residues bind calcium ions. In contrast to factor XII, the vitamin K-dependent blood clotting

factors are synthesized as prepro-proteins (Kurachi et al, 1982; Degen et al., 1983; Jaye et

al, 1983; Long et al, 1984; MacGillivray and Davie, 1984; Fung et al, 1984; 1985). In

each of these proteins, formation of the plasma protein results from the cleavage of a bond

carboxy-terminal to an arginyl residue. Since signal peptidase does not cleave such bonds

(Strauss et al, 1978; Gordon et al, 1983) it has been proposed that the leader peptides of

these proteins may be cleaved by signal peptidase to form proproteins, and a second

protease(s) then converts the proproteins to the forms found in plasma. Other plasma

proteins are also synthesized as prepro-proteins, including albumin (Straus et al, 1977;

MacGillivray et al, 1979; Lawn et al, 1981), apolipoprotein A-II (Gordon et al, 1983) and

PAs (Pennica etal, 1983; Freizner-Degen etal, 1986; Riccio et al, 1985). The pro-proteins

Discussion / 111

of prothrombin, factor X, factor IX, and protein C share a high level of sequence homology

(Fung et al., 1985) that is not found in the pro-regions of albumin, apolipoprotein A-II or

plasminogen activators. The pro region has now been shown to be important for

carboxylation of the vitamin K-dependent proteins (Jorgensen et al., 1987).

B. HOMOLOGIES WITH OTHER PROTEINS

Comparison of residues 1-276 of factor XII with protein sequences in the National

Biomedical Research Foundation (Washington, D.C.) Protein Data Base reveals extensive

sequence homology with both fibronectin and plasminogen activators. Based on these

sequence homologies, a schematic representation of the structure of factor XII is shown in

Fig. 18. This structure is based on five different regions that share homology with regions

of both fibronectin and plasminogen activators. In addition, factor XII contains a

proline-rich region that does not share sequence homology with any known protein

sequence. Each of these six regions of factor XII are discussed in detail in the following

sections.

1. Amino-Terminal End of the Zymogen Form of Factor XII

The amino terminal region of factor XII (region N H 2 - A , Fig. 18) contains the signal peptide

amino acid sequence with its characteristic hydrophobic core (Blobel et al., 1979). The

signal peptide sequence is followed by a region that is rich in charged amino acids (amino

acids 1-20, Fig. 8). There are 9/19 amino acids that are charged at neutral pH, five of

which have a positive charge and four of them appear clustered near the middle of the

domain. This region is a candidate for the anionic surface binding property of factor XII

Figure 18: Proposed Model for Factor XII

The model was based on amino acid sequence homologies with tPA (Pennica et al., 1983; Ny

et al., 1984) and bovine fibronectin (Petersen et al., 1983). Thin arrows demarcate the

proposed protein structural domains found in human factor XII. Regions A-B represents a

fibronectin typell homology; B-C and D-E are growth factor-like reigons; C-D region is a

fibronectin type I homology; region E - F is a kringle structure and F - G is a proline rich

region whose structure is undefined, and G-COOH represents the catalytic region (see text

for details). Disulfide bonds have been inserted according to their arrangement in the

homologous domains of other proteins; the disulfide bond arrangement of the fibronectin

type II region has been determined by Patthy et al. (1984). The disulfide bonds in the

catalytic region were based on the proposed model of 0-factor X l l a (see Discussion).

Kallikrein cleavage sites that give rise to /3-factor X l l a are indicated by the heavy arrows.

Discussion / 114

although both positively and negatively charged amino acids are represented.

2. Fibronectin Homology

The next region of factor XII (region A-B, Fig. 18) shares sequence homology with the type

II homology regions of fibronectin. The type II homologies are each composed of about 60

residues including four half-cysteine residues (Petersen et al., 1983), and share

approximately 54% sequence identity with each other (31/57 residues in corresponding

positions are identical). Residues 13-69 of factor XII share 39% sequence identity (22/57

residues are identical) and 40% sequence identity (23/57 residues are identical) with the two

fibronectin sequences, respectively, including the four half-cysteine residues (Fig. 19-A).

The type II homologies are present in a M r 45,000 fragment of fibronectin that binds to

collagen (see Yamada, 1983; Hermans, 1985); by analogy, the region of factor XII that

shares sequence homology to the type II regions may be responsible for the putative

collagen-binding properties of factor XII (Wilner et al., 1968). Evidence for collagen

activation of factor XII has been strongly disputed, however, since purified collagen added to

plasma does not initiate coagulation as expected for surface-dependent activation of the

cascade (Griffin et al., 1975; Fujikawa et al., 1980a). The proposed disulfide-bridging

pattern shown in the model for factor XII corresponds to that described by Patthy et al.

(1984) for fibronectin and has a kringle-like structure in which the first and third and the

second and fourth cysteine residues form disulfide bridges.

Separating the two growth factor-like regions of factor XII (described below) is a 43 amino

acid peptide (regions C-D, Fig. 18) that shares limited sequence homology with the type I

regions of fibronectin (Fig. 19-C). This domain in fibronectin is characterized by two

Figure 19: Homologies Between the Amino Acid Sequence of Factor XII, Bovine

Fibronectin (Petersen et al., 1983) and Human tPA (Pennica et al., 1983)

Identical residues in corresponding positions are boxed; gaps have been inserted in the

sequences to maximize homology. A: Comparison of Segment S3, residues 14-73 (type

II-1), and residues 74-130 (type II-2) of fibronectin with residues 13-69 of factor XII (FXII).

B: Comparison of residues 51-84 of tissue-type plasminogen activator (tPA) and residues

79-111 (FXII-1) and residues 159-190 (FXII-2) of factor XII. Arrows denote glycine and

cysteine residues that are invariant in this type of homology. C: Comparison of segment

S l , residues 17-57 (type 1-1) and segment S l residues 66-104 (type 1-2) of fibronectin with

residues 116-151 of factor XII (FXII). D: Comparison of residues 87-173 of tissue-type

plasminogen activator (tPA) with residues 193-276 of factor XII (FXII).

A Fibronectin Type II Homology:

Type I I - l Type II -2 FXII

T T T T T T

G E P C|V L|P F G|A L|C H F P F

V T|G E P C H F P F

t T T l E j Q D Q K N Y D ( A ) D Q K

N { T | D Q D QpTw

Y N I G K T F Y S

Y N J N H N [ 7 | T D

Y ( H R Q L | Y | H K

C T C T C T G P

H L W c M K W c Q P w c

|3 Growth Factor Homology:

tPA FXII -2 FXII -1

E P •si F N G G 1 P C Q Q A L Y F S D F V C Q C P E G F A G K C C T N P c L H G G [ | Oc L E V E G H R L - - C H C P v G . Y T G P F C H S P c Q K G G 1 ? c V N M P S G P H - - £ L C P Q H L T G N H C_

t t t t t t t t

C Fibronectin Type I Homology

- T T Y P E I - l

T Y P E 1-2

FXII : 0

K H Y Q I

N T [ Y J R V

R F F H K

7 J Y L G S A ] L - V C T c Y G G S R G - F N C

P K D S - - M _ I W D C T c I G A G R G R I S C

T ) E Q - A Aj V A R cjol |c| K G _ P D A H - - - £_

Q Kringle Homology:

tPA FXII

D T D T

P D R N

A I R W - -

L G L G

R [ G J L

N " H J N

G _HjA

R G R G

T W L A

Y C R N P D

F C R N P D

R I D " S

N [ D J I

K P W C

R P W c CTi

Discussion / 117

disulfide bonds giving a two loop structure that has been named a "finger" domain (Petersen

et al., 1983). The functions of the finger domains in fibronectin may be varied and involve

the fibrin, heparin and perhaps gelatin binding properties of the protein (see Yamada, 1983;

Petersen and Skorstengaard, 1985).

Fibronectin is a macromolecular dimer of nearly identical polypeptide chains ( M r

220,000-250,000) which are held together at their carboxy termini by two disulfide bonds

(Petersen and Skorstengaard, 1985). The protein is found inside the cell, in the

extracellular matrix and in plasma (McDonagh et al., 1985). Plasma fibronectin is

synthesized in the liver and is secreted. Fibronectin is also synthesized by megakaryocytes

and found in platelets (Plow et al., 1979). A single gene codes for human fibronectin

although three different proteins have been described which result from alternative splicing

of the mRNA transcript (Schwarzbauer et al., 1983). The protein is comprised of multiple

structural and functional domains consisting of three repeated units, 12 with type I

homology, two with type II homlogy and 15 with type III homology (Petersen and

Skorstengaard, 1985). As determined by electron microscopy studies (Hirano et al., 1983)

the chicken fibronectin gene consists of at least 48 exons with each being about 150 bp in

length and correspond to the lengths of the type I and type II homologies.

The functions of fibronectin are varied due to its binding to many different biological

substances, especially to those on cell surfaces (Petersen and Skorstengaard, 1985).

Interactions with collagen, fibrin and cells implicates fibronectin in the process of wound

repair (Clark and Colvin, 1985) either as a result of severe injury or in inflammation. After

fibrin, fibronectin is the major protein found in the blood clot (4-5% of the total protein) and

has been shown to be cross-linked to fibrin by factor XHIa in vitro (Mosher, 1975).

Discussion / 118

Fibronectin also binds to collagen as well as being chemotactic for fibroblasts, neutrophils

and macrophages. Therefore, it appears to be associated with various stages of tissue

repair in organisms including the initial stage, cellular infiltration and the phagocytic

removal of debris (McDonagh et al., 1985). Importantly, fibronectin may be a central

component in cell migration, attachment and differentiation although formal proof is lacking

(Clark and Colvin, 1985).

3. Epidermal Growth Factor Homology

Two regions of factor XII (regions B-C and D-E , Fig. 18) are homologous to an epidermal

growth factor-like sequence that has been found in many proteins including the 19K protein

from Vaccina virus, transforming growth factor type 1, tPA, and several clotting factors

(Blomquist et al., 1984; Doolittle et al., 1984). In each of these proteins, there is a highly

conserved region of 50 amino acids with nine invariant cysteine and glycine residues. After

the insertion of three gaps in the factor XII sequences, all cysteine residues align perfectly

(Fig.l9-B). The carboxy-terminal growth factor domain also contains the invariant glycine

residues; in the amino-terminal domain, however, one invariant glycine residue has been

replaced with a histidine.

4. Kringle Homology

Another type of homology found in factor XII is the kringle domain (region E - F , Fig. 18).

Kringle domains are typically 80 amino acids in length and form three characteristic

disulfide bonds. Kringles have been found in prothrombin (Magnusson et al., 1975),

plasminogen (Sottrup-Jensen et al., 1978), tPA (Pennica et al., 1983) and urokinase

Discussion / 119

(Gunzler et al., 1982) and share approximately 40% sequence identity with each other

including six invariant half-cysteine residues (see Jackson and Nemerson, 1980). An

alignment of one of the kringle regions of tPA with factor XII is shown in Fig.l9-D. If three

gaps are inserted into the factor XII sequence, the kringles share 41% sequence identity (36

out of 87 residues are identical). The function of the kringle regions is unclear but it may be

involved in binding factor XII to fibrinogen and fibrin as has been described for a tPA

kringle (van Zonneveld et al., 1986). The presence of both a finger domain and a kringle

suggests that the fibrin binding capacity of factor XII may be important in vivo.

5. Proline-Rich Domain

The kringle structure is followed by a region (residues 279-330, Fig 8; region F - G , Fig. 18)

in which 33% of the residues are proline (17 out of 52). Despite the high proline content,

this region does not share any sequence homology with other proline-rich proteins such as

those from salivary glands (Ziemer et al., 1984). However, residues 299-308 of the

proline-rich region of factor XII share sequence homology with residues 29-38 of calf

thymus HMG-17 (Walker et al., 1977) in which eight out of ten residues in corresponding

positions are identical. The significance of this homology and the function of the proline-rich

region of factor Xn are unclear.

6. Serine Protease Domain

Downstream of the proline-rich region in factor XII is the catalytic region (region G to the

COOH-terminus, Fig. 18) that shares amino acid sequence homology with other serine

proteases. A n alignment of the amino acid sequences of the catalytic regions of /3-factor

Discussion / 120

X l l a , plasminogen activators and the three pancreatic serine proteases is given in Figure

20. An analysis of the sequence homology between these enzymes is summarized in Table

V. It is notable that the sequence identity between /3-factor X l l a and the pancreatic

enzymes (average value of 35%) is comparable to the identity present between the

pancreatic enzymes themselves (average value of 42%).

A computer generated structural model of /3-factor X l l a was developed by Dr. Brayer and

G.V. Louie (Department of Biochemistry,University of British Columbia) (see Cool et al.,

1985), based on the known three dimensional structures of the pancreatic enzymes. The

model provided structural information about factor XII which may affect its catalytic

activity. For example, it is known that all 14 cysteine residues of /3-factor X l l a are involved

in disulfide bridging (Fujikawa and McMullen, 1983). From the sequence alignment of

/3-factor X l l a (Figure 20), it is apparent that four disulfide bridges (involving residues

42-58, 136-201, 168-182 and 191-220) are conserved in all three pancreatic enzymes, and a

further homologous disulfide bridge is shared with chymotrypsin (residues 1-122). The

disulfide bridging pattern of the remaining four cysteine residues of /3-factor X l l a cannot be

determined from primary structure alignments alone. Within the tertiary structure model

of /3-factor X l l a , however, pairing of these remaining cysteines residues were proposed and

are Cys-50 to Cys-111 and Cys-77 to Cys-80. This accounts for the seven possible disulfide

bonds in the serine protease domain.

Also evident from the tertiary structural model of /3-factor X l l a are the positions of major

insertions that occur in the amino acid sequence of /3-factor X l l a [residues 61 A - C , 109 A - E

and 205 A-C (Figure 20)]. All three of these insertions occur towards the end of existing

surface /3-loops in the pancreatic enzymes; two insertions are positioned on the forward

Figure 20: Amino Acid Sequence Alignment of the Catalytic Regions of 0-Factor

Xlla (FXII), Bovine Trypsin (TRYP), Porcine Elastase (ELAS), bovine chymotrypsin

(CHYM) and Human tPA

The sequences of trypsin, elastase, chymotrypsin and tPA are taken from Marquart et al.

(1983), Sawyer et al. (1978), Cohen et al. (1981) and Pennica et al. (1983), respectively.

The numbering system used is based on that of chymotrypsinogen A (Hartley and

Kauffman, 1966), with insertions in the sequences of related enzymes denoted by letters

(36A, 36B, etc.). Deletions are indicated by dashed lines. Identical residues in

corresponding positions of all five proteins are boxed. The standard single letter code for

amino acids is used.

