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Xerox University Microfilm#300 NorVt ZMb RoadAnn Arbor, Michigan 40106

MASTERS THESIS M-6S45

SFEAIMAN, Al«n d ’Avesne, 1948- ABNMMAL HLMAN HB40GLOBINS INVOLVING AMINO ACID SUBSrnUTIONS: RANDOM OR NON-RANDOM PROTEIN MJTATIONS? A CURRENT INVENTORY AND STATISTICAL ANALYSIS.

The American U n iv e rs ity , M .A., 1974 G enetics

Xerox University Microfilms , Ann Arbor. Michigan 4B106

^ Copyrighted by Alan d'Avesne Spealman

1974

THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED.

ABHOBMAL HUMAN HEMOGLOBINS INVOLVING AMINO ACID SUBSTITUTIONS ;

RANDOM OR NON-RANDOM PROTEIN MUTATIONS?A CURRENT INVENTORY AND STATISTICAL ANALYSIS,

by

Alan d'Avesne Spealman

Submitted to the

Faculty of the College of Arts and Sciences

of The American University

in Partial Fulfillment of

the Requirements of the Degree

of

Master of Arts

Anthropology

Signatures of Como^tee:

Chairman :

Dean of th& College Date : Date:

1974

The American University Washington, D.C.

THE AMEHICM UNIVERSITY LIBRARY

ACKNOWLEDGEMENTS

I w o u ld l i k e t o e x p r e s s my g r a t e f u l t h a n k s t o Dr. Gary

Hume f o r s e r v i n g t h e t r i p l e r o l e s o f g r a d u a t e a d v i s o r , t h e s i s

c o m m it te e c h a i r m a n , a n d r e s i d e n t p h y s i c a l a n t h r o p o l o g i s t i n

t h e d e p a r tm e n t o f a n t h r o p o l o g y .

I am a l s o i n d e b t e d t o t h e o t h e r m em bers o f my c o m m i t t e e ,

D r s . Edward P i e r c e and C h a r l e s M cN ett , t h e f o r m e r f o r f o s t e r

i n g my i n t e r e s t i n b i o c h e m i c a l g e n e t i c s an d t h e m ech an ism s o f

human e v o l u t i o n , a n d t h e l a t t e r f o r h i s u s e f u l s u g g e s t i o n s i n

s t a t i s t i c a l a n a l y s i s .

G r a t i t u d e ' i s a l s o e x te n d e d to t h e s t a f f o f t h e N a t i o n a l

L i b r a r y o f M e d ic in e , B e th e s d a , M a ry la n d , f o r t h e i r a b l e a s

s i s t a n c e i n c o m p i l i n g t h e d a t a .

TABLE OP CONTENTS

P age

^ACKNOWLEDGEMENTS i

TABLE OF CONTENTS Ü

LIST OF TABLES i v

LIST OP FIGURES y ü

CHAPTER

I . INTRODUCTION 1

B a c k g ro u n d 1

The R e s e a r c h P rob lem 4

D a ta 6

I n v e n t o r y 7

I I , HEMOGLOBIN 21

P r o t e i n s : S t r u c t u r e and F u n c t io n 21

H e m o g lo b in o p a th y : The U n s ta b leH em oglob ins 23

P h y s i o l o g y o f H em oglobin 27

H e m o g lo b in o p a th y : H em oglobins W ithA l t e r e d Oxygen A f f i n i t y 28

I I I . GENETICS OP HEMOGLOBIN 37

One G ene: One P r o t e i n 37

Random an d Non-Random P r o t e i n M u t a t i o n s : P ro to m e rs and P o s i t i o n 40

Random a n d Non-Random M u ta t io no f P ro to m e rs 43

Random a n d Non-Random M u ta t io nt h e Amino A c id s 46

i i

i l l

CHAPTER Page

Random a n d Non-Random M u t a t i o n :t h e Codon B a s e s 48

IV , a n a l y s is and r e s u l t s 75

R andom ness Among C h a in s 75

R andom ness o f Amino AcidR e p la c e m e n ts 75

RandoxOiiess o f N u c l e o t id eB ase S u b s t i t u t i o n s 75

T r a n s i t i o n s a n d T r a n s v e r s i o n s 76

LITERATURE CITED 77

LIST OF TABLliS

T a b l e P age

1• Ghi^ T e s t t o D e te rm in e i f Hemo- P o i n t M u ta t io n s a r e Random ly D i s t r i b u t e d O ver t h e A lp h a ,B e t r , Gamma a n d D e l t a C h a in s 20

2# C h i^ T e s t t o d e t e r m i n e i f Hemog l o b i n P o i n t M u t a t i o n s a r e C a u se d b y N u c l e o t i d e R e p la c e m en t Random ly D i s t r i b u t e d Over t h e F i r s t , S e c o n d a n d T h i r d P o s i t i o n o f Codon T r i p l e t s 50

3# K o lm o g o ro v -S m irn o v One Sam ple T e s t o f M u ta t io n From a G iven Amino A c id A ssum ing Random Amino A c id R e p la ce m en t* A lp h a C h a in 51

4* K o lm o g o ro v -S m irn o v One Sam ple T e s t o f M u ta t i o n From a G iven Amino A c id A ssum ing Random Amino A c id R e p la c e m e n t . B e ta C h a in 52

5 . K o lm o g o ro v -S m irn o v One Sam ple T e s to f M u ta t io n From à G iven Amino A c id A ssum ing Random Amino A c id R e p la c e m e n t . Gamma C h a in 53

6 , K o lm o g o ro v -S m irn o v One Sam ple T e s to f M u ta t io n From a G iv en Amino A c id A ssum ing Random Amino A cidR e p la c e m e n t . D e l t a C h a in 54

7 . K o lm o g o ro v -S m im o v One Sam ple T e s to f M u ta t io n To a G iven Amino A c id A ssum ing Random Amino A c id r e p l a c e m e n t . A lpha C h a in . 55

8 , K o lm o g o ro v -S m irn o v One Sam ple T e s to f K iu ta t io n To a G iven Amino A c id A ssum ing Random Amino A c id r e p l a c e m e n t . B e t a C h a in 56

; . K o lm o g o ro v -S m irn o v One Sam ple T e s to f 1%t a t i o n To a G iven Amino A c id A ssum ing Random Amino A cid R e p la c e m e n t , Gamma C h a in 57

1 0 , K o lm o g o ro v -S m irn o v One Sam ple T e s to f M u ta t io n To a G iven Amino A c id A ssum ing Random Amino A c id R e p la c e m e n t . D e l t a C h a in 58

i v

T a b le Page

1 1 . K o lm o g o ro v -S m irn o v One Sam ple T e s to f M u t a t i o n f ro m a G iv e n Amino A c id A ssum ing Random B a se Subs t i t u t i o n . T o t a l 59

1 2 . K o lm o g o ro v -S m irn o v One Sam ple T e s to f M u t a t i o n f ro m a G iven Amino A c id A ssu m ing Random B ase Subs t i t u t i o n . A lp h a C h a in 60

1 ^ K o lra o g o ro v -S m irn o v One Sam ple T e s t o f M u t a t i o n f ro m a G iven Amino A c id A ssum ing Random B a se Subs t i t u t i o n . B e ta C h a in 61

1 4 . K o lm o g o ro v -S ra irn o v One Sam ple T e s t o f M u t a t i o n from a G iv en Amino A c id A ssu m in g Random B ase Subs t i t u t i o n . Gamma C h a in 62

15# K o lm o g o ro v -S m irn o v One Sam ple T e s t o f M u t a t i o n f ro m a G iven Amino A c id A ssum ing Random B ase Subs t i t u t i o n . D e l t a C h a in 63

1 6 , K o lm o g o ro v -S m irn o v One Sam ple T e s to f M u t a t i o n t o a G iven Amino A cid A ssum ing Random Base S u b s t i t u t i o n .T o t a l 64

1 7 . K o lm o g o ro v -S m irn o v One Sam ple T e s to f I ' i u t a t i o n t o a G iven Amino A cid a s s u m in g Rfjidom B ase S u b s t i t u t i o n ,A lp h a C h a in 65

1 8 . K o lm o g o ro v -S m irn o v One Sam ple T e s to f M u t a t i o n to a G iv en Amino A c id A ssu m in g Random B a se S u b s t i t u t i o n ,B e ta C h a in 66

1 9 , K o lm o g o ro v -S m irn o v One Sam ple T e s to f M u t a t i o n t o a G iven Amino A cid A ssu m in g Random B ase S u b s t i t u t i o n ,Gamma C h a in 67

2 0 , K o lm o g o ro v -S m irn o v One Sam ple T e s to f m u t a t i o n t o a G iven Amino A cid A ssu m in g Random B ase S u b s t i t u t i o n . D e l t a C h a in 68

2 1 . E x p e c t e d F r e q u e n c i e s o f Amino A c id sb y t h e T o t a l Number o f P o s s i b l e S i n g l e B ase S u b s t i t u t i o n s an d Exp e c t e d E b e q u e n c ie s o f T r a n s i t i o n s a n d T r a n s v e r s i o n s gg

vi

T a b le P ag e

2 2 , B in o m ia l T e s t o f T r a n s i t i o n sand T r a n s v e r s i n s 72

▼ii

LIST UP FIGURES

yF i g u r e -tage

1 , P o s s i b l e Amino A c id r e p l a c e m e n t s R e s u l t i n g BVora a S i n g l e B ase C hange 29

2* The Common xjnino A c id s 30

3# The H e l i c a l and N o n - H e l i c a l P o r t i o n s o f t h e A lp ha a n d B e ta C h a in s o f Human H em oglob in an d o f Human M y o g lo b in 31

4» T o t a l O b s e rv e d Amino A c id S u b s t i t u t i o n son Human H em oglob in C h a in s 32

5 . A lp h a C h a in : O b se rv e d Amino A c idS u b s t i t u t i o n s 35

6 , B e ta C h a in : O b se rv e d Amino A cidS u b s t i t u t i o n s 34

7* D e l t a C h a in : O b se rv e d Amino A c idS u b s t i t u t i o n s 35

8 , Gamma C h a in : O b s e rv e d amino A c ids u b s t i t u t i o n s 36

9 . O b s e rv e d T r a n s i t i o n s and T r a n s v e r s i o n s 70

1 0 , P e d i g r e e o f K i l e n g e (New B r i t a i n )F a m ily w i t h Hb J - T o n g a r i k i 73

11 , l e d i g r e e o f H u n g a r ia n F a m ily w i t htwo A bnorm al H em o g lo b in s i n A d d i t i o n to Hb A, 74

1

CRAPTEH I

INTRODUCTION

B a c k g round

The p r i m a r y a im o f p h y s i c a l a n t h r o p o l o g y i s t o e x p l a i n t h e

p a s t and j i r e s e n t e v o lu t i o n - o f t h e human o r g a n i s m by e x a m in in g

t h e c h a n g e s i n gene f r e q u e n c i e s b r o u g h t a b o u t by t h e a d a p t a t i o n

o f o u r s p e c i e s t o i t s v a r i o u s e c o l o g i c a l s i t u a t i o n s t h r o u g h t h e

p r o c e s s e s o f m u t a t i o n and n a t u r a l s e l e c t i o n .

S in c e e a c h a s p e c t o f human v a r i a t i o n i s t h e r e s u l t o f e i t h

e r o n g o in g o r c o m p le te d n a t u r a l s e l e c t i o n , and b e c a u s e t h e num

b e r o f v i a b l e p ro g e n y i s a m e a s u re o f t h e f i t n e s s o f a p o p u l a t i o n ,

s e l e c t i o n c a n be i n t e r p r e t e d a s t h e r e s u l t o f m o r t a l i t y and f e r

t i l i t y ; d i " f e r e n c e s i n f i t n e s s can be i n t e r p r e t e d i n t e r m s o f

d i f f e r e n t i a l m o r t a l i t y and d i f f e r e n t i a l f e r t i l i t y (M o tu l s k y , 1 9 6 0 ; 2 9 ) .

One o f t h e m o s t i m p o r t a n t f a c t o r s l e a d i n g t o t h e d i f f e r

e n c e s i n m o r t a l i t y r a t e s o f human p o p u l a t i o n s i s t h e s e l e c t i v e

o c c u r r e n c e o f g e n e t i c a l l y i n h e r i t e d d i s e a s e o r d i s e a s e s u s c e p t

i b i l i t y , i n d e e d t h e f a c t t h a t some p a t h o l o g i c a l t r a i t s w e re fo u n d

to e x i s t o n ly i n c e r t a i n g e o g r a p h i c a r e a s was one o f t h e f i r s t

phenom ena p h y s i c a l e j i t h r o p o l o g i a t s b e g a n to c o n s i d e r a s h a v in g

t h e pow er to e x p l a i n t h e m ech an ism s o f e v o l u t i o n .

The c l a s s i c exam ple o f "chis a j jp ro a c h b e g a n w i t h t h e d i s

c o v e r i e s o f t h e t r a i t r e s p o n s i b l e f o r t h e s y n t h e s i s o f t h e ab

n o rm a l h e m o g lo b in r e s p o n s i b l e f o r s i c k l e c e l l a n e m ia ( N e e l , 194-9;

6 4 -6 6 ) a n d t h e s t r u c t u r a l d i f f e r e n c e b e tw e e n t h i s v a r i a n t an d

norm .cl h e m o g lo b in ( P a u l i n g , 1949 ; 5 4 3 ) . T h is p a r t i c u l a r v a r i

a n t , h e m o g lo b in S (Hb S , o r a l p h a - 2 - A b e t a - 2 - S ) , i s t h e r e s u l t

o f t h e s u b s t i t u t i o n o f a m o le c u le o f v a l i n e f o r one o f g lu ta m ic

a c i d a t t h e s i x t h r e s i d u e p o s i t i o n o f t h e b e t a c h a i n s . The a l -

l e i e s i n v o lv e d ( i . e . t h o s e r e s p o n s i b l e f o r b e t a c h a i n p r o d u c t i o n )

a r e i n h e r i t e d c o d o m i n a n t l y , b o th m a n i f e s t i n g t h e m s e l v e s w i t i i e -

q u a l power i n t h e p h e n o t y p e , t h u s from t h e m a t in g o f two h e t e r o -

z y g o te s ( a l p h a - 2 - A b e t a - l - A - b e t a - 2 - S ) t h e r e a r e t h r e e r e s u l t a n t

g e n o ty p e s w h ic h w i l l o c c u r i n a 1 i 2 ; 1 r a t i o . T h o se hom ozy

gous f o r S r a r e l y r e a c h m a t u r i t y a s t h e y s u f f e r f ro m s e v e r e hemo

l y t i c a n e m ia , t h o s e h e t e r o z y g o u s f o r Hb-S e x h i b i t t h e s i c k l i n g

t r a i t b u t n o t t h e , a t h o l o g i c a l c o n d i t i o n , and t h o s e h om ozygous

f o r Hb-A p ro d u c e o n l y n o rm a l h e m o g lo b in .

U s u a l l y s u c h d e l e t e r i o u s t r a i t s a r e b r e d o u t o f a g e n e p o o l

q u i c k l y s i n c e t h o s e i n d i v i d u a l s homozygous f o r them a r e g e n e r a l

l y l e s s h e a l t h y a n d / o r l e s s f e r t i l e t h a n o t h e r g e n o t y p e s . Y e t

i n some iV fr ica n p o p u l a t i o n s th e t r a i t f r e q u e n c y r e a c h e s 40Jo a n d

among some M e d i t e r r a n e a n p o p u l a t i o n s o b s e rv e d f r e q u e n c i e s r a n g t ,

from 5% t o 30% ( B u e t t n e r - J a n u s c h , 1 9 6 7 ) . The m ec h an ism m a i n t a i n

i n g a s i t u a t i o n o f t h i s s o r t ( a b a l l a n c e d p o ly m o rp h ism ) i s o f t e n

p o s i t i v e s e l e c t i o n a c t i n g on t h e h e t e r o a . , g o t e ; c o n d i t i o n s m u s t

e x i s t ouch t h o t t h e d e a t h s o f t h e homozygous m u t a n t s ( i n t h i s e x

am ple from o x y g en t r a n s p o r t d e f f i c i e n c y ) an d t h e hom ozygous norm

a l s (from m a l a r i a ) a r e c o m p e n sa te d by t h e s u r v i v a l a n d b r e e d i n g

p o t e n t i a l o f t h e h e t e r o z y g o t e s .

E x p e r im e n ta l s t u d i e s h ave shown t h a t t h e h e t e r o z y g o t e s a r e

m ore r e s i s t a n t t o a t y p e o f m a l a r i a c a u s e d by H a sm o d iu m f a l c i

pa ru m , a p a r , s i t e t r a n s m i t t e d by t h e i j i o p h e l e s m o s q u i to ( A l l i s o n ,

1954; 3 1 2 ) . F u r t h e r e v id e n c e c o n f i rm e d t h a t t h e num ber o f p a r a

s i t e s was lo w e r i n t h o s e e x h i b i t i n g t n e s i c k l i n g t r a i t t h a n i n

n o rm al i n d i v i d u a l s , p re s u m a b ly due to t h e i r b l o o d b e i n g a p o o r e r

h o s t medium. I t i s a l s o known t h a t a r e a s o f h ig h i n c i d e n c e o f

t h i s m o s q u i to c o i n c i d e w i t h a r e a s o f h i g h f r e q u e n c i e s o f t h e

s i c k l i n g t r a i t . The c o n c l u s i o n was t h a t s i n c e m a l a r i a i s m ore

3d e t r i m e n t a l to t h e g en e p o o l s u n d e r e x a m in a t io n t h a n i s th e s i c k -

l i n g , p o s i t i v e s e l e c t i o n f o r t h e t r a i t i s i n e v i d e n c e . I f t h i s

i s t r u e and t h e t r a i t i s i n e q u i l i b u i u m , i . e . t h e ch an g e i n gene

f r e q u e n c y w i t h r e s p e c t t o t im e i s z e r o , t h e r e l a t i v e f i t n e s s o f

t h e h e t e r o z y g o t e s c a n be c a l c u l a t e d v i a s e l e c t i o n c o e f f i c i e n t s :

^ q = p q ( s ^ p - S g q ) / ! - ( s ^ p f - S g q ) = 0

w here p and q a r e t h e r e s p e c t i v e f r e q u e n c i e s of a and h, a p p ro x

i m a t e l y 0 . 8 and 0 . 2 . F o r :.he p u r p o s e o f t h i s e x a m p le , t h e r e l

a t i v e f i t n e s s o f t h e ,(S ;hom ozygo te c an be e s t i m a t e d t o b e 0 .2 5 ;

t h o s e s u f f e r i n g from a n e m ia w i l l p ro d u c e o n ly 25% a s many c h i l d

r e n a s t h e h e t e r o z y g o t e ( B u e t t n e r - J a n u s c h , 1 9 6 7 ; 4 2 1 ) . The s e l

e c t i o n a g a i n s t th e n o rm a l (A) hom ozygo te c a n t h e n b e e x p r e s s e d a s ;

S,, = S g q /p « ( Q . 2 ) ( l - 0 . 2 5 ) / ( 0 , 8 ) = 0 .1 8 8

and t h e r e l a t i v e f i t n e s s o f t h e h e t e r o z y g o t e a s 1 / 1 - s ^ = 1/ 4*4188

» 1 . 2 3 . The f i g u r e 1 ,2 3 r e p r e s e n t s a 23% r e l a t i v e a d v a n t a g e .

The a n t h r o p o l o g i c a l i m p l i c a t i o n s o f t h e s e u a t a f o l l o w e d s h o r t

l y , L i v i n g s t o n e (1 9 5 8 ) p r o p o s e d t h a t s i n c e P . f a l c i p a r u m i s a v e r y

r e c e n t v a r i e t y o f t h i s g e n u s to h ave d e v e lo p e d a p a r a s i t i c r e

l a t i o n s h i p w i th man an d s i n c e A n o p h e le s i s n o t a n o rm a l e le m e n t

o f t r o p i c a l r a i n f o r e s t f a u n a , r e q u i r i n g open s t a g n a n t p o o l s i n

w h ic h to b r e e d , t h i s r e l a t i o n s h i p c o u ld n o t h av e e v o lv e d b e f o r e

t h e i n v e n t i o n o f s l a s h and b u r n a g r i c u l t u r e , t h e o p e n in g o f t h e

r a i n f o r e s t , t h e p r o v i s i o n o f i d e a l b r e e d i n g g ro u n a s f o r A n o p h e le s

a n d t h e i n c r e a s e d d e n s i t y o f a v a i l a b l e h o s t s f o r P . . f a l c i p a r u m .

The " a g r i c u l t u r a l r e v o l u t i o n " i n ^ i f r i c a was t h u s a c u l t u r a l mod

i f i c a t i o n o f t h e p h y s i c a l e n v iro n m e n t p r o d u c in g a new e c o l o g i c a l

n i c h e t o w h ic h t h e human p o p u l a t i o n h a s a d a p t e d a t l e a s t p a r t i a l l y

v i a a m u ta n t p r o t e i n .

H ere t h e n i s one exam ple o f an a b n o rm a l h e m o g lo b in whose

e v o l u t i o n c a n b e t r a c e d i n d e t a i l and whose s e l e c t i v e a d v a n ta g e

h a s b e e n d i s c o v e r e d .

4Tiig. R e s e a r c h P ro b le m

W ith in t h i s a r e a o f s t u d y w h ic h h a s come to be k^iown a s

" g e n e t i c a n th r o p o l o g y " ( J o h n s t o n , 1 9 7 0 ; 9 9 ) , d i r e c t e x a m in a t io n

o f human p r o t e i n s , enzym es .nd t h e i r s t r u c t u r a l v a r i a n t s i s th e

l e v e l o f i n v e s t i g a t i o n m o s t e a s i l y e x p l o i t e d s i n c e e a c h o f t h e s e

m o le c u l e s i s th e p r im a r y r e s u l t .>f gene a c t i o n , t h e amino a c i d

s e q u e n c e c o rv e s u o n d in g to a s e r i e s o f codon t r i p l e t s on a s e g

m en t o f DNA, a n d s i n c e s t r u c t u r a l a n a l y s i s h a s become r e l a t i v e l y

r o u t i n e l a b o r a t o r y p r o c e d u r e b y p a r t i a l h y d r o l y s i s ( M i y a j i , o t . a l , ,

1 9 6 7 ) and Edman d e g r a d a t i o n ( C h e r n o f f an d L iu , 1 9 6 1 ) ,

D i f f e r e n t i a l d i s t r i b u t i o n o f s u c h p o ly m o rp h ism s i n p o p u la

t i o n s w h ic h h ave e x i s t e d i n v a r y i n g e c o l o g i c a l s i t u a t i o n s s h o u ld

s u p p l y i n f o r m a t i o n c o n c e r n i n g t h e i r p o s s i b l e a d a p t i v e v a l u e s , e -

v e n when f r e q u e n c i e s a r e q u i t e low a s b o t h B i r d s e l l (195 2 ) and

M o tu lsk y ( i9 6 0 ) h a v e n o t e d , t h a t i s , even i f a t r a i t h a d a f r e

q u en cy o f o n ly 1%, t h e f a c t t h a t i t e x i s t s i n e q u i l i b r i u m i m p l i e s

some a d v a n ta g e f o r i t s c a r r i e r s .

Of t h e many fo rm s o f m u ta n t h e m o g lo b in s t h u s f a r d i s c o v e r

e d , t h e m a j o r i t y a r e t h e r e s u l t o f a s i n g l e t r a n s i t i o n o r t r a n s

v e r s i o n ( s e e b e lo w ) i n uhe codon sé q u e n c e t h a t i s t r a n s l a t e d i n

s y n t h e s i s a s a s u b s t i t u t i o n o f a s i n g l e amino a c i d on one o r

m ore o f t h e n o rm a l p o l y p e p t i d e s . The r e l a t i v e l y h ig h f r e q u e n c i e s

a n d d i f f e r e n t i a l d i s t r i b u t i o n o f some o f t h e s e h e m o g lo b in v a r

i a n t s among t h e human p o p u l a t i o n o f f e r th e p h y s i c a l a n t h r o p o l o g i s t

and human g e n e t i c i s t an o p p u r t u n i t y to s t u d y t h e m echan ism s o f

fiene f lo w and n a t u r a l s e l e c t i o n i n m a i n t a i n i n g p o ly m o rp h ic t r a i t s

a s w e l l a s p r e s e n t i n g s e v e r a l i n t e r e s t i n g r o b le m s .