F X I I TRYP ELAS CHYM tPA

10 11 12 13

16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 36 36 36 36 F X I I V V G G L V A L R G A H P Y I A A L Y TRYP I V G G Y T C G A N T V P Y Q V S L N ELAS V V G G T E A Q R N S w P S Q I S L Q Y R S G S -CHVH I V N G E E A V P c s w P W Q V s L C 0 X - - - -tPA I K G G L r A 0 1 A S H P W Q A A I F A X H R R S

E 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

F X I I W G H S F C A G S L 1 A P c W V L T A A H C L Q TRYP - S G Y H F c G G S L I N S Q W V V S A A H C Y K ELAS - 5 W A H T c G G T L I R 0 H w V M T A A H C V D CH KM - T G r K F c G G S L I N E H M V V T A A H c G V CPA P G E R F L c G G I L I S S C W I L S A A H c P Q

A B C A 61 61 61 61 62 63 64 65 65 66 67 6B 69 70 71 72 73 74 75 76 77 78 79 80 81

r X I I D R P A P E D h T V V L G 0 B R R N H S C E P c Q TRYP S - - . - G I Q V R L - - G E 0 N I H V V E G N E Q ELAS R - - E L T F, R V V V G E H N L N Q 14 N G T Z Q CHYM T • - - T S D V - V V A G E F D Q G S S S E X I Q tPA E R F P P H . H L T V I L G R T Y R V V P G E E E Q

62 83 84 85 86 87 38 89 , .90 91 92 93 94 95 96 97 98 99 A B

99 99 100 101 102 103 104 r x i i T L A V R S Y R L H E A F S P V S Y - - Q H D L A TRYP F 1 S A 5 X S I V H P S Y N S N T L - - N N D X H ELAS Y V G V Q K X V V H P Y W N T D D V A A G Y D I A CHYM K L K I A K V F K N s X Y . N S L T I - - N N D I T tPA K r E V e .K Y I V H X E F D D D T t D N D 1 A

r X I I TRYP. ELAS CHYM tPA

105 106 107 108 109 109 109 109 109 109 110 111 112 113 114 115 116 117 118 119 120" 121 122 123 124 I E D A D G S C A L L S P '

125 126 127 128 129 130 131 132 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 F X I I S G A A R P S E T T L C Q V A G H G H Q F E G A E TRYP T - S c A S - A G T 0 C L I S G N G N T X S S G T ELAS R A G T I L A N N S P c Y I T G W G L T R T N G CHYM S A S 0 D F A A G T T c V T T G H G L T R Y T N A tPA P A D L C L P D W T E c E L S G Y G K H E A L S P

150.151 152 153 154 155 156 157 15B 159 160 161 162 163 164 165 166.167 168 169 170 170 170 171 172

173 174 175 176 177 178 179 180 181 182 183 184 184 185 186 187 188 168 166 188 168 168 168 188 169 L E G G T - - - - - - D

F XII TRYP ELAS CHYM tPA

190 191 192 193 194 195 196 197 196 199 200 201 202 203 204 205 205 205 205 206 207 208 209 210 211 C " ] E D Q A A E R R L T L Q

- ' - - C K L f i V N G - - - Q Y A V H

: K N G - - - A W T L V N . D G - - - R M T L V

F XII TRYP ELAS CHYM tPA

212 213 214 215 216 217 217 218 219 220 221 221 222 223 224 225 226 227 228 229 230 231 232 233 234 C

Cj G 0. 235 236 23J 238 239 240 241 242 243 244 245

0 - f a c t or X l l a

Chymo t r yp s In

E l a s t a s e

Chymo t r y p s i n

(243 )

86 (35%)

E l a s t a s e

( 240)

79 (33%)

94 (41%)

T r yp s In

( 223 )

84 (38%)

101 (45%)

87 (39%)

T a b l e V . C o m p a r a t i v e A n a l y s i s o f the Amino A c i d S e q u e n c e A l i g n m e n t

Between the C a t a l y t i c R e g i o n s of / 3 - F a c t o r X l l a and the

P a n c r e a t i c S e r i n e P r o t e a s e s .

The t o t a l number of r e s i d u e s in each p r o t e i n Is g i v e n in p a r e n t h e s e s be l ow

i t s name in the h e a d i n g . Fo r each p a i r o f p r o t e i n s a l i g n e d in F i g u r e 20 , the

m a t r i x c o n t a i n s the number of i d e n t i c a l r e s i d u e s f o l l o w e d by the o v e r a l l

s e q u e n c e homology ( g i v e n as a p e r c e n t a g e of the s m a l l e r p r o t e i n ) in

p a r e n t h e s e s .

Discussion / 124

active site face of the enzyme (residues 61 A-C and 109 A-E), and the other is positioned

towards the back surface of the enzyme (residues 205 A-C). Thus, the insertions observed

for /3-factor X l l a occur at sites easily accommodated within the overall structural

framework of the pancreatic enzymes.

The insertion of five residues at position 109 is found in a region in other proteases which

shows no amino acid sequence variability amongst them and includes the pancreatic

proteases, thrombin, factor Xa and factor IXa (Furie et al., 1982). Interestingly, this insert

is also found in tPA (Figure 20). Since the insertion occurs on the surface of the enzyme it

is most probably related to the ability of /3-factor X l l a to recognize the physiological

molecular surfaces with which it must interact. It is notable that the carbohydrate moiety

of /3-factor X l l a (bound to Asn-74; Fujikawa and McMullen, 1983) is positioned near this

insertion and according to the teritiary structural model, is on the substrate binding surface

of the enzyme. Specialized surface features such as these and the variable regions already

discussed provide a mechanism by which complex cascade pathways like those involved in

blood coagulation and fibrinolysis can occur by the specific recognition of substrate molecules

(Jackson and Nemerson; Nemerson and Furie, 1980).

7. Plasminogen Activator Homology

From these sequence comparisons, it can be seen that factor XII shares remarkable

sequence homology with tPA (Ny et al., 1984), a protease that converts plasminogen to

plasmin in the fibrinolytic pathway (see Collen, 1980). The complete amino acid sequence

of human tPA has been predicted from the cDNA sequence (Pennica et al., 1983; Fisher et

al., 1985) and shows that tPA consists of a finger domain, a growth factor-like domain, two

Discussion / 125

kringle structures and the catalytic serine protease domain. This organization is similar to

regions C to the COOH-terminus of factor XII except that one of the tPA kringles is replaced

by the proline-rich region (Fig. 18). However, the tPA transcript contains a leader sequence

with not only the signal peptide sequence but also a pro-region which is removed prior to

secretion (Ny et al., 1984). No corresponding pro-region is found in factor XII but it is

present in other proteases involved in coagulation such as, prothrombin, factor X , factor IX

and protein C (discussed above).

8. Physiological Significance of the Homologous Protein Domains

Complex proteins such as the coagulation and fibrinolysis serine proteases and fibronectin

consist of multiple structural and functional domains which have been assembled during

evolution to produce the molecules found today. When a function has been defined for a

particular domain such the epidermal growth factor homology, it is tempting to attribute an

EGF-like function to the acceptor protein. This argument is flawed, however, since proteins

with homologous structures can possess different functions [for example, hemoglobin, a

protein in red blood cells and myoglobin (Blanchetot et al., 1983) have homologous domains;

or, a-crystallin which is an eye lens protein, is similar to a Drosophila heat shock protein

(Doolittle, 1985)]. Evidence for functionality of a protein domain within a particular protein

will have to be obtained directly. For example, functional assays have been performed on

the kringle domains of tPA (van Zonneveld et al., 1986). These studies showed that the

kringle binds to fibrin which would be important in the degradation of fibrin by plasmin (see

Collen, 1980). However, similar studies have not been performed on the putative functional

domains of FXII.

Discussion / 126

C. A PERSPECTIVE ON THE ROLE OF THE CONTACT SYSTEM IN

HOMEOSTASIS

Our understanding of the complex process of wound repair and the regenerative process

(reviewed in Clark and Colvin, 1985) is becoming clearer as the components of the plasma

systems involved are being defined. The plasma systems, blood coagulation, fibrinolysis

and inflammation have already been described in detail (see "Introduction"). Other plasma

systems involved in wound repair include the complement system and the immunoglobulin

molecules and are described elsewhere (Bennett and Ogston, 1981; Pesce and Dosekun,

1983). Also important to the healing process is the regeneration of new tissue at the site of

injury. Epithelium cells at the wound margin are stimulated to elongate and divide and

migrate to cover the thick mat of fibrin and fibronectin. Blood vessels sprout branches and

migrate to the wound bed and also begin to divide. Fibroblasts and macrophages are

summoned to the wound and newly synthesized glycosaminoglycans (GAG), fibronectin and

collagen, probably supplied by the fibroblasts are used to form the new extracellular matrix.

Central to these events is fibronectin which binds to fibrin, G A G , collagen, can stimulate cell

migration (epithelium) and is chemotactic for neutrophils and macrophages.

The convergence of the plasma systems involved in wound repair and the regenerative

process occurs as a result of the assembly of the essential components at the site of injury.

Plasma and tissue factors initiate the coagulation cascade producing a network of fibrin.

Trapped in this mesh are the molecules that are vital for repair. These include plasminogen

activators, plasminogen, fibronectin and perhaps factor XII. The proteins are all related

structurally although it is not known if each homologous domain exhibits similar function.

The plasminogen activator, factor XII and plasminogen have homologous kringles (possible

Discussion / 127

fibrin binding property) and serine protease domains. Plasminogen activator, factor XII

and fibronectin are related through the type I homology (possible fibrin binding property).

Factor XII also contains a type II homology (possible collagen binding property) found in

fibronectin.

Interestingly, like fibronectin, factor XII appears central in the processes of wound repair

(see "Introduction") in that factor XII is important in the contact activation of coagulation,

fibrinolysis, inflammation and kinin generation through the activation of prekallikrein.

Also, the epidermal growth factor domains in factor XII may be premordial growth factors

which may still have stimulatory properties since at the site of injury, cell proliferation

takes place and it may be possible that these regions in factor XII (and tissue-type

plasminogen activator) may play a minor role. Factor XII and fibronectin are structurally

unalike except in the fibronectin type I and type II homologous domains found in factor XII.

It is possible that during the process of evolution, factor XII acquired these two domains (see

below), and since factor XII was also functional in wound repair, the newly acquired

domains may have assumed similar function. The amino acid sequence of the factor XII

protein has changed during the process of evolution but may still have retained essential

amino acids required for the binding of collagen or fibrin. Finally, this evolutionary

strategy may also be applied to the other proteins important in wound repair (plasminogen

and tissue-type plasminogen activator) which have also acquired the homologous domains

(kringles and type I homology).

It should be emphasized that there is only circumstantial evidence to implicate fibronectin in

the wound repair and the regenerative processes. In addition, factor XII and prekallikrein

are not essential for wound repair since individuals deficient in either of these proteins are

Discussion / 128

assymptomatic. However, it is likely that the plasma systems are interrelated in such a

way that if an initiation factor is not present then the reaction will still occur via another,

perhaps less efficient, mechanism. Secondary activation mechanisms of any one of these

plasma systems involved in wound repair are crucial since inactivation of the system would

probably be lethal to the organism.

An understanding of the mechanism of gene evolution may elucidate the importance and

relevance of the exchange of protein domains within a family and between families of

proteins. This study of the factor XII gene structure has provided more data concerning the

evolution of the serine proteases and their structural and functional relationships.

D. DNA SEQUENCE ANALYSIS OF THE HUMAN FACTOR XII GENE

Two recombinant phage containing the entire factor XII gene were isolated from a human

genomic phage library. The gene is approximately 12 kb in length and consists of 13

introns and 14 exons. Restriction enzyme mapping and D N A sequence analysis were used

to localize all exon sequence for factor XII mRNA in the gene (Fig. 12). An A A T A A A

sequence is often found approximately 20 bp 5' to the polyadenylation site in eukaryotic

genes (Birnstiel et al., 1985). This sequence was located at nucleotides 11693-11699 in the

FXII gene. A consensus sequence C A Y T G is another possible recognition site downstream

of the polyadenylation site (Berget, 1984). A similar sequence was found at position 11703

as C T T T G . Al l the splice junction sequences agree with the consensus sequence of Mount

(1982) except for the splice donor of intron J which has a G C instead of a G T dinucleotide.

However, other genes have been described with similar junction sequences (Wieringa et al.,

1984; Dush et al, 1985; Irwin, 1986) and Aebi et al. (1986) have shown that GC is a

Discussion / 129

reasonable splice donor sequence which is spliced accurately in vivo.

An unusual feature of the FXII gene is that it has many very small introns such that exons

3 through 14 are contained in a 4.2 kbp fragment of genomic D N A with three introns less

than 100 bp in length. However, due to the presence of the first two large introns, the total

length of the gene is 12 kb which is an expected size when compared to other genes with

similar lengths of coding sequence (Naora and Deacon, 1982).

E. ANALYSIS OF THE PROMOTER REGION IN THE HUMAN FXII GENE

Promoters of many genes have a C C A A T or a T A T A element (reviewed in Corden et al.,

1980; Breathnach and Chambon, 1981; Hansen and Sharp, 1984; Dynan and Tjian, 1985;

Bucher and Trifonov, 1986) which are positioned at -70 to -80 bp and -27 to -34 bp from the

transcriptional start site, respectively. The primer extension and nuclease S i mapping

results for the human FXII gene show that these elements are not present in the 5' end of

the gene although a C C A A G sequence at -105 and a C T A T T T C at -26 bp are present (see

Table VI for frequency of nucleotides at each position).

1. The C A A T Element

These promoter elements are not essential for the expression of all R N A Polymerase

II-transcribed genes. For example, the C C A A T region in the globin gene family is required

for expression but there is no corresponding sequence in many genes expressed in the liver

such as the blood clotting factors, retinol binding protein (Laurent et al., 1985) and a-1

antitrypsin (Table VI). Deletion studies on the a-1 antitrypsin (Ciliberto et al., 1985) and

C o n s e n s u s (Cor den et a l . , 1980)

( -33 to - 2 7 ) ( -27 to - 2 1 )

T ( 3 7 ) A ( 3 9 ) T ( 3 7 ) A ( 3 5 ) A ( 2 3 ) A ( 3 7 ) T ( 2 0 ) C (3) T (1) A (4) T (3) T ( 1 8 ) T (4) A ( 1 4 )

G (6) C (1)

a -1 An t I t r yp s i n - 2 6 (CI l i b e r to et a l . 1 985 ) T T A A A T A

B o v i n e P r o t h r o m b i n - 2 9 ( I r w i n , 1986) C A T T A A C

F a c t o r V I I I - 2 8 ( G i t s c h l e r et a l . , 1984) TAAAAAG

F a c t o r IX - 4 4 ( Y o s h i t a k e et a l . , 1985) TAAATAC

P r o t e l n C - 3 4 ( P l u t z k y et a l . , 1986) CAAATAT

E - a Immune R e s p o n s e - 3 0 ( M a t h i s et a l . , 1983) T T T T A A T

F a c t o r XI I - 2 6 C T A T T T C

T a b l e V I . Summary o f T A T A - 1 Ike E l e m e n t s F o u n d In L i v e r S p e c i f i c

G e n e s , C o a g u l a t i o n F a c t o r G e n e s and the E - a Immune R e s p o n s e G e n e .

The c o n s e n s u s s e q u e n c e f o r TATA l i k e s e q u e n c e s was d e t e r m i n e d f r o m

40 e u k a r y o t i c P o l y m e r a s e II t r a n s c r i b e d genes and were s u m m a r i z e d

in C o r d e n et a l . The numbers a b o v e the s e q u e n c e e l e m e n t s d e s c r i b e

the p o s i t i o n in the gene f r o m the t r a n s c r i p t i o n a l s t a r t s i t e s in

where t h e s e e l e m e n t s a re commonly f o u n d .

Discussion / 131

albumin (Gorski et al., 1986) promoter regions have shown that both genes have strong

promoters upstream of C A A T or T A T A regions. Albumin has a C C A A T element at -89 bp,

but it was shown that deletion of this region reduced the transcription rate by only 10%

whereas sequences further 5' reduced the rate by 85%. In addition, promotion of both the

albumin and a-1 antitrypsin genes required liver specific factors since the promoters did not

function significantly when the deletion mutants were expressed in tissues that did not

normally produce the gene products. Therefore, it appears that there are strong promoters

in these liver genes that are independent of the C A A T regions. Although similar studies

have not been described in the literature for clotting factor promoters, it is possible that the

genes which are highly expressed (prothrombin and antithrombin III) probably will also

have non-CAAT-like promoter sequences recognized by liver specific factors.

2. The TATA Box

The blood clotting factor genes as well as many others have atypical or no canonical T A T A

elements (Table VI). Other genes such as the housekeeping genes

[3-hydroxy-3-methylglutaryl CoA (Reynolds et al., (1984); dihydrofolate reductase (Crouse

et al., (1982); hypoxanthine-guanine phosphoribosyl transferase (Melton et al., 1984), and

adenosine deaminase (Valerio et al., (1985)], Thy-1 (Giguere et al., (1985), E G F receptor

(Ishii et al., 1985) and viral promoters (Baker et al., 1979; Ghosh et al., 1979) do not have

T A T A elements but do have G + C rich regions 5' to the transcriptional start site. These

genes also often have S p l binding sequences (GGGCGG) (reviewed in Dynan and Tjian,

1985). Another class of non-TATA like promoters includes those which have no apparent

D N A features that are particularly distinguishable. These genes are human ^-tubulin (Lee

et al., 1983), T-cell receptor/T3 complex (van den Elsen et al., 1986) and perhaps factor XII .

Discussion / 132

In most of the cases listed, all the mRNA transcripts appear to have heterogeneous 5' ends

suggesting that the major role of the T A T A sequence in these genes is to direct the position

of the 5' end of the transcript.

Variability in the nature and function of the T A T A sequence is demonstrated in in vivo

deletion studies. The T A T A sequences in the SV40 (Benoist and Chambon, 1980) and sea

urchin H2A (Grosschedl et al., 1980) gene promoters showed that the T A T A sequence was

required for precision in transcript initiation but did not affect transcription rates.

Sequences around the mRNA capping site may also contribute to the determination of the

transcription initiation site as illustrated in a mutant rabbit 0 globin gene which lacks a

proper T A T A sequence (Charnay et al., 1985; Dierks et al, 1983) and yet the majority of

mRNA transcripts contained the correct 5'end. On the other hand, both rate and placement

of transcript initiation required T A T A sequence elements in expression of the thymidine

kinase and globin genes (McKnight and Kingsbury, 1982; Charnay et al., 1985; reviewed in

Hansen and Sharp, 1984). Certainly the lack of T A T A or presence of weaker TATA-like

sequences (Table VI) is exceptional as most eukaryotic RNA Polymerase II genes do have

the consensus sequence (Breathnach and Chambon, 1981). However, many genes function

normally without the T A T A sequence and probably possess other factors pertinent to

transcription initiation and regulation.

Until further studies are performed with the FXII 5' flanking region, the identity of the

regulatory sequence(s) of the factor XII gene remains unknown. The nuclease S l and

primer extension products for FXII strongly suggest that FXII is lacking a typical T A T A

sequence possibly resulting in transcription initiation at more than one site. However,

analysis of the 5' flanking regions of the genes listed in Table VI and those discussed above

Discussion / 133

indicates that many genes are lacking or have atypical T A T A sequences. The absence of

true C A A T or T A T A sequences may be a means of maintaining a low level of transcription

for those genes coding for low abundant mRNAs (Bucher and Trifonov, 1986). Genes which

require stronger promotion but do not have typical promoter sequences (for example, a-1

antitrypsin and prothrombin, Table VI) may possess unique promoter elements such as

those recognized by liver specific factors (Ciliberto et al., 1985; Gorski et al., 1986) resulting

in high levels of gene expression.

F. PUTATIVE PROTEIN DOMAINS ARE FOUND ON SEPARATE EXONS

The positions of the introns in the factor XII gene divide the coding region into discrete

protein modular units (demarcated by arrows in Fig. 18) which share amino acid sequence

identity with similar regions in tPA (Ny et al., 1984) and fibronectin (Peterson etal., 1983).

Comparisons of the FXII amino acid sequence with those of tPA and fibronectin show that

there is only approximately 30-40% (Figure 19) sequence identity but the positions of

half-cysteine residues in each unit are conserved.

The schematic representation of the FXII coding sequence, shown in Fig. 21, was based on

sequence homologies found in tPA and fibronectin. The lengths of each exon are given in

Table IV. A putative signal peptide sequence (Blobel et al., 1979) at the N-terminus of the

zymogen is located on the first exon (NH2-A, Fig. 21) followed by a region of unknown

homology (A-B, Fig. 21) that is encoded by the second exon. A "type II" homology with

apparent collagen binding properties in fibronectin (Petersen et al., 1983; Yamada, 1983) is

represented by exons three and four (B-D, Fig. 21). Next is the EGF-like domain (D-E,

Fig.21) on exon five (Blomquist et al, 1984; Doolittle et al., 1984) followed by the "type I"

Figure 21: Schematic Diagram of the FXII Protein Coding Domains and the

Positions of Introns.

The standard one-letter code of each amino acid is given in the open circles. The solid black

bars, indicating possible disulfide bonding between cysteine residues is based on protein

homologies of other proteins (Magnusson et al., 1975; Yamada, 1983; Blomquist et al.,

1984; Patthy et al, 1984; Cool et al., 1985). The arrows A - M indicate the positions of

introns in the coding sequence. He at position 1 indicates the first residue of the zymogen

and is marked by a curved arrow. The short, closed arrows show cleavage activation sites

by kallikrein which produce j3-factor Xl la .

Discussion / 136

homology, or, the fibrin finger in fibronectin (E-F, Fig. 21) (Petersen et al., 1983) found on

exon six. Exon seven encodes another EGF-like domain (F-G, Fig.21) preceeding a kringle

structure and a proline rich region (G-I, Fig. 21) on exons eight and nine.

FXII activation requires cleavage by kallikrein, carboxy terminal to two Arg residues (thick

arrows in Fig. 21), generating one form of the serine protease, (3-FXIIa (I-COOH, Fig. 21).

The serine protease consists of five exons with the catalytic triad residues, His, Asp and Ser

separated by introns. Exon 14 is the largest (320 bp) and encodes 55 amino acid residues of

the serine protease and 150 bp of the 3' untranslated end of the mRNA.

Although the protein domains described above may be functional in other proteins, it is not

yet known whether similar functions can be attributed to FXII or tPA. It has been

speculated that exons may represent functional building blocks in a protein and that these

elements can be exchanged between existing genes and generate new kinds of proteins

(Gilbert, 1978; Blake, 1978). Patthy (1985) has suggested that the proteins involved in

coagulation and fibrinolysis may have been altered through this action of exon shuffling to

account for their present day structures.

G. EVOLUTION OF THE FACTOR XII GENE

The number and position of introns can be used to describe the evolutionary relatedness of

proteins (Craik et al., 1983) since proteins with conserved structure and function have

common gene organizations and have probably arisen through gene duplication events

(Patthy, 1985; Rogers, 1985). The urokinase and tPA genes have been described (Ny et al.,

1984; Riccio, et al., 1985; Friezner Degen et al., 1986) and are a family of serine proteases

Discussion / 137

separate from the clotting factors (Patthy, 1985; Rogers, 1985; Irwin, 1986). It was

anticipated that since FXII and tPA had many regions of protein homology which were

organized in the same manner in both proteins and that half-cysteine residues were

conserved, then the number and position of introns within their genes would likely be

similar. The positions of introns of the plasminogen activators were compared to those of

FXII and were found to be more conserved in the carboxy terminal half of the molecules

than in the amino terminal region of the proteins (Figs. 22 and 23).

1. Evolution of the Amino Terminal Half of Factor XII

Protein coding sequences of the amino terminal regions of the plasminogen activators and

FXII are interrupted by introns in identical codon reading phases although there is

considerable variation in the coding sequence itself. For example, there are only two exons

(those encoding the EGF-like domain and the kringle) which are common to all three

proteins but the lengths of each exon is conserved and varies by only 10 bp. These exons,

whose functions have been described above, have similar sequence identity and conserved

half-cysteine residues (Fig. 22). The fibronectin type I homology is found amino terminal to

the EGF-like domain in FXII and tPA but not in human urokinase. The fibronectin

homology appears in the FXII and tPA genes on separate exons of almost identical length

and with conserved half-cysteine residues.

Further variation in gene organization was found in the connecting regions in which there is

a kringle in tPA (85 amino acids), a proline-rich region in FXII (55 amino acids), but no

corresponding region in urokinase. The porcine urokinase protein has a 27 bp insert at this

position relative to the human urokinase protein (Nagamine et al., 1984). Analysis of the

Figure 22: Comparison of the Exon Organization in the ^N-Terminal Chains of

Human FXII, tPA (Ny et al., 1984) and uPA (Riccio et al., 1985).

Exons are represented by open bars; 5' untranslated regions are represented by solid bars

and signal or pre-regions by dotted bars. The sizes of exons are drawn to scale. Triplet

codon phase in which the exon is interrupted by the intron is designated as 0, I or II. The

sizes of the introns are not to scale. The vertical lines below the open bars represent

positions of cysteine residues in the exons. The lettering in the open boxes refer to the type

homology encoded by the exon: Fnl l , type II homology in fibronectin; E G F , epidermal

growth factor; F F , type I homology in fibronectin, the "fibrin finger"; K , kringle; and P,

proline-rich region. The scale represents 50 bp.

Urokinase ••B-ll-jM^~J-0-£2> EGF h H [ K h l H ^ ( H

tPA HIIIIHhl-BIIEiKKISh 1-̂ Fnl H - ^ j & F ^ I - T K HH ^ HH K r- l l -fg-yi

Factor XII sssTT̂ r̂C-J h H FnllT-lHTnTTr-H EGF H H Fnt h H EGF h I H K >IH K r~P~r-l

1 JI i 111 II i T r i i i r I—I 50 bp

Discussion / 140

gene shows that the intron/exon junction has shifted downstream 27 bp keeping the protein

in the correct reading frame (Nagamine et al., 1985). The insert is not long enough to be a

result of an inserted exon with subsequent loss of an intron in the porcine gene (Blake,

1983).

Kringles are encoded by two exons (reviewed in Patthy, 1985), interrupted by an intron in

the II codon reading frame. The second exon does not usually contain more than about 30

amino acids of coding sequence in the plasminogen activators, plasminogen or prothrombin

(Patthy, 1985). Unlike these genes, the FXII gene contains a 55 amino acid proline-rich

region attached to the second exon of the kringle. However, the length of the first exon of

the FXII kringle and the placement of the middle intron are almost identical to the

plasminogen activator kringles and therefore, the kringle in the FXII gene was probably

present prior to the time of FXII and plasminogen activator divergence. It is possible that,

by exon shuffling or another mechanism of gene evolution, FXII acquired a proline-rich exon

between the second exon of the kringle and the first exon of the serine protease domain with

subsequent loss of an intron. Other genes such as collagen, insulin and actin exhibit loss of

introns when their intron/exon gene organzitions are compared to other members in their

respective families (see Blake, 1983).

Sequences amino terminal to the fibronectin type I homology domain in the plasminogen

activators and FXII share no common homologies although codon phase interruption is

conserved (Fig. 2 2). The FXII gene has two exons which bear amino acid sequence

homology to the type II collagen binding domain in fibronectin and an E G F domain. These

domains are not present in the plasminogen activators. However, the plasminogen

activators have a prepro region which is cleaved prior to secretion, generating the single

Discussion / 141

chain form of the plasminogen activators found in plasma (Ny et al., 1984). This region is

found in the gene as part of the second exon which also contains 26 bp of the 5' untranslated

end of the transcript and nucleotide sequence for the signal peptide domain. The prepro

region is interrupted by an intron in the plasminogen activators and shares the third exon

with amino acid sequence of the zymogens. This third exon is very small (27 nucleotides) in

urokinase and (45 nucleotides) in tPA, and is flanked by O and I triplet codon intron

insertions in both genes. The second exon of FXII encodes the first 19 amino acids of the

zymogen, which follows the signal peptide domain and is flanked by introns in the 0 and I

reading frames, similar to the third exon of the plasminogen activator genes. Since it is not

known whether exons are lost or are acquired through mechanisms such as exon shuffling

in the genome, it is difficult to assess the actual structure of the ancestral gene prior to the

divergence of the plasminogen activator and FXII genes.

The untranslated 5' end of the plasminogen activators and FXII genes are also different.

There is an intron in the plasminogen acitivators which is not found in FXII. Urokinase and

tPA differ also in that the intron is inserted in different reading frames in the genes and the

lengths of the first exons vary. The first exon of FXII contains coding sequence for the 5'

untranslated end (47 bp) and the signal peptide sequence. Interestingly, the first intron in

the FXII gene is positioned immediately following the last residue of the signal peptide in

the O triplet codon reading frame. The 5' untranslated region of the gene is subject to more

variation than the coding regions as observed in the plasminogen activators themselves.

Furthermore, the clotting factors, human FIX (Yoshitake et al., 1985), bovine prothrombin

(Irwin, 1986) and human protein C (Foster et al., 1985; Plutzky et al., 1986) which belong to

the same family of serine proteases (Rogers, 1985; Irwin, 1986) also exhibit variation in

that there is an intron in the 5' untranslated region of the protein C gene but not in the

corresponding regions of the FIX or prothrombin genes.

Discussion / 142

2. Evolution of the Serine Protease Domain of Factor XII

The serine protease domains of FXII, tPA and uPA consist of five exons, four of which are

flanked by introns that have inserted into the same triplet codon reading frame in all three

genes (Fig. 23). This organization was compared to other serine proteases (Fig. 23) in order

to determine the evolutionary history of the FXII gene. Serine proteases can be divided into

five different families of genes based on their intron/exon gene organization (Rogers, 1985;

Irwin, 1986). Three of these families are shown in Figure 23 and are: first, the pancreatic,

kallikrein, nerve growth factors, plasminogen activators and factor XII family; second, the

factor IX and protein C family; and third (but closely related to the FIX and Protein C

family), the prothrombin family. Two other distantly related families belonging to the

serine protease superfamily of proteins (not shown in the figure) are represented by

haptoglobin and complement factor B (Rogers, 1985). Haptoglobin has lost its serine

protease activity although it has retained its activation by proteolysis property common to

the serine proteases. Also, the haptoglobin gene does not have introns in the equivalent

domain (Doolittle, 1985; Rogers, 1985). Complement factor B has serine protease activity

upon activation by proteolysis and has two more introns compared to the FXII/PA family of

proteins.