W orking from t h e l e v e l o f t h e p o p u l a t i o n down, t h e f i r s t

p ro b le m i s t h a t o f dem ography ond e c o l o g y , a s i n t h e c a s e o f hemo-

5g l o b i n , s p e c i f i c a l l y , w hat a r e t h e a g e n t s o f n a t u r a l s e l e c t i o n ,

t h r o u g h w h a t p r e s s u r e s ( i f a n y ) a r e t h e v a r i a n t s s e l e c t e d f o r o r

a g a i n s t . U n f o r t u n a t e l y th e s o l u t i o n to t h i s q u e s t i o n h a s been

f o u n d o n l y f o r a v e r y few o t h e r m u t a t i o n s ( e , g ,H b - C ; due p r im

a r i l y t o t h e l a c k o f t h e n e e d e d p o p u l a t i o n s a m p l i n g d a t a . Al

t h o u g h L i v i n g s t o n e ' s work (1 9 6 ?) c o n s t i t u t i s a m i l e s t o n e i n t h e

d e m o g ra p h y o f a b n o rm a l h e m o g lo b in s , t h e d a t a a r e s t i l l f a r to o

s p a r s e i n m o s t c a s e s to g e n e r a t e h y p o t h e s e s c o n c e r n i n g t h e i r s e l

e c t i v e a d v a n t a g e s .

On a n o r g a n ! s m i c /m o l e c u la r l e v e l - a n d t h e c e n t z a l p rob lem

t o b e e x p a n d e d h e r e - a r e t h e q u e s t i .ns o f t h e e f f e c t o f amino

a c i d s u b s t i t u t i o n s on t h e n o rm a l f u n c t i o n i n g o f t h e h e m o g lo b in

m o l e c u l e , t h e c h a i n s and p o s i t i o n s a f i e c t e d a n d t h e am ino a c i d s

i n v o l v e d . I t i s g e n e r a l l y assum ed t h a t a l l m u t a t i o n s a r e r a n

dom, y e t t h i s a s s u m p t io n h a s r e c e n t l y b e e n b r o u g h t u n d e r s c r u

t i n y ( r e r u t z and Lehmann, 1 9 6 8 ; O a r r e l l a n d L ehm ann , 1 9 6 9 ; J o n e s ,

e t , a l , , 1 9 7 2 ) , and w i l l be exam ined by s u b j e c t i n g th e f o l l o w i n g

q u e s t i o n s t o s t a t i s t i c a l a n a l y s i s ;

1 , Are t h e num ber o f a l ,yha, b e t a , gamma an d d e l t a m u t a t i o n s i n t h e e x p e c t e d r a t i o ?

2 , j i r e t h e t r a n s v e r s i o n s i n p r o p o r t i o n t o t h e t r a n s i t i o n s ?

3 , Are t h e m u ta t io n s ran d o m ly d i s t r i b u t e d o v e r e a c h c h a in ?

4 , vire t h e m u ta t io n s random w i t h r e g a r d t o t h e am ino a c i d s i n v o l v e d , a n d s i n c e t h e t h e g e n e g ic c o d e i s d e g e n e r a t e ,

5 , J ire t h e p o s s i b l e b a s e c h a n g e s random ?

I n t h e i n t e r e s t o f c l a r i t y , a l l m u t a t i o n s w i l l be d e s c r i b e d

a c c o r d i n g t o t h e f o l l o w i n g m o d e l : a lp h a 54 g l n - a r g (Hb S h im o n o se k i)

d e s c r i b e s t h e m u ta n t h e m o g lo b in S h im o n o sek i i n w h ic h a s u b s t i t u t i o n

o f a r g i n i n e f o r g lu ta m in e h a s o c c u r r e d a t t h e 5 4 ^^ r e s i d u e p o s i t i o n

o f t h e a l p h a p o l y p e p t i d e c h a i n .

6D a ta

The d a t a t o b e u s e d i n t h e a b o v e a n a l y s i s w i l l c o n s i s t o f

a c o m p re h e n s iv e i n v e n t o r y o f a l l r e p o r t e d h e m o g lo b in v a r i a n t s pub

l i s h e d i n t h e m e d i c a l l i t e r a t u r e a s o f F e b . 1 , 1 9 7 4 . T h i s i n v e n t

o r y w i l l b e p r e s e n t e d i n a f o r m a t s i m i l a r t o t h e f o u r e a r l i e r p u b

l i s h e d c o m p i l a t i o n s ( L i v i n g s t o n e , 1 9 6 7 ; n u i s m a n , 1 9 6 9 ; D a y h o f f ,

1 9 7 0 ; M cK usick , 1 9 7 1 ) and w i l l c o n t a i n 266 human h e m o g lo b in m u ta t

i o n s , a l t e r n a t i v e nam es g iv e n to e a c h , n e t c h a r g e a t j . h y B i o l o g i c a l

pH, r e s i d u e an d h e l i c a l p o s i t i o n t n d c o m p o s i t i o n o f am ino a c i d

s u b s t i t u t i o n an d a g e n e r a l d i s c u s s i n o f g e o g r a p h i c o c c u i r e n c e

a n d c l i n i c a l m a n i f e s t a t i o n s w h e re a p p l i c a b l e .

R e s id u e

5

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Inventory From To

of IRuaan Hemoglobin Point NatationsALPHA CHAIN

Y

a la

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9 o r 12 ? ?

12 a l a a s p

15 g i y a s p

16 l y s g lu

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21 a l a a s p

22 g i y a s p

23 g lu l y s

23 g l u g i n

23 g lu v a l

23 ? ?

27 g lu g i y

30 g lu g i n

43 phe v a l

47 a s p g i y

47 a s p h i s

50 h i s a s p

51 g i y a r g51 g i y a s p

54 g i n a r g

54 g i n g lu

c a m e ls j

J - T o r o n t o

Saw ara

G -P e s t

J - P a r i s

I - I n t e r l a k e n J - a l p h a - O x f o r d II - P h i l a d e l p h i a I - T e x a s I - B u r l i n g t o n Ube I I I

J -N y a n z a

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Chad

Memphis

G -A u d h a li

D- a l p h a - C y p ru s

F t , W orth

G -C h in eseH o n o lu luHongkongS in g a p o reT o r in o

L - F e r r a r a UmiK okura Tagawa I I M ich ig an I Y ukuhash i I B e i l e n s o n S i n a i S e a ly H a sh a ro n J - S a r d e g n a

R ussJ - A b i d j a n

S h im o n o sek i H iro sh im a U p s a l l a J -M e x ic o P a r i s I I

S o u r c e ( s )

C r o o k s to n , e t . a l . 1965

S um ida , e t . a l , 1973

H o l l a n , e t . a l . 1972

R o sa , e t . a l . 1965 R a h b a r , e t . a l . 1972 M a r t i , e t . a l , 1964

B e a le an d Lehmann 1965

I m a i , e t . a l , 1970

K e n d a l l , e t . a l . 1973

G o t t l i e b , e t . a l . 1964

B o y e r , e t . a l . 1969

K ra u s , e t . a l . 1966

M arengo-R cw e, e t . a l . 1968

Ingram 1963

S c h n e i d e r , e t . a l . 1971

Sw enson, e t . a l , 1962

B e r e t t a , e t . a l . 1968 P r # t o , e t . a l . 1970 B ia n c o , e t . a l . 1963 D e V r ie s , e t . a l . 1963

G r e e r , e t . a l . 1971 C h a ra c h e and Mondzak 1967

G a l lo e t . a l . 1972 P e r u t z a n d Lehmann 1968 R e y n o ld s and Huisman 1966 G a b a n n ts e t . a l , 1972

H anada and R u c k n a g e l 1963

F e s s a s , e t . a l . 1969

8R a h b a r , e t . a l , 196957 giy a r g L - P e r s i a n G u lf

57 giy a s p J - N o r f o l k G - I b id a n K agoshim a N i s h i k i I , I I , I I I

B a g l io n i e t . a l . 1963

58 h i s t y r M -B oston O saka G o th e n b u rg L e i p z i g I I K o ln

G e ra ld and r f r o n 1961

60 l y s a s n Z am bia B a r c la y e t . a l . 1969

61 l y s a s n J -B u d a H o l la n e t . a l . 1972

64 a s p a s n G-W aimanalo B la c k w e l l e t . a l . 1975

64 a s p h i s w - I n d ia Sukumaran e t . a l . 1972

6 4 ,7 4 ,8 5 a s p l y s G - P a r i s L a b ie and a c h a p i r a 1966

68 a s n a s p Ube I I M iy a j i e t . a l . 1967

68 a s n l y s G - P h i l a d e l p h i a C h e rn o f f and P e t t i t 1965

70

74

75

75

7879

78

80

84

85

85

87

v a l

a s p

a s p

a s p

a s na l a

a s n

l e u

s e r

a s p

a s p

h i s

a s p

h i s

h i s

t y r

a s pgiy

l y s

a r g

a r g

a s n

t y r

t y r

G -K n o x v i l le G - B r i s t o l G -A z a k o u l is D -W ash ing ton D - S t .L o u i s D -B a l t im o re S t a n l e y v i l l e I G— S t —I S e a t t l e

M a h id o l G -TaiChung Q -S .E . A s ia W a l p h a

W in n ipeg

J - S i n g a p o r e

A nderson e t . a l . 1973

P o o t r a k u l , e t . a l . 1970 B la c k w e l l and L iu 1970

L o r k i n , e t . a l . 1970

V e l la e t . a l . 1973

B la c k w e ll e t . a l . 1972

S t a n l e y v i l l e I I vanRos e t . a l , 1968

Ann A rb o r

E to b ic o k e

G -N o rfo lk

At ago

M-Iwate 01 d e n b u rg K ankakee

Adams, e t . a l . 1972

C ro o k s to n e t . a l . 1969

Huntsman e t . a l . 1963

F a j i w a r a , e t . a l . 1970

S h i b a t a , e t . a l , 1964

90 l y s a s n J - B r o u s s a i s î ü j i t a e t . a l . 1972Tagawa I V e l l a e t . a l . 1970

90 l y s t h r J - R a j a p p e n H yde, e t . a l . 197192 a r g l e u Che s a p e . .k e C h a r a c h e , e t . a l . 1966

92 a r g g i n J -O a p e to w n b o t h a , e t . a l , 1966

95 n ro l e u G -G e o rg ia S m i th , e t . a l , 1972

95 p ro s e r Rampa S m i th , e t . a l . 1972

95 p ro a r g S t ,L u k e s B l a c k w e l l , e t . a l . 1972

95 pro a l a uenm ark H i l l W i l t s h i r e e t . a l , 1972

99 a s p h i s Yakim a J o n e s , e t . a l . 1967

99 a s p t y r Y p s i l a n t i G ly n n , e t . a l . 1968

102 s e r a r g M a n i to b a C r o o k s t o n , e t . a l . 1970

106 l e u P ro S o u th a m to n H yde, e t . a l , 1972G a sp e r J o n e s , e t . a l , 1972

112 h i s g in D akar L a b ie ; no R o sa 1968

112 h i s a s p H o iid in s 11 S c h r o e d e r a n d J o n e s 19 65

114 p ro a r g C h ia p a s J o n e s e t . a l , 1968

115 a l a a s p J - T o n g a r i k i G a jd u s e k , e t . a l , 1966

116 g l u l y s 0 - I n d o n e s i a Eng a n d s a d o n e 1958X -B u g in e s e

136 l e u p ro B ibba K l e i n h a u e r , e t . a l . 1968

141 a r g p r o S i n g a p o r e C l e g g , e r # a l . 1969

141 a r g n i l K o l l i k e r M a r t i , e t . a l . 1967

n fc S o e u l I Kim and S'nih 1969

n fc M -K is k u n a la s H o l l a n and S z e l e n y i 1965

n f c J -M a c e d o n ia S a d i k a r i o , e t . a l . 1968

n f c M -R ese rve O v e r l y , e t . a l . 1966

n f c Ii-Bombay H a l l - C r a g g s e t . a l . 1 9 6 4

n fc N i c o s i a F e s s a s e t . a l . 1965

n f c K-M adras Lehmann 1962

n fc J - I n d i a Lehmann 1962

10

n f c

n f c

n f c

n f c

n f c

155 o r 156 r e s i d u e s

172 r e s i d u e s

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C o lum bia

T h a i

Kumamoto

Koya D ora

v i l l b r o o k , e t . a l , 1965

A l l b r o o k , e t . a l . 1965

N a g e l , e t . a l . 1964

W asi , e t . a l . 1972

K usom oto , 1967

B e r n i n i , e t . a l . 1970

C o n s t a n t S p r i n g C le g g , e t . a l . 1971

11

R e s id u e2

2

673

6

6

6

7

7

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16

17

19

20

22

22

22

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24

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25

26

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h i s a r g

g l u v a la s p a s n

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BETA CHAINN am e(sJ T o k u c h i

D e e r Lodge

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3

0

G-M akassair

C - o i r i r a j G-Honan

G -oan J o s e

P o r t o A le g r e

Sogn

J - B a l t i m o r e J - T r i n i . d a d J - I r e l a n d N-New Haven D-Bushman

N a g a s a k i

D -O u led Hagba

O lym pia

G - T a ip e i

G -S a s k a to o n G -C h o u s h a t t a G -H sin Ghu G -T a igu D - l r a n

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S a v a n n a h

R i v e r d a l e - B r o n x

G -T aiw an iuai

E

S o u r c e ( s )S h i b a t a , e t . a l , 1963

L a b o s s i e r e , e t . a l . 1972

B o o k e h in , e t . a l . 1967

In g ram 1957

I t a n o ; n<i K e e l 1950

B i a c k w i l l , e u . a l , 1970

'T u c h in a d a , e t . a l , 1965 L l a c k w e l l , e t . a l . 1970

R i l l , e t . a l . I9 6 0

B o n a v e n tu r a and R ig e s 1967

Monn, e t . a l . 1968

W e a t h e r a l l , e t . a l , 1964

Wade, e t . a l , 1967

Maekav/a, e t . a l , 1970

E l i o n , e t . a l . 1975

o ta m a to y a n n o p o u lo B e t . a l .1973

B l a c k w e l l , e t . a l . 1969

Bowman, e t . a l . 1967 B l a c k w e l l , e t . a l . 1967

E l i o n , e t . a l . 1973

Eng, e t . a l . 1970

J o n e s , e t . a l . 196b

H u ism an , e t . a l , 1971

R a n n ey , e t . a l . 1968

P e r u t z an c Lehmann 1968 B l a c k w e l l , e t . a l . 1968 B l a c k w e l l , e t . a l . 1970

28 l e u p r o Genova

12

S a n s o n e , e t . a l . 1967

30 a r g s e r Taoom a B a u r a n d R ow ley 1 9 6 4

32 l e u pro Abraham L i n c o l n H o n i g , e t . a l , 1 973

35 t y r phe P h i l l y R e i d e r , e t . a l . 1969

37 t r p s e r H i r o s e

C h e r n o f f a n d P e t t i t 196!

Yamoka 1971

42 phe s e r H am m ersm ith W ajcm an, e t . a l , 1973

42 phe l e u L o u i s v i l l e K e e l i n g , e t . a l . 1971

43 g lu a l a

B u c c u r e s t i

G - G a lv e s to n

B r a t u , e t . a l , 1971

Bowman, e t . a l , 1962

46 gly g lu

G -T ex as G - P o r t A r t h u r

K - I b i d a n A l l a n , e f . a l . 1965

47 a s p a s n G -G openhagen S i c k , e t . a l . 1967

50 t h r l y s E dm onton L a b o s s i e r e , e t . a l . 1971

52 a s p a s n O s u - G h r i s t i a n s b o r g K o n o t e y - a h u l u , e t .

52 a s p a l a O c h o -R io s

a l . 1971

B e r e s f o r d , e t . a l . 1972

56 g ly a s p J -B a n g k o k C l e g f , e t . a l . 1966

58 p ro a r g

J - l I a n a d oJ -M e in u n gJ - K o r a tL h o f a r P u j i t a , e t . a l . 1972

59 l y s g lu I - H i g h Wycombe B o u l to n e t . a l . 1970

59 l y s t h r K a o h s iu n g B l a c k w e l l , e t . a l . 1971

61 l y s g luJ - H o n o l u l u N - B e a t t i e H u eh ns an d S h o o te i ’ 1966

61 l y s a sn H i k a r i S h i b a t a , e t . a l . 1964

63 h i s t y r M -B a sk a to o n G e r a l d an d E f r o n 1961

63 h i s a r g

FknoryHamburgArhusC h ic a g oL e i p z i gRandomHi d aKurmeZ u r i c h M u l l e r a n d K ingm a 1961

66 l y s g lu T o u lo u s e L a b i e , e t . a l . 1971

' % "

67 V8-1 a s p B r i s t o l S teadm an e t . a l . 1970

67 v a l a l a Bydney G a r r e l l , e t . a l . 1967

67 v a l g lu M-M ilwaukee I G e ra ld emd E fro n 1961

69 g l y a s p J -G a m b r id g eJ-Rambam

S i c k , e t . i - f . 1965

69 ' l y a r g A -2 I n d o n e s i a Eng, e t . a l . 1971

71 phe s e r C h r i s t c h u r c h G a r ; e l l , e t . a l , 1971

73 a s p a s n K o rle -B u K o n o te y -J ih u lu , e t . a l . 1969

74 g l y a s p Bhe a r d s Bush W h ite , e t . a l . 1970

76 a l a g lu S e a t t l e Huehns and S h o o te r 1966

77 h i s a s p J - I r a n R a h b a r , e t . a l . 1967

79 a s p g l y G—H s-Tsou B l a c k w e l l , e ; , . a l . 1969

79 a s p a s n G -A ccra Lehmann e t . a l , 1964

80 a s n l y s G-SzuhuG ifu

B l a c k w e l l , e t . a l . 1969 M atsum oto 1971

83 g l y c y s T i - L i B l a c k w e l l , e t . a l . 1971

87 t h r l y s D - I b id a n W a ts . ,n -W il l ia m s , e t . a l . 1965

88 l e u a r g B o ras H o l l e n d e r , e t . a l . 1969

88 l e u p ro S a n ta a n n a H o l l e n , e t . a l . 1970

90 g lu l y s A genogi M i y a j i , e o . a l , 1966

91 l e u p ro S a b in e S c h n e i d e r , e i . a l . 1969

92 h i s t y r M-Hyde l a r k A k i t a

H e l l e r , e t . r d , 1966 S h i b a t a , e t . a l , 1968

93 c y s n i l Ube I Hanada 1965

94 a s p a s n Oak R id g e S c h r o e d e r and J o n e s 1965

95 l y s g l u N - B a l t im o re N-Memphis H opkins I J e n k i n s WMalmo

C le g g , e t . a l . 1965

97 h i s g i n F a i r b a n k s , e t . a l . 1971

98 v a l m et K oln G a r r e l l , e t . a l . 1966

99 a s p h i s Yakima O sgood , e t . a l . 1967

14

99 ao p a s n Kempsey R e ed , e t . a l . 1968

100 p ro l e u Brigham L o k ic h , e t . a l . 1973

102 a s n l y s Richmond G re e r 1971

102 a s n t h r K ansas R ig g s and B o n a v e n tu ra 1967

103 phe l e u H eath row W h ite , e t . a l , 1973

108 a s n a s p Y o s h iz u k a Iraam ura, e t . a l . 1969

1 i l v a l phe r e t e r b o r o u g h K in g , e t . a l , 1972

113 v a l g lu New York R anney , e t . a l , 1967

117 h i s a r g P -U a lv e s t o n s c h n e i d e r , e t . a l . 1969

120 l y s g lu H i j iy a m a M y a j i , e t . a l , , 1968 T a k a o k i , e t . a l . 1968

121 g lu g in D-Los im g e le s P u n ja b D -G hicago D - P o r tu g a l D -N o rtn C a r o l i n a

B a g l i o n i , e t . a l , 1962

121 g lu l y s 0 - A r a b ia 0-New York 0 - B u l g a r i a 0 - E gypt

B a g l i o n i and Lehmann 1962 Kamel, e t . a l . 1^67

124 p ro a r g K hartoum G le g , e t . a l . 1969

126 v a l g lu Hofu i- i iy a j i e t . a l . 1968

129 a l a a s p J -T a i -G h u n g B l a c k w e l l , e t . a l , 1 9 6 l

129 a l a g lu K-Gameroon iv l l a n , e t . a l . 1965

130 t y r a s ■: Wien V ogel 1969

131 g in g lu Gaiiiden C ohen, e t . a l . 1975

132 l y s g i n K-V/oolwich A l l a n , e t . a l . 1965

136 g ly a s p Hope M n n i c h , e t . a l . 1965

141 l e u a r g O lm stea d L o r d i n , e t . a l . 1970

143 h i s g in L i t t l e Hock B rom berg , e t . a l . 1973

143 h i s a r g A bruszo ï e n t e r i , e t . a l . 1972

143 h i s a s p Kenwood B a y r a k c i , e t . a l . 1964145 t y r cy s R a n ie r B ta m a to y a n n o p o u lo s , e t . a l .

1 9 6 7 , 1969N a g a i , e t . a l . 1972

15

145 t y r h i s

146 h i s a s p

B e th e s d a

H iro s h im a

O ls e n , e t . a l , 1972 Bunn, e t . a l . 1972 H a m il to n , e t . a l . 1969

7 4 -7 4 d e ; e t e d T o u rs '.Vajcman, e t . a l . 19 73

87 d e l e t e d T o u r s Wajcman, e t . a l . 1973

6 o r 7 g lu d e l e t e d L e id e n D eJong, e t . a l . 1968

5 6 -59 d e l e t e d T o c h ig i Yamada, e t . a l . 1971

95 -99 d e l e t e d Gunn H i l l B r a d l e y , e t . a l . 1967

(83 to 1 2 0 ) s i n g l e s u b , n f c T u b ingen K l e i n h a u e r , e t . a l , 1971

n f c S t . M a r y ' s S c h r o e d e r ; no J o n e s 3 965

n fc K i s h i k i Iv Hanada 1965

n fc T s u k i j i Hanada 1985

n fc C h ica g o I C h e r n o f f a n d iC L i . i t 1964

n fc 1 - C a l a b r i a S i l v e s t r o n i , e t . a l . 1963

n fc M-VLiles Lehmann 1962

n fc 0 - T e l Hasho-ter Lehmann 1962

n fc J - J ' jT ia ic a S c h r o e d e r and J o n e s 1965

n fc T - t ie o rg ia S c h r o e d e r and J n e s 1965

n fc M ilwaukee I I S c h r o e d e r an d J o n e s 1965

n fc L - B o r fo lk H untsm an , e . a l . 1963

n fc ù o e u l 11 Kim a n a ohim 1969

n f c G -S oeu l Chon a n a whim 1969

h fc S o e u l I I I Chon a n a Shim 1969

n fc R,Durham C h e r n o f f an d l e t t i t 1 96 4

p lu s t h r - l y s - l e u - l e u - a l a - Tak ï l a t z , e t . a l . ,19 71( s e r , l e u , a s n ) - p h e - t y r

DELTA CHAIM

16

R e s id u e J’rom To Harae(s) b o u r o e ( s )2 h i s a r g E u h a k ia J o n e s , e t . a l , 1966

12 a s n l y s a - 2 NYU Hannt.y, e t . a l . 1965

16 g iy a r g B-2A '- 2

V e l l a a n a Graham 1969

22 a l a g lu i ' l a t b u s h J . n e s , e t . a l . 1966

69 g iy a r g A-2 I n d o n e s i a .Eng, e t . a l , 1971

156 g iy a s p a- 2 B a b In g a H uisraan, e t . a l . 1969

GAi'Û̂ lA CHAIN

17

R e s id u e5

Fromgly

Tol y s

N am e(s) T ex as I

6 o u r c e ( s JS c h n e id e r and J n é s 1965

6 g lu l y s T e x a s I I V ogel 1969

12 t h r l y s F - A le x a n d ra J e l l i f f e e t . a l . 1961

61136

l y sgly

g l ua l a

F - J a m a ic a A h e rn , e t . a l . 1970

94 a s p t y r S e t i f Wajcman, e t . a l . 1973

97 h i s a r g F - D ic k in s o n S c h r o e d e r , e t . a l . 1968

117 h i s a r g F - M a l ta G a u c h i , e t . a l , 1969

121136

g lugly

l y sa l a

F - H u ll A h e rn , e t . a l . 1973

n f c F - W arren F-Hou s t o n

H uism an, e t . a l , I9 b 5

n f c F-Roma S i l v e s t r o n i a n d l i ianco 1'

n f c F - ^ e g in a F e s s a s , e t . a l , 1961

n f c B r i s t o l - b i n g a p o r e R â p e r , e t , a l . I 9 6 0

18

Name

L e p o r e C y p ru s I

L e p o r e B a l t i m o r e

L e p o r e H o l l a n d i a

L e p o r e B o s to n L e p o r e l y l o s

L e p o r e W a sh in g to n L e p o r e A u g u s ta I

L e p o r e A enya

M iy a d i

P -C ongo

r - N i l o t i c

H

B a r t s

A u g u s ta I I

N i o r t

A -4

Gower I

D-4

n f c

n f c

n f c

n f c

n f c

n f c

n f c

n f c

n f c

n f c

OTHER VARIi\NTS

D e s c r i p t i o n

d e l t a - b e t a f u s i o n

d e l t a - b e t a f u s i n





b e t a - d e l t a f u s i o n



b e t a t e t r a m e r

gamma t e t r a m e r

Hb C b e ta t e t r a m e r

d e l t a a b s e n t

a l p h a t e t r a m e r

e p s i l o n t e t r a m e r

d e l t a t e t r a m e r

New Haven I

K ing s B ounty

A tw a te r

Tokyo

B ud-V ie tnam

H-I'iLlv;aukee I I

M Novi Bad

B endai

F r a n k f u r t

P i e r c e

B o u rc e

H uehns and b h o o te r 1965

u s t e r t a g and Bm ith 1969

H u eh ns ;.i.nd S h o o te r 1965

B a b l i o n i , 1962

L a b i e , e t . a l * 1966

H uism an e t . a l , 1972

U h ta , e t . a l . 1971

L ehm ann, e t . a l . 1970

B a d r , ( t . a l , 1975

In g ra m 1963

In g ra m 1965

H uism an and B y d e n s t r y c k e r 1962

W ajcm an, e t . a l . 1970

C h e r n o f f 1964

M eK usick 1971

H u eh n s a n d S h o o te r 1965

G h e r n o f f anu P e t t i t 1964

s a t h i a p a l a n , e t . a l . 1968

A t w a t e r , e t . a l . 1962

F u k u ta k e and K ato 1961

A l b a h a r y , e t . a l . I9 6 0

P i s c i o t t a e t . a l . 1959

S t a n v l o v i c 1971

O n o d e r a , e t . a l . 1975

N o w ic k i , e t . a l . 1971

P i e r c e , e t . a l . 1963

■{ ' r ' f • ' î t

19

n f c

n f c

n f c

n f c

n f c

S c o t t

N -S a rd e g n a

G a l l i e r i Genova

G - P a r i s

M -L e ip z ig I

S c o t t , e t . a l . I 9 6 0

s i l v e s t r o n i n d B ian c o 1961

S a n s o n e , e t . a l . 1965

u a b i e and S c h a p i r a 1966

B e tk e , e t . a l . I9 6 0

* n fc = n o t f u l l y c h a r a c t e r i z e d .