Although there are differences, FXII is closely related to the plasminogen activator,

pancreatic, nerve growth factor and kallikrein family of serine proteases since each of these

genes contain introns 3' to codons for both the active site histidine and aspartate and a 5'

intron to the codon for the active site serine (Fig. 23). However, the placement of all four

Figure 23: Comparison of the Serine Protease Exon Organization in the

Trypsinogen (Craik et al., 1984), Chymotrypsinogen (Bell et al., 1984), Proelastase

(Swift et al., 1984), Kallikrein (Mason et al., 1983; van Leeuwen, 1986), a and 7

Subunits of Nerve Growth Factor (Evans and Richards, 1985), Tissue-type

Plasminogen Activator (Ny et al., 1984; Degen et al., 1986), Urokinase (Nagamine et

al., 1984; Riccio et al., 1985), Factor IX (Anson et al., 1984; Yoshitake et al., 1985),

Protein C (Foster et ah, 1985; Plutzky et al., 1986), Prothrombin (Irwin, 1986) and

Factor XII genes.

Exons are shown as open boxes; the scale at the bottom represents 100 bp. Triplet codon

phase in which the intron is positioned is represented as 0, I or II. The activation sites of

the zymogens are shown by an arrow (the gamma subunit of nerve growth factor is not

activated this way). The codons for the histidine, aspartic acid and serine residues

comprising the catalytic triad are indicated by H , D and S, respectively. The 3'

untranslated ends of factor IX, tPA and urokinase have been abbreviated and are 1935, 914

and 1119 bp in length, respectively. Dotted bars represent exons coding for signal peptides

and slashed bars represent 3' untranslated regions of the mRNAs.

H D s Trypsinogen D - 1 H V ll H h i H r-O-t

Chymotrypsinogen • H D s Chymotrypsinogen ILJ-i-l _ _ r - o - L J - i i H \o\ r • H hCH

Proelastase • H

Proelastase D H H H lol h i H rO-T

Kallikrein H D S

Kallikrein N h 1 H H H h i H r-0-i' V/A H 1,

D s « N G F Rv| h 1 H H H h i H rOH VA « N G F

H 0 S T N G F N h 1 H ' h l H h i H 1-0-.' VA T N G F

D s tPA H H H 'HH T H r-0-T V///AY/////////A

H D s Urokinase H V i H r H H H l-OH V//AV/////////A Urokinase

It H D s Factor XII H ' H H Vi H •hlH Factor XII

D s Factor IX H • ^ o r H V//AV////////X

• H t

D s Protein C H 3 o - r h 1 A V////////////A Protein C

D s • _ Prothrombin 1 H HH h ' H V///A

5' —-3 ' 100 bp

Discussion / 145

introns in almost identical locations and in the same codon phase in the plasminogen

activators and FXII genes suggests that these genes may be more closely related to one

another than to the other serine proteases of this family. Figure 23 also shows that the

intron/exon gene organization of FXII and the plasminogen activators is unlike that found in

the FIX and protein C family and the prothrombin gene.

The evolution of the serine protease family of genes has been intensively studied as the

amino acid sequence and tertiary structure of many of the proteins involved have been

determined as well as their respective intron/exon gene organizations. In addition, these

proteins represent a very old family of genes, present in both prokaryotic and eukaryotic

species. The presence of the blood proteins probably arose in organisms when the

vertebrates evolved (Doolittle, 1984). At this time a primordial protease gene was

amplified by gene duplication events and the family was generated (Young et al., 1978;

Doolittle, 1984a; Neurath, 1984; Doolittle, 1985; Patthy, 1985).

Important to gene evolution is the emergence of introns. It is not known and is heavily

debated as to whether introns invaded coding sequences or introns from ancient genes were

lost during the evolution of gene families (Blake, 1983; Gilbert, 1979; Rogers, 1985).

Analysis of the serine proteases in Figure 23 shows that the introns could have been present

prior to gene duplication and subsequently lost. Thus, the older the protein, the fewer

introns which are present. For example, the most distantly related protein is haptoglobin

which has no introns and may have been present in invertebrate species prior to the clotting

factors. Also, invertebrates generally have fewer introns (Gilbert, 1985; Gilbert et al.,

1986) due to loss of introns or less insertion. The role of introns is not known but may be

important in providing raw genetic material (as in intron/exon sliding events observed in the

Discussion / 146

porcine gene and described below) or they allow recombination to occur between genes but

not within coding sequences so that important protein domains are not interrupted (Gilbert,

1979). Evidence for insertion of introns or loss of introns will be provided as we gain more

information about the intron/exon organization of related proteins in lower species and a

time scale of intron loss or gain in a protein family can be assigned.

3. Intron/Exon Sliding

The mechanism of intron/exon junction sliding could explain any small variation in lengths

of exons found in homologous domains (Craik et al., 1983; Nagamine et al., 1985) in the

plasminogen activators and FXII (Fig. 23). According to the tertiary structural model

proposed for /3-FXIIa (Cool et al., 1985), there are three insertions in the serine protease

domain which occur on the surface of the molecule. Two of these inserts (also found in tPA)

appear at or close to a junction (positions A r g 3 , 8 to A l a 4 0 0 and G l u 4 5 0 to G l y 4 5 4 , Fig. 14)

and are at positions 61 and 109 of the serine protease domain (Figure 20). Craik et al.

(1983) proposed that intron/exon sliding, which generates new coding sequence occurs on

the surfaces of molecules. Such modifications in 0-factor X l l a also map to the surface of the

molecule (Cool et al., 1985). However, the third insertion at position 205 is 13 amino acids

away from the junction (position A l a 5 5 s to A r g 5 5 7 , Fig. 14) and could be acquired through

other mechanisms. These length variations could confer new function to a molecule since

the insert at 109 is near an Asn-74 residue to which a carbohydrate moeity is bound

(Fujikawa and McMullen, 1983) and, according to the tertiary structural model, the insert is

on the substrate binding face of the enzyme.

Discussion / 147

H. A MODEL FOR THE EVOLUTION OF THE HUMAN FACTOR XII GENE

According to the model of evolution of the serine proteases proposed by Platthy (1985) and

based on the amino acid sequence homology in the serine protease domain, FXII and the

plasminogen activators evolved separately from the coagulation family of proteins (Figure

24). The model also illustrates that the plasminogen activator family diverged from the

clotting members after the EGF(A) domains were acquired but before the kringles. It is

interesting that after this divergence, both the FXII and the clotting factor genes were

invaded by another E G F domain of the B type but the plasminogen activators were not,

unless this exon was lost during evolution. The uPA and tPA/FXII genes separated at some

time prior to the acquisition of the fibrin finger domain in tPA and FXII. Since the amino

terminal region of the plasminogen activators and FXII proteins are remarkably different in

terms of their structure and function, it is very difficult to apply the model of exon shuffling

completely to the evolution of the FXII gene as it relates to either the plasminogen activator

or the family of coagulation proteins. Perhaps by some recombination event, all or parts of

the amino terminal region of an ancient gene (represented by the 5' untranslated end, signal

peptide sequence, fibronectin type II homology and possibly the E G F domains in FXII) were

spliced onto or fused with a primordial plasminogen activator gene (Doolittle, 1985) followed

by the insertion of exon domains as described in the exon shuffling model. A recombination

event involving gene fusion has also been proposed for the generation of the FXI and

prekallikrein genes (Chung et al., 1986). The model suggests that the amino terminal chain

(heavy chain consisting of the four tandem repeat units) was fused with the serine protease

domain of a FIX related ancestral gene (Chung et al., 1986) producing FXI or prekallikrein.

Analysis. of plasminogen activators and clotting factors of lower vertebrates and

invertebrates may verify this model and provide a time scale to the events.

Figure 24: A Model for the Evolution of the Plasminogen Activators and FXII.

Rectangles with S represent the serine protease domain. Triangles with Gg or G ^

represent the epidermal growth factor type B or type A homology, respectively. Diamonds

with I or II represent fibronectin homologies with the fibrin finger or collagen properties,

respectively. Circles with a K indicates a kringle domain or with an N , the amino terminal

domains for the plasminogen activators. Circles with an X represents the unknown

homology in FXII with possible surface binding properties. A rectangle with N indicates the

factor XII amino terminus or with Pro, the proline-rich domain, (see text for details)

gene fusion

N w u v r K

Duplication

uPA gene f u s i o n ^ - <^>

00<O> gene fusioiW^

gene fusion

©<£^©©CZI tPA

gene fusion

EK^><0>7^<I>7M^ p - S

intron loss

CONCLUDING REMARKS

The complete amino acid sequence of human factor XII, including the signal peptide

sequence, has been predicted from the D N A sequence of the cDNA and the gene. The

human gene has been identified and the intron/exon gene organization and the 5' and 3'

flanking regions determined. Complete characterization of the human factor XII protein

and its gene has expanded our understanding of the role of factor XII in the contact system.

The presence of putative functional domains which bear homology with fibronectin and

plasminogen activators suggests that the overlapping functions in wound repair for all these

proteins may be a result of these common protein domains. The major plasma systems

involved in wound repair, coagulation, fibrinolysis, complement and regeneration require the

components of the contact system (factor XII, prekallikrein, H M W kininogen), plasminogen

activator and fibronectin. Since the components involved have overlapping functions (at

least in vitro) they may be the means through which the plasma systems are interrelated

and homeostasis maintained during injury.

Analysis of the gene for factor XII shows that it is a member of the plasminogen activator

gene family. Residual function may still be present in the modern factor XII molecule so

that it can act, perhaps weakly, in plasma systems other than coagulation. The complexity

of these serine protease and fibronectin molecules seems to depend on the acquisition of

multiple protein domains. Whether this mechanism is merely an evolutionary strategy to

rapidly increase sequence variation in proteins or whether it is a strategy to confer new

function as well on a protein remains to be determined for human factor XII.

V. LITERATURE CITED

Abelson, J . (1979). RNA Processing and the Intervening Sequencing Problem. Ann. Rev.

Biochem. 48; 1035-1069.

Aebi, M . , Hornig, H . , Padgett, P.A., Reiser, J . and Weissman, C. (1986). Sequence

Requirements for Splicing Higher Eukaryotic Nuclear Pre mRNA. Cell 47, 555-565.

Astrup, T. (1978). Fibrinolysis-An Overview in "Progress in Chemical Fibrinolysis and

thrombolysis" (Davidson, J . F . , Rowan, R . M . , Samama, M . M . , Desynoyers, P.C.,

Eds.) Vol.3 Raven Press, New York, pp. 1-57.

Atkinson, T. & Smith, M . (1984) Solid Phase Synthesis of Oligodeoxyribonucleotides by the

phosphite-Triester Method, in Oligonucleotide Synthesis, a practical approach (Gait,

M . J . , ed.) pp. 35-81, IRL Press Ltd., Oxford.

Aviv, H . and Leder, P. (1972). Purification of Biologically Active Globin Messenger RNA

by Chromatography on Oligothymidylic Acid-Cellulose. Proc. Natl. Acad. Sci.

U S A 69; 1408-1412.

Bachmann, F . and Kruithof, I.E. (1984). Tissue Plasminogen Activator: Chemical and

Physiological Aspects. Semin. Thromb. Hemost. 10; 6-17.

Baker, C , Herisse, J . , Courtois, G. , Galibert, F . and Ziff, E . (1979). Messenger RNA for

the AD2 D N A Binding Protein:DNA Sequences Encoding the First Leader and

Heterogeneity at the mRNA 5' End. Cell 18, 569-580.

Baker, C C . and Ziff, E .B. (1981). Promoters and Heterogeneous 5' Termini of Messenger

RNAs of Adenovirus Serotype 2. J . Mol. Biol. 149; 189-221.

Literature Cited / 152

Beckmann, R.J . , Schmidt, R.J . , Santerre, R.F. , Plutzky, J . , Crabtree, G.R. and Long, G .L .

(1985). The Structure and Evolution of a 461 Amino Acid Human Protein C

Precursor and Its Messenger RNA Based Upon the D N A Sequence of Cloned Liver

cDNA. Nucl. Acids Res. 13; 5233-5247.

Benoist, C , O'Hare, K. , Breathnach, R. and Chambon, P. (1980). The Ovalbumin

Gene-Sequence of Putative Control Regions. Nucl. Acids Res. 8; 127-142.

Bennett, B. and Ogston, D. (1981). Role of Complement, Coagulation, Fibrinolysis and

Kinins in Haemostasis and Thrombosis (Bloom, A . L . and Thomas, D.P., Eds.)

Churchill Livingstone, New York, pp. 236-251.

Benoist, C. and Chambon, P. (1981). In vivo Sequence Requirements of the SV40 Early

Promoter Region. Nature (Lond.) 290, 304-310.

Benton, W.D. & Davis, R.W. (1977). Screening Xgt Recombinant Clones by Hybridization

in situ. Science, 196, 180-182.

Berget, S .M. (1984). Are U4 Small Nuclear Ribonucleoproteins Involved in

Polyadenylation? Nature (Lond.) 309, 179-182.

Birboim, H .C . and Doly, J . (1979). A Rapid Extraction Procedure for Screening

Recombinant Plasmid DNA. Nucl. Acids Res. 7; 1513-1523.

Birnstiel, M . L . , Busslinger, M . and Strub, K. (1985). Transcription Termination and 3'

Processing: The End is in Site. Cell 41, 349-359.

Blake, C C F . (1978). Do Genes-In-Pieces Imply Proteins-In-Pieces? Nature (Lond.) 273,

Blake, C. (1983). Exons- Present From the Beginning? Nature (Lond.) 306, 535-537.

Blake, C. (1983a). Exons and the Evolution of Proteins. Trends. Biochem. Sci. 8; 11-13.

Blanchetot, A . , Wilson, V. , Wood, D. and Jeffreys, A . J . (1983). The Seal Myoglobin Gene:

An Unusually long Globin Gene. Nature (Lond). 301; 733-734.

Blattner, F.R., Williams, B.G. , Blechl, A . E . , Denniston-Thompson, K . , Farber, H . E . ,

Furlong, L . - A . , Grunwald, D.J . , Kiefer, D.O., Moore, D.D., Schamm, J.W., Sheldon,

E . L . and Smithies, O. (1977). Charon Phages: Safer Derivatives of Bacteriophage

Lambda from D N A Cloning. Science 196; 161-169.

Blobel, G. , Walter, P., Chang, C.N. , Goldman, B . M . , Erickson, A . H . , and Lingappa, R.

(1979). Translocation of Proteins Across Membranes: the Signal Peptide Hypothesis

and Beyond in Secretory Mechanisms (Hopkin, C.R., and Duncan, C.J.,Eds.) Vol. 33,

pp. 9-36, Cambridge University Press, London.

Blomquist, M . C , Hunt, L . T . & Barker, W.CU984). Vaccinia Virus 19-Kilodalton Protein:

Relationship to Several Mammalian Proteins Including Two Growth Factors. Proc.

Natl. Acad. Sci. USA 81, 7363-7367.

Bloom, A . L . (1981). Inherited Disorders of Blood Coagulation, in Heamostasis and

Thrombosis (Bloom, A . L . and Thomas, D.P., Eds.) Churchill Livingstone, Edinburgh,

pp. 321-370.

Bock, P .E . , Srinivasan, K.R. and Shore, J .D. (1981). Activation of Intrinsic Blood

Coagulation by Ellagic Acid: Insoluble Ellagic Acid-Metal Ion Compexes are the

Activating Species. Biochemistry 20; 7258-7266.

Bock, P .E. , Shore, J .D. , Tans, G. and Griffin, J . H . (1985). Protein-Protein Interactions in

Contact Activation of Blood Coagulation. J . Biol. Chem. 260; 12434-12443.

Bouma, B.N. and Griffin, J . H . (1977). Human Blood Coagulation Factor XI: Purification,

Properties, and Mechanism of Activation by Activated Factor XII. J . Biol. Chem.

252; 6432-6437.

Bouma, B .N. and Griffin, J . H . (1981). Activation of the Intrinsic Fibrinolytic System via

the Factor XII Dependent Pathway. Prog. Fibrinolysis. 5; 54-64.

Bouma, B.N. , Miles, L . A . , Beretta, G. and Griffin, J . H . (1980). Human Plasma

Prekallikrein. Studies of its Activation by Activated Factor XII and of its Inactivation

by Diisopropyl Phosphoflouridate. Biochemistry 19; 1151-1160.

Bouma, B .N. , Vlooswijk, R .A.A. and Griffin, J . H . (1983). Immunological Studies of

Human Coagulation Factor XI and its Complex With High Molecular Weight

Kininogen. Blood 62; 1123-1131.

Breathnach, R., Mandel, J . L . and Chambon, P. (1977). Ovalbumin Gene is Split in

Chicken DNA. Nature (Lond.) 270; 314-319.

Breathnach, R. and Chambon, P. (1981). Organization and Expression of Eukaryotic Split

Genes Coding for Proteins. Ann. Rev. Biochem. 50, 349-383.

Brinster, R .L . , Chen, H.Y. , Warren, R., Sarthy, A. and Palmiter, R. (1982). Regulation of

Metallothionein-Thymidine Kinase Fusion Plasmids Injected Into Mouse Eggs.

Nature (Lond.) 296; 39-42.

Buchner, P. and Trifonov, E . (1986). Compilation and Analysis of Eukaryotic POL II

Promoter Sequences. Nucl. Acids Res. 14; 10009-10026.

Burgess, A . L and Esnouf, M.P. (1985). Blood Clotting Systems. Enzymol. Posttransl.

Mod. 2; pp. 301-337.

Calos, M.P. and Miller, J . H . (1980). Transposable Elements. Cell 20; 579-595.

Casadaban, M . J . and Cohen, S.N. (1980). Analysis of Gene Control Signals by D N A

Fusion and Cloning in Escherichia coli. J . Mol. Biol. 138; 179-207.

Chaconas, G. , and van de Sande, J . H . (1980).5'-3 2 P Labeling of RNA and D N A Restriction

Enzyme Fragments. Methods Enzymol. 65, 75-85.

Charnay, P., Mellon, P. and Maniatis, T. (1985). Linker Scanning Mutagenesis of the 5'

Flanking Region of the Mouse Beta-Major-Globin Gene-Sequence Requirement for

Transcription in Erythroid and Nonethrythoid Cells. Mol. Cell. Biol. 5, 1498-1151.

Chirgwin, J . M . , Pryzbyla, A . E . , MacDonald, R.J. & Rutter, W.J . (1979). Isolation of

Biologically Active Ribonucleic Acid from Sources Enriched in Ribonuclease.

Biochemistry 18, 5294-5299.

Chung, D. W., Fujikawa, K . , McMullen, B.A. and Davie, E .W. (1986). Human Plasma

Prekallikrein, a Zymogen to a Serine Protease That Contains Four Tandem Repeats.

Biochemistry, 25; 2410-2417.

Ciliberto, G . , Dente, L . and Cortese, R. (1985). Cell-Specific Expression of a Transfected

Human a 1-Antitrypsin Gene. Cell 41, 531-540.

Claeys, H . and Collen, D. (1977). Purification and Characterization of Bovine Coagulation

Factor XII (Hageman Factor). Eur. J . Biochem. 87; 69-74.

Clark, R.F. and Colvin, R.B. (1985). Wound Repair, in "Plasma Fibronectin: Structure

and Function" (McDonagh, J . Ed.) Marcel Dekker, Inc. New York, pp. 197-262.

Cochrane, C .G. , Revak, S.D. and Wuepper, K . D . (1973). Activation of Hageman Factor in

Solid and Fluid Phases. A Critical Role of Kallikrein. J . Exp. Med. 138;

1564-1583.

Cochrane, C.G. and Revak., S.D. (1980). Dessemination of Contact Activation in Plasma

by Plasma Kallikrein. J . Exp. Med. 152; 608-619. «>

Cochrane, C.G. (1980) Biochemistry and Pathophysiologic Effects of the Hageman Factor

System in Immunopathology: VHIth International Symposium, Academic Press, Inc.,

pp. 443-457.

Cochrane, C.G. and Griffin, J . H . (1982). The Contact System of Plasma. Adv. Immunol.

33; 241-306.

Cohen, G .H. , Silverton, E.W. and Davies, D.R. (1981). Refined Chrystal Structure of

7-Chymotrypsin at 1.9 A Resolution. Comparison with Other Serine Proteases. J .

Mol. Biol. 148; 449-479.

Collen, D. (1980) On the Regulation and Control of Fibrinolysis. Thrombosis and

Hemostasis 43; 77-89.

Colman, R.W. (1969). Activation of Plasminogen by Kallikrein. Biochem. Biophys. Res.

Commun. 35; 273-279.

Colman, R.W. (1984). Surface-Mediated Defense Reactions. J . Clin. Invest. 73;

1249-1253.

Cool, D .E . , Edgell, C.-J.S., Louie, G.V. , Zoller, M . J . , Brayer, G.D. and MacGillivrary,

R.T.A. (1985). Characterization of Human Blood Coagulation Factor XII cDNA. J .

Biol. Chem. 260, 12666-13676.

Corden, J . , Wasylyk, A. , Buchwalder, A. , Sassone-Corsi, P.,Kedinger, C. and Chambon, P.

(1980). Promoter Sequences of Eukaryotic Protein-Coding Genes. Science 209,

1406-1414.

Craik, C.S., Rutter, W.J. and Fletterick, R. (1983). Splice Junctions: Asociation with

Variation in Protein Structure. Science 220, 1125-1129.

Creighton, T . E . (Ed.) (1984). "Proteins. Structures and Molecular Principles". W. H .

Freeman and Company, New York, pp. 427-439.

Crouse G. F . , Simonsen, C C , McEwan, R.N. and Schimke, R.T. (1982). Structure of

Amplified Normal and Variant Dihydrofolate Reductase Genes in Mouse Sarcoma

S180 Cells. J . Biol. Chem. 257, 7887-7897.

Curtis, C .G. (1981). Plasma Factor XIII, in Haemostasis and Thrombosis (Bloom, A . L .

and Thomas, D.P., Eds.) Churchill Livingstone, Edinburgh, pp. 192-197.

Dahlback, B., Lundwall, A. and Stenflo, J . (1986). Primary Structure of Bovine Vitamin

K-Dependent Protein S. Proc. Natl. Acad. Sci. U S A 83; 4199-4203.

Davie, E .W. and Ratnoff, O.D. (1964). Waterfall Sequence for Intrinsic blood Clotting.

Science 145; 1310-1312.

Davie, E .W. , Fujikawa, K . , Kurachi, K . and Kisiel, W. (1979). The Role of Serine

Proteases in the Blood Coagulation Cascade. Adv. Enzymol. 48; 277-318.

Davis, R.W., Botstein, D. and Roth, J.R. (1980). A Manual for Genetic Engineering.

Advanced Bacterial Genetics. Cold Spring Harbour Laboratory, Cold Spring

Harbour, New York.

de Agostini, A. , Lijnen, H.R., Pixley, R.A., Colman, R.W. and Schapira, M . (1984).

Inactivation of Factor XII Active Fragment in Normal Plasma. J . Clin. Invest. 73;

1542-1549.

Degen, S.J.F., MacGillivray, R.T.A. and Davie, E .W. (1983). Characterization of the

Complementary Deoxyribonucleic Acid and Gene Coding for Human Prothrombin.

Biochemistry 22; 2087-2097.

Deininger, P .L. (1983). Random Subcloning of Sonicated DNA: Applicatoin to Shotgun

DnA Sequence Analysis. Anal. Biochem. 129, 216-223.

Delaney, A .D . (1982). A D N A Sequence Handling Program. Nucl. Acids Res. 129;

216-223.

Derkx, F . H . M . , Bouma, B.N. , Schalekamp, M.P.A. and Schalekamp, M . A . D . H . (1979).

An Intrinsic Factor XH-dependent Pathway Activates the Human Plasma

Renin-Angiotensin System. Nature 280; 315-316.

Dierks, P., von Ooyen, A. , Cochran, M.D. , Dobkin, C , Reiser, J . and Weissman, C. (1983).

Three Regions Upstream from the Cap Site are Required for Efficient and Accurate

Transcription of the Rabbit (3-Globin Gene in Mouse 3T6 Cells. Cell 32, 695-706.

Diz, D.I. (1985). Bradykinin and Related Peptides in Central Control of the Cardiovascular

System. Peptides 6(Suppl. 2); 57-64.

Doolittle, R.F. (1981). Fibrinogen and Fibrin in Thrombosis and Haemostasis (Bloom, A . L .

and Thomas, D.P.) Churchill Livingstone, pp. 163-191.

Doolittle, R.F. (1984). Fibrinogen and Fibrin. Ann. Rev. Biochem. 53; 195-229.

Doolittle, R.F. , Feng, D.F. & Johnson, M.S. (1984). Computer-Based Characterization of

Epidermal Growth Factor Precursor. Nature (Lond) 307; 558-560.

Doolittle, R.F. (1984a). in The Plasma Proteins (Putnum, S.W., Ed.) 2nd Edn., Vol. 4,

Academic Press, pp. 317-360.

Doolittle, R.F. (1985). the Geneology of Some Recently Evolved Vertebrate Proteins.

Trends Biochem. Sci. 10; 233-237.

Dunn. J .T . and Kaplan, A.P.(1982a). Formation and Structure of Human Hageman

Factor Fragments. J . Clin. Invest. 70, 627-631.

Dunn, J .T . , Silverberg, M . & Kaplan, A.P. (1982b). The Cleavage and formation of

Activated Human Hageman Factor by Autodigestion and by Kallikrein. J . Biol.

Chem. 257; 1779-1784.

Dush, M . K . , Sikela, J . M . , Kahn, S.A., Tischfield, J .A . and Stambrook, P.J. (1985).

Nucleotide Sequence and Organization of the Mouse Adenine

Phosphoribosyltranserase Gene. Proc 'Nat l . Acad. Sci. U S A 32, 2731-2735-

Dynan, W.S. and Tjian, R. (1985). Control of Eukaryotic Messenger R N A Sythesis by

sequence-Specific DNA-Binding Proteins. Nature (Lond.) 316, 774-778.

Edgell, M . H . , Hardies, S.C., Brown, B., Voliva, C , Hill, A . , Phillips, S., Comer, M . , Burotn,

F . , Weaver, S. and Hutchison III, C A . (1983). Evolution of the Mouse 7-Globin

Complex Loci, in Evolution of Genes and Proteins (Nei, M . and Koehn, R.K. , Eds.),

Sinauer Associates Inc., Sanderland, Mass., pp. 1-13.

Edmonds, M . , Vaughn, M . H . and Nakazato, H . (1971). Polyade,nylic Acid Sequence in the

Heterologous Nuclear RNA and Rapidly-Labeled Poly ribosomal RNA of HeLa Cells:

Possible Evidence for a Precursor Relationship. Proc. Natl. Acad. Sci. U S A 68;

1336-1340.

Erdos, E . (ed) (1979). Bradykinin, Kallikin, and Kallikrein in Handbook of Experimental

Pharmacology X X V (Suppl.). Springer-Verlag, Berlin.

Evans, B.A. and Richards, R.I. (1985). Genes for the Alpha-Subunit and gamma-Subunit

of Mouse Nerve Growth-Factor are Contiguous. E M B O J . 4, 133-138.

Fisher, R., Waller, E . K . , Grossi, G. , Thompson, D., Tizard, R. and Schleuning, W-D.

(1985). Isolation and Characterization of the Human Tissue-type Plasminogen

Activator Structural Gene Including Its 5' Flanking Region. J . Biol. Chem. 260;

11223-11230.

Foster, D . C , Yoshitake, S. and Davie, E.W. (1985). The Nucleotide Sequence of the Gene

for Human Protein C. Proc. Natl. Acad. Sci. U S A 82, 4673-4677.

Friezner-Degen, S.J., Rajput, B. and Reich, E . (1986). The Human Tissue Plasminogen

Activator Gene. J . Biol. Chem. 261, 6972-6985.

Fujikawa, K. , Kurachi, K . , and Davie, E .W. (1977a). Characterization of Bovine Factor

X l l a (Activated Hageman Factor). Biochemistry 16; 4182-4188.

Fujikawa, K . , Walsh, K . A . and Davie, E .W. (1977b). Isolation and Characterizatoin of

Bovine Factor XII (Hageman Factor). Biochemistry, 16; 2270-2278.

Fujikawa, K. , Heimark, R.L. , Kurachi, K. and Davie, E.W. (1980a). Activation of Bovine

Factor XII (Hageman Factor) by Plasma Kallikrein. Biochemistry 19; 1322-1329.

Fujikawa, K . , Heimark, R.L. , Kurachi, K. , and Davie, E.W. (1980c). Activation of Bovine

Factor XII (Hageman Factor) by Plasma Kallikrein. Biochemistry 19; 1322-1330.

Fujikawa, K. , McMullen, B.A. , Heimark, R.L. , Kurachi, K. and Davie, E.W. (1980b). The

role of Factor XII (Hageman Factor) in Blood Coagulation and a Partial Amino Acid

Sequence of Human Factor XII and Its Fragments, in Protides Biol. Fluids Proc.

Colloq. 28; 193-196.

Fujikawa, K. and Davie, E .W. (1981). Human Factor XII (Hageman Factor). Meth.

Enzym. 80; 198-211.

Fujikawa, K. and McMullen, B.A. (1983). Amino Acid Sequence of Human /3-Factor Xl la .

J . Biol. Chem. 258; 10924-10933.

Fujikawa, K. , Chung, D.W., Hendrickson, L . E . and Davie, E.W. (1986). Amino Acid

Sequence of human Factor XI, a Blood Coagulation Factor with Four Tandem Repeats

That Are Highly Homologous with Plasma Prekallikrein. Biochemistry, 25;

2417-2424.

Fung, M.R., Campbell, R .M. and MacGillivray, R .T .A. (1984). Blood Coagulation Factor X

mRNA Encodes a Single Polypeptide Containing a Pre-Pro Leader Sequence. Nucl.