20

T a b le 1 : Chi s q u a r e d t e s t t o d e t e r m i n e i f h e m o g lo b in p o i n t m u t a t i o n s a r e ra n d o m ly d i s t r i b u t e d o v e r t h e a l p h a , b e t a , gamma, a n d d e l t a p o l y p e p t i d e c h a i n s .

0 E ( 0 - E ) (0 -E > ^

Alpha 62 43 .01 1 8 ,9 9 3 6 0 .6 2

B e ta 100 4 4 .6 0 5 5 .4 0 3 0 6 9 .1 6

D e l t a 6 4 4 .6 0 3 8 .6 0 1 4 8 9 .9 6

Gamma 9 4 4 .6 0 3 5 .6 0 1 2 6 7 .3 6

177 176 ,81 6 1 8 7 .1 0

= 6 1 8 7 /1 7 7 = 3 4 .9 5

d . f . = 3

P < < .001

c h a p t e r I I

HEMOGLOBIN

P r o t e i n s : S t r u c t u r e a n d F u n c t i o n

As m e n t io n e d a b o v e , p r o t e i n s a n d t h e i r s t r u c t u r a l v a r i a n t s

p r o v i d e a v e r y p o v /e r fu l t e s t i n g g ro u n d f o r th e s tu d y o f b i o lo g

i c a l v a r i a t i o n a n d a d a p t a t i o n s i n c e t h e y r e p r e s e n t th e d i r e c t

m o l e c u l a r e x p r e s s i o n o f g e n e t i c i n f o r m a t i o n . Hem oglobin i s p a r

t i c u l a r l y u s e f u l , s i n c e i t i s b o t h p l e n t i f u l (m aking up a b o u t

90% b y w e i g h t o f m am m alian e r y t h r o c y t e s ) an d e a s i l y s e p a r a t e d

an d p u r i f i e d .

P r o t e i n m o l e c u l e s a r e s y n t h e s i z e d by e a c h c e l l i n a v a r i e t y

o f s i z e s , s h a p e s a n d c o m p o s i t i o n s , e a c h o f w h ich h a v in g e v o lv e d

t o p r e f o r m s p e c i f i c f u n c t i o n s . The m a in f e a t u r e a l l p r o t e i n s

s h a r e i s a p r i m a r y s t r u c t u r e c o n s i s t i n g o f t h e amino a c i d s common

t o a l l l i f e fo rm s ( f i g , 1 ) a r r a n g e d i n a h e ad t o t a i l f a s h i o n and

j o i n e d b y p e p t i d e b o n d s w h ic h r e s u l t f ro m t h e s p l i t t i n g o u t o f a

m o le c u l e o f w a t e r b y t h e j o i n i n g o f t h e c a r b o x y l g ro up o f one a -

m ino a c i d t o t h e am ino g ro u p o f t h e n e x t . Thus each p r o t e i n mol

e c u l e i s e s s e n t i a l l y an am ino a c i d .o ly m e r , o r p o ly p e p t i d e c h a i n .

The s p e c i f i c am ino a c i d s e q u e n c e , u n i q u e to eac h p r o t e i n , i s te rm

e d i t s p r i m a r y s t r u c t u r e a n d u l t i m a t e l y d e te r m in e s norm al n a t i v e

c o n f o r m a t i o n . I n a d d i t i o n , a p r o t e i n may o r may n o t be bound to

n o n -a m in o a c i d o r g a n i c o r i n o r g a n i c m a t e r i a l ( p r o s t h e t i c g r o u p ) ;

t h o s e c o n t a i n i n g a p r o s t h e t i c g ro u p a r e t h e c o n ju g a te d p r o t e i n s ,

t h o s e w i t h no p r o s t h e t i c g ro u p a r e t h e s im p le p r o t e i n s .

The b i o l o g i c a l a c t i v i t y o f a p r o t e i n depends n o t o n ly on

i t s amino a c i d b a c k b o n e a n d p r o s t h e t i c g r o u p s , b u t a l s o (among

o t h e r f a c t o r s ) on t h e a c t u a l s h a p e i t a ssu m es i n s p a c e , i . e , on

t h e p o s s i b l e r e a c t i v e s i t e s i t e x p o s e s , p o s i t i o n o f p r im a ry r e

a c t i v e s i t e , e t c , ( G r e e r , 1 9 7 1 a , 1 9 7 1 b ) , T h is i s a v e ry com plex

21

22

phenomenon . n d a g a i n i s d e p e n d e n t on t h e am ino a c i d s e q u e n c e .

F o r e x a m p le , p r o l i n e p r o d u c e s a k i n k i n t h e h e l i x due to t h e

b u l k o f i t s im in o g ro u p ( C le g g , e t . a l . , 1 9 6 9 ) , two c y s t e i n e m o le

c u l e s Can c o n d e n s e t o f o n n a c y s t i n e d i s u l f i d e b r i d g e ( a s f o u n d

i n I n s u l i n ) , d i s t r i b u t i o n o f c h a r g e d and p o l a r am ino a c i d s a c

c o u n t s f o r t h e b e h a v i o r o f r o t e i n s i n s o l u t i o n and so o n .

In t h e c a s e o f h e m o g lo b in , t h e m a j o r i t y o f t h e R g r o u p s

( c a r b o h y d r a t e p o r t i o n o f am ino a c i d s ) on t h e e x t e r i o r o f t h e m o le

c u l e a r e h y d r o p h i l i c ( e i t h e r p o l a r o r c h a r g e d ) , w h i l e Lhe R g r o u p s

h id d e n i n t h e i n t e r i o r a r e p r e d o m i n a n t l y h y a r o p h o b ic ( n o n - p o l a r ) ,

m ak ing h e m o g lo b in v e r y s o l u b l e i n p o l a r s o l v e n t s s u c h . s w a t e r

b u t r e l a t i v e l y l i p i d i n s o l u b l e , a c h a r a c t e r i s t i c o f m o s t t r a n s

p o r t p r o t e i n s .

D e s c r i p t i o n s o f t h e c o n f o r m a t i o n o f a p r o t i e n u s u a l l y a l s o

r e f e r to i t s s e c o n d a r y e n d t e r t i a r y s t r u c t u r e s ; i t s h e l i c a l c o i l

i n g and f o l d i n g i n s p . n e r e s p e c t i v e l y . The s e c o n d a r y s t r u c t u r e

o f human h e m o g lo b in i s a p p r o x i m a t e l y 7 0 % a l p h a h e l i c a l tUid i t s

t e r t i a r y s t r u c t u r e i s g l o b u l a r ( a s o p p o s e d t o f i b r o u s ; , a n o t h e r

c h a r a c t e r i s t i c o f t r a n s p o r t p r o t e i n s su c h a s m y o g lo b in a n d se ru m

a lb u m in , m e a n in g i t i s r e l a t i v e l y t i g h t l y f o l d e d b a c k on i t s e l f

g i v i n g i t a s p h e r i c a l ( o r i n t h i s p a r t i c u l a r c a s e , t e t r a h e d r a l )

a p p e a r a n c e .

A f i n a l a s p e c t o f p r o t e i n c o n f o r m a t i o n i s i t s q u a t e r n a r y

s t r u c t u r e ; i f a p r o t e i n c o n t a i n s m o re t h a n on e p o l y p e p t i d e , i t s

q u a t e r n a r y s t r u c t u r e i s o l i g o m e r i c , e a c h o f t h e p o l y p e p t i d e s b e

in g a p r o t o m e r .

Thus h e m o g lo b in c a n b e d e s c r i b e d a s an oxy g en t r a n s p o r t i n g

c o n ju g a te d o l i g o m e r i c p r o t e i n c o n t a i n i n g f o u r p r o t o m e r s a r r a n g e d

s y m e t r i c a l l y a b o u t t h e d y ad a x i s ( a l p h a y - b e t a ^ / a l p h a g - b e t a g i n

23s u c h a way t h a t c.11 o f t h e h e l i c a l p o r t i o n s e x p o s e a t l e a s t p a r t

o f t h e m s e lv e s t o t h e e x t e r i o r a n d su c h t h a t d im e r s ( a l . , h a - b e t a

s u b u n i t s ) w i l l d i s s o c i a t e b e f o r e s e p a r a t e monomers ( p r o t o m e r s ) .

T o g e t h e r , t h e s e f o u r p r o to m e r s a r e r e f e r r e d to a s t h e g l o -

b i n p o r t i o n o f h e m o g lo b in a n d e a c h o f them i s bou nd t o a p r o s

t h e t i c heme g ro u p w h ic h s e r v e a s a c t i v e s i t e s f o r oxygen u p t a k e .

The heme g ro u p I t s e l f c o n s i s t s o f one m o le c u le o f i r o n an d one

o f ^ jo rp h o r in , a p ig m e n t , w h ic h i n t u r n i s com posed o f f o u r p y r r o l e

r i n g s w i t h a t o t a l o f f o u r m e t h y l , two v i n y l a n u two p r o p i o n i c

a c i d g ro u p s r e p l a c i n g ei.i.h t h y d r o g e n s .

The m a j o r i t y (97-98% ) o f n o rm a l h e m o g lo b in , t h e " m a jo r com

p o n e n t " , (Ifb A o r Hb Aq ) c o n t a i n two i d e n t i c a l p a i r s o f p o ly p e p

t i d e s , a l p h a (141 r e s i d u e s , W - te rm in a l s e .p e n c e v a l - l e u - p r o - s e r

a i s ) and b e t a (146 r e s i d u e s , M - t e r m in a l s e q u e n c e v a l - h i s - l e u - t h r -

p r o ) ; t h e r e m a in d e r (Hb A^) i s com posed o f two a l p h a and two d e l

t a c h a i n s . The b e t a a n d d e l t a c h a i n s d i f f e r i n p r im a r y s t r u c t u r e

a t o n ly t e n r e s i d u e s . F e t a l h e m o g lo b in (hb F) i s com posed o f two

a l p h a a n d two gamma c h a i n s , t h e l a t t e r c h a r a c t e r i z e d by t h e p r e s

e n c e o f i s o l e u c i n e , t h e o n l y o f t h e common amino a c i d s n o t norm

a l l y p r e s e n t on t h e o t h e r c h a i n s .

H e m o g lo b in o p a th y : The U n s ta b l e H em o g lo b in s

j r a t h o l o g i c u l c o n d i t i o n s o f p r o t e i n s y n t h e s i s i n v o l v e e i t h e r

a c h a n g e i n t h e r a t e o f p r o t e i n s y n t h e s i s o r t h e p r o d u c t i o n o f an

abnormEil p r o t e i n m o l e c u l e . S in c e t h i s p a p e r i s c o n c e r n e d p r i

m a r i l y w i t h p o i n t m u t a t i o n s , o n l y h e m o g l o b i n o p a t h i e s o f a s t r u c t

u r a l n a t u r e w i l l b e d i s c u s s e d .

The s t r u c t u r a l h e m o g l o b i n o p a t h i e s c a n b e d i v i d e d g r o s s l y

i n t o two c a t e g o r i e s ; m u tu n t h e m o g lo b is whose c o n f o r m a t i o n i s r e n

d e r e d u n s t a b l e b y t h e s u b s t i t u t i o n a n d s t a b l e h e m o g lo b in s w hose

o x y g en b i n d i n g an d r e l e a s i n g f u n c t i o n s h av e b e e n a l t e r e d (H u eh n s ,

24

1 9 7 0 ) .

The u n s t a b l e h e m o g lo b in s a r e t h e r e s u l t o f a s u b s t i t u t i o n

o f one amino a c i d b y a n o t h e r w h ic h c a u s e s d i s r u p t i o n o f t h e n o r

m al s t a b i l i z i n g f o r c e s a t t h a t p o i n t o f t h e c h a i n ( P e r u t z and

Lehm ann, 1 9 6 8 ) and a r e one o f t h e m a jo r c a u s e s o f c o n g e n i t a l h e

m o l y t i c a n e m ia . T hese a i s r u p t i o n s c a n b e f u r t h e r b r o k e n down i n

t o t h o s e a f f e c t i n g t h e s p a c e i m m e d ia te l y s u r r o u n d i n g t h e p o i n t

o f m u t a t i o n u i d t h o s e a f f e c t i n g t h e n e t c h a r g e o f t h e m o l e c u l e .

F o r e x a m p le , i n Hb T o r in o ( a l p h a 45 p h e - v a l ) a l . r g e p h e n y l

a l a n i n e i s r e p l a c e d by a s m a l l e r v a l i n e w h ich c a n n o t make t h e

n e c e s s a r y i n t e r a c t i o n s ( P r a t o , e t . a l . , 1 9 7 0 ) . The s i t u a t i o n i s

r e v e r s e d i n Hb O lym pia ( b e t a 20 v a l - m e t ) w h e re a s m a l l e r v a l i n e

i s r e p l a c e d by a l a r g e r m e t h i o n in e c a u s i n g a l o c a l s w e l l i n g a t

t h i s u o i n t (S ta m a to y a n n o p o u lo s , e t . a l . , 1 975 )* Even g r o s s e r c o n

f o r m a t i o n a l c h a n g e s o c c u r when a n y r e s i d u e i s s u b s t i t u t e d b y p r o

l i n e a s i n Hb Genova ( b e t a 28 l e u - p r o ) s i n c e t h e am ino h y d ro g e n

o f p r o l i n e e x i s t s a s p a r t o f t h e im in o r i n g a n d c a n n o t p a r t i c i

p a t e i n h y d r o g e n b o n d in g w i t h t h e c a r b o x y l oxygen o f t h e amino

a c i d f o u r r e s i d u e s rem o v e d , t h u s b r e a k i n g t h e h e l i x a t t h a t p o i n t

( S a n s o n e , e t . a l , , 1 9 6 7 ) .

R e g a r a in g c h a r g e c h a n g e s , a t p h y s i o l o g i c a l pH, 19 o f t h e

common amino a c i d s c a n y no n e t c h a r g e ; two c a r r y a n e t n e g a t i v e

c h a r g e a n d a r e t h u s a c i d i c ; t h r e e ( i n c l u d i n g h i s t i d i n e , w h ic h i s

b o r d e r l i n e i n t h i s r e s p e c t ) c a r r y a n e t p o s i t i v e c h a r g e a n d a r e

t h u s b a s i c . F u r t h e r , h a l f o f t h e n e u t r a l amino a c i d s a r e p o l a r ,

t h a t i s t h e y a r e a l s o h y d r o p h i l i c , w h i l e t h e o t h e r h a l f a r e n o n

p o l a r ( f i g , 2 J . T h is r e s u l t s i n s i x p o s s i b l e c h a n g e s i n t h e n e t

c h a r g e o f a h e m o g lo b in m o le c u le b y t h e s u b s t i t u t i o n o f a s i n g l e

25

am ino a c i d . A n e g a t i v e c h a r g e c a n b e g a i n e d by s u b s t i t u t i o n o f

a n a c i d i c amino a c i d ( a s p o r g lu ) f o r a n e u t r a l am ino a c i d o r

b y t h e s u b s t i t u t i o n o f a n e u t r a l am ino a c i d f o r a b a s i c am ino

a c i d ( l y s , a r g , o r h i s ) , a p o s i t i v e c h a r g e c a n be g a i n e d by t h e

s u b s t i t u t i o n o f a b a s i c f o r a n e u t r a l o r a n e u t r a l f o r an . - c i d i c .

Two n e g a t i v e c h a r g e s c an be g a in e d o n ly by The s u b s t i t u t i o n o f

a n a c i d i c f o r a b a s i c , and two p o s i t i v e c h a r g e s c a n b e g a in e d o n

l y by t h e s u b s o i t u t i a n o f a b a s i c f o r a n a c i d i c . E xam p les o f

t h e s e c h a n g e s a r e , r e s p e c t i v e l y ; Hb J - T o r o n t o ( a l p h a 5 a l a - a s p ) ,

Hb M -B oston ( a l p h a 5b h i s - t y r ) , Hb G h i a p i s ( a l p h a 114 p r o - a r g ) ,

Hb S ( b e t a 6 g l u - v a l ) , Hb N a g a s a k i ( b e t a 17 l y s - g l u ) a n d Hb C-

B i r i r a j ( b e t a 7 g l u - l y s ) .

The above c h a r g e c h a n g e s c a n o c c u r on a n y p a r t o f any p o l y

p e p t i d e o f h e m o g lo b in , w i t h v a r y i n g c o n s e q u e n c e s . On t h e e x t e r i o r

s u r f a c e o f t h e m o le c u le , i . e . t h a t p a r t i n c o n t a c t w i t h t h e s u r

r o u n d i n g medium, t h e s u b s t i t u t ! n o f e i t h e r a p o s i t i v e o f n e g

a t i v e c h a r g e f o r a n e u t r a l , e . g . lib E to b i c o k e ( a l p h a 48 s e r - a r g ) ,

Hb Hofu ( b e t a 126 v a l - g l u ) , may, i n t h e h e t e r o z y g o u s c o n d i t i o n ,

p r o d u c e no c l i n i c a l symptômes aS w h i l e s t r u c t u r a l c h a n g e s h av e

t a k e n p l a c e , a n o th e r h y d r o p h i l i c r e s i d u e w i l l t e n d t o s t a b i l i z e

t h e e x t e r i o r h e l i x and i n c r e a s e s o l u b i l i t y , , o r may p ro d u c e o n ly

m i l d p a t h o l o g y . F o r e x am p le , Hb E to b i c o k e s t a b i l i z e s t h e F a n d

G h e l i c e s w i t h no c l i n i c a l sym ptom s, a n d Hb D a k a r s t a b i l i z e s t h e

G h e l i x a n d p ro d u c e s o n ly m i ld h e m o l y t i c a n e m ia ( L a b i e a n d R o s a ,

1 9 6 8 )* vHien th e s i t u a t i o n i s r e v e r s e d , i . e . when a p o l a r r e s i d u e

i s r e p l a c e d by a n o n - p o l a r r e s i d u e , s o l u b i l i t y i s g e n e r a l l y r e

d u c e d l e a d i n g to some p r e c i p i t a t i o n w h ic h i s o b s e r v e d i n t h e c e l l

a s H e in z b o d i e s . T h ere a r e e x c e p t i o n s t o t h i s , i n t h e c a s e o f

Hb L - F e r r a r a ( a l p h a 47 a s p - g l y ) i n w h ic h no sym ptoms w e re o b s e r -

26

Yed ( B l a n c o , e t . a l . , 1 9 6 3 ) . More c o m p le te d i s c u s s i o n s o f c l i n

i c a l sym ptom ology a r e g iv e n b y O a r r e l l , Lehmann a n d L o r k in (1 9 6 7 )

a n d b y P e r u t z and Lehmann ( 1 9 6 8 ) .

U n l ik e t h e e x t e r i o r , t h e i n t e r i o r p o r t i o n o f e a c h p o ly p e p

t i d e a n d t h e i n t e r n a l c a v i t y s e p a r a t i n g them a r e com posed a lm o s t

e x c l u s i v e l y o f h y d r o p h o b ic am ino a c i d s a n d s e v e r e i n s t a b i l i t y r e

s u l t s from t h e i n t r o d u c t i o n o f a c h a r g e d o r p o l a r g ro u p a s i n Hb

iutm A rb o r ( a l p h a 80 l e u - a r g ) , Hb Bogn ( b e t a 14 l e u - a r g ) , a n d Hb

Wien ( b e t a 130 t y r - a s p ) . C l i n i c a l symptoms r a n g e f ro m non e (Hb

Sogn) (Monn, e t . a l . , 1 9 6 7 ) t o f a i r l y s e v e r e (Hb W ien) ( V o g e l , 1 9 6 9 ) .

Some o f t h e m o s t s e v e r e p a t h o l o g i c a l c o n d i t i o n s a r e o b s e r v e d

when a s u b s t i t u t i o n o c c u r s a t one of t h e heme c o n t a c t s , a s o x y g e n

a f f i n i t y w i l l a lm o s t c e r t a i n l y be a f f e c t e d i n a d d i t i o n to p r o

n o u n c e d m o l e c u l a r i n s t a b i l i t y , a s c a n be d e m o n s t r a t e d by nb x o r i n o

( a l p h a 43 p h e - v a l ) , Hb B ib b a ( a l p h a 136 l e u - p r o ) , Hb B o ra s ( b e t a

88 l e u - a r g ) , Hb K o ln ( b e t a 98 v a l - m e t ) , Hb Bydney ( b e t a 67 v a l -

a s p ) , Hb Ham mersm ith ( b e t a a2 p h e - s e r ) , Hb S a b in o ( b e t a 91 l e u -

p r o ) , Hb B a n ta Anna ( b e t a 88 l e u - p r o ) , Hb Z u r i c h ( b e t a 63 h i s - a r g ) ,

Hb B r i s t o l ( b e t a 67 v a l - a s p ) , Hb B h e p a rd s Bush ( b e t a 74 g l y - a s p ) ,

and Hb T o u lo u s e ( b e t a 66 l y s - a r g ) ,

B u b s t i t u t i o n s a t t h e a l p h a - b e t a c o n t a c t p o i n t s a r e co m p ar

a t i v e l y m i l d and g e n e r a l l y r e s u l t i n a l t e r e d d i s s o c i a t i o n p r o p e r

t i e s . Some ( e . g . Hb G - G h in e s e ( a lp h a 30 g l u - g i n ) a n d Hb K -w oo lw ich

( a l p h a 132 l y s - g l u ) show no a b n o rm a l p r o p e r t i e s o r c l i n i c a l symp-

om s, w h i l e o t h e r s ( e . g . Hb E ( a l p h a 26 g l u - l y s ) ) p r o d u c e m i l d p o l y

c y th e m ia o n ly i n t h e hom ozygous c o n d i t i o n . ( W a jc m a n , 1 9 7 3 c ) .

The s e c o n d m a jo r d i v i s i o n o f h e m o g l o b i n o p a t h ie s i s com posed

o f h e m o g lo b in s w i t h e i t h e r i n c r e a s e d o r d e c r e a s e d ox y g en a f f i n i t y

b u t r e t a i n t h e i r m o l e c u l a r s t a b i l i t y . B e f o re t h e y a r e d i s c u s s e d

2 7

i t i s i n p o i n t t o d i s c u s s t h e n o rm a l f u n c t i o n o f h e m o g lo b in .