Acids Res. 12; 4481-4492.

Fung, M.R., Hay, C W . and MacGillivray, R .T .A. (1985). Characterization of an Almost

Full-Length cDNA Coding for Human Blood Coagulation Factor X. Proc. Natl.

Acad. Sci. U S A 82; 3591-3595.

Furie, B., Bing, D . H . , Feldmann, R.J. , Robison, D.J . , Burnier, J.P. and Furie, B.C. (1982).

Computer-Generated Models of Blood Coagulation Factor Xa , Factor IXa and

Thrombin Based on Structural Homology with Other Serine Proteases. J . Biol.

Chem. 257; 3875-3882.

Gaffney, P.J . (1981). The Fibrinolytic System, in Thrombosis and Haemostasis (Bloom,

A . L . and Thomas, D.P.) Churchill Livingstone, pp. 198-224.

Ghosh, P., Reddy, V. , Swinscoe, J . , Lebowitz, P. and Weissman S. (1979). Heterogeneity

and 5'-Terminal Structures of the Late RNAs of Simian Virus 40. J . Mol. Biol.

126, 813-846.

Giguere, V . , Isobe, K. and Grosveld, F . (1985). Structure of the Murine Thy-1 Gene.

E M B O J . 4, 2017-2024.

Gilbert, W. (1978). Why Genes in Pieces? Nature (Lond.) 271, 501.

Gilbert, W. (1979). Introns and Exons: Playgrounds of Evolution, in Eukaryotic Gene

Regulation (Axel, R., Maniatis, T., and Fox., C .F . , Eds.), Academic Press, New York,

pp. 1-12.

Gilbert, W. (1985). Genes-In-Pieces Revisited. Science 228; 823-824.

Gilbert, W., Marchionni, M . , and McKnight, G. (1986). On the Antiquity of Introns. Cell

46; 151-154.

Gordon, J . L , Budelier, K . A . , Sims, H . F . , Edelstein, C , Scanu, A . M . and Strauss, A .W.

(1983). Biosynthesis of human Preproalbumin A-II. J . Biol. Chem. 258;

14054-14059.

Gorski, K . , Carneiro, M . and Schibler, U . (1986). Tissue-Specific In Vitro Transcription

from the Mouse Albumin Promoter. Cell 47, 767-776.

Grabowski, P.J . , Seiler, S.R. and Sharp, P.A. (1985). A Multicomponent Complex is

Involved in the Splicing of Messenger RNA Precursors. Cell 42; 345-353.

Griffin, J . H . (1978). The Role of Surface in the Surface-Dependent Activation of Hageman

Factor (Factor XII). Proc. Natl. Acad. Sci. U S A 75; 1998-2002.

Griffin, J . H . (1981). The Contact Phase of Blood Coagulation, in Heamostasis and

Thrombosis (Bloom, A . L . and Thomas, D.P., Eds.) Churchill Livingstone, Edinburgh,

pp. 84-97.

Griffin, J . H . and Cochrane, C .G. (1976). Human Factor XII. Methods Enzymol. 45;

56-65.

Griffin, J . H . and Cochrane, C.G. (1976a). Mechanisms for Involvement of High Molecular

Weight Kininogen in Surface-Dependent Reactions of Hageman Factor (Coagulation

Factor XII). Proc. Natl. Acad. Sci. U S A 73; 2554-2558.

Griffin, J . H . and Cochrane, C .G. (1979). Recent Advances in the Understanding of

Contact Activation Reactions. Semin. Thromb. Hemostasis 5, 254-273.

Griffin, J . H . , Harper, A . and Cochrane, C.G. (1975). Studies on the Activation of Human

Blood Coagulation Factor XII (Hageman Factor) by Soluble Collagen. Proc. Fed.

Am. Soc. Exp. Biol. 34; 860.

Grosschedl, R. and Birnstiel, M . L . (1980). Identification of Regulatory Sequences in the

Prelude Sequences of an H2A Histone Gene by the Study of Specific Deletion

Mutations in vivo. Proc. Natl. Acad. Sci. U S A 77; 1432-1436.

Gunzler, W.A. , Steffens, G.J. , Otting, F . , Kim, S.M.A. , Frankus, E . and Flohe,L. (1982).

The Primary Structure of High Molecular Mass Urokinase from Human Urine. The

Complete Amino Acid Sequence of the A Chain. Hoppe-Seyler's Z. Physiol. Chem.

363, 1155-1165.

Guyton, A . C . (1977) Basic Human Physiology: Normal Function and Mechamisms in

Disease, Second Ed. W.B. Saunders, Philadephia.

Hagen, F.S., Gray, C .L . , O'Hara, P., Grant, F . J . , Saari, G.C. , Woodbury, R.G. , Hart, C .E . ,

Insley, M . , Kisiel, W., Kurachi, K. , and Davie, E .W. (1986). Characterization of a

cDNA Coding for Human Factor VII. Proc. Natl. Acad. Sci. . U S A 83; 2412-2416.

Hall, L . , Craig, R.K. , Edbrooke, M.R. and Campbell, P.N. (1982). Comparison of the

nucleotide Sequence of cloned Human and Guinea-Pig Pre-a-Latalbumin cDNA With

That of Chicken Pre-Lysozyme cDNA Suggests Evolution From a Common Ancestral

Gene. Nucl. Acids Res. 10; 3503-3515.

Hanahan, D. & Meselson, M . (1980). Plasmid Screening at High Colony Density. Gene

10, 63-67.

Hansen, U . and Sharp, P.A. (1984). Transcription by R N A Polymerasell. Compr. Virol.

19, 65-97.

Harpel, P.C. (1972). Studies on the Interaction Between Collagen and Plasma

Kallikrein-Like Activity. J . Clin. Invest. 51, 1813-1822.

Hartley, B.S. and Kauffman, D .L . (1966). Corrections to the Amino Acid Sequence of

Bovine Chymotrypsinogen A. Biochem. J . 101; 229-231.

Hartley, B.S. and Kauffman, D.L. (1966). Biochem. J . 101; 229-231.

Heimark, R.L. and Davie, E .W. (1981). Bovine and Human Plasma Kallikrein. Methods

Enzymol. 80; 157-172.

Heimark, R.L. , Kurachi, K. , Fujikawa, K. & Davie, E .W. (1980). Surface Activation of

Blood Coagulation, Fibrinolysis and Kinin Formation. Nature (London) 286;

456-460.

Hewett-Emmett, D., Czelusniak, J . and Goodman, M . (1981). The Evolutionary

Relationships of the Enzymes in Blood Coagulation and Haemostasis. Ann. N.Y.

Acad. Sci. 370; 511-527.

Hirano, H . , Yamada, Y. , Sullivan, M . , de Crombrugghe, B., Pastan, I. and Yamada, K . M .

(1983). Isolation of Genomic D N A Clones Spanning the Entire fibronectin Gene.

Proc. Natl. Acad. Sci. U S A 80; 46-50.

Hojrup, P., Jensen, M.S. and Petersen, T . E . (1985). Amino Acid Sequence of Bovine

Protein Z: A Vitamin K-Dependent Serine Protease Homolog. F .E.B.S . Lett. 184;

333-338.

Hood, L . , Kronenberg, M . and Hunkapiller, T. (1985). T Cell antigen Receptor and

Immunoglobulin Supergene Family. Cell 40; 225-229.

Hoskins, J . , Norman, D.K. , Beckman, R.J. and Long, G .L . (1987). Cloning and

Characterization of Human Liver cDNA Encoding a Protein S Precursor. Proc. Natl.

Acad. Sci. 84; 349-353.

Ichinose, A . , Kisiel, W. and Fujikawa, K. (1984). Proteolytic Activation of Tissue

Plasminogen Activator by Plasma and Tissue Enzymes. F E B S Lett. 175; 412-418.

Irwin, D . M . (1986). The Structure and Evolution of the Bovine Prothrombin Gene. Ph.D.

diss., University of British Columbia, Vancouver

Ishii, S., Xu, Y . , Stratton, R .H. , Roe, B.A., Merlino, G.T. and Pastan, I. (1985).

Characterization and Sequence ofthe Promoter Region of the Human Epidermal

Growth Factor Receptor Gene. Proc. Natl. Acad. Sci. U S A 82, 4920-4924.

Jackson, C M . & Nemerson, Y . (1980). Blood Coagulation. Ann. Rev. Biochem. 49,

765-811.

James, M . N . G . , Delbaere, L . T . J , and Brayer, G.D. (1978). Amino Acid Sequence

Alignment of Bacterial and Mammalian Pancreatic Serum Proteases Based on

Topological Equivalents. Can. J . Biochem. 56; 396-402.

Jaye, M . , de la Salle, H . , Schamber, F . , Balland, A . , Kohli, V . , Findeli, A. , Tolstoshev, P.

and Lecocq, J.P. (1983). Isolation of Anti-Haemophilic Factor IX cDNA Using a

Unique 52-Base Synthetic Oligonucleotide Probe Deduced from the Amino Acid

Sequence of Bovine Factor IX. Nucl. Acids Res. 11; 2325-2335.

Jorgensen, M . J . , Cantor, A .B . , Furie, B.C. , Brown, C .L . , Shoemaker, C B . and Furie, B.

(1987). Recognition Site Directing Vitamin K-Dependent Gammma-Carboxylation

Resides on the Propeptide of Factor IX. Cell. 48, 185-191.

Kan, Y.W. & Dozy, A . M . (1978). Polymorphism of D N A Sequence Adjacent to Human

7-Globin Structural Gene: Relationship to Sickle Mutation. Proc. Natl. Acad. Sci.

(USA) 75, 5631-5635.

, Kaplan, A.P. (1978). Initiation of the Intrinsic Coagulation and Fibrinolytic Pathways of

Man: The Role of surfaces, Hageman Factor, Prekallikrein, High Molecular Weight

Kininogen and Factor XI. Prog. Hemostasis Thromb. 4, 127-175.

Kaplan, A.P. & Austen, K . F . (1971). A Prealbumin Activation of Prekallikrein. J . Exp.

Med. 133, 696-712.

Kerbiriou, D . M . and Griffin, J . H . (1979). Human High Molecular Weight Kininogen.

Studies of Structure-Function Relationships and of Proteolysis of the Molecule

Occurring During Contact Activation of Plasma. J . Biol. Chem. 254; 12021-12027.

Kessel, M . and Khoury, G. (1983). Induction of Cloned Genes After Transfer Into

Eukaryotic Cells in "Expression of Cloned Genes in Prokaryotic and Eukaryotic

Cells", Vol. 3 (T.S. Papas, M . Rosenberg, and J . G . Chirikjian, eds.), Elsevier, New

York, pp. 233-260.

Kingston, R .E . , Kaufman, R.J. and Sharp, P.A. (1984). Regulation of Transcription of the

adenovirus EII Promoter by E1A Gene Products-Absence of Sequence Specificity.

Mol. Cell. Biol. 4, 1970-1977.

Kisiel, w., Fujikawa, K. and Davie, E .W. (1977). Activation of Bovine Factor VII

(Proconvertin) by Factor X l l a (Activated Hageman Factor). Biochem. 16;

4189-4194.

Koschinsky, M . K . , Funk, W.F. , van Oost, B.V. and MacGillivray, R.T.A. (1986).

Complete cDNA Sequence of Human Preceruloplasmin. Proc. Natl. Acad. Sci.

U S A 83, 5086-5090.

Kozak, M . (1984). Compilation and Analysis of Sequences Upstream From the

Translational Start Site in Eukaryotic mRNAs. Nuc. Acids Res. 12, 857-873.

Kurachi, K . and Davie, E .W. (1977). Activation of Human Factor XI by Factor Xl la .

Biochemistry, 16; 5831-5839.

Kurachi, K . and Davie, E.W. (1982). Isolation and Characterization of a cDNA Coding for

Human Factor IX. Proc. Natl. Acad. Sci. U S A 79; 6461-6464.

Kurachi, K . , Fujikawa, K. and Davie, E.W. (1980). Mechanism of Activation of Bovine

Factor XII by Factor XII and Factor Xl la . Biochemistry 19, 1330-1338.

Laurent, B .C. , Nilsson, M . H . L . , Bavik, C , Jones, T .A . , Sundelin, J . and Peterson, P.A.

(1985). Characterization of the Rat Retinol-binding Protein Gene and Its Comparison

to the Three-Dimensional Structure of the Protein. J . Biol. Chem. 260,

11476-11480.

Lawn, R . M . , Adelman, J . , Bock, S.C., Franke, A . E . , Houck, C M . , Najarian, R . C , Seeburg,

P .H. and Wion, K . L . (1981). The Sequence of Human Serum Albumin cDNA and Its

Expression in E. Coli. Nucl. Acids Res. 9; 6103-6114.

Lee, M . G . , Lewis, S.A., Wilde, C D . and Cowan, N .J . (1983). Evolutionary History of a

Multigene Family: An Expressed Human 0-Tubulin Gene and Three Processed

Pseudogenes. Cell 33, 477-487.

Lehrach, H . , Diamond, D., Wozney, J.R. and Boedtker, H . (1977). RNA Molecular Weight

Determination by Gel Electrophoresis Under Denaturing Conditions, a Critical

Examination. Biochemistry 16; 4743-4751. of Disease, Second Edn., W.B.

Saunders, Philadephia.

Lerner, M.R. and Steitz, J . A. (1979). Antibodies to Small Nuclear RNAs Complexed

With Proteins are Produced by Patients With Systemic Lupus Erythematosus.

Proc. Natl. Acad. Sci. USA 76; 5495-5499.

Lewin, B. (Ed.) (1983). Mechanisms of RNA Splicing in Genes. John Wiley and Sons,

New York, pp. 405-416.

Leytus, S.P., Chung, D.W., Kisiel, W., Kurachi, K. and Davie, E .W. (1984).

Characterization of a cDNA Coding for Human Factor X. Proc. Natl. Acad. Sci.

U S A 81; 3699-3702.

Leytus, S.P., Foster, D . C , Kurachi, K. and Davie, E.W. (1986). Gene for Human Factor

X. A Blood Coagulation Factor Whose Gene Organization is Essentialy Identical with

that of Factor IX and Protein C. Biochem. 25, 5098-5102.

L i , W- .H. , Luo, C-.C. and Wu, C-. I. (1985). Evolution of DNa Sequence, in Molecular

Evolutionary Genetics (Maclntyre, R.J . Ed.) Plenum Press, New York, pp. 1-94.

Lindquist, S. (1986). Heat Shock Response. Ann. Rev. Biochem. 55; 1151-1191.

Long, G . L . , Belagaje, R .M. & MacGillivray, R .T .A . (1984). Cloning and Sequencing of

Liver cDNA Coding for Bovine Protein C. Proc. Natl. Acad.Sci. U S A 81;

5653-5656.

Lucas, M.A. , Fretto, L . J . & McKee, P.A. (1983). The Binding of Human Plasminogen to

Fibrin and Fibrinogen. J . Biol. Chem. 258; 4249-4256.

Lundwall, A . , Dackowski, W., Cohen, E . , Shaffer, M . , Mahr, A., Dahlback, B. (1986).