P h y s i o lo g y s i h e m o g lo b in

The p r im a r y p h y s i o l o g i c a l f u n c t i o n o f h e m o g lo b in i s oxy g en

t r a n s p o r t f ro m t h e l u n g s t o v a r i o u s t i s s u e s . Oxygen u p t a k e i s a

r e v e r s i b l e r e a c t i o n w h e reb y e a c h o f t h e p o l y p e p t i a e s w i l l a c c e p t

o x y g e n i n a r e a s w h e re t h e oxygen t e n s i o n i s h i g h , t h a t i s , a t

t h e l u n g s . The o x y g e n a te d fo rm o f h e m o g lo b in , o r o x y h e m o g lo b in ,

i s t h e n c a r r i e d t o t h e t i s s u e s w h e r e , i n t h e p r e s e n c e o f low oxy

g e n t e n s i o n , t h e o x y g e n i s r e l e a s e d . The u p t a k e o f oxygen a t

t h e l u n g s i s s u b j e c t t o t h e same a l l o s t e r i c a f f e c t c h a r a c t e r i s t i c

o f m u l t i - e n z y m e s y s te m s w i t h oxygen i t s e l f a c t i n g a s t h e m o d u la

t o r . T h i s i s an i m p o r t a n t e v o l u t i o n a r y t r e n d w h ic h c o n s e r v e s

c h e m ic a l e n e rg y b y b r e a k i n g down t h e r e a c t i o n i n t o s e v e r a l s t e p s .

In t h i s c a s e , one m o le c u le o f oxyg en w i l l com bine w i t h t h e heme

g ro u p on one o f t h e f < u r c h a i n s , c h a n g in g t h e c o n f o r m a t i o n i n

s u c h a way t h a t t h e oxygen a f f i n i t y i n i n c r e a s e d i n d i r e c t p r o

p o r t i o n t o t h e d e c r e a s e i n e n e r g y o f a c t i v a t i o n o f t h e o x i d a t i o n

r e a c t i o n . I n a l l , t h e r e a r e f o u r s t e p s to t h i s r e a c t i o n , e a c h

s u c c e e d i n g s t e p h a v i n g a l o w e r e n e r g y b a r r i e r t o o v e rco m e . The

p r o d u c t , o x y h e m o g lo b in (HbOg) i s much l e s s s t a b l e t h a n r e d u c e d

h e m o g lo b in (H b ) , s i n c e i n n o rm a l h e m o g lo b in , t h e i r o n a tom r e

m a in s a s a f e r r i c c a t i o n (Pe +++) a n d c a n n o t fo rm a t r u e s t a b l e

o x i d e . B e c a u se o f t h e o x i d a t i o n s t a t e o f t h e i r o n , oxygen i s on

l y l o o s e l y bound t o o x y h e m o g lo b in i n a d e l i c a t e s t a t e o f e q u i l i b

r i u m ,

./hen a m o le c u le o f HbO^ r e a c h e s an e n v i ro n m e n t w h e re t h e

p a r t i a l p r e s s u r e o f oxygen i s lo w , t h e oxygen m o le c u l e s immed

i a t e l y b e g i n t o d i s s o c i a t e a t w h ic h t im e some c a r b o n d i o x i d e i s

a b s o r b e d d i r e c t l y by t h e p a r t i a l l y r e d u c e d h e m o g lo b in , w h i l e

28

o t h e r m o le c u le s o f c a r t o n d i o x i d e go i n t o s o l u t i o n a s c a r b o n i c

a c i d b y e n z y m a tic a c t i o n o f c a r b o n i c a n h y e r a s e . B oth t h e d e

c r e a s e i n pH and t h e c n f o r m a t i o n a l c h a n g e s c a u s e d by COg u p t a k e

i n c r e a s e t h e i n s t a b i l i t y o f Hb a l l o w i n g more and more oxygen t o

d i s s o c i a t e , B ince t h e i n i t i a l d i s s o c i a t i o n i s p r o p o r t i o n a l t o

t h e oxyg en t e n s i o n o f t h e t i s s u e f l u i d , a c e l l r e g u l a t e s i t s o x y

gen u p t a k e by i t s e x p e n d i t u r e ,

H ^ P ^ P ^ . in o p a t j : iy r H em o g lob in s W ith A l t e r e d Oxygen A f f i n i t y

H igh oxygen a f f i n i t y d e s c r i b e s a s i t u a t i o n i n w h ic h t h e f o r

c e s b i n d in g oxygen to t h e heme a r e g r e a t e r t h a n n o r m a l , i n e f f e c t

m ak ing i t more d i f f i c u l t f o r t h e h e m o g lo b in to r e l e a s e t h e o x y g en

to t h e t i s s u e s i t s u p p l i e s . T h i s i s a m a jo r c a u s e o f p o l y c y t h e m i a

( C h a ra c h e , W e a th e r a l l an d C l e g g , 1 9 6 6 ) and i s e x e m p l i f i e d b,> Hb

J -C a p e to w n ( a l p h a 92 a r g - g i n ) , Hb C h e sa p ea k e ( a l u h a 92 l e u - a r g ) ,

Hb Tacoma ( b e t a 30 a r g - s e r ) , Hb Yakima ( b e t a 99 a s p - h i s ; , Hb

kempsey ( a e t a 99 a s p - a s n ) a n d Hb K o ln ( a l p h a 98 v a l - m e t ) . The

"M" h e m o g lo b in s a l s o b e lo n g t o t h i s g r o u p , s i n c e t h e heme g r o u p

i s s t a b i l i z e d i n t h e m e th e m o g lo b in ( r e d u c e d ) fo rm , s t a b i l i z i n g

t h e i r u n i n t h e f e r r o u s (F e+ + ) s t a t e ( B e tk e , t e , s i , , I 9 6 0 ) ,

H em oglobin v a r i a n t s w i t h r e d u c e d oxygen a f f i n i t y a r e com

p a r a t i v e l y r a r e b u t a t l e a s t o n e s t a b l e fo rm , Hb K an sas ( b e t a

102 a s n - t h r ) d o e s e x i s t . R e d u c e d o x y g e n a f f i n i t y l e a d s t o c y a n

o s i s due to t h e l a r g e am oun ts o f r e d u c e d h e m o g lo b in ^ r e s e n t

(R ig g s a n d B o n a v e n tu r a , 1 9 6 7 ) .

u h i l e c l i n i c a l s e v e r i t y may v a r y c o n s i d e r a b l y b e tw e e n v a r i

a n t s w i t h i n c r e a s e d o r d e c r e a s e d o xy g en a f f i n i t y , t h e n e t e f f e c t

i s much t h e sam e; th e am ount o f o x y g e n s u p p l i e d to t h e t i s s u e s

i s d e c r e a s e d ( e i t h e r due t o i n a b i l i t y t o b i n d o r t h e i n a b i l i t y

t o r e l e a s e ) , an d th e m o le c u le b e c o m e s l e ^ s e f f i c i e n t .

2 9

F i g u r e 1 : The p o s s i b l e amino a c i d r e p l a c e m e n t s r e s u l t i n g fro m a s i n g l e b a s e c h a n g e .

iV laV alL eu Ile i-T o lheT rp M etG ly S erT hrC ysT y rA sn G ln A sp G lu L ysA rgH lsA la X X X X X X XVal X X X X X X X XLeu X X X X X X X X X Xl i e X X X X X X X X XPro X X X X X X XPhe X X X X X XT rp X X X X XMet X X X X X XGly X X X X X XS e r X X X X X X X X X X X XT hr X X X X X X X XGys X X X X X XTyr X X X X X XAsn X X X X X X XGin X X X X X XAsp X X X X X X XGlu X X X X X XLys X X X X X X XArg X X X X X X X X X X X XH is X X X X X X X

V e r t i c a l = p o s s i b l e m u t a t i o n t o a g iv e n amino a c i d H o r i z o n t a l = p o s s i b l e m u t a t i o n from a g iv e n amino a c i d

♦ N o te : t h e f i n a l f i v e am ino a c i d s ( i . e . . A sp , G lu , L y s , A rg , H is ) a r e t h o s e e x i s t i n g i n c h a r g e d fo rm a t pH 7*

F i g u r e 2The Common Amino A c id s

30

Amino A c id

G ly c in e

A la n in e

S e r i n e

i r o l i n e

V a l in e

T h r e o n in e

C y s t e i n e

L e u c in e

I s o l e u c i n e

A s p a r a g in e

A s p a r t i c Acid

L y s in e

G lu ta m in e

G lu ta m ic A cid

A r g i n i n e

M e th io n in e

H i s t i d i n e

P h e n y l a l a n i n e

T y r o s i n e

T ry p to p h a n

A b b r e v i a t i o n

g l y

a l a

s e r

p ro

v a l

t h r

c y s

l e u

l i e

a s n

asp

l y s

g i n

g lu

a r g

m et

h i s

phe

t y r

t r p

P o l a r i t y

P

w .P .

P.

N .P .

N .P ,

P.

P .

N .P ,

N .P ,

P.

Neg,

P o s ,

P .

Neg,

P o s ,

N ,P .

P o s ,

N ,P .

P,

N ,P .

M ole , Wt.

75

89

105

114

117

119

121

131

131

132

133

146

146

147

148

149

155

165

181

190

P ,* P o l a r

N .P , a N o n - P o la r

P o 8 .= N et p o s i t i v e c h a r g e a t p h y s i o l o g i c a l pH

N eg ,= N e t p o s i t i v e c h a r g e a t p h y s i o l o g i c a l pH

31

F i g u r e 3T h e h e l i c a l a n d n o n - h e l i c a l p o r t i o n s o f t h e a l p h a a n d b e t a c h a i n s o f human h e m o g lo b in and o f human m y o g lo b in .

H e l i c a l N o n - H e l i c a l ' A lpha B e t a Myoblol

F re -A 1-2 1 -3 1 -2

A 3 -1 8 4 -1 9 3 -1 8

AH 19 19

B

BG

2 0 -3 5 2 0 - 3 4 2 0 -3 5

C 36-42 35-41 3 6 -42

CD 43-51 4 2 - 4 9 4 3 -50

D

DE

5 0 - 5 6 51-57

£ 52-71 5 7 - 7 6 58-77

EP 7 2 -7 9 7 7 - 8 4 7 8 -8 5

P 8 0 -8 8 8 5 - 9 3 8 6 -9 4

FG 8 9 -9 3 9 4 - 9 8 9 5 -9 9

G 94 -112 9 9 -1 1 7 100-118

GH 1 13-118 1 1 8 -1 2 3 119-124

H

P o s t -H

119-141 1 2 4 -1 4 6 125-148

149-153

N o t e : H e l i c a l r e s i d u e s a r e l i s t e d by A t h r u H f o l l o w e d by t h e p o s i t i o n on t h a t s e g m e n t , e . g . r e s i d u e 55 o n t h e a l p h a c h a i n h a s h e l i c a l p o s i t i o n M . N o n - h e l i c a l r e s i d u e s a r e l i s t e d by 2 l e t t e r s o f t h e h e l i c a l s e g m e n ts i m m e d ia te l y b e f o r e and a f t e r i t , e . g . r e s i d u e 42 o f t h e b e t a c h a i n h a s n o n - h e l i c a l p o s i t i o n GDI.

32

F ig u r e 4 : T o t a l o b s e rv e d amino a c i d s u b s t i t u t i o n s on hum ;n h e m o g lo b in c h a i n s .

A la V a lL eu IIe rro P h eT rp M e tG ly S e rT h rC y sT y rA sn G ln A sp G lu L y sA rg H i3 JVla 1 5 3Val 1 1 2 2 3Leu 6 4H ePro 1 2 1 4Phe 1 2 2T rp 1MetGly 1 1 10 1 8S e r 1 2T hr 3CysT y r 1 1 1 1Asn 1 4 7Gin 2 1Asp 2 2 4 9 1 6Glu 4 3 3 4 11Lys 2 4 1 8Arg 1 1 1 1H is 5 4 5 7

N s 177

V e r t i c a l = mutf. t i o n t o a g i v e n amino a c i dH o r i z o n t a l = m u t a t i o n from a g i v e n amino a c i d

33

F i g u r e 5 : A lpha c h a i n o b s e r v e d am ino a c i d s u b s t i t u t i o n s

A la V a lL eu Ile ï tro irh eT rp M etG ly S e rT h rC y sT y rA sn G ln A so G lu L y sA rg H is Ala 1 4'Vàl 1Leu 2 1H ePro 1 1 1 2Phe 1T rpMetG ly 2S e r 2T hrCysT y rAsn ^ .Gin 1 1Asp 1 1 3 2 1Glu 1 1 2 2

S ■ ' '

N » 62

V e r t i c a l « m u t a t i o n t o a g i v e n amino a c i dH o r i z o n t a l = m u t a t i o n f rom a g i v e n amino a c i d

34

F i g u r e 6 : B e ta c h a i n o b s e r v e d amino a c i d s u b s t i t u t i o n s .

A la V a lL eu I leP ro P h eT rp M etG ly S e rT h rG y sT y rA sn G ln A sp G lu L y sA rg H is A la 1 2V al 1 1 2 1 3Leu 4 3H ePro 1 2t h e 2 2T rp 1MetG ly 1 1 5 1 4S e r 1T hr 2CysT y r 1 1 1 1Asn 1 1 4Gin 1Asp 1 1 7 1Glu 3 2 2 2 6Lys 1 1 1 6Arg 1H is 3 3 3 4

N = 100

V e r t i c a l = m u t a t i o n t o a g i v e n amino a c i dH o r i z o n t a l = m u t a t i o n f rom a g i v e n amino a c i d

35

D e l t a c h a i n o b s e r v e d amino a c i d s u b s t i t u t i o n s

Al a Val L eu 11 e P r o I'h e TrpMe t Gl y b e rT hrC y s T y r A sn G1 n A k p Glu Ly s i i r g H i s Al.a 1Val Leu H e Pro Phe Trp MetGly 1 2S erThrCysTyrAsnGinAsp 1GluLysArgHis 1

N = 6

V e r t i c a l = m u t a t i o n t o a g i v e n amino a c i dH o r i z o n t a l = m u t a t i o n f ro m a g i v e n amino a c i d

36

F i g u r e 8 : Gamma c h a i n o b s e r v e d amino a c i d s u b s t i t u t i o n s

A la V a lL e u I le P r o rh eT rp l 'le tG ly S erT hrC ys 'T yrA snG lnA spG luL ysA rgH isA laValLeuH eProPheT rpMetGluS e rT h r 1CysT y rAsnGinAsp 1Glu 1 3Lys 1ArgH is 2

N = 9

V e r t i c a l = m u t a t i o n t o a g i v e n amino a c i dH o r i z o n t a l = m u t a t i o n f rom a g i v e n amino a c i d

37C h a p te r I I I

G e n e t i c s o f H em og lo b in

On̂ âsae: £y.a,1;,ej.nOnce t h e e x i s t e n c e o f h e m o g lo b in b h a d b e e n shown t o b e

due t o a v a r i a t i o n i n p r o t e i n s t r u c t u r e ( P a u l i n g , 1 9 4 9 ) , In g ra m

(1 9 5 1 ) h a d d e m o n s t r a t e d t h e s t r u c t u r a l d i f f e r e n c e t o be a su b

s t i t u t i o n i n am ino a c i d s e q u e n c e a n d R h i n e s m i t h a n d h i s cow o rk

e r s (1 9 5 7 ) h a d d e s c r i b e d t h e two c h a i n s o f t h e m a jo r com p onen t

o f n o rm a l a d u l t h e m o g lo b in , i t becam e e v i d e n t t h a t a t l e a s t two

s e p a r a t e g e n e s w e re i n v o l v e d i n h e m o g lo b in i n h e r i t a n c e a s t h e s e

g e n e s seem ed t o f o l l o w s i m p le M e n d e l ia n s e g r e g a t i o n . A c t u a l l y

t h i s was f i r s t d i s c u s s e d t h e o r e t i c a l l y by R anney (1 9 5 4 ) b a s e d on

o b s e r v a t i o n s o f t h e i n h e r i t a n c e o f h e m o g lo b in s S a n d C. I t w as

a f o r t u n a t e c o i n c i d e n c e t h a t t h e s e two m u t a t i o n s w e re t h e f i r s t

t o b e s t u d i e d v i g o r o u s l y , s i n c e b o t h i n v o l v e s u b s t i t u t i o n s a t

t h e s i x t h r e s i d u e p o s i t i o n o f t h e b e t a c h a i n . The f a c t t h a t t h e s e

s u b s t i t u t i o n s a r e made by d i f f e r e n t am ino a c i d s a t t h e same p o s

i t i o n a n d c a n o c c u r i n t h e same i n d i v i d u a l l e d H unt a n d In g ram

(1 9 5 8 ) t o c o n c l u d e t h a t t h e b e t a c h a i n h a d a t l e a s t two a l l e l e s ,

c o n f i r m i n g i n p a r t I t a n o ' s h y p o t h e s i s t h a t e a c h o f t h e two c h a i n s

a r e c o n t r o l l t d by a l l e l i c g e n e s ( I t a n o , 1 9 5 7 ) . C o n c lu s iv e p r o o f

o f a l l e l i s m f o r b o t h t h e a l p h a a n d t h e b e t a g e n e s came i n t h e

fo rm o f d o u b le h é t é r o z y g o t e s r e p o r t e d b y I t a n o a n d R o b in s o n ( I 9 6 0 ) ,

B a g l i o n i an d In g ra m ( 1 9 6 1 ) , R a p e r e t . a l . ( i 9 6 0 ) a n d A tw a te r e t ,

a l , ( 1 9 6 0 ) , e a c h o f w h ic h e x h i b i t e d f o u r d i s t i n c t e l e c t r o p h o r e t -

i c a l l y s e p a r a b l e c o m p o n e n ts , i . e , , n o rm a l a l p h a - n o r m a l b e t a , n o r

m al a l p h a - m u t a n t b e t a , m u ta n t a l p h a - n o r m a l b e t a , a n d m u ta n t a l p h a -

m u ta n t b e t a . To c o m p le t e t h e I c t u r e , i t r e m a in e d o n l y t o p r o v e

t h a t t h e gpmma c h a i n s o f f e t a l h e m o g lo b in a n d t h e d e l t a c h a i n s

58o f t h e m in o r com ponent w ere a l s o u n d e r s e p a r a t e g e n e t i c c o n t r o l *

Gamma c h a i n s w ere shown t o b e g e n e t i c a l l y d i s t i n c t f ro m a l p h a o r

b e t a b y M in n ic h and h i s c o l l e a g u e s ( 1 9 6 2 ) , o b s e r v i n g a c h i l d w i t h

n o rm a l gamma c h a i n s an d m u ta n t a l p h a D -B t , L o u i s c h a i n s b o r n t o

two h e t e r o z y g o u s a l p h a D -B t. L o u is p a r e n t s , B i m i l a r l y , H uehns

and o h o o t e r (1965) fo u n d a m in o r co m p o n en t m u ta n t r e s u l t i n g f ro m

n o rm a l d e l t a and m u ta n t a lp h a G - I b id a n c h a i n s . T hus t h e f o u r

c h a i n s o f human h em og lob in w ere shown to be c o n t r o l l e d by a t l e a s t

f o u r g e n e s , s u p p r o t i n g t h e one g e n e - o n e p r o t e i n t h e o r y o f R e n n e t

an d D r e y e r ( 1 9 6 4 ) .

T h i s was th e s t a t e o f t h e a r t u n t i l q u i t e r e c e n t l y when e v

i d e n c e s t r o n g l y s u g g e s t i n g t h a t i n man t h e r e a r e two a l p h a c h a i n

g e n e s ( H o l l a n , e t , a l . , 1 9 7 0 ) , I t s h o u l d b e n o t e d a t t h e o u t s e t

t h a t w h i l e e q u a l l y p o w erfu l e v id e n c e e x i s t s f o r a s i n g l e a l p h a

c h a i n l o c u s m odel (^ibramson, e t , a l , , 1 9 7 0 ) i f i n f a c t t h e r e a r e

two a l - j h a - l o c i i n a s i g n i f i c a n t p o r t i o n o f t h e human p o p u l a t i o n ,

t h e e x p e c t e d m u ta t io n r a t i o b e tw e e n a l p h a a n d b e t a w o u ld b e 2 : 1 ,

d o u b l in g t h e d i s c r e p a n c y b e tw ee n t h e o b s e r v e d f r e q u e n c i e s ( t a b l e 1 ) .

The s t r o n g e s t a rgum en t f o r two a l p h a l o c i com es f ro m a s t u d y

i n v o l v i n g a H u n g a r ia n f a m i ly h a v in g n o r m a l b e t a c h a i n s a n d a n a l

p h a c h a i n f r a c t i o n w i t h t h r e e e l e c t r o p h o r e t i c a l l y s e p a r a b l e com

p o n e n t s ; a s lo w m in o r com ponent (Hb G - t e s t ) , n o r m a l a l p h a c h a i n s

a n d a f a s t m in o r com ponent (Hb J -B u d a ) o c c u r r i n g i n a 23% : 54% :

2 I/O r a t i o ( f i g , 11 ) ( H o l l a n , e t . a l . , 1 9 7 0 ) , The p r e s e n c e o f two

m u ta n t a l p h a c h a i n s i n a u d i t i o n t o n o r m a l c h a i n s ( e s p e c i a l l y i n

t h e o b s e r v e d r a t i o ) would s u g g e s t t h e e x i s t e n c e o f f o u r a l l e l e s

a t two l o c i , V/ith r e g a r d to a l t e r n a t i v e e x p l a n a t i o n s , t h e y n o t e :

" A n a ly s i s o f t h e p e d i g r e e w i t h s p e c i a l r e f e r e n c e t o t h e h e m o g lo b in s i n h e r i t e d b y t h e c h i l d r e n o f I I 7

39a n d h i s two a f f e c t e d b r o t h e r s , I I 5 an d I I 10 i n d i c a t e s t h a t g e n e s c o d in g f o r t h e two a b n o rm a l a l p h a c h a i n s a r e a t s e p a r a t e l o c i . I f t h e y w ere a l - - l e i e s a t a s i n g l e l o c u s , t h e two n o rm a l o f f s p r i n g ,I I I 13 a n d I I I 1 7 , c o u ld n o t h a v e b e e n p r o d u c e d .M o re o v e r , i t i s p o s s i b l e t o t e s t f o r l i n k a g e b e tw een t h e two a l p h a c h a i n l o c i . . . T h e o c c u r r e n c e o f two n o rm a l o f f s p r i n g among s e v e n f a v o r s i n d e p e n d e n t s e g r e g a t i o n o f m u ta n t a n d n o rm a l a l l e l e s a t two l o c i . A l t e r n a t i v e l y , i f t h e s e two r e s u l t e d from c r o s s i n g o v e r b e tw e e n two l o c i on t h e same chrom osom e, t h e r e c o m b i n a t i o n f r a c t i o n o f 2 /7 a r g u e s a g a i n s t c l o s e l i n k a g e . T h e r e f o r e , t h e s i m p l e s t e x p l a n a t i o n c o n s i s t e n t w i t h a l l t h e d a t a i n t h i s f a m i l y i s t h a t t h e r e a r e two a l p h a c h a i n l o c i , t h a t t h e m u ta n t g en e f o r Hb J -B u d a o c c u r s a t a se c o n d l o c u s an d t h a t t h e l e s t l o c u s a r e n o t c l o s e l y l i n k e d . " ( H o l l a n , e t . a l . , 1 9 7 0 ; 4 9 )

The f a c t t h a t t h e a l p h a c o m p o n e n ts o f t h i s i n d i v i d u a l o c c u r

i n a 1 : 2 I 1 r a t i o w o u ld a l s o t e n d t o e x . l a i n t h e l a r g e num ber

o f a l p h a m u ta n t s c o n t a i n i n g a b ' u t 25% a b n o rm a l m o le c u l e s a n a Lhe

f a c t t h a t s e v e r a l a l p h a ra u t io i t s a r e known to o c c u r a t b o t h a p

p r o x i m a t e l y 25% a n d a p p r o x i m a t e l y 50% l e v e l s ( s e e L i v i n g s t o n e , 1 9 6 7 ) .

T hese o b s e r v a t i o n s c o u ld t h e n b e e x p l a i n e d a s hom ozygous n o rm a l

a t o n e l o c u s a n d h e t e r o z y g o u s m u ta n t a t t h e s e c o n d , a n d homo

z y g o u s n r m a l a t one l o c u s an d hom ozygous m u ta n t a t t h e o t h e r r e

s p e c t i v e l y . I n d e e d , t h i s was t h e o r i g i n a l e x p l a n a t i o n o f f e r e d

b y Lehmann and G a r r e l l (1 9 6 8 ) f o r t h e ab o v e m e n t io n e d o b s e r v e d

f r e q u e n c i e s , a n d h a s b e e n u s e d to a r g u e f o r m u l t i p l e gamma l o c i

i n man ( b c h r o e d e r , e t . e l . , 1 9 6 8 ) .

E v id e n c e to t h e c o n t r a r y , i . e , f a v o r i n g a s i n g l e a l p h a g en e

h a s a l s o b e e n p u b l i s h e d r e c e n t l y (A bram son , e t . a l , , 1 9 7 0 ) , T h e i r

d a t a c o n s i s t e d o f s a m p le s f ro m 67 i n d i v i d u a l s i n a r e l a t i v e l y i n -

b r e d v i l l a g e i n New B r i t a i n , ühe p e d i g r e e o f one s u c h f a m i l y i s

g i v e n i n f i g u r e 1 0 , T h i s p e d i g r e e shows t h e c l a s s i c d i s t r i b u t i o n

o f two a l l e l e s a t one l o c u s ; t h o s e e x h i b i t i n g t h e m u ta n t a l p h a

c h a i n i n t h e hom ozygous s t a t e c o m H e t e l y l a c k n o rm a l a l p h a c h a i n s ,

a n d h e t e r o z y g o t e s e x h i b i t a p p r o x i m a t e l y 50% n o rm a l a n d 50% m u ta n t

40

c h a i n s . I f two l o c i a r e i n v o l v e d , th e y b o th w ou ld h a v e t o h a v e

m u ta t e d i n an i d e n t i c a l m a n n e r . The p r o b a b i l i t y o f s u c h an o c c u r

r e n c e w ould b e v e r y c l o s e t o z e r o .

The o n ly log iC E il c o n c l u s i o n t o b e draw n from t h e s e c o n f l i c t

i n g r e p o r t s i s t h a t t h e human p o p u l a t i o n i s v e r y p r o b a b ly h e t e r o

g e n e o u s i n t h e num ber o f a l p h a c h a i n l o c i . F o r t h e p u r p o s e o f

c a l c u l a t i o n s , i t i s a ssum ed t h a t o n ly a s i n g l e a l p h a gene e x i s t s ,

a s t h i s w i l l h e l p a r g u e f o r r a n d o m n e s s .

Random and Non-Random I r o t e i n M u t a t i o n : p r o to m e r s an d P o s i t i o n

I t i s a b a s i c t e n e t o f g e n e t i c t h e o r y t h a t a l l m u t a t i o n s

o c c u r r a n d o m ly , h o w e v e r i t c a n be shown t h a t t h i s i s n o t a v a l i d

a s s u m p t io n i n l i g h t o f p r e s e n t d a t a on human h e m o g lo b in .

T h e re a r e two l e v e l s a t w h ic h t h i s p ro b lem c a n be a p p r o a c h

e d , a t t h e l e v e l o f t h e p r o t e i n i t s e l f and a t t h e l e v e l o f t h e

g e n e t i c m a t e r i a l c o d in g f o r t h a t p r o t e i n . On t h e p r o t e i n l e v e l

t h e r e a r e v a r i o u s c r i t e r i a t h a t c an b e u s e d to t e s t f o r random

n e s s , a lm o s t a l l o f w h ic h a r e t h e p h y s ic ; J . a n d c h e m ic a l p r o p e r

t i e s o f t h e amino a c i d s . G a r r e l l , Lehmann ;uid L o r k in ( 1 9 6 7 ;6 2 7 )

h a v e n o t e d a p r e p o n d e r a n c e o f n o n - c h a r g e d s u b s t i t u t i o n s w h ic h

t h e y a t t r i b u t e t o an a s su j i ip t io n t h a t a n i n t e r n a l r e p l a c e m e n t o f

a h y d ro p h o b ic r e s i d u e by a c h a r g e d r e s i d u e w ou ld n e c e s s a r i l y r e

s u l t i n a " t o t a l l y n o n - v i a b l e " m o l e c u l e , t h u s e s c a p i n g d e t e c t i o n .

They f , . i l t o g iv e t h e i r c a l c u l a t i o n s o r ev en an e x p l a n a t i o n o f

t e s t i n g p r o c e d u r e ^.nd i n f a c t m ore r e c e n t d a t a s u g g e s t s t h e r e

v e r s e i s i n e v i d e n c e ( f i g , 4 ) . I n any e v e n t , t h e i r a s s u m p t io n

c a n b e shown t.-, h a v e s e v e r a l e x c e p t i o n s , a t l e a s t one o f w h ic h ,

iib We i n ( b e t a 130 t y r - a s p ) , p r o u u c e s o n ly m i l d h e m o ly t i c a n e m ia

i n t h e h é t é r o z y g o t e , ( r e r u t z and Lelimann, 1 9 6 8 ; V o g e l , 1 9 6 9 ) , an d

a n o t h e r , Hb Bogn ( b e t a I 4 l e u - a r g ) p o r d u c e s no c l i n i c . . . ] a b n o rm a l-

\

41i t y w h a t s o e v e r (Monn e t . a l . , 1 9 6 8 ) .

a b a s i c w e ak n e ss o f d i s c u s s i o n s a t th e p r o t e i n l e v e l i s

t h a t t h e y a r e o f t e n done w i t h o u t c o n s i d e r a t i o n o f t h e g e n e t i c

c o d e , n e g l e c t i n g to o much e v i d e n c e t o be p a r t i c u l a r l y s i g n i f i c

a n t . T h is i s n o t to s a y s u c h i n v e s t i g a t i o n s h av e no v a l u e , m e r e l y

t j i a t ' a m ore a c c u r a t e i n t e r p r e t a t i o n c an be draw n from t h e l e v e l

o f codon b a s e s u b s t i t u t i o n ,

The e x i s t e n c e o f u n c h a l l e n g e d p r i o r e v id e n c e f o r n o n - r a n

dom ness o f i iiuxataon o f an y p r o t e i n i s n i l , how ever o n ly a v e r y

few o b s e r v e r s re m a in s t e a d f a s t i n t h e i r a s s u m p t io n o f a b s o l u t e

ran d o m n e ss ( e . g . King a n a J u k e s , 1 9 6 9 ) , and t h e s e a rg u m e n ts g e n

e r a l l y a d d r e s s t h e m s e lv e s t o t h e q u e s t i o n o f random m u t a t i o n by

. o s i t i o n . The K ing a n d J u k e s s t u d y \;as i n t e n d e d p r i m a r i l y a s a n

a rg u m e n t f o r random g e n e t i c d r i f t ( a s o p p o se d co n a t u r a l s e l e c t

i o n ) a s t h e p r im a ry a g e n t f o r m o s t a ia ino a c i d s u b s t i t u t i o n s b e t

ween s p e c i e s . T h e i r c o n t e n t i o n t h a t s i n c e t h e oxygen d i s s o c i a

t i o n c o n s t a n t s o f m o s t m am m alian h e m o g lo b in s .^re f a i r l y c l o s e ,

t h e i n t e r s p e c i e s v a r i a t i o n s f o u n d i n t h e p r im a ry s t r u c t u r e s o f

t h e i r h e m o g lo b in s a r e f u n c t i o n a l l y n e u t r a l , i . e . w ere n o t su b

j e c t to n a t u r a l s e l e c t i o n i n t h e p r o c e s s o f s p é c i a t i o n . T h is i s

an o v e r s i m p l i f i c a t i o n o f c h e m ic a l t h e o r y . To b e g in w i t h , many

m o le c u le s e x h i b i t s i m i l a r physic;TL p r o p e r t i e s , a s f o r exam ple

human h e m o g lo b in and m y o g lo b in , b u t I d o u b t t h a t anyone w o u ld a r

gue t h a t n a t u r a l s e l e c t i o n w as n o t i n v o l v e d i n t h e d i f f e r e n t i a t i o n

o f t h e s e p r o t e i n s . A l s o , t h e o x y g e n d i s s o c i a t i o n c o n s t a n t , o r f o r

e a s e o f c o m p a r is o n t h e p K 's o f many o r g a n i c a c i d s a r e q u i t e s im -

H a r ( e . g p K i^ o ta l ; . " P ^ o a r b o n a t e " 5 .7 7 J y e t may s e r v e e n -

t i r e l y d i f f e r e n t f u n c t i o n s .

As a t e s t f o r t h i s h y p o t h e s i s t h e y s u b j e c t t h e am ino a c i d

42

s u b s t i t u t i o n s o f human h e m o g lo b in t o a P o i s s o n d i s t r i b u t i o n ( a

b in o m i a l a p p r o x i m a t i o n f o r l a r g e s a m p le s ) o n ly a f t e r th e y r e d u c e d

t h e num ber o f o b s e r v e d o c c u r r e n c e s i n t h e z e r o m u t a t i o n c a t e g o r y ,

w h ic h C la r k e (1 9 7 0 ; 1 0 09 ) h a s a r g u e d i s s t a t i s t i c a l l y i l l e g i t i

m a te . 'f/tfhat C l a r k e f a i l s t o m e n t io n i s t h a t by r e d u c i n g t a e z e r o

m u t a t i o n c a t e g o r y , K ing a n d J u k e s h ave t o t a l l y i g n o r e d one o f t h e

two p r e v a i l i n g t h e o r i e s o f d i f f e r e n t i a l m u ta b i l i t ; ^ o f s e g m e n ts

on p o l y p e p t i d e s . The " c o l d s p o t " t h e o r y b a s e s i t s e l f on t h e

f a c t t h a t some o f t h e b e t t e r ktiown p r o t e i n s h a v e c e r t a i n p o s i

t i o n s f o r w h ic h no s u b s t i t u t i o n s a r e fo u n d o t o c c u r ; t h e s e a r e

t h e so c a l l e d " i n v a r i a n t r e s i d u e s " fo u n d i n m y o g lo b in , h e m o g lo b in ,

c y to c h ro m e C a n d o t h e r m o l e c u l e s , w h ic h show a h i g h d e g r e e o f

b o t h i n t e r s p e c i e s iuid i n t r a s p e c i e s c o n f o r m i t y . The " c o l d s p o t s "

o r a r e a s o f lo w m u t a b i l i t y r e s t r i c t m u t a t i o n a n d s e l e c t i o n t o t h e

r e m a in i n g p o s i t i o n s . I n f a c t , t h e K ing an d J u k e s m is u s e o f t h e

P o i s s o n d i s t r i b u t i n s u p p o r t s t h i s t h e o r y c o n s i d e r a b l y , a s t h e

m u t a t i o n s o f h e m o g lo b in do f i t t a e d i s t r i b u t i o n r a t h e r w e l l ( a s

t h e y d e m o n s t r a t e d ) i f a num ber a p p r o x i m a t i n g t h e num ber o f i n

v a r i a n t r e s i d u e s i s rem o ved .

C l o s e l y a k i n t o t h e t h e o r y o f a r e a s o f lo w m u t a b i l i t y i s

t h e c o n v e r s e t h e o r y o f a r e a s o f h i g h m u t a b i l i t y , o r " h o t s p o t "

t h e o r y b a s e d f o r t h e m ost p a r t on B e n z e r ' s c l a s s i c b a c t e r i a l chrom

osom al s t u d i e s , how ever i t h a s b e e n n o t e d t h a t t h i s i s r a r e l y ap

p l i e d to h i g h e r o r g a n is m s (B o y e r , e t . a l . , 1 9 7 1 ; 4 3 8 ) .

A c t u a l l y , v ie w e d i n t h e c l a s s i c a l D a rw in ia n s e n s e a n d o v e r

l a r g e t im e p e r i o d s , t h e two t h e o r i e s a r e n o t m u t u a l l y e x c l u s i v e ;

b o t h a t t e m p t to e x p l a i n o b s e r v â t ! -ns t h a t some a r e a s o f some p r o -

td L n s a r e m ore s u s c e p t i b l e to m u t a t i o n t h a n o t h e r s . I f t h e a s

s u m p t io n i s made t h a t n a t u r a l s e l e c t i o n and n o t random d r i f t i s

43t h e p r i n c i p a l a g e n t o f p r o t e i n e v o l u t i o n , t h e n a r e a s o f h i g h m u t

a b i l i t y w i l l e x i s t o n ly f o r r e l a t i v e l y s h o r t p e r i o d s o f t i m e , t h a t

i s t h e y w i l l t e n d t o e l i m i n a t e t h e m s e lv e s a s t h e p r o t e i n ' f i n d s "

t h e am ino a c i d s e q u e n c e i n t h a t a r e a w h ic h w i l l p ro d u c e t h e m ost

e f f i c i e n t b i o l o g i c a l a c t i v i t y , a t w h ic h t im e n a t u r a l s e l e c t i o n

w o u ld r e i n f o r c e i t s " c h o i c e " , S i m i l a r l y , an a r e a o f low m u t a b i l

i t y c o u l d b e e x p l a i n e d a s a n a r e a w hose f u n c t i o n h a s e v o lv e d to

t h e p o i n t w h e re i t s am ino a c i d s e q u e n c e i s so e s s e n t i a l to t h e

a c t i v i t y o f t h e p r o t e i n t h a t a n y c h an g e w o u ld p ro d u c e a n o n - v i a b l e

m o l e c u l e . F o r e x a m p le , t h e s i m i l a r i t y i n am ino a c i d s e q u e n c e b e

tw een h e m o g lo b in a n d m y o g lo b in c o u ld b e e x p l a i n e d by t h e e x i s t e n c e

o f a common a n c e s t r a l p r o t e i n w h ic h , v i a g en e d u p l i c a t i o n , r e s u l t

e d i n t h e p r o d u c t i o n o f two p r o t e i n s w i t h ( a t f i r s t ) i d e n t i c a l

s t r u c t u r e . T h is i s g e n e r a l l y a c c e p t e d a s t h e e x p l a n a t i o n f o r t h e

e x i s t e n c e o f t h e a l p h a , b e t a an d d e l t a c h a i n s fo u n d i n t h e h i g h e r

New W o rld p r i m a t e s an d a p e s . Random m u t a t i o n c a u s i n g s l i g h t l y

d i v e r g e n t s t r u c t u r e s w o u ld a l l o w t h e o rg a n is m t o i n c r e a s e t h e d i

v e r s i t y o f t h e p a r e n t p r o t e i n ’ s o r i g i n a l f u n c t i o n , i n t h i s ex

a m p le a l t e r i n g t h e f u n c t i o n f ro m g e n e r a l oxyg en t r a n s p o r t t o c i r

c u l a t o r y oxyg en t r a n s p o r t a n d m u s c u la r t r a n s p o r t . F i n a l l y , a s

t h e s e d i v e r g e n t p r o t e i n s h a d assum ed d i f f e r e n t f u n c t i o n s , d i f f e r

e n t " h o t " an d " c o l d " s p o t s w o u ld a r i s e i n r e s p o n s e t o s e l e c t i v e

p r e s s u r e to w a rd i n c r e a s i n g e f f i c i e n c y o f t h e i r new r o l e s . Thus

a g iv e n c h a i n o f h e m o g lo b in a n d a m o le c u l e o f m y o g lo b in h a v e r e

t a i n e d v e r y s i m i l a r s t r u c t u r e s i n t e r m s o f am ino a c i d s e q u e n c e s

a n d s i m i l a r f u n c t i o n s i n t e r m s o f o xy g en t r a n s p o r t , b u t e x h i b i t

d i f f e r e n t a r e a s o f h i g h a n d lo w m u t a b i l i t y .

Random and Non-Random M u ta t io n o f P r o to m e r s

A s e c o n d c r i t e r i o n on w h ic h a rg u m e n ts o f ra n d o m n e ss c an be

b a s e d i s o c c u r r e n c e o f am ino a c i d s u b s t i t u t i o n s on e a c h o f t h e

4 4

s p e c i f i c p o l y p e p t i d e s r a t h e r t h a n p o s i t i o n on a g iv e n c h a i n .

B e c a u s e t h e d e l t a c h a i n o f th e m in o r com ponent (Hb Ag) n o r

m a l l y e x i s t s i n r e l a t i v e l y s m a l l c o n c e n t r a t i o n s ( a b o u t y/o) a n d

b e c a u s e t h i s c o n c e n t r a t i o n i s fo u n d to be c o n s t a n t i n a l l c e l l s

( i . e . t h e r e i s no a n a t o m i c a l d i f f e r e n t i a t i o n ) , i t h a s b e e n hypo

t h e s i z e d t h a t t h e p r im a r y p h y s i o l o g i c a l f u n c t i o n o f h e m o g lo b in

i s c a r r i e d o u t b y Hb A end t h a t Hb Ag s e r v e s a l e s s e r f u n c t i o n ,

m ak in g i t r e l a t i v e l y i n s e n s i t i v e to n a t u r a l s e l e c t i o n ( B o y e r , e t .

a l . , 1 9 6 9 ) . T h e i r c o n c l u s i o n c an be s u b s t a n t i a t e d t o a p o i n t by

t h e o b s e r v a t i o n t h a t t h e d e l t a and b e t a c h a i n s h a v e a c c u m u la t e d

a b o u t t h e came numbL.r o f amino a c i d c h a n g e s s i n c e t h e o r i g i n a l

d u p l i c a t i o n , b y t h e e x i s t e n c e o f a t e t r a m e r o f a l p h a c h a i n s o f

f e t a l h e m o g lo b in c o m p l e t e ly l a c k i n g d e l t a c h a i n s (Hb N i o r t ; (W ajc-

man e t . a l , , 1 9 7 0 ) , a n d by t h e f a c t t h a t o ld w orld p r i m a t e s ( e x

c e p t a p e s and m an) seem to g e t a lo n g f i n e w i t h o u t i t . B u t t h i s

h y p o t h e s i s c a n b e e q u a l l y d i s c r e d i t e d by t h e r e p o r t o f Huehns and

S h o o t e r (1 9 6 5 ) o f a v i a b l e d e l t a t e t r a m e r (Hb D^) l a c x i n g a n a l

p h a c o m p o n en t a n d by th e c h e m ic a l d a t a o f K i n d e r l e r e r , Lehmann

a n d T i p t o n (1 9 7 0 ) vho have fo un d Hb Ag c o u n t e r a c t s t h e r m a l dé

n a t u r a t i o n o f Hb A, s u g g e s t i n g a s t a b i l i l i z i n g f u n c t i o n n o t ob

s e r v e d i n a l p h a o r b e t a c h a i n s . At t h e p r e s e n t t im e t h e q u e s t i o n

o f s e n s i t i v i t y t o n a t u r a l s e l e c t i o n o f th e m in o r com ponen t o f a d

u l t h e m o g lo b in r e m a in s u n r e s o l v e d , a l t h o u g h i t w o u ld seem m ore

p r o b a b l e t h a t d e l t a c h a i n s s e r v e a u n iq u e f u n c t i o n a n d w ou ld t h u s

b e q u i t e s e n s i t i v e s i n c e i t s c o n c e n t r a t i o n i s so low t h a t a m u ta

t i o n p r o d u c i n g a t o t a l l y n o n - v i a b l e m o le c u le w ou ld l e a v e t h e hemo

g l o b i n a t l e a s t 37% e f f i c i e n t . T h is b e in g t h e c a s e , s e l e c t i o n on

d e l t a w o u ld be v e r y s m a l l ( a b o u t 3% o f b e t a a ssu m in g e q u a l f u n c t

i o n a l i m p o r t a n c e ) a n d t h e number o f d e l t a m u t a t i o n s s h o u l d b e c o r

45

r e s p o n d i n g l y l a r g e . I t i s n o t . ( t a b l e l ) . T h e re a r e f o u r p o s s i b l e

e x p l a n a t i o n s f o r t h i s s i t u a t i o n ; ( 1 ) d e l t a m u s t s e r v e some f u n c

t i o n o t h e r t h a n b e t a o r a l p h a su c h t h a t m u t a t i o n s a r e s u b j e c t t o

p o w e r f u l n e g a t i v e s e l e c t i o n , o r ( 2 ) m o s t o f t h e d e l t a c h a i n i s

i n t r i n s i c a l l y an a r e a o f lo w m u t a b i l i t y , w h ic h a m o u n ts t o a r e

s t a t e m e n t o f (1 ) i f one a c c e p t s t h e D a rw in ia n a rg u m e n t f o r p r o

t e i n m u t a t i o n ( a b o v e ) , o r ( 3 ) Hb Ag r e a l l y i s i n s e n s i t i v e t o s e l

e c t i v e a g e n t s c a u s i n g a l a c k o f c l i n i c a l symptoms r e s u l t i n g i n a

r e d u c t i o n o f d i s c o v e r a b l e m u t a t i o n s , o r {Aj t h e d i s c r e p a n c y i s

du e to s a m p l in g e r r o r a s s o c i a t e d w i t h s m a l l s a m p le s . S a m p lin g t e c h

n i q u e s h a v e im p ro v e d a n d t h e num ber o f r e p o r t s h a s grown t o s u c h

an e x t e n t t h a t s a m p l in g e r r o r w ou ld n o t be t h e m o s t p r o b a b l e ex

p l a n a t i o n , n o r w o u ld l a c k o f d i s c o v e r y , so i t seem s l i k e l y t h a t

d e l t a h a s m ore t h a n one f u n c t i o n .

The num ber o f gamma c h a i n m u ta n t s i s a l s o much s m a l l e r t h a n

e x p e c t e d , a n o b s e r v a t i o n g e n e r a l l y a s c r i b e d t o f e t a l l o s s (Lehmann

1 9 6 2 ) . A g a in , i f t h e r e a r e two gamma l o c i ( a b o v e ) t h e d i s c r e p a n c y

i s d o u b le d , how ever o n l y one l o c u s i s a ssu m ed f o r p u r p o s e s o f c a l

c u l a t i o n s .

The d i f f e r e n c e i n f r e q u e n c i e s o f m u t a t i o n b e tw e e n a l p h a an d

b e t a i s som ewhat m ore d i f f i c u l t t o e x p l a i n , e s p e c i a l l y i f t h e a r

gum ent f o r two a l p h a l o c i i s p r e s e n t e d . J o n e s and h i s c o w o rk e r s

( J o n e s , e t . a l , , 1 9 7 2 ) h a v e a r g u e d i n t h e f o l l o w i n g m a n n e r :

" U n d e r e s t i m a t i o n w o u ld r e s u l t f o r a n y u n s t a b l e a l p h a c h a i n m u ta n t w h ic h com prom ised i n t r a u t e r i n e d e v e lo p m e n t a n d r e s u l t e d i n f e t a l l o s s . I t c o u l d a l s o o c c u r b e c a u s e m o s t a l p h a c h a i n m u ta n t s r e s u l t i n t h e p r o d u c t i o n o f l e s s t h a n 23% o f t h e a f f e c t e d c h a i n ; t h u s t h e y m ig h t a s a g ro u p be l e s s s e v e r e an d m ore l i k e l y to e s c a p e d e t e c t i o n . " (8 2 )

T h e re a r e s e v e r a l a lp h a m u t a t i o n s w h ic h a r e i n d e e d l e s s s e v

e r e t h a n t h e m a j o r i t y o f b e t a m u t a t i o n s ( e . g . Hbs H a s h a r o n , H o p k in s

46

I I , D a k a r , E t o b i c o k e , G - C h in e s e , C h i a p i s ) , h o w ever t h e r e a r e

s t i l l s u f f i c i e n t a l p h a m u t a t i o n s o f g r e a t s e v e r i t y ( e . g . Hbs

KfOisas, C h e s a p e a k e , B ib b a , T o r in o ) t o c a s t some d o u b t on t h i s

h y p o t h e s i s . A l s o , i f i n t e r f e r e n c e i n u t e r o was t h e s o l e c a u s e ,

t h e num ber o f a l p h a a n d gamma m u t a t i o n s s h o u ld be much c l o s e r

t h a n o b s e r v e d f r e q u e n c i e s .

The f a c t r e m a in s t h a t t h e b e t a c h a i n s a r e t h e o n ly o n e s to

a p p e a r o n ly a f t e r t h e i n d i v i d u a l i s f a i r l y w e l l d e v e lo p e d , su g

g e s t i n g a t l e a s t some c o r r e l a t i o n b e tw e e n e a r l y c e a t h an d low

o b s e r v e d m u t a t i o n s .

Random a n d Non-Random M u ta t io n ; The Amino A c id s

As i t o a h b s shown t h a t m u t a t i o n s a r e f a r from random on

t h e c r i t e r i a o f c h a i n o r p o s i t i o n , t h e q u e s t i o n a r i s e s i f t h e mu

t a t i o n s i n v o l v e random amino a c i d r e p l a c e m e n t s . A n a l y s i s o f t h e

d a t a c a p a b l e o f s u p p l y i n g an a n s w e r c a n b e made i n two w a y s ; by

t h e a n a l y s i s o f t h e amino a c i d c o m p o s i t i o n o f t h e p r o t e i n i t s e l f

a n d a n a l y s i s o f t h e b a s e t r i p l e t s , c o d in g f o r t h e p r o t e i n .

I t i s known t h a t n o t a l l am ino a c i d s c a n r e p l a c e e a c h -

o t h e r i n m u t a t i o n s r e s u l t i n g f ro m a s i n g l e b a s e s u b s t i t u t i o n .

P o s s i b l e s u b s t i t u t i o n s a r e g i v e n i n f i g u r e 1 , E x p e c te d f r e

q u e n c i e s o f m u t a t i o n s f ro m a n d t o a g i v e n amino a c i d c a n n o t be

e x p r e s s e d s im p ly a s t h e num ber o f m u t a t i o n s d i v i d e d b.y t h e num

b e r o f amino a c i d s a n d m u s t t a k e in o c o n s i d e r a t i o n s e v e r a l f a c

t o r s , a l t h o u g h i t i s d i f f i c u l t t o a r r i v e a t an e x a c t f i g u r e u n

l e s s c a l c u l a t i o n s a r e made on t h e g e n e t i c l e v e l ( . i . e . , i n t e r m s

o f b a s e t r a n s i t i o n s and t r a n s v e r s i o n s ) , a good a p p r o x i m a t i o n c a n

n e v e r t h e l e s s b e m ade . The e x p e c t e d f r e q u e n c y o f m u t a t i o n s from

a g i v e n amino a c i d i s c e r t a i n l y a f u n c t i o n o f t h e num ber o f t im e s

t h a t am ino a c i d n a t u r a l l y o c c u r s on t h e p r o t e i n . F o r e x a m p le .

47

i f l y s i n e n o r m a l l y a p p e a r s o n l y o n c e a n d a r g i n i n e a p p e a r s 20

t i m e s , o n e w o u ld c e r t a i n l y e x p e c t m ore m u t a t i o n s from a r g i n i n e

t h a n f ro m l y s i n e . T h i s i s m o s t e a s i l y e x p r e s s e d a s a p e r c e n ta g e

b y d i v i d i n g t h e n u m b er o f o c c u r r e n c e s b y H I ( a l p h a ) o r 146 ( b e t a ,

d e l t a , gam m a). S e c o n d , t h e t o t a l num ber o f p o s s i b l e m u ta t io n s

m u s t b e t a k e n i n t o a c c o u n t , s i n c e some amino a c i d s c a n be r e

p l a c e d o n l y b y 5 o r 6 o t h e r s ( e . g . , t r y p t o p h a n , c y s t e i n e ) , w h i le

o t h e r s c a n be r e p l a c e d by a s many a s 12 ( e . g . , a r g i n i n e , s e r i n e ) .

F i n a l l y t h e num ber o f d i f f e r e n t am ino a c i d s m u st oe i n c l u d e d

s i n c e n o t a l l ^ r o t e i n s c o n t a i n a l l 20 o f t h e "common" amino a c i d s

w h i l e o t h e r s i n c o r p o r a t e r a r e r am ino a c i d s su c h a s 4 -h y d ro x y -

p r o l i n e a n d 5 - h y d r o x y l y s i n e . The e x p e c t e d f r e q u e n c y o f m u ta t io n

f ro m a p a r t i c u l a r am ino a c i d c a n t h e n be e x p r e s s e d a s th e number

n a t u r a l l y o c c u r r i n g d i v i d e d by t h e num ber o f r e s i d u e s on t h e

c h a i n ( « f r e q u e n c y o f o c c u r r e n c e ) , t i m e s t h e num ber o f ai,:ino a c i d s

i t c a n m u t a t e t o d i v i d e d b y 1 / 2 0 o f t h e t o t a l . n s s i b l e m u ta t io n s

o f a l l am ino a c i d s on t h a t c h a i n ( « f r e q u e n c y o f m u t a t i o n ) . O r;

f (E)fj,om = f ( o c c u r r e n c e ) ( x / t o t a l / 2 0 )

B e c a u s e t h i s i n v o l v e s t h e m u l t i p l i c a t i o n o f a f r e q u e n c y by a f r e

q u e n c y , t h e sum t o t a l o f am ino a c i d s n e e d n o t e -

q u a l 1 , h o w e v e r i n t h i s c a s e t h e m o s t p ro n o u n c e d d e v i a t i o n was

o n l y 17 p a r t s p e r t h o u s a n d , w e l l w i t h i n n o rm a l ro u n d in g o f f a c -

c e p t i b i l i t y .

C a l c u l a t i o n s o f e x p e c te d f r e q u e n c i e s o f m u ta t i o n s to a g iv

en am ino a c i d c a n b e ex r e s s e d s im p ly a s th e num ber o f amino

a c i d s n a t u r a l l y o c c u r r i n g t h a t c a n p o s s i b l y m u ta te t o i t i n one

s t e p d i v i d e d by t h e t o t a l num b er o f p o s s i b l e m u t a t i o n s . B o th o f

t h e s e f r e q u e n c i e s a r e g iv e n i n t a b l e s tw o t h r o u g h tO d ^ .

48

Random and Non-Random M u t a t i o n : The Codon B a se s

A more p o w e r fu l t e s t c a n h e done i f o n l y t h e b a s e t r a n s

i t i o n s and t r a n s v e r s i o n s a r e c o n s i d e r e d . t r a n s i t i o n i s a p u r

i n e - p u r i n e o r a p y r i m i d i n e - p y r i m i d i n e s u b s t i t u t i o n ; a t r a n s v e r

s i o n i s a p u r i n e - p y r i m i d i n e o r a p y r i m i d i n e - p u r i n e s u b s t i t u t i o n ;

t h e p u r i n e s a r e a d e n i n e ( 6 - a m i n o p u r in e ) a n d g u a n in e ( 2 - a m i n o - 6 -

h y d r o x y p u r in e ) ; t h e p y r i m i d i n e s a r e c y t o s i n e ( 2 - o x y - 4 - a m i n o p y r -

im id in e ) v a n d a l s o u r a c i l ( 2 , 4 - d i o x y p y r l m i d i n e ) i n RNA. T h i s t e s t

i s more p o w e r f u l s i n c e DNa ( i n t h i s c a s e , t h e t r a n s c r i b e d t-RNA

i s u s e d ) i s t h e a c t u a l s i t e o f m u t a t i o n a n d b e c a u s e random b a s e

s u b s t i t u t i o n s leed n o t r e s u l t i n random am ino a c i d r e p l a c e m e n t s

on t h e p r o t e i n a n d v i c e - v e r s a . Thus t h i s t e s t e l i m i n a t e s t n e

w e l l knov/n b i a s o f d i s c o v e r y to v /a rd s c h a n g e s i n n e t c h a r g e and

to w a rd s c l i n i c a l a o n o r m a l i t i e s , a s t h e s e phenom ena o c c u r o n ly on

th e p r o t e i n l e v e l .

The f i r s t s t e p i n t h i s p r o c e d u r e i s t o c a l c u l a t e a c c u r a t e l y

t h e number o f e x o e c t e d t r a n s i t i o n s and t r a n s v e r s i o n s . One o f

t h e f i r s t s t u d i e s i n w h ic h c a l c u l a t i o n was made was t h a t by

Lehamnn and G a r . e l l (1 9 0 9 ) who, f o r r e a s o n s unknow n, a s su m e d an

e x p e c te d r a t i o o f 30%-30%, S in c e f o r a n y g i v e n b a s e t h e r e i s

o n ly one p o s s i b l e t r a n s i t i o n ( e . g . , U c a n o n l y be r e p l a c e d b y C

v i a t r a n s i t i o n ) b u t two p o s s i b l e t r a n s v e r s i o n s ( e . g . , U c a n be

r e p l a c e d by A o r G v i a t r a n a v e r s i o n ) , s e v e r a l a u t h o r s h a v e a s

sumed a r a t i o o f 33%-67%, v ;h ich i s c l o s e r a n d w o u ld b e c o r r e c t

f o r a p r o t e i n c o d e d f o r b y a DNA se g m e n t c o n t a i n i n g e q u a l n u m b ers

o f a l l b a c e s . T h i s a s s u m p t io n d o e s n o t c o r r e c t f o r t h e c h e m ic a l

c o m p o s i t io n o f t h e p r o t e i n , o r m ore e x a c t l y , t h e c h e m ic a l com

p o s i t i o n o f t h e DNA. iin e x a c t f i g u r e c a n b e c a l c u l a t e d b y c o u n t

in g a l l p o s s i b l e b a s e c h a n g e s l e a d i n g to m u t a t i o n ( n e u t r a l m u t-

49

a t i o n s , e . g . , g l u - g l u by GAA to GaG, and m u t a t i o n s l e a d i n g to

n o n s e n s e t r i p l e t s a r e e x c lu d e d s i n c e t h e f o r m e r i n n o t d e t e c t

a b l e and t h e l a t t e r i n n o t known t o o c c u r on homo, l o b i n ) f o r

e a c h amino a c i d , summing them a n d d i v i d i n g e a c h by t h e su m m atio n .

T hese f r e q u e n c i e s a r e g i v e n i n t a b l e 2 2 , bhen e i t h e r a t r a n s -

v e r s i o n o r t r a n s i t i o n c o u l d e x p l a i n t h e m u t a t i o n ( e . g . , g l y -

a r g ) , t h e m u t a t i o n was o m i t t e d f ro m t h e s a m p le ( s e e i n v e n t o r y ) .

To t e s t w h e th e r t h e b a s e s w e re r e p l a c i n g e a c h o t h e r randoïily,

e x p e c t e d f r e q u e n c i e s o f m u t a t i o n f ro m a g i v e n b a s e w e re c a l c u

l a t e d by d i v i d i n g t h e num ber o f p o s s i b l e m u t a t i o n s l e a d i n g to

a co d e f o r a n o t h e r amino a c i d by th e num ber o f p o s s i b l e m u t a t i o n s

o f o i l amino a c i d s . S i m i l a r l y , t h e e x p e c t e d f r e q u e n c i e s o f m ut

a t i o n to a g iv e n am ino a c i d w ere c a l c u l a t e d by d i v i d i n g t h e t o t a l

num ber o f p o s s i b l e s u b s t i t u t i o n s l e a d i n g to t h e code f o r t h a t

amino a c i d by t h e t o t a l num ber o f a l l p o s s i b l e m u t a t i o n s on t h e

c h a i n .

50

T a b l e 2 i Uhi s q u a r r e d t e s t t o d e t e r m i n e i f h e m o g lo b in p o i n t m u t a t i o n s a r e c a u s e d b y n u c l e o t i d e r e p l a c e m e n t s r a n d o m ly d i s t r i b u t e d o v e r t h e f i r s t ,s e c o n d a n d t h i r d p o s i t i o n s on c o d o n t r i p l e t s .

P o s i t i o n 0 E (0 -E ) ( 0 - E J ^

F i r s t 81 5 7 .3 5 2 3 .6 7 5 6 0 .2 7

S eco n d 76 5 7 .3 3 1 8 .6 7 3 4 8 .5 7

T h i r d 15 5 7 .3 3 4 2 .3 3 1 7 9 1 .8 3

T72 1 7 1 .9 9 2 7 0 0 .^ 7

. 2 7 0 0 /1 7 2 » 1 5 .7 0

d . f . = 2

P « . 0 0 1

51

T a b le 3 : K o lm ogo ro v -S m im ov one sam ple t e s t o f m u t a t i o n f ro m ag i v e n amino a c i d a ssu m in g random am ino a c i d r e p l a c e m e n t .A lpha c h a i n .

f F (x ; sum F(X) S(X) sum S(XJ /F (X )

Gly 6 • 055 .0 5 5 .0 9 7 .0 9 7 .0 4 2

A la 5 .157 .212 .081 .1 7 8 .0 3 4

S e r 2 .1 1 3 .32 5 .0 3 2 .2 1 0 .1 1 5

Phe 5 .0 6 6 .391 .081 .291 .1 0 0

Val 1 .1 1 0 .501 .0 1 6 .3 0 7 .1 9 4

Thr 0 .067 .5 6 8 .0 0 0 .3 0 7 .261

Cys 0 .0 0 4 .5 7 2 .0 0 0 .3 0 7 .2 6 5

Leu 3 .1 2 6 .6 9 8 .0 4 8 .3 5 5 .3 4 3

l i e 0 .0 0 0 .6 9 8 .0 0 0 .3 5 5 .3 4 3

Asn 5 .0 2 6 .7 2 4 .081 .4 3 6 .2 8 8

Asp 13 .0 6 3 .7 8 7 .2 1 0 .6 4 6 .141

Lys 5 .032 .8 1 9 ,081 .7 2 7 .0 9 2

Gin 2 .007 .8 2 6 . 0 3 2 .7 5 9 .0 6 7

Glu 6 .031 .857 .0 9 7 .8 5 6 .001

Arg 3 .029 .885 .0 4 8 .9 0 4 .0 1 8

Met 0 .0 1 4 .9 0 0 .UOO .9 0 4 .V.04

H is 5 .0 5 6 .955 .081 .9 8 5 .0 2 9

Phe 1 .041 .997 .0 1 6 1 .0 01 .0 0 4

T yr 0 .u15 1 .0 12 ,u 0 0 1 .0 01 .0 1 1

Trp 0 .005 1 .0 17 .0 0 0 1 .00 1 .0 1 6

N 62

D = .3 4 3

C r i t i c a l v a l u e * .173

52

T a b le 4 ; K o lm o g o ro v -S m irn o v one sam p le t e s t o f m u t a t i o n from ag i v e n am ino a c i d a s s u m in g random am ino a c i d r e p l a c e m e n t .B e ta c h a i n .

f P(X ) sum F(X) S(X) sum S(X ) /F (X

G ly 12 .0 9 6 .0 9 6 ,1 2 0 ,1 2 0 .0 2 4

A la 3 .1 2 0 .2 1 6 .0 3 0 .1 5 0 ,0 6 6

S e r 1 .0 5 0 .2 6 6 ,0 1 0 ,1 6 0 .1 0 6

P ro 3 .051 .3 1 7 .0 3 0 ,1 9 0 ,1 2 7

Val 8 .1 0 2 .4 1 9 ,0 8 0 .2 7 0 .1 4 9

T hr 2 .0 4 0 .4 5 9 ,0 2 0 ,2 9 0 .1 6 9

Cys 0 .0 0 9 ,4 6 8 ,0 0 0 ,2 9 0 .1 7 8

Leu 7 .1 2 4 .5 9 2 .0 7 0 .3 6 0 .2 3 2

l i e 0 .0 0 0 .5 9 2 ,0 0 0 .3 6 0 ,2 3 2

Asn 6 .031 .6 2 3 .0 6 0 .4 2 0 .2 0 3

Asp 10 .0 6 5 ,6 8 8 ,1 0 0 .5 2 0 .1 6 8

Lys 9 .051 .7 3 9 .0 9 0 ,6 1 0 .1 2 9

G in 1 ,021 .7 6 0 ,0 1 0 .6 2 0 .1 4 0

Glu 15 .0 6 6 ,8 2 6 é150 .7 7 0 .0 5 6

A rg 1 ,0 2 9 ,8 5 5 .0 1 0 ,7 8 0 .0 7 5

Met 0 .0 0 7 ,8 6 2 .0 0 0 .7 8 0 ,v 8 2

H is 13 .0 5 7 .9 1 9 .1 3 0 .9 1 0 .0 0 9

Phe 4 .0 4 5 .9 6 4 .0 4 0 .9 5 0 .0 1 4

T yr 4 .0 1 4 .9 7 8 .0 4 0 .9 9 0 .01 2

T rp 1 .0 1 0 .9 8 8 ,0 1 0 1 ,0 0 0 ,0 1 2

N 100

D « ,2 3 2

C r i t i c a l v a l u e = .1 3 6

53

T a b le 5 : K o lm o g o ro v -S m irn o v one sa m p le t e s t o f m u ta t i o n fro m ag i v e n am ino a c i d a s su m in g random amino a c i d r e p l a c e m e n t ,Gamma d i a i n .

f FIX ) sum F(X) S(X) sum S(X ; /F (X )

Gly 0 ,0 8 9 .0 8 9 ,0 0 0 ,uOO .u 8 9

A la u .075 .1 6 4 ,u 0 0 .uOO .1 6 4

S e r 0 *u75 .2 3 9 ,0 0 0 .0 0 0 .23 9

Pro 0 .0 2 7 .2 6 6 .uoo ,uOO .2 6 6

Val u .0 8 9 .3 5 5 ,0 0 0 ,0 0 0 .3 5 5

x h r 1 .0 6 8 .4 2 3 ,111 ,111 .3 1 2

Cys 0 .0 0 7 .4 3 0 .0 0 0 .111 .3 1 9

Leu 0 .1 1 6 .5 4 6 ,0 0 0 .111 .4 3 5

l i e 0 .0 2 7 .5 7 3 ,0 0 0 .111 .4 6 2

Asn 0 .0 3 4 .6 0 7 ,0 0 0 .111 ,4 9 6

Asp 1 .0 5 5 .6 6 2 .111 ,2 2 2 .4 4 0

Lys 1 .082 .7 4 4 .111 .3 5 3 .4 3 8

Gin 0 .0 2 7 .771 ,0 0 0 .3 3 3 .4 3 8

Glu 4 .0 5 5 ,8 2 6 .4 4 4 .7 7 7 .0 4 9

Arg 0 .u21 .8 4 7 .0 0 0 .7 7 7 .0 7 0

Met 0 .0 1 4 .861 ,0 0 0 .7 7 7 .0 8 4

H is 2 .0 4 8 .9 0 9 . 2 2 2 .9 9 9 ,0 9 0

Phe 0 .0 5 5 .9 6 4 ,0 0 0 .9 9 9 .0 3 5

T yr 0 .0 1 4 .9 7 8 .0 0 0 .9 9 9 .021

T rp 0 ,021 .9 9 9 ,0 0 0 .9 9 9 .0 0 0

N 9

D * ,4 9 6

C r i t i c a l v a l u e » .4 3 2

54

T a b le 6 : K o lm ogorov-S m irnov o n e s a m p le t e s t o f m u ta t i o n f ro m ag iv e n am ino a c i d a s s u m in g random amino a c i d r e p l a c e m e n t . D e l t a c h a i n .

f F(X) sum P (X ; s ( x ; sum S(X ; / F U ;

G ly 3 .091 .091 ♦ 500 ,5 0 0 .4 0 9

Ala 1 .111 .2 0 2 .1 6 7 .66 7 .4 6 5

S e r 0 .061 .2 6 3 .0 0 0 ,6 6 7 .4 0 4

I'To 0 .0 4 3 .3 0 6 ,0 0 0 .6 6 7 .3 6 1

Val 0 .1 4 3 .4 4 9 .0 0 0 .6 6 7 .2 1 8

Thr 0 ,u31 .4 8 0 ,0 0 0 .6 6 7 .1 8 7

Cys 0 .00 9 .4 8 9 ,0 0 0 .66 7 ,1 7 8

Leu 0 .1 2 0 ,6 0 9 ,0 0 0 .6 6 7 .0 5 8

l i e 0 .uOO .6 0 9 ,0 0 0 .6 6 7 .0 5 8

Asn 1 .0 4 0 .6 4 9 .1 6 7 .8 3 3 ,1 8 4

Asp 0 ,U61 .7 1 0 .0 0 0 .8 3 3 .1 2 3

Lys 0 .041 .751 .0 0 0 .8 3 3 ,0 8 2

Gin 0 .0 3 2 .7 8 3 ,0 0 0 .8 3 3 ,0 0 8

Glu 0 ,0 5 8 .841 .0 0 0 .8 3 3 ,0 0 8

Arg 0 .0 3 6 .8 7 7 .0 0 0 .8 3 3 .0 4 4

Met 0 .0 1 4 .8 9 1 ,0 0 0 .8 3 3 .0 5 8

Hie 1 ,045 .9 3 6 .1 6 7 1 ,0 0 0 ,0 6 4

Phe 0 .0 4 7 .9 8 3 ,0 0 0 1 ,uOO ,0 1 7

Tyr 0 .0 2 3 1 ,0 0 6 ,0 0 0 1 ,0 0 0 ,0 0 6

T rp 0 ,0 0 0 1 .0 0 6 ,0 0 0 1 .0 0 0 ,u 0 6

N 6

D « ,465

C r i t i c a l v a l u e m ,521

55

x a b l e 7 : K o lm o gorov -S m irno v on e sam p le t e s t o f m u t a t i o n to ag i v e n am ino a c i d a s su m in g random amino a c i d r e p l a c e m e n t .A lp h a ch a i n .

f F(X) sum F(x; s (x) sum S(x) /p(x;

Gly 3 .0 5 5 .0 5 5 .0 4 8 ,0 4 8 .0 0 7

A la 2 .0 5 2 .1 0 7 .0 3 8 ,0 8 0 .0 2 7

S e r 1 ,0 7 2 .1 7 9 .0 1 6 .0 9 6 ,0 8 3

Pro 3 .0 6 6 .2 4 5 ,0 4 8 ,1 4 4 ,101

Val 2 .0 6 0 .3 0 5 .0 3 2 ,1 7 6 ,1 2 9

Thr 1 .0 5 3 .3 5 8 ,0 1 6 .1 9 2 , 166

Cys 0 .0 2 9 .3 8 7 ,000 ,1 9 2 .1 9 5

Leu 2 .0 4 9 .4 3 6 .0 3 2 .2 2 4 .2 1 2

H e 0 .0 7 0 .5 0 6 ,u 0 0 .2 2 4 ,2 8 2

Asn 5 ,0 4 6 .5 5 2 .081 ,3 0 5 ,2 4 7

Asp U .0 5 5 .6 0 7 ,2 2 6 .531 .076

Lys 5 .021 ,6 2 6 ,081 ,6 1 2 ,0 1 6

Gin 4 .0 4 7 .6 7 5 ,0 6 5 .6 7 7 ,0 0 2

Glu 2 .0 5 4 .7 2 9 ,0 3 2 .7 0 9 ,0 2 0

Arg 8 .0 7 0 .7 9 9 .1 2 9 .8 3 8 .0 3 9

Met 0 f048 .8 4 7 ,000 .8 3 8 ,0 0 9

H is 5 .0 3 9 .8 8 6 ,081 .9 1 9 .033

Pro 0 .041 .9 2 7 ,0 0 0 .9 1 9 ,u 0 8

Tyr 5 .0 3 7 .9 6 4 ,081 1 ,0 0 0 .036

T rp

N

D * .2 8 2

0

62

.036 1 .0 0 0 ,000 1 .0 0 0 .0 0 0

56

T a b le 8 : K o lm o g o ro v -S m irn o v o n e sam p le t e s t o f m u t a t i o n t o aa g i v e n amin'^ a c i d a s s u m in g random amino a c i d r e p l a c e m e n t . B e ta c h a i n .

f P U ) sum P(X) S(XJ sum S U ) /P ( X )

Gly 3 ,0 5 4 .054 .0 3 0 .0 3 0 .0 2 4

A], a 5 .0 5 8 .1 1 2 .0 5 0 ,u 8 0 .031

S e r 4 .0 7 4 ,1 8 6 .0 4 0 ,1 2 0 ,u 6 6

P ro 4 .0 5 4 .2 4 0 .0 4 0 ,1 6 0 ,0 8 u

Val 5 .041 ,^81 .0 3 0 .1 9 0 ,091

Thr 2 .041 .3 2 2 .0 2 0 .2 1 0 ,1 1 2

Cys 3 .031 .3 5 3 .0 3 0 ,2 4 0 .1 1 3

l e u 3 .0 5 0 .4 0 3 .0 3 0 .2 7 0 ,1 3 3

l i e 0 .0 6 9 .4 7 2 .0 0 0 ,2 7 0 .2 0 2

Asn 8 .0 3 8 ,5 0 0 .0 8 0 .3 5 0 .1 5 0

Asp 12 .0 6 7 .5 6 7 .1 2 0 .4 7 0 .0 9 7

Lys 12 .0 3 4 .601 .1 2 0 .5 9 0 .011

Gin 6 .0 5 0 .651 .0 6 0 .6 5 0 .001

Glu 13 .0 6 0 .711 .1 3 0 .7 8 0 .0 6 9

Arg 13 .0 7 0 .781 .1 3 0 .9 1 0 .0 2 9

Met 2 .051 .8 3 2 .0 2 0 .9 3 0 .0 9 8

H is 2 .0 4 6 .8 7 8 .0 2 0 .9 5 0 .0 7 2

Phe 2 .041 .9 1 9 ,0 2 0 .9 7 0 .051

T yr 3 .0 3 3 .9 5 2 .0 3 0 1 .0 0 0 .0 4 8

T rp 0 .0 3 7 .9 8 9 ,0 0 0 1 .wOO .011

N 100

1) = ,2 0 2

C r i t i c a l v a l u e = .1 3 6

57

T a b l e 9 : K o lm o g o ro v -S m irn o v one sa m p le t e s t o f m u t a t i o n t o ag i v e n a m in o a c i d a s su m in g random am ino a c i d r e p l a c e m a i t .Gamma c h a i n *