Isolation and Sequence of the Complementary D N A for Human Protein S, A

Regulator of Blood Coagulation. Proc. Natl. Acad. Sci. U S A 83; 6716-6720.

MacFarlane, R.G. (1964). An Enzyme Cascade in the Blood Clotting Mechanism and Its

Function as a Biological Amplifier. Nature 202; 498-499.

MacGillivray, R.T.A. , Chung, D.W. and Davie, E.W. (1979). Biosynthesis of Bovine

Plasma Protein in a Cell-Free System: Amino-Terminal Sequence of Preproalbumin.

Eur. J . Biochem. 98; 477-485.

MacGillivray, R.T.A. , Degen, S.J.F., Chandra, T. , Woo, S.L.C. and Davie, E .W. (1980).

Cloning and Analysis of a cDNA Coding for Bovine Prothrombin. Proc. Natl. Acad.

Sci. U S A 77; 5153-5157.

MacGillivray, R.T.A. and Davie, E .W. (1984). Characterization of Bovine Prothrombin

mRNA and Its Translation Product. Biochemistry 23; 1626-1634.

MacGillivray, R.T.A. , Irwin, D . M . , Guinto, E.R. and Stone, J .C . (1986). Recombinant

Genetic Approaches to Functional Mapping of Thrombin. N .Y. Acad. Sci. Ann.

485, 73-79.

Magnusson, S., Petersen, T.E.,Sottrup-Jensen, L . and Claeys, H . (1975). Complete

Primary Structure of Prothrombin: Isolation, Structure and Reactivity of Ten

Carboxylated Glutamic Acid Residues and Regulation of Prothrombin Activation by

Thrombin.in Proteases and Biological Control (Reich, E . , Rifkin, D.B. and Shaw, E . ,

Eds.) pp. 123-149, Cold Spring Harbor Laboratories, Cold Spring Harbor, N.Y.

Malinowski, D.P., Sadler, J . E . and Davie, E .W. (1984). Characterization of a

Complementary Deoxyribonucleic Acid Coding for Human and Bovine Plasmingen.

Biochemistry 23; 4243-4250.

Mandle, R., Jr. , Colman, R.W. and Kaplan, A.P. (1976). Identification of Prekallikrein and

High Molecular Weight Kininogen as a Complex in Human Plasma. Proc. Natl.

Acad. Sci. USA. 73; 4179-4183.

Mandle, R., Jr . and Kaplan, A.P. (1977). Hageman Factor Substrates. Human Plasma

Prekallikrein Mechanism of Activation by Hageman Factor and Participation in

Hageman Factor-Dependent Fibrinolysis. J . Biol. Chem. 252; 6097-6104.

Maniatis, T., Jeffrey, A. & Kleid, D . G . (1975). Nucleotide Sequence of the Rightward

Operator of Phage X. Proc. Natl. Acad. Sci. U S A 72;1184-1188.

Maniatis, T., Fritsch, E .F . & Sambrook, J . (1982) Molecular Cloning. A Laboratory

Manual, Cold Spring Harbor Laboratories, Cold Spring Harbor, N .Y.

Manley, J . L . (1983). Analysis of the Expression of Genes Encoding Animal mRNA by in

vitro Techniques. Prog. Nucl. Acid Res. Mol. Biol. 30, 195-244.

Margolis, J . (1958). Activation of Plasma by Contact With Glass: Evidence for a Common

Reaction Which Releases Plasma Kinin and Initiates Coagulation. J . Physiol.

(Lond.) 144, 1-22.

Marquart, M . , Walter, J . , Deisenhofer, J . , Bode, W. and Haber, R. (1983). Acta

Crystallogr. Sect. B, 39; 480.

Mason, A . J . , Evans, B.A., Cox, D.R., Shine, J . and Richards, R.J. (1983). Structure of

Mouse Kallikrein Gene Family Suggests a Role in Specific Processing of Biologically

Active Peptides. Nature (Lond.) 303, 300-307.

McDonagh, J . , Hada, M . and Kaminski, M . (1985). Plasma Fibronectin and Fibrin

Formation in "Plasma Fibronectin: Structure and Function" (McDonagh, J . Ed.)

Marcel Dekker, Inc. New York, pp. 121-148.

McGraw, R., Frazier, D., Deserres, M . , reisner, H . , Stafford, D. (1986). Antigenic

Determinants in Human Coagulation Factor IX Immunoglobulin Screening and D N A

Sequence Analysis of Recombinant Phage Map-A Monoclonal Antibody to Residue

111 Through Residue 132 of the Zymogen. Blood 67, 1344-1348.

McKnight, S.L. and Kingsbury, R. (1982). Transcriptional Control of Signals of a

Eukaryotic protein-Coding Gene. Science 217, 316-324.

McMillin, C.R., Saito, H . , Ratnoff, O.D. & Walton, A . G . (1974). The Secondary Structure

of Human Hageman Factor (Factor XII) and its Alteration by Activating Agents. J .

Clin.Invest. 54, 1312-1322.

McMullen, B.A. and Fujikawa, K. (1985). Amino Acid Sequence of the Heavy Chain of

Human a-Factor X l l a (Activated Hageman Factor). J . Biol Chem. 260, 5328-5341.

Melton, D.W., Konecki, D.S., Brennand, J . and Caskey, C.T. (1984). Structure,

Expression, and Mutation of the Hypoxanthine Phosphoribosyltranserase Gene.

Proc. Natl. Acad. Sci. U S A 81, 2147-2151.

Meier, H . L . , Pierce, J . V . , Colman, R.W. & Kaplan, A.P. (1977). Enhancement of the

Surface Dependent Hageman Factor Activation by High Molecular Weight Kininogen.

J . Clin. Invest. 60; 18-31.

Messing, J . (1983). New M13 Vectors for Cloning. Methods Enzymol. 101, 20-78.

Messing, J . , Crea, R. and Seeburg, P .H. (1981). A system for Shotgun D N A Sequencing.

Nucl. Acids Res. 9; 309-321.

Montell, C , Fisher, E . E . , Caruthers, M . H . and Berk, A . J . (1983). Inhibition of RNA

Cleavage But Not Polyadenylylation by a Point Mutation in mRNA Concensus

Sequence A A U A A A . Nature 305; 600-608.

Mori, K. and Nagasawa, S. (1981). Studies on High Molecular Weight (HMW) Kininogen

II. Structural Change of H M W kininogen by the Action of Human Plasma Kallikrein.

by the J . Biochem. (Tokyo) 89; 1465-1473.

Mosher, D.F . (1975). Cross-Linking of Cold-Insoluble Globulin by Fibrin-Stabilizing

Factor. J . Biol. Chem. 250; 6614-6621.

Mount, S.M. (1982). A Catalogue of Splice Junction Sequences. Nuc. Acids Res. 10,

459-472.

Movat, H.Z. and Ozge-Anwar, A . H . (1974). The Contact Phase of Blood Coagulation:

Clotting Factors XI and XII, Their Isolation and Interaction. J . Lab. Clin. Med.

84; 861-877.

Mullertz, S. (1984). Fibrinolysis: An Overview. Semin. Throm. Hemostas. 10; 1-5.

Nagamine, Y . , Pearson, D., Altus, M.S. and Reich, E . (1984). cDNA and Gene Nucleotide

Sequence of Porcine Plasminogen Activator. Nucl. Acids Res. 12; 9525-9541.

Nagamine, Y . , Pearson, D. and Grattan, M . (1985). Exon-Intron Boundary Sliding in the

Generation of Two mRNAs Coding for Porcine Urokinase-Like Plasminogen

Activator. Biochem. Biophys. Res. Comm. 132, 563-569.

Nakayasu, T. and Nagasawa, S. (1979). Studies on Human Kininogens. J . Biochem.

(Tokyo) 85; 249-258.

Naora, H . and Deacon, N.J . (1982). Relationship Between the Total Size of Exons and

Introns in Protein-Coding Genes of High Eukaryotes. Proc. Natl. Acad. Sci. U S A

79, 6196-6200.

Nemerson, Y . and Furie, B. (1980). Zymogens and Cofactors of Blood Coagulation. CRC

Crit. Rev. Biochem. 9; 45-85.

Neurath, H . (1984). Evolution of Proteolytic Enzymes. Science 224; 350-357.

Neurath, H . (1985). Proteolytic Enzymes, Past and Present. Fed. Proc. 44; 2907-2913.

Neurath, H . and Walsh, K . A . (1976). The Role of Proteases in Biological Regulation, in

Proteolysis and Physiological Regulation (Robbins, D.W. and Brew, K. Eds.),

Academic Press, new York, pp. 29-42.

Nevins, J.R. (1983). The Pathway of Eukaryotic mRNA Formation. Ann. Rev.

Biochem. 52; 441-466.

Noller.H.F. (1984). Structure of Ribosomal RNA. Ann. Rev. Biochem. 53; 119-162.

Ny, T., Elgh, F . & Lund, B. (1984). the Structure of the Human Tissue-Type Plasminogen

Activator Gene: Correlation of Intron and Exon Structures to Functional and

Structural Domains. Proc. Natl. Acad. Sci. U S A 81; 5355-5359.

Ohkubo, I., Fujikawa, K. and Kurachi, K. (1982). Mechanism of Activation of Human

Factor XI by Human Factor XII, a-Factor X l l a and 0-Factor Xl la . Fed. Proc. Fed.

Am. Soc. Exp. Biol. 41; 656.

Palmiter, R.D., Gagnon, J . , Ericsson, L . H . and Walsh, K . A . (1977). Precursor of Egg

White Lysozyme. J . Biol. Chem. 252; 6386-6893.

Patthy, L . , Trexler, M . , Vali, Z. , Banyai, L and Varadi (1984). Kringles: Modules

Specialized for Protein Binding. Fed. Eur. Biochem. Soc. 171; 131-136.

Patthy, L . , (1985). Evolution of the Proteases of blood Coagulation and Fibrinolysis by

Assembly From Modules. Cell 41, 657-663.

Pennica,D., Holmes, W . E . , Kohr, W.J . , Harkins, R.N. , Vehar, G.A. , Ward, C.A. , Bennett,

W.F . , Yelverton, E . , Seeburg, P .H. , Heyneker, H . L . Goeddel, D.V. and Collen, D.

(1983). Cloning and Expression of Human Tissue-Type Plasminogen Activator cDNA

in E . coli. Nature (Lond.) 301, 214-221.

Perry, R.P. (1976). Processing of RNA. Ann. Rev. Biochem. 45, 605-629.

Pesce, A . J . and Dosekun, A . K . (1983). Interrelationships Between the Immune Systems,

Complement, Coagulation and Inflammation. Clin. Physiol. Biochem. 1; 92-116.

Petersen, T . E . and Skorstengaard, K. (1985). Primary Structure in "Plasma Fibronectin:

Structure and Function" (McDonagh, J . Ed.) Marcel Dekker, Inc. New York,

pp.7-26.

Petersen, T . E . , Thogersen, H.C. Skorstengaard, K . , Vibe-Pedersen, K . , Sahl,P.,

Sottrup-Jensen, L . & Magnusson, S. (1983). Partial Primary Structure of Bovine

Plasma Fibronectin: Three Types of Internal Homology. Proc. Natl. Acad. Sci.

U S A 80,137-141.

Plow, E . F . , Birdwell, C. and Ginsberg, M . H . (1979). Identification and Quantification of

Platelet-Associated Fibronectin Antigen. J . Clin. Invest. 63; 540-543.

Plow, E . F . (1980). The Major Fibrinolytic Proteases of Human Leukocytes. Biochim.

Biophys. Acta 630; 47-56.

Plow, E . F . (1982). Leukocyte Elastase Release During Blood Coagulation. J . Clin.

Ivest. 69; 564-572.

Plutzky, J . , Hoskins, J . , Long, G.L. and Crabtree, G.R. (1986). Evolution and

Organization of the Human Protein C Gene. Proc.Natl. Acad. Sci. U S A (1986) 83,

546-550.

Prochownik, E . V . , Markham, A . F . & Orkin, S.H. (1983). Isolation of a cDNA Clone for

Human Antithrombin III. J . Biol. Chem. 258; 8389-8394.

Proudfoot, J . M and Brownlee, G.G. (1976). 3' Non-Coding Region Sequences in Eukaryotic

Messenger RNA. Nature (Lond.) 265, 211-214.

Que, B.G. and Davie, E.W. (1986). Characterization of a cDNA Coding for Human Factor

XII (Hageman Factor). Biochem. 25, 1525-1528.

Radcliffe, R., Bagdasarian, A. , Colman, R. and Nemerson, Y. (1977). Activation of Bovine

Factor VII by Hageman Factor Fragments. Blood 50; 611-617.

Ragg, H . and Weissman, C. (1983). Not More Than 117 Base Pairs of 5' Flanking

Sequence are Required for Inducible Expression of a Human IFN-a gene. Nature

(Lond.) 303; 439-442.

Ratnoff, O.D. and Colopy, J . H . (1955). A Familial Hemorrhagic Trait Associated With a

Deficiency of a Clotting Promoting Fraction of Plasma. J . Clin. Invest. 34;

602-613.

Ratnoff, O.D. & Rosenblum, J . M . (1958). Role of Hageman Factor in the Initiation of

Clotting by Glass. American J . Medicine 25, 160-168.

Ratnoff, O.D. (1969). Some Relationships Among Hemostasis, Fibrinolytic Phenomena,

Immunity and the Inflammatory Response. Adv. in Immunol. 10; 146-228.

Ratnoff,O.D. and Saito, H . (1979). Surface-Mediated Reactions. Curr. Top. Hematol. 2,

Reich, E . (1975). Plasminogen Activator: Secretion by Neoplastic Cells and Macrophages

in "Proteases and Biological Control" (Reich, E . , Rifkin, D.B., Shaw, E . , Eds.). Cold

Spring Harbor Laboratory, pp. 333-341.

Revak, S.D. and Cochrane, C .J . (1976). The Relationship of Structure and Function in

Human Hageman Factor. The Association of Enzymatic Binding and Activities With

Separate Regions of the Molecule. J . Clin. Invest. 57; 852-860.

Revak, S.D., Cochrane, C.J . and Griffin, J . H . (1977). The Binding and Cleavage

Characteristics of Human Hageman Factor During Contact Activation. A

Comparison of Normal Plasma With Plasmas Deficient in Factor X i , Prekallikrein, or

High Molecular Weight Kiniogen. J . Clin. Invest. 59; 1167-1175.

Revak, S.D., Cochrane, C .G. , Johnston, A.R. and Hugli, T . E . (1974). Structural Changes

Accompanying Enzymatic Activation of Human Hageman Factor. J . Clin.Invest.

54, 619-627.

Reynolds, G.A. , Basu, S.K., Osborne, T . F . , Chin D.J . , Gil, G. , Brown, M.S. , Goldstein, J . L .

and Luskey, K . L . (1984). H M G CoA Reductase: A Negatively Regulated Gene With

Unusual Promoter and 5' Untranslated Reigons. Cell 38, 275-285.