X F(X ) sum ü (X j S U ; sum S (X ; / n x j

G ly 0 ,0 5 0 .0 5 0 ,u0Ü .0 0 0 .0 5 0

A la 1 .0 5 8 ,1 0 8 .111 .1 1 1 .0 3 3

S e r 0 .0 7 0 .1 7 8 ,0 0 0 ,111 .0 6 7

P ro 0 .0 5 5 .2 3 3 .0 0 0 .1 1 1 ,1 2 2

V a l 0 ,0 6 2 .2 9 5 ,0 0 0 .1 1 1 .1 8 4

T hr 0 .0 4 5 .3 4 0 ,0 0 0 .1 1 1 .2 2 9

Oys 0 .0 3 5 . 3 / 5 ,0 0 0 .1 1 1 .2 6 4

Leu 0 .051 .4 2 6 ,0 0 0 .1 1 1 .3 1 5

H e 0 .0 7 0 .4 9 6 ,0 0 0 ,111 . 3 8 5

Asn 0 .0 4 7 .5 4 5 ,0 0 0 ,1 1 1 .4 3 2

A sp 0 .051 .5 9 4 ,0 0 0 .1 1 1 .4 8 3

Lys 4 ,u31 .6 2 5 .4 4 4 .5 5 5 .0 7 V,

G in 0 .0 4 4 .6 6 9 ,0 0 0 .5 5 5 .1 1 4

G lu 1 .0 5 3 .7 2 2 ,111 ,6 6 6 .0 5 6

A rg 2 .0 7 6 .7 9 8 ,2 2 2 ,8 8 8 .0 9 0

Met 0 .051 .8 4 9 ,0 0 0 ,8 8 8 .0 3 9

H is 0 .0 3 5 .8 8 4 ,0 0 0 .8 8 8 .0 0 4

Phe 0 .0 4 2 .9 2 6 ,uOO .8 8 8 .0 3 8

T y r 1 .0 3 5 .961 ,111 .9 9 9 ,v 3 8

T r p 0 .0 3 9 1 ,0 0 0 ,0 0 0 .9 9 9 ,0 0 1

N 9

D = ,4 8 3

C r i t i c a l v a l u e = .4 3 2

57

T a b le 9 : K o lm o g o ro v -S m irn o v one sa m p le t e s t o f m u t a t i o n t o ag iv e n am ino a c i d a s s u m in g random amino a c i d r e p l a c e m a i t .Gamma c h a i n .

f Jî-(X) sum ü (X j SU; sum S (X ; /F(X;G ly 0 ,0 5 0 •050 ,uOO ,0 0 0 .0 5 0

A la 1 ,0 5 8 .1 0 8 ,111 .111 .0 3 3

S e r 0 •070 ,1 7 8 ,0 0 0 ,111 ,0 6 7

Pro 0 ,0 5 5 .2 3 3 ,0 0 0 .111 ,1 2 2

Val 0 ,0 6 2 .2 9 5 ,0 0 0 • 111 ,1 8 4

Thr 0 .0 4 5 .3 4 0 ,0 0 0 • 111 ,2 2 9

Cys 0 .0 3 5 .3 7 5 ,0 0 0 .111 ,2 6 4

Leu 0 .051 ,4 2 6 ,0 0 0 .111 .3 1 5

H e 0 .0 7 0 .4 9 6 ,0 0 0 •111 .3 8 5

Asn 0 .0 4 7 .5 4 5 .0 0 0 ,111 ,4 3 2

Asp 0 .051 .5 9 4 ,0 0 0 ,111 .4 8 3

Lys 4 .w31 .6 2 5 ,4 4 4 .5 5 5 .07w

Gin 0 .0 4 4 .6 6 9 ,0 0 0 .5 5 5 .1 1 4

Glu 1 .0 5 3 ,7 2 2 • 111 , 666 .0 5 6

Arg 2 ,0 7 6 .7 9 8 ,2 2 2 ,8 8 8 .0 9 0

Met 0 .051 .8 4 9 .0 0 0 ,8 8 8 .0 3 9

H is 0 .0 3 5 ,8 8 4 ,0 0 0 ,8 8 8 .0 0 4

Phe 0 .0 4 2 .9 2 6 • uOO ,8 8 8 ,0 3 8

T y r 1 .0 5 5 ,9 6 1 • 111 • 999 ,u 3 8

T rp

N

D = ,4 8 3

0

9

.0 3 9 1 ,0 0 0 ,0 0 0 ,9 9 9 .001

58

T a b le 1 0 : K o lm o g o ro v -S m irn o v one sa m p le t e s t o f m u t a t i o n to ag iv e n amino a c i d a s s u m in g random am ino a c i d r e p l a c e m e n t ,D e l t a c h a i n .

f F(X) sum P(X) S(X ) sum S(X) /F(xj

G ly 0 ,051 .051 ,0 0 0 .0 0 0 ,051

A la 0 .0 5 4 .1 0 5 ,0 0 0 ,0 0 0 ,1 0 5

S e r 0 ,0 7 4 .1 7 9 ,0 0 0 .0 0 0 .1 7 9

Pro 0 .0 5 3 .2 3 2 ,w00 ,0 0 0 .2 3 2

Val 0 .0 6 2 .2 9 4 ,0 0 0 .0 0 0 .2 9 4

T h r 0 ,0 4 6 .3 4 0 .0 0 0 .0 0 0 .3 4 0

Cys 0 .0 3 2 .3 7 2 ,0 0 0 ,0 0 0 ,3 7 2

Leu 0 .0 4 9 .421 ,0 0 0 .0 0 0 ,421

H e 0 .0 7 0 .491 ,0 0 0 .0 0 0 .491

Asn 0 .0 3 6 .5 2 7 ,0 0 0 .0 0 0 .5 2 7

Asp 1 .0 6 4 .591 .1 6 7 .1 6 7 .4 2 4

Lyj 1 .0 2 7 ,6 1 8 .1 6 7 * .3 3 3 ,2 8 5

Gin 0 .0 4 7 .6 6 5 ,0 0 0 .3 3 3 .332

Glu 1 .0 6 0 . / 2 5 .1 6 7 .5 0 0 .2 2 5

Arg 3 .0 6 6 .791 ,5 0 0 1 ,0 0 0 ,2 0 9

Met 0 .0 4 9 ,8 4 0 ,0 0 0 1 ,0 0 0 .1 6 0

H is 0 .0 4 7 .8 8 7 ,0 0 0 1 ,0 0 0 ,1 1 3

Phe 0 ,0 4 2 .9 2 9 ,0 0 0 1 ,0 0 0 ,071

T y r 0 .0 3 4 .9 6 3 ,0 0 0 1 .0 0 0 ,0 3 7

T rp 0 .0 3 8 1 .001 ,0 0 0 1 ,0 0 0 .001

N 6

D = .5 2 7

C r i t i c a l v a l u e = .521

59

T a b l e 1 1 : K o lm o g o ro v -S m irn o v one s a m p le t e s t o f m u ta t io n from a g i v e n am ino a c i d a s s u m in g ran dom b a s e s u b s t i t u t i o n . T o t a l ,

f F(XJ sum P(X) S(XJ sum S(X) / F ( X |

G ly 21 ,0 6 2 .0 6 2 ,1 1 9 ,1 1 9 .0 5 7

A la Q ,0 6 5 ,1 2 7 .051 .1 7 0 .043

S e r 3 ,1 0 0 .2 2 7 .0 1 7 ,1 8 7 ,0 4 0

Itro 8 .0 6 5 .2 9 2 ,0 4 5 .2 3 2 ,U60

Val 9 ,0 6 5 .3 5 7 .051 .2 8 5 .0 7 4

Thr 3 ,0 6 0 .4 1 7 ,0 1 7 .3 0 0 .117

Cys 0 ,u 3 8 .4 5 5 ,0 0 0 .300 .155

Leu 10 ,0 8 9 .5 4 4 .0 5 6 .3 5 6 .1 8 8

l i e 0 .0 0 0 .5 4 4 ,0 0 0 .35 6 ,1 8 8

Asn 12 .0 4 3 .5 8 7 ,0 6 8 .4 2 4 .1 6 3

Asp 24 ,0 4 3 .6 3 0 .1 3 6 ,56 0 .070

Lys 15 .0 3 8 ,6 6 8 .0 8 5 ,645 .0 2 3

Gin 3 .0 5 8 .7 0 6 ,0 1 7 ,6 6 2 ,0 4 4

Glu 25 .0 3 8 .7 4 4 .141 .8 0 3 ,0 5 9

A rg 4 .0 9 2 .8 3 6 .0 2 3 ,8 2 6 .0 1 0

Met 0 .0 2 4 ,8 6 0 ,0 0 0 ,8 2 6 .0 3 4

His 21 .0 4 3 .9 0 3 ,1 1 9 .945 ,042

Phe 5 .0 4 3 .9 4 6 ,0 2 8 .9 7 3 .0 2 7

T y r 4 .0 3 3 .9 7 9 .0 2 5 .9 9 6 .0 1 7

T rp

N

D =

1

177

,1 6 8

.0 1 9 .9 9 8 .0 0 6 1.001 ,0 0 4

C r i t i c a l v a l u e = ,1 0 2

60

T a b le 12 : K o lm o g o ro v -S m irn o v one s a m p le t e s t o f m u t a t i o n f ro m ag iv e n am in o a c i d a s s u m in g random b a s e s u b s t i t u t i o n .A lp h a c h a i n .

f FIX ; sum PCX) S(X ) sum S (X ; /P ( X )

Gly 6 ,0 6 2 .0 6 2 .0 9 7 ,0 9 7 .0 3 5

A la 5 • 065 .1 2 7 ,081 .1 7 8 .051

S e r 2 ,1 0 0 ,2 2 7 .0 3 2 .2 1 0 .0 1 7

Pro 5 •065 .2 9 2 ,081 .29 1 ,001

Val 1 .0 6 5 ,3 5 7 .0 1 6 .3 0 7 ,0 5 0

Thr 0 ,0 6 0 .4 1 7 .0 0 0 ' .3 0 7 ,1 1 0

Cys 0 .0 3 8 .4 5 5 ,0 0 0 .3 0 7 .1 4 8

Leu 3 .0 8 9 .5 4 4 .0 4 8 ,3 5 5 .1 8 9

H e 0 .0 0 0 ,5 4 4 ,0 0 0 .3 5 5 .1 8 9

Asn 5 .0 4 3 .5 8 7 .081 .4 3 6 .151

As y 13 .0 4 3 .6 3 0 ,2 1 0 .6 4 6 ,0 1 6

Lys 5 .0 3 8 ,6 6 8 .081 .7 2 7 .0 5 9

Gin 2 .0 3 8 .7 0 6 .0 3 2 .7 5 9 .0 5 3

Glu 6 .0 3 8 .7 4 4 .0 9 7 .8 5 6 .1 1 2

Arg 3 ,0 9 2 .8 3 6 .0 4 8 .9 0 4 .0 6 8

Met 0 .0 2 4 ,8 6 u .0 0 0 .9 0 4 .0 4 4

His 5 ,0 4 3 .9 0 3 .081 .9 8 5 ,0 8 2

Phe 1 .0 4 3 .9 4 6 .0 1 6 1 .001 .0 5 5

Tyr 0 ,0 3 3 .9 7 9 , uOO 1,001 ,0 2 2

T rp 0 ,0 1 9 .9 9 8 ,0 0 0 1 .0 0 1 .0 0 5

N 62

D « ,189

C r i t i c a l v a lu e = .1 7 3

61

T a b le 1 3 : K o lm o g o ro v -S m im o v on e sa m p le t e s t o f i . u t a t i o n f ro m aa g iv e n am ino a c i d a s s u m in g random b a s e s u b s t i t u t i o n .B e ta c h a i n ,

f P(X) sum P(X) s ( x ) sum S(X) /F (X )

G ly 12 ,0 6 2 ,0 6 2 .1 2 0 ,1 2 0 .v 5 8

A la 3 ,0 6 5 .1 2 7 ,0 3 0 .1 5 0 .0 2 3

S e r 1 ,1 0 0 .227 .0 1 0 .1 6 0 ,0 6 7

Pro 3 ,0 6 5 .2 9 2 ,0 3 0 .1 9 0 ,1 0 2

Val 8 ,0 6 5 .3 5 7 ,0 8 0 .2 7 0 .0 8 7

T h r 2 ,0 6 0 .4 1 7 ,0 2 0 .2 9 0 .1 2 7

Cys 0 ,u 3 8 .4 5 5 .0 0 0 .2 9 0 .1 6 5

Leu 7 ,0 8 9 .5 4 4 •v70 .3 6 0 .1 8 4

H e 0 .0 0 0 .54 4 ,0 0 0 .5 6 0 .1 8 4

Asn 6 .0 4 3 .5 8 7 ,0 6 0 ,4 2 0 .1 6 7

Asp 1C .0 4 3 .6 3 0 ,1 0 0 .5 2 0 ,1 1 0

Lys 9 .0 3 8 . 668 .0 9 0 ,61 0 .0 5 8

Gin 1 .0 3 8 .7 0 6 .0 1 0 .6 2 0 ,0 8 6

Glu 15 .0 3 8 .7 4 4 .1 5 0 .7 7 0 ,0 2 6

Arg 1 .0 9 2 .8 3 6 .0 1 0 .7 8 0 .0 5 6

Met 0 .0 2 4 ,8 6 0 ,0 0 0 .7 8 0 ,0 8 0

His 13 .0 4 3 .9 0 3 .1 3 0 .9 1 0 ,0 0 7

Phe 4 .0 4 3 .9 4 6 ,u 4 0 .9 5 0 ,0 0 4

T y r 4 .0 3 3 .9 7 9 .0 4 0 .9 9 0 ,011

T rp 1 .0 1 9 .9 9 8 ,0 1 0 1 .0 0 0 ,0 0 2

N 100

D = ,1 8 4

C r i t i c a l w l u e = * 136

62

T a b le 14 : K o lm og orov -S m im o v o n e sam ple t e s t o f m u t a t i o n fro m ag iv e n am ino a c i d a s su m in g random b a s e s u b s t i t u t i o n .Gamma c h a i n .

f F(X) sum F(X ; S(X ; sum S(X ) / F ( X ;

Gly 0 ,u 6 2 ,0 6 2 ,0 0 0 ,0 0 0 ,0 6 2

A la 0 ,u 6 5 .1 2 7 ,0 0 0 ,uOO .1 2 7

S e r 0 ,1 0 0 .22 7 ,0 0 0 ,vOO .2 2 7

Pro 0 ,0 6 5 ,2 9 2 ,0 0 0 ,0 0 0 .2 9 2

Val 0 ,0 6 5 .3 5 7 ,0 0 0 ,0 0 0 .3 5 7

T hr 1 ,0 6 0 ,4 1 7 .111 .111 .3 0 6

Cys 0 .0 3 8 ,4 5 5 .0 0 0 .111 .3 4 4

Leu 0 .0 8 9 .5 4 4 ,0 0 0 ,111 .4 3 3

H e 0 ,0 0 0 .5 4 4 ,0 0 0 .111 .4 3 3

Asn 0 .04 3 .5 8 7 ,0 0 0 .111 ,4 7 6

Asp 1 .0 4 3 .6 3 0 .111 ,2 2 2 .4 0 8

Lys 1 .0 3 8 . 668 .111 .5 3 3 .3 3 5

Gin 0 .0 3 8 ,7 0 6 ,0 0 0 .3 3 3 ,3 7 3

Glu 4 .0 3 8 .7 4 4 .4 4 4 .7 7 7 .0 3 3

Arg 0 .0 9 2 .8 3 6 .0 0 0 .7 7 7 .0 5 9

Met 0 ,0 2 4 ,8 6 0 ,0 0 0 .7 7 7 .0 8 3

H is 2 .0 4 3 ,9 0 3 ,2 2 2 ,9 9 9 .0 9 6

T y r 0 .0 3 3 .9 7 9 ,0 0 0 .9 9 9 .0 2 0

T rp 0 .0 1 9 ,9 9 8 ,0 0 0 ,9 9 9 .001

N 9

D » .4 7 6

C r i t i c a l v a l u e = .4 3 2

63

T ab le 15 î K o lm o g o r rv -S m im o v one sam p le t e s t o f m u t a t i o n from a g i v e n amino a c i d a ssu m in g random b a s e s u b s t i t u t i o n . D e l t a c h r i n .

f FCX) sum F(X) S(X ) sum S(X ) /F (X J

Gly 3 ,062 ,0 6 2 .5 0 0 ,5 0 0 .4 3 8

Ala 1 •065 .127 .1 6 7 .6 6 7 .5 4 7

S e r 0 ,1 0 0 .227 ,0 0 0 .6 6 7 ,4 4 0

Pro 0 .065 .2 9 2 .0 0 0 .6 6 7 .3 7 5

Val 0 .065 .35 7 ,0 0 0 .6 6 7 .3 1 0

T hr 0 ,0 6 0 .4 1 7 .0 0 0 .6 6 7 .2 5 u

Cys 0 .0 3 8 .45 5 ,0 0 0 ,6 6 7 .2 1 2

Leu 0 .0 8 9 .54 4 ,0 0 0 .6 6 7 .1 2 3

H e 0 ,000 .5 4 4 ,0 0 0 ,6 6 7 .1 2 8

Asn 1 .0 4 3 .587 ,1 6 7 .8 3 3 .2 4 6

Asp 0 .0 4 3 .6 3 0 ,0 0 0 ,8 3 3 .2 0 3

Lys 0 .u 3 8 ,6 6 8 ,0 0 0 ,8 3 3 .1 6 5

Gin 0 .0 3 8 .7 0 6 ,0 0 0 .8 3 3 ,1 2 7

Glu 0 .U38 .7 4 4 ,0 0 0 .8 3 3 .0 8 9

Arg 0 .038 .8 3 6 ,0 0 0 .8 3 3 .0 0 3

Met 0 .0 3 8 ,836 .0 0 0 .8 3 3 .0 0 3

H is 1 .043 .9 0 3 ,0 0 0 1 ,0 0 0 .0 5 4

Tyr 0 .0 3 3 .9 7 9 .0 0 0 1 .0 0 0 .021

T rp 0 .019 .9 9 8 ,0 0 0 1 ,0 0 0 .0 0 2

N 6

D = ,5 7 4

C r i t i c a l V a lu e = .521

64

T a b le 1 6 ; K o lm o g o ro v -S m irn o v one sa m p le t e s t o f m u t a t i o n t o a g iv e n amino a c i d a s su m in g random b a s e s u b s t i t u t i o n . T o t a l ,

f sum i ’(X; SIX) sum S(X ;

Gly 6 .0 5 9 .0 5 9 .0 3 4 .0 3 4 ,0 2 5

A la 8 ,0 6 2 ,121 .0 4 5 .0 7 9 ,04 2

S e r 5 .0 9 5 .2 1 6 .0 2 8 .1 0 7 ,1 0 9

P ro 7 .0 6 2 .2 7 8 .0 4 0 .1 4 7 .131

Val 5 .0 6 2 .3 4 0 ,0 2 8 .1 7 5 .1 6 5

T hr 3 ,0 5 6 .3 9 6 ,0 1 7 .1 9 2 ,2 0 4

Cys 5 .0 3 6 .4 3 2 .0 1 7 .2 0 9 ,2 2 3

Leu 5 .0 8 3 .5 1 5 .0 2 8 .2 3 7 ,2 7 8

l i e 0 ,0 5 4 .5 6 9 ,0 0 0 .2 3 7 .3 3 2

Asn 13 .041 .6 1 0 .0 7 3 .3 1 0 .3 0 0

Asp 27 .041 .651 .1 5 3 ,4 6 3 ,1 8 8

Lys 22 .0 3 6 .6 8 7 .1 2 4 ,5 8 7 ,1 0 0

Gin 10 .0 3 6 .7 2 3 .0 5 6 ,6 4 3 ,w80

Glu 17 .0 3 6 .7 5 9 .0 9 6 .7 3 9 .0 2 0

Arg 26 .0 8 7 .8 4 6 .1 4 7 ,8 8 6 .0 4 0

Met 2 .0 2 3 .8 6 9 .011 .8 9 7 .0 2 8

H is 7 .041 .9 1 0 .0 4 0 .9 3 7 ,0 2 7

Tyr 9 .031 .9 8 2 ,051 .9 9 9 .0 1 7

Trp

N

D =

0

177

.33 2

.0 1 8 1 ,0 0 0 ,0 0 0 ,9 9 9 ,0 0 0

65

T a b le 1 7 : K o lm o g o ro v -S m irn o v o ne sam p le t e s t o f m u t a t i o n s to ag iv e n am ino a c i d a s s u m in g random b a s e A lp h a c h a i n .

s u b s t i t u t i o n

f P(X> sum P(XJ S(XJ sum SIX ) / P ( X |

Gly 3 •Ü59 .0 5 9 .0 4 8 .'->48 .0 0 9

A la 2 .0 6 2 .121 .0 3 2 ,0 8 0 .041

S e r 1 .0 9 5 .^ 1 6 .0 1 6 .0 9 6 .1 2 u

Pro 3 ,u 6 2 .2 7 8 .0 4 8 .1 4 4 .1 3 4

Val 2 .u 6 2 .34 0 .0 3 2 .1 7 6 .1 6 4

T hr 1 .0 5 6 .3 9 6 .0 1 6 .1 9 2 .2 0 4

Cys 0 .0 3 6 .4 3 2 .0 0 0 .1 9 2 .2 4 0

Leu 2 ,w85 .5 1 5 .0 3 2 .2 2 4 .291

I l e 0 .0 5 4 .5 6 9 ,U00 .2 2 4 .3 4 5

Asn 5 .041 .6 1 0 .081 .3 0 5 .3 0 5

Asp 14 .041 .651 .2 2 6 .531 .1 2 0

Lys 5 .0 5 6 .6 8 7 .081 .51 2 .0 7 5

Gin 4 .0 3 6 .7 2 3 .u 6 5 .6 7 7 .0 5 4

Glu 2 .0 3 6 .7 5 9 .0 3 2 .7 0 9 ,w50

Arg 8 .0 8 7 .8 4 6 .1 2 9 .8 3 8 ,u 0 8

Meu 0 .0 2 3 .8 6 9 .0 0 0 .8 3 8 .0 2 9

H is 5 .041 .9 1 0 .081 .9 1 9 .0 0 9

T y r 5 .031 .9 8 2 .081 1 ,0 0 0 .0 1 8

T rp

N

D = .5 4 5

0

62

.0 1 8 1 .0 0 0 ,0 0 0 1 .000 ,000

66

T a b le 1 8 : K o lm ogorov-S m irnov o n e s a m p le t e s t o f m u ta t i o n t o ag iv e n amino a c i d a s s u m in g random b a s e s u b s t i t u t i o n .B e ta c h a i n .

f P(X , sum s(x) sum SIX) A ’(X)

Gly 5 .0 5 9 .0 5 9 .0 3 0 .0 3 0 .0 2 9

A la 5 .0 6 2 .121 .0 5 0 .0 8 0 .041

S e r 4 .0 9 5 .2 1 6 .0 4 0 .120 .0 9 6

ITO 4 .0 6 2 .2 7 8 .0 4 0 .16 0 .1 1 8

Val 3 ,0 6 2 .3 4 0 .0 3 0 .1 9 0 .1 5 0

T h r 2 .0 5 6 .3 9 6 .0 2 0 .2 1 u .1 8 6

C3B 3 .0 3 6 .4 3 2 .0 3 0 .2 4 0 .1 9 2

Leu 5 .0 8 5 .5 1 5 .0 3 0 .2 7 0 .2 4 5

l i e 0 .0 5 4 .5 6 9 .0 0 0 .2 7 0 .2 9 9

Asn 8 .041 .6 1 0 ,0 8 0 .3 5 0 .2 6 0

Asp 12 .041 .651 .1 2 0 . 4 % .181

Lys 12 .0 3 6 .6 8 7 .1 2 0 .5 9 0 .0 9 7

Gin 6 .0 3 6 .7 2 3 .0 6 0 .6 5 0 .0 7 3

Glu 13 .0 3 6 .7 5 9 .1 3 0 .7 8 0 .0 2 1

Arg 13 .0 8 7 .8 4 0 .1 3 0 .9 1 0 .0 7 0

Met 2 .0 2 3 .8 6 9 .0 2 0 .930 .061

H is 2 .041 .9 1 0 .0 2 0 .9 5 0 .0 4 0

Phe 2 .041 .951 .0 2 0 .9 7 0 .0 1 9

T yr 3 .031 .9 8 2 .u 3 0 1 .000 . 0 1 9

T rp u .0 1 8 1 .0 0 0 .uOO 1 .0 0 0 .0 0 0

N 100

D = ,299

C r i t i c a l v a lu e = .1 3 6

67

T a b le 1 9 : K o lm o g o ro v -S m irn o v one sam p le t e s t o f m u t a t i o n t o ag iv e n amino a c i d a s s u m in g r ; ndom b a s e s u b s t i t u t i o n .Gamma c h a i n .

f sum S(X) sura S (X ; /F IX

G ly 0 .0 5 9 .0 5 9 .0 0 0 ,u 0 0 .0 5 9

i l a 1 .0 6 2 .121 .111 .1 1 1 .0 1 0

S e r 0 .0 9 5 .2 1 6 .0 0 0 .111 .1 0 5

Pro 0 .0 6 2 .2 7 8 .0 0 0 .111 .1 6 7

Val 0 .u 6 2 .3 4 0 ,uOO .111 .2 2 9

Thr 0 .0 5 6 .3 9 6 .0 0 0 .111 .2 8 5

Gys 0 .0 3 6 » .4 3 2 .0 0 0 ,111 .521

I) eu 0 .0 8 3 .5 1 5 ,0 0 0 ,111 .4 0 4

I l e 0 .0 5 4 .5 6 9 ,0 0 0 .111 .4 5 8

Asn 0 .041 .6 1 0 ,0 0 0 .111 .4 9 9

Asp 0 .041 .651 .0 0 0 .111 .5 4 0

Lys 4 .0 5 6 .6 8 7 .4 4 4 .5 5 5 .1 3 2

Gin 0 .0 3 6 .7 2 3 .0 0 0 .5 5 5 .1 6 8

Glu 1 .0 3 6 .7 5 9 .111 .6 6 6 .0 9 3

Arg 2 .0 8 7 .8 4 0 .2 2 2 ,8 8 8 .0 4 8

Met 0 .0 2 3 .8 6 9 .0 0 0 .8 8 8 .0 1 9

H is 0 .041 ,9 1 0 .0 0 0 ,8 8 8 ,0 2 2

Phe 0 .041 .951 .0 0 0 ,8 8 8 .0 6 3

T yr 1 .031 .9 8 2 .111 .9 9 9 .0 1 7

T rp 0 .0 1 8 1 .0 0 0 ,0 0 0 .9 9 9 .0 0 1

N 9

D « ,'540

C r i t i c a l v a l u e = .4 3 2

68

T a b le 2 0 : K o lm o g o ro v -S m im o v one sam ple t e s t o f m u t a t i o n to ag iv e n am ino a c i d a s s u m in g random b a s e s u b s t i t u t i o n .D e l t a c h a i n .

f F(XJ sum F(X) s ( x ; sum S(X) / F ( x ;

G ly 0 .0 5 9 .0 5 9 .0 0 0 .0 0 0 .0 5 9

A la 0 .0 6 2 .121 .0 0 0 .uOO .121

S e r 0 .0 9 5 .2 1 6 .0 0 0 .0 0 0 .2 1 6

i-TO 0 .0 6 2 .2 7 8 .0 0 0 .0 0 0 .2 7 8

Val 0 .0 6 2 .3 4 0 .0 0 0 ,U00 .3 4 0

T h r 0 .0 5 6 .3 9 6 .0 0 0 ,U00 .3 9 6

Cys 0 .0 3 6 .4 3 2 .uOO ,0 0 0 .4 3 2

Leu 0 .0 8 3 .5 1 5 .0 0 0 .0 0 0 .515

l i e 0 .0 5 4 .5 6 9 .0 0 0 .wOO .5 6 9

Asn 0 .041 .6 1 0 .0 0 0 .0 0 0 .6 1 0

Asp 0 .041 .651 .0 0 0 .0 0 0 ,651

Lys 1 ,u 3 6 .6 8 7 .1 6 7 .1 6 7 .5 2 0

Gin 1 .0 3 6 .7 2 3 .1 6 7 .3 3 3 .3 9 0

Glu 1 .0 3 6 .7 5 9 .1 6 7 .5 0 0 .2 5 9

Arg 3 .0 8 7 . 8 4 6 .5 0 0 1 ,0 0 0 .1 5 4

Met 0 .0 2 3 .8 6 9 ,0 0 0 1 ,uOO .131

H is 0 .041 .9 1 0 .0 0 0 1 .0 0 0 .0 9 0

i h e 0 .041 .951 .0 0 0 1 . Of'O .0 4 9

T y r 0 .031 .9 8 2 ,uuO 1 ,0 0 0 .0 1 8

T rp 0 ,0 1 8 1 .0 0 0 ,0 0 0 1 .0 0 0 ,0 0 0

N 6

D = ,651

C r i t i c a l Vi l u e = .521

69

T a b l e 21 : E x p e c t e d f r e q u e n c y o f amino a c i d r e p la c e m e n t b y t o t a l n u m b er o f p o s s i b l e s i n g l e b a s e s u b s t i t u t i o n s a n d ex p e c t e d f r e q u e n c y o f t r a n s i t i o n s and t r a n s v e r s i o n s .

T i & Tv f Ti Tv

A la 24 .062 .3 3 3 ,667

V al 24 .062 .3 3 3 .6 6 7

Leu 33 .085 ,242 .7 5 8

l i e 21 .0 5 4 .190 .8 1 0

P ro 24 ,062 .3 3 3 .6 6 7

Phe 16 .041 .250 ,7 5 0

T rp 7 ,018 .2 8 6 .7 1 4

Met 9 .0 2 3 .3 3 3 .6 6 7

G ly 23 ,059 ,3 4 8 .6 5 2

S e r 37 .095 .3 7 8 .6 2 2

T hr 22 .0 5 6 .273 .7 5 7

Cys 14 .0 3 6 .2 8 6 .7 1 4

T y r 12 ,031 .3 3 3 .6 6 7

Asn 16 ,041 .2 5 0 .7 5 0

Gin 14 .036 .14 3 .8 5 7

Asp 16 .041 .2 5 0 .7 5 0

Glu 14 ,036 .2 8 6 .7 1 4

Lys 14 ,036 .2 8 6 .7 1 4

Arg 34 ,087 .3 2 4 .6 7 6

H is 16 .0 4 1 .2 2 0 .ISO

390 1.000 5 .7 0 7 1 4 .2 9 3

E x p e c te d f r e q u e n c y o f t r a n s i t i o n s » 5 ,7 0 7 /2 0 ■ 0 ,2 8 5

E x p e c te d f r e q u e n c y o f t r a n s v e r s i o n s = 1 4 .2 9 3 /2 0 » 0 .7 1 5

70

F i g u r e 9# O b se rv e d t r a n s i t i o n s a n d t r a n s v e r s i o n s

AminoA cidS ub ,

P'rom

A la

Val

Leu

P ro

Phe

T rp

G ly

S e r

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75CHAPTER IV

AIUiLYSIS RESULTS

Randomness Among C h a in s

The no n -ran d o m d i s t r i b u t i o n o f p o i n t m u t a t i o n s among

t h e f o u r p o l y p e p t i d e s h a s a l r e ady b uen d i s c u s s e d and a s t e n -2

t i o n n e e d be draw n o n l y t o t h e c h i t e s t show ing a p r o b a b i l

i t y a s s o c i a t e d w i t h o b s e r v e d d a t a b e in g c a u s e d by ran d o m mu

t a t i o n o f l e s s th a n .0 0 1 ( t a b l e 1 ) .

Randomness W m A c id R e p la c e m e n t

The K o lm o g o ro v -S m irn o v o n e sa m p le t e s t was em p loy ed t o

t e s t f o r ran d o m n e ss a t b o t h t h e p r o t e i n and th e RNA l e v e l s

u s i n g a .0 5 l e v e l o f s i g n i f i c a n c e .

On t h e p r o t e i n l e v e l , a lp ,h a , b e t a and gamma c h a i n s show

ed d e v i a t i o n g r e a t en o u g h t o r e j e c t t h e h y p o t h e s i s o f ran d o m

m u t a t i o n , w h i l e t h e d i s t r i b u t i o n o f m u t a t i o n s f ro m a g i v e n

amino a c i d on t h e d e l t a c h a i n c o u ld b e due t o random r e s i d u e

s u b s t i t u t i o n . B e c a u se t h e d e l t a sam p le was t h e s m a l l e s t (Na

6 ) , t h i s c h a i n o f f e r e d t h e l e a s t p o w e r fu l t e s t an d c a n n o t

n e g a t e t h e o t h e r f i n d i n g s , n e s u l t s a r e g iv e n Jn t a b l e s 3 - 6 ,

In a l l c a s e s o f m u t a t i o n t o a g iv e n amino a c i d ( t a b l e s

7 - 1 0 ) , o b s e r v e d d e v i a t i o n w as s u f f i c i e n t to r e j e c t t h e ran dom

h y p o t h e s i s . A g a in , t h e d e l t a c h a i n showed t h e g r e a t e s t t e n

d en cy to w a r d s r a n d o m n e s s , w i t h a maximum d e v i a t i o n o f o n l y

,0 0 6 o u t s i d e t h e c r i t i c a l v a l u e ( t a b l e 1 0 ) ,

Randomness o f n u c l e o t i d e n a s e S u b s t i t u t i o n

As d i s c u s s e d a b o v e , c a l c u l a t i o n s a ssu m in g random RHA b a s e

s u b s t i t u t i o n s r a t h e r t h a n ran dom amino a c i d r e p l a c e m e n t s s h o u l d

g i v e m ore a c c u r a t e r e s u l t s ( s e e p . 4 1 ) .

ün e a c h o f t h e a l p h a , b e t a , d e l t and f amwa c h a i n s , c o n -

76

s i d e r e d s e p a r a t e l y ana c o l l e c t i v e l y , o b s e r v e d d e v i a t i o n g r e a t

e r t h a n t h e c r i t i c a l v a l u e a t t h e ,0 5 l e v e l o f s i g n i f i c a n c e

was f o u n d , an d th e h y p o t h e s i s o f random s u b s t i t u t i o n was r e j e c t

e d . ( t a b l e s 1 1 - 2 1 ) ,

T r a n s i t i o n s an d T r a n s v e r s i o n s

E x p e c te d f r e q u e n c i e s o f t r a n s i t i o n s and t r a n s v e r s i o n s w ere

c a l c u l a t e d f o r e a c h amino a c i d c o d e and w ere summed i n o r d e r to

f i n d an o v e r a l l r a t i o f o r t h e h e m o g lo b in m o le c u l e .

The b in o m ia l t e s t was u s e d to com pare t o t a l e x p e c te d f r e

q u e n c i e s a g a i n s t o b s e r v a t i o n s ( f i g . 9 j and no c o r r e l a t i o n (p

l e s s t h a n ,0 0 0 0 6 ) was fo u n d , w i t h t r a n s i t i o n s b e tw e e n a d e n i n e

and g u a n in e (a t o G a n d G t o a ) sh ow ing f r e q u e n c i e s e v e n g r e a t

e r th a n any t r a n s v e r s i o n . The l a t t e r o b s e r v a t i o n s u g g e s t s t h a t

m u t a b i l i t y o f a g iv e n seg m en t o f DNA i s m ore a f u n c t i o n o f com

p o s i t i o n o f i n d i v i d u a l n u c l e o t i d e s t h a n o f c o d o n s .

I t c an a l s o b e d e m o n s t r a t e d t h a t t h e m u t a t i o n s o f human

h e m o g lo b in r e s u l t from f i v e t i m e s a s many b a s e s u b s t i t u t i o n s

i n t h e f i r s t an d se c o n d p o s i t i o n o f a t r i p i e t t h a n i n t h e t h i r d

( t a b l e l a ) .

An h y p o t h e s i s c a n t h e n b e made t h a t t h e r e i s a p o s i t i v e

c o r r e l a t i o n b e tw e e n c o n c e n t r a t i o n s o f a d e n i n e an d g u a n in e i n

t h e f i r s t and se c o n d p o s i t i o n s o f cod on t r i p l e t s and t h e r a t e

o f m u t a t i o n , o r m u t a b i l i t y , o f DNa s e g m e n ts . T h is s h o u ld be

t e s t e d a g a i n s t p o i n t m u t a t i o n s o f o t h e r p r o t e i n s b o t h i n humans

and i n o t h e r s n e c i e s .

■ ■■'■ - ■"T(ÿiy ':îv:'

L iterature Cited

A bram son , R .K . , D .L .R u c k n a g e l , D . C . S h r e f f l e r a n d J . J . S a a v e 1970 Homozygous h e m o g lo b in T o n g a r i k i ; E v id e n c e f o r o n l y

o n e a l p h a c h a i n s t r u c t u r a l l o c u s i n M e l a n e s i a n s ,S c i e n c e , 1 69 ; 1 9 4 -1 9 6 ,

Adams, J . G , , W .P .W in te r , L .L .R u c k n a g e l a n d H .H .S p e n c e r1972 B i o s y n t h e s i s o f h e m o g lo b in Ann A r b o r ; E v id e n c e f o r

c a t a b o l i c a n d f e e d b a c k r e g l u a t i o n . S c i e n c e , 1 7 6 ; 1 4 2 7 -1 4 2 9 .

A g e r , J .A .M . , a n d H.Lebmann1957 H em oglob in L ; A new h e m o g lo b in f o u n d i n P u n ja b i

H in d u . B r i t . M e d . J . , 2; 1 4 2 -1 4 3 .A h e m , E . J . , R . T . J o n e s , B .B r i m h a l l a n d R .H ,G a ry

1970 H em oglob in P - J a m a ic a ( a l p h a - 2 - gamma-2 61 l y s - g l u ;136 a l a ) . B r i t , J , H a e m a t o l . , 1 8 ; 3 6 4 -3 7 5 .

A h e rn , Ë . J . , V .A h e rn , B . G , W i l t s h i r e a n d H.Lehmann1973 F u r t h e r c h a r a c t e r i z a t i o n o f h e m o g lo b in P - H u l l ; 121

g l u - l y s ; 136 a l a . B i o c h i m .B io p h y s .A c ta , 3 0 3 ; 2 4 2 - 2 4 5 .

A dsoy , M ,, S .h rd e m , G ,D ,i . f re m o v , J . B . W i l s o n , T .H .J .H u is m a n , W .A .S c h ro e d e r , J . R , S h e l t o n , J , B , S h e l t o n , O . N . U l i t i n and A, M u f tu o g lu

1972 H em og lob in I s t a n b u l ; S u b s t i t u t i o n o f g l u t a m in e f o r h i s t i d i n e i n a p r o x im a l h i s t i d i n e (F -8 92 b e t a ) .J . C l i n , I n v e s t , , 5 1 ; 2 3 8 0 -2 3 8 7 .

A l b a h a r y , G . , J , C , D r e y f u s , D .L a b ie , G .S c h a p i r a a n d L .TramI 9 6 0 H e m o g io b in is a n o r m a le s au S u d -V ie tn a m . H em o b lo b in -

o s e C h o m o z y g o te . T r a i t E , H ém og lo b ine N o u v e l le R ev . H e m a t o l . , 13 ; 1 6 3 -1 7 0 .

A l c i a t i , G,1968 C a r to g r a p h y o f a b n o rm a l h e m o g lo b in s . 2 . M orph.A n-

t h r o p , , 6 0 ; 1 8 4 -1 9 4 .A l l a n , N . , D ,B e a l e , D , I r v i n e a n d H.Lehmann

1965 T h r e e h e m o g lo b in s ; K -W oolw ich , a n a b n o rm a l , Cameroon a n d I b i d a n , two u n u s u a l v a r i a n t s o f Hb a . N a t u r e ,2 0 8 ; 6 5 8 -6 6 1 .

A l l b r o o k , D , , N . A . B a m i c o t , N .D an c e , S .D .L a w le r , R .M a r s h a l l , a n d J .M u n g a i

1965 B lo o d g r o u p s , h a e m o g lo b in a n d serum f a c t o r s o f t h e K aram ofo . B u m .B i o l , , 3 7 ; 2 1 7 -2 3 7 .

/ i l l i s o n , A.A.1954 The d i s t r i b u t i o n o f t h e s i c k l e c e l l t r a i t i n E a s t

A f r i c a a n d e l s e w h e r e a n d i t s a p p a r e n t r e l a t i o n s h i p t o t h e i n c i d e n c e o f s u b t e r t i a n m a l a r i a . T r a n s a c t i o n s o f t h e R o y a l S o c i e t y o f T r o p i c a l M e d ic in e a n d H ygene. 4 8 ,

A n d e r s o n , N .L . an d M ,F .P e r u tz1973 S i t e o f t h e am ino a c i d s u b s t i t u t i o n i n h e m o g lo b in

S e a t t l e (2 A-2 70 a s p ) . N a t u r e , 2 4 3 ; 2 7 4 - 2 7 5 .A t w a t e r , J . , I . R . S c h w a r t z and L ,M .T o c a n t in s

1960 A v a r i e t y o f human h e m o g lo b in w i t h f o u r d i s t i n c t e l e c t r o p h o r e t i c c o m p o n e n ts . B lo o d , 1 5 ; 9 0 1 ,

Atwater, J , , C .B a g l i o n i a n d L .M ,T o c a n t in s1962 A v a r i e t y o f human h e m o g lo b in w i t h a f a s t com ponen t

b u t u n a l t e r e d t r y p t i c d i g e s t f i n g e r p r i n t , i r o c ,9 C o n g ré I n t e r n , S o c . t t e m a t o l . , 1 1 5 -1 1 9 ,

B a d r , F,M,1973 H em oglob in P - N i l o t i c . N a tu r e New B i o lo g y , 242 ; 1 0 7 - 1 1 0 .

78

B a g l i o n i , G,1 9 62 A c h e m ic a l s tu d y o f h e m o g lo b in N o r f o l k . J . B i o l .

Ghem ., 2 3 7 ; 6 9 - 7 4 .1 9 63 C o r r e l a t i o n s b e tw ee n g e n e t i c s an d c h e m i s t r y o f human

h e m o g lo b in . I n ; J . t i , T a y l o r ( E d . ) M o le c u la r G e n e t i c s . P a r t One. N .Y ., Random H ouse .

B a g l i o n i , C , , an d V .M ,Ingram1 9 6 1 Abnorm al human h e m o g lo b in s V, C h e m ic a l i n v e s t i g

a t i o n s o f h e m o g lo b in s A, G, C, X f ro m o n e i n d i v i d u a l , 3 i o c h i m ,B io p h y s ,A c ta , 4 8 ; 2 5 3 .

B a n n e rm a n n , M ., an d H. Lehmann1 9 7 0 H em oglob in A -2 , What i s i t f o r ? J . M e d . K x u . C l i n , , 1;

1 2 9 -1 3 1 .B a n n i s t e r , W .H,, J .L .G r o c h , C . F . P l e s e , L .L ,S m i t h , B . I . B a r t o n ,J . B , W i l s o n , C ,A ,R e y n o ld s an d T .H .J .H u is m a n

19 72 H em oglob in S t . L u k e ' s o r a l p n a 2 95 a r g (G2) b e t a 2 , E u r . j . B i o c h e m . , 2 9 ; 3 0 1 -3 0 7 .

B a r c l a y , G . P . T . , D .C h a r ie s w o r th and H,Lehmann1 9 6 9 Abnormal h e m o g lo b in s I n Z am bia ; A new h e m o g lo b in

Zam bia a l p h a 60 (E9) l y s - a s n , B r i t . M e d .J . 5683» 5 9 5 -5 9 6 ,

B a u r , E,W, an d N.M.Rowley1 9 6 4 H em oglob in Tacoma; a b e t a c h a i n v a r i a n t a s s o c i a t e d

w i t h i n c r e a s e d Hb A -2 , H u m a n g e n e t ik , 1 ; 6 2 1 -6 3 4 .B a y r a k c i , C . , A .J o s e p h s o n , L , S i n g e r , M . S .H e l l e r a n d R .D .Colem an

1 9 6 4 A new f a s t h e m o g lo b in , P r o c .1 0 C o n g r . I n t e r n . S o c , H a e m a to l . , S to c k h o lm ,

B e a l e , D . , a n d H.Lehmann19 65 Abnorm al h e m o g lo b in s tmd t h e g e n e t i c c o d e . N a tu r e ,

2 0 7 ; 2 5 9 -2 6 1 .B e n n e t t , J . C , and W .J .D re y e r

1 9 6 4 G e n e t i c c o d in g f o r p r o t e i n s t r u c t u r e . A nn ,R ev . B io c h e m ,, 3 3 ; 2 0 5 .

B e r e s f o r d , C .H . , J . B . C l e / g and B . J , W e a t h e r a l l1972 H m o g lo b in Ocho R io s ( b e t a 52 (B3) a s p - a l a ) ; A new

b e t a c h a i n v a r i a n t o f Hb A fo u n d i n c o m b in a t io n w i th Hb S , J .H e d .G e n . , 9 ; 1 5 1 -1 5 3 .

B e r e t t a , A . , V ,P r a t o , E .G a l lo an d H.Lehamnn1968 H em oglob in T o r in o a l p h a 43 (CD1) p h e n y l a l a n i n e t o

v a l i n e . N a tu re 2 1 7 ; 1016-1U 18.B e r n i n i , L , V . , W.W.DeJong and P.M.Kohn

1 9 7 0 H em oglob in v a r i a n t s i n t h e t r i b a l p o p u l a t i o n o f A ndhra P r a d e s h . E v id e n c e f o r t h e d u p l i c a t i o n o f t h e a l p h a h e m o g lo b in l o c u s i n man. A t t i . A s s . B e n e t . I t a l . , 15; 1 9 1 -1 9 4 .

B e t k e , K , , E .G ro s c h n e r a n d K.BockI 9 6 0 P r o p e r t i e s o f a f u r t h e r v a r i a n t o f h e m o g lo b in M,

N a t u r e , 188; 8 6 4 -8 6 5 .B i a n c o , T . , G .M odiano, E . B o t t i n i a n d R .L u c c i

19 63 A l t e r a t i o n i n t h e a l p h a c h a i n o f h e m o g lo b in L P a r r a r a , N a t u r e , 198 ; 3 9 5 -3 9 6 .

B i r d s e l l , J . B ,19 52 On t h e v a r i o u s l e v e l s o f o b j e c t i v i t y i n g e n e t i c a l

a n t h r o p o l o g y . A m . J .P h y s . i i n t h r o , , 1 0 ; 3 5 5 - 3 6 2 .B l a c k w e l l , R .Q .

1 9 6 8 H em oglob in G Taiw an Ami; a l p h a 2 b e t a 2 g l y - a r g , B io c h im .B io p h y s .A c ta , 1 8 8 ; 5 9 -6 4

79

1973 H em oglob in G -W aim analo , a l p h a 64 a s p - a s n , B io c h lm . B i o p h y s .A c t a , 3 2 2 ; 2 7 - 3 3 .

B l a c k w e l l , R . Q . , W.H.Boon, C .S .L iu a n d M .I .w en g1972 H em oglob in J S i n g a p o r e ; a l p h a 78 a s n - a s p ; a l p h a i3

a l a - g l y * B i o c h im .B io p h y s .A c ta , 2 7 8 ; 4 8 2 - 4 9 0 ,B l a c k w e l l , R .W .» J .T .H u a n g a n d I .H ,R o

1967 H em oglob in v a r i a n t s i n K o re a n s ; Hb T a e g u , s c i e n c e ,15 8 ; 1 0 5 6 -1 0 5 7 .

B l a c k w e l l , R . Q . , R . T . S . J i m , C .S .L i u , M .I .W eng , C .L.W ang and T .B .S h ih

1972 F a s t h e m o g lo b in v a r i a n t fo u n d i n H a w a i i a n - G h in e s e - G a u c a s ia n f a m i l y i n H av /a ii a n d a C h in e s e s u b j e c t i n T a iw a n , V o x ,S a n g , , 2 2 ; 4 6 9 -4 7 3 .

B l a c k w e l l , n . Q . , a n d C .S .L iu1970 H em oglob in G j. a l e h u n g ; a l p h a 74 a s p - h i s . B io c h im ,

B i o p h y s . A c t a , , 2 0 0 ; 7 0 - 7 5 .B l a c k w e l l , R .Q . , C . S . L i u , and T .B .S h ih

1971 H em oglob in J K a d s iu n g , b e t a 59 l y s - t h r . B io c h im ,B i o p h y s .A c t a . , 2 2 9 ; 3 4 3 -3 4 8 .

B l a c k w e l l , R . 0 . , C .S . L i u , a n d C.L.W ang1971 H em oglob in T i - L i ; b e t a 83 g l y - c y s . B io c h im .B io p h y s .

A c ta , 2 4 3 ; 4 6 7 -4 7 4 .1972 H em oglob in S i r i r a j ; b e t a 7 g l u - l y s , i n a C h in e s e su b

j e c t i n T a iw a n , V o x ,S a n g . , 2 3 ; 4 3 3 - 4 3 8 ,B l a c k w e l l , R . O . , S . O e r a i j a t i , W .P r i b a d i , M.E.Weng a n d C .S .L iu

1970 H em oglobin G I^Jakassar; b e t a 6 g l u - a l a . B io ch im ,B i o p h y s .A c t a , 214 ; 3 9 6 -4 0 1 ,

B l a c k w e l l , R . O . , T . B . S h i s , C.L.W ang a n d C .S .L iu1972 H em oglobin G H s i T so u ; b e t a 79 a s p - g l y . B io c h im ,

B io p h y s .A c ta , 2 5 7 ; 4 9 - 5 3 .B l a c k w e l l , R . y , , M .I .W eng , C . S . L i u , T .B .S h ih and C .L .W ang

1972 H em oglobin