Riccio, A . , Grimaldi, G. , Verde, P., Sebastio, G. , Boast, S. and Blasi, F. (1985). The

Human Urokinase-Plasminogen Activator Gene and its Promoter. Nuc. Acids Res.

13, 2759-2771.

Rogers, J . (1985). Exon Shuffling and Intron Invasion in Serine Protease Genes. Nature

(Lond.) 315, 458-459.

Rosing, H . , Tans, G. and Griffin, J . H . (1985). Surface-Dependent Activation of Human

Factor XII (Hageman Factor) by Kallikrein and Its Light Chain. J . Biochem. 151;

531-538.

Saito, H . (1977). Purification of High Molecular Weight Kininogen and the Role of This

Agent in Blood Coagulation. J . Clin. Invest. 60; 584-594.

Salerno, G. , Verde, P., Nolli, M . L , Corti, A . , Szots, H . , Meo, T., Johnson, J . , Bullock, S.,

Cassani, G and Blasi, F . (1984). Proc. Natl. Acad. Sci. U S A 81; 110-114.

Sanger, F . , Nicklen, S. & Coulson, A.R. (1977). D N A Sequencing With Chain-Terminating

Inhibitors. Proc. Natl. Acad. Sci. U S A 74; 5463-5467.

Sawyer, L . , Shotton, D . M . , Campbell, J .W., Wendell, P .L. , Muirhead, H . , Watson, H.C. ,

Diamond, R. and Ladner, R.C. (1978). The Atomic Structure of Crystalline Porcine

Pancreatic Elastase at 2.5 A Resolution: Comparisons With the Structure of

a-Chymotrypsin. J . Mol. Biol. 118; 137-208.

Schapira, M . , Scott, C.F. and Colman, R.W. (1982). Contribution of Plasma protease

Inhibitors to the Inactivation of Kallikrein in plasma. J . Clin. Invest. 69; 462-468.

Schwarzbauer, J .D. , Tamkun, J .W., Lemischka, I.R. and Hynes, R.D. (198). Three

Different Fibronectin mRNAs Arise By Alternative Splicing Within the Coding

Region. Cell 35; 421-431.

Schapira, M . , Despland, E . , Scott, C , Boxer, L . A . and Colman, R.W. (1982). Purified

Human Plasma Kallikrein Aggregates Human Blood Neutrophils. J . Clin. Ivest.

69; 1191

Schmid, C W . and Jelinek, W.R. (1982). The Alu Family of Dispersed Repetitive

Sequences. Science 216; 1065-1070.

Scott, C.F . , Schapira, M . , James, H . L . , Cohen, A.B. and Colman, R.W. (1982).

Inactivation of Factor XIa by Plasma Proteae Inhibitors. J . Clin. Invest. 69;

844-852.

Sealy, J . E . , Atlas, S.A., Laragh, J . H . , Silverberg, M . and Kaplan, A.P. (1979). Initiation

of Plasma Prorenin Activation by Hageman FactorDependent Coversion of Plasma

Prekallikrein to Kallkrein. Proc. Natl. Acad. Sci. U S A 76; 5914-5918.

Seligsohn, U . , Osteoid, B., Griffin, J . H . and Rapaport, S.I. (1978). Evidence for the

Participation of Both Activated Factor XII and Activated Factor IX in Cold-Promoted

Activation of Factor VII. Thrombosis Res. 13; 1049-1056.

Seligsohn, U . , Osteoid, B., Brown, S.F., Griffin, J . H . and Rapaport, S.I. (1979).

Activation of human Factor VII in Plasma and in Purified Systems: Roles of

Activated Factor IX, Kallikrein and Activated Factor XII. J . Clin. Invest. 64;

1056-1065.

Shatkin, A . J . (1985). On the Origin of Splicing and Introns. Cell 42; 397-400.

Silverberg, M . , Nicoll, J . E . and Kaplan, A.P. (1980). The Mechanism by Which the Light

Chain of Cleaved H M W kininogen Augments the Activation of Prekallikrein, Factor

XI and Hageman Factor. Thromb. Res. 20; 173-189.

Sottrup-Jensen, L . , Claeys, H . , Zajdel, M . , Petersen, T . E . , Magnusson, S. (1978a) The

Primary Structure of Human Plasminogen: Isolation of Two Lysine-Binding

Fragments and One "Mini-" Plasminogen (MW, 38,000) by Elastase-Catalyzed

Specific Limited Proteolysis, in Progress in Chemical Fibrinolysis and Thrombolysis

(Davidson, J .F . , Rowan, R . M . , Samama, M . M . , Desnoyers, P.C., Eds.) Raven Press,

New York, pp. 191-209.

Sottrup-Jensen, L . , Petersen, T . E . , Magnusson, S. (1978b) In Atlas of Protein Sequence

and Structure 5; 91.

Southern, E . M . (1975).Detection of a Specific Sequence Among D N A Fragments Separated

by Gel Electrophoresis. J . Mol. Biol. 98, 503-517.

Staden, R. (1982). Automation of the Computer Handling of Gel Reading Data Produced

by the Shotgun Method of D N A Sequencing. Nucl. Acids Res. 10, 4731-4751.

Stenflo, J . and Suttie, J .W. (1977). Vitamin K-Dependent Formation of

7-Carboxyglutamic Acid. Ann. Rev. Bochem. 46; 157-172.

Strauss, A .W. , Bennett, C D . , Donohue, A . M . , Rodkey, J .A . and Alberts, A .W. (1977).

Rat Liver Pre-Proalbumin: Complete Amino Acid Sequence of the Pre-Piece. J . Biol.

Chem. 252; 6846-6855.

Strickland, S. (1978). Studies on the Role of Plasminogen Activator in Ovulation and

Early Embryogenesis. In "Regulatory Proteolytic Enzymes and Their Inhibitors"

(Magnusson, S., Ottesen, M . , Foltman, B. , Dano, K . , Neurath, H . , Eds.) Pergamon

Press, Oxford, pp. 181-185.

Sundsmo, J.S. and Fair, D.S. (1983). Relationships Among the Complement, Kinin,

Coagulation and Fibrinolytic Systems in the Inflammatory Reaction. Clin. Physiol.

Biochem. 1; 225-284.

Suttie, J .W. (1985) Vitamin K-Dependent Carboxylase. Ann. Rev. Biochem. 54;

459-477.

Swift, G . H . , Craik, C.S., Stary, S.J., Quinto, C , Lahaie, R.G. , Rutter, W.J . and

MacDonald, R.J. (1984). Structure of the Two Related Elastase Genes Expressed in

the Rat Pancreas. J . Biol. Chem. 259, 14271-14278.

Tans, G. and Griffin, J . H . (1982). Properties of Sulfatides in Factor XII Dependent

Contact Activation. Blood 59; 69-75.

Thompson, R.E. , Mandle, R., Jr. and Kaplan, A .P . (1977). Association of Factor XI and

High Molecular Weight Kinogen in Human Plasma. J . Clin. Invest. 60; 1376-1380.

Thompson, R .E . , Mandle, R. Jr. , and Kaplan, A.P. (1979). Studies of the Binding of

Prekallikrein and Factor XI to High M W Kininogen and Its Light Chain. Proc. Natl.

Acad. Sci. U S A 76; 4862-4866.

Tripodi, M . , Citarella, F . , Guida, S., Galeffi, P., Fantoni, A. and Cortese, R. (1986). cDNA

Sequence Coding for Human Coagulation Factor XII (Hageman Factor). Nucl. Acids

Res. 14, 3146.

Valerio, D., Duyvesteyn, M . G . C . , Dekker, B . M . M . , Weeda, G. , Berkvens, Th. M . , van der

Voorn, L . , van Ormandt, H . and van der Eb, A . J . (1985). Adenosine Deaminase:

Characterization and Expression of a Gene With a Remarkable Promoter. E M B O J .

4, 437-443.

van den Elsen, P., Georgopoulos, K. , Shepley, B., Orkin, S. and Terhorst, C. (1986).

Exon/Intron Organization of the Genes Coding for the A Chains of the Human and

Murine T-Cell Receptor/T3 Complex. Proc. Natl. Acad. Sci. U S A 83, 2944-2948.

van der Graaf, F . , Keus, F . J . , Vlooswijk, R.A. and Bouma, B.N. (1982). The Contact

Activation Mechanism in Human Plasma: Activation Induced by Dextran Sulfate.

Blood 59; 1225-1233.

van der Graaf, F . , Tans, G. , Bouma, B .N. and Griffin, J . H . (1982). Isolation and

Functional Properties of the Heavy and Light Chains of Human Plasma Kallikrein.

J . Biol. Chem. 257, 14300-14305.

van der Graaf, F . , Koedam, J .A. and Bouma, B.N. (1983). Inactivation of Kallikrein in

Human Plasma. J . Clin. Invest. 71; 149-158.

van der Graaf, F . , Greengard, J.S., Bouma, B.N., Kerbiriou, D . M . and Griffin, J . H .

(1983b). Isolation and Functional Characterization of the Active Light Chain of

Activated Human Blood Coagulation Factor XI. J . Biol. Chem. 258; 9669-9675.

van Leeuwen, B . H . , Evans, B.A. , Tregear, G.W. and Richards, R.I. (1986). Mouse

Glandular Kallikrein Genes: Identificaton, Structure, and Expression of the Renal

Kallikrein Gene. J . Biol. Chem. 261, 5529-5535.

van Zonneveld, A . - J . , Veerman, H . and Pannekoek, H . (1986). Autonomous Functions of

Structural Domains on Human Tissue-Type Plasminogen Activator. Proc. Natl.

Acad. Sci. U S A 83, 4670-4674.

Verde, P., Stoppelli, M.P. , Galeffi, P., Di Nocera, P., and Blasi, F . (1984). Identification

and Primary Sequence of an Unspliced Human Urokinase Poly A + RNA. Proc. Natl.

Acad. Sci. U S A 81; 4727-4731.

Vieira, J . and Messing, J . (1982). The pUC Plasmids, an M13mp7 Derived System for

Insertion Mutagenisis and Sequencing With Synthetic Universal Primers. Gene 19;

259-268.

Wachtfogel, Y . T . , Kucich, U . , James, H . J . , Scott, C F . Schapira, M . , Zimmerman, M . ,

Cohen, A . B . and Colman, R.W. (1983). Human Plasma Kallikrein releases

Neutrophil Elastase During Blood Coagulation. J . Clin. Invest. 72, 1672-1679.

Wachtfogel, Y . T . , Pixley, R.A., Kucich, U . , Abrams, W., Weinbaum, G. , Schapira, M . and

Colman, R.W. (1986). Purified Plasma Factor X l l a Aggregates Human Neutrophils

and Causes Degranulation. Blood 67, 1731-1737.

Wallen, P., Pohl, G. , Bergsdorf, N . , Randby, M . , Ny, T., and Jornvall, H . (1983).

Purification and Characterization of a Melanoma Cell Plasminogen Activator. Eur.

J . Biochem. 132; 681-686.

Walker, J . M . , Hastings, J.R.B. & Johns, E.W. (1977). The Primary Structure of a

Non-Histone Chromosomal Protein. Eur. J . Biochem. 76; 461-468.

Watson, M . E . E . (1984). Compilation of Published Signal Sequences. Nucl.Acids. Res.

12, 5145-5164.

Wieringa, B., Hofer, E . and Weissmann, C. (1984). A Minimal Intron Length But No

Specific Internal Sequence is Required For Splicing the Large Rabbit 0-Globin Intron.

Cell 37; 915-925.

Wiggins, R.C. , Giclas, P.C. and Henson, P.M. (1981). Chemotactic Activity Generated

from the Fifth Component of Complement by Plasma Kallikrein of the Rabbit. J .

Exp. Med. 153; 1391-1404.

Wilner, G.D., Nossel, H . L . & Leroy, E . C . (1968). Activation of Hageman Factor by

Collagen. J . Clin. Invest. 47; 2608-2615.

Wilson, A . C . , Carlson, S.S. and White, T .J . (1977). Biochemical Evolution. Ann. Rev.

Biochem. 46; 573-639.

Wiman, B. (1978) Biochemistry of the Plasminogen to Plasmin Coversion in "Fibrinolysis.

Current Fundamental and Clinical Aspects" (Gaffney, P.J. , Balkuv-Ulutin, S., Eds.)

Academic Press, London, pp. 47-60.

Wiman, B. and Collen, D. (1977). Purification and Characterization of Human

Antiplasmin, the Fast-acting Plasmin Inhibitor in Plasma. Eur. J . Biochem. 78;

19-26.

Wiman, B. and Collen, D. (1978). On the Kinetics of the Reaction Between Human

Antiplasmin and Plasmin. Eur. J . Biochem. 84; 573-578.

Wright, D .G. (1982). The Neutrophil as a Secretory Organ of Host Defense. Adv. in Host

Defense Mechan. 1; 75-110.

Wun, T .C . , Ossowski, L . , Reich, E . (1982). A Proenzyme Form of Human Urokinase. J .

Biol. Chem. 257; 7262-7268.

Yamada, K . M . (1983). Cell Surface Interactions with Extracellular Materials. Ann.

Rev. Biochem. 52, 761-799.

Yamamoto, T. and Cochrane, C.G. (1981). Guinea Pig Hageman Factor as a Vascular

Permeability Enhancement Factor. Am. J . Pathol. 105; 164

Young, C .L . , Barker, W . C , Tomaselli, C M . , and Dayhoff, M.O. (1978). Serine Proteases,

in Atlas of Protein Structure (Dayhoff, M.O. Ed.) vol. 5 (suppl. 3), National

Biomedical Research Foundation, Silver Spring, Maryland, pp. 73-93.

Yoshitake, S., Schach, B .G. , Foster, D . C , Davie, E.W. and Kurachi, K. (1985). Nucleotide

Sequence of the Gene for Human Factor IX. (Antihemophiliac Factor B). Biochem.

24, 3736-3750.

Young, R.A. and Davis, R.W. (1983a). Efficient Isolation of Genes by Using Antibody

Probes. Proc. Natl. Acad. Sci. U S A 80; 1194-1198.

Young, R.A. and Davis, R.W. (1983b). Yeast RNA polymerase II Genes: Isolation With

Antibody Probes. Science 222; 778-282.

Ziccardi, R .J . (1981). Activation of the Early Components of the Classical Complement

Pathway Under Physiologic Conditions. J . Immunol. 126; 1769-1773.

Ziemer, M . A . , Swain, W.F. , Rutter, W.J . , Clements, S., Ann, D.K. and Carlson, D . M .

(1984). Nucleotide Sequence Analysis of a Proline-Rich cDNA and Peptide

Homologies of Rat and Human Proline-Rich Proteins. J . Biol. Chem. 259,

10475-10480.

Zuckerkandl, E . and Pauling, L . (1965). Evolutionary Divergence and Convergence in ,

Plasma Proteins in Evolving Genes and Proteins (Bryson, V . and Vogel, H . J . , Eds.),

Academic Press, New York, pp. 97-166.

Zur, M . and Nemerson, Y. (1981). Tissue Factor Pathways of Blood Coagulation in

Haemostasis and Thrombosis (Bloom, A . L . and Thomas, D.P., Eds.) Churchill and

Livingston, Edinburgh, pp. 124-139.

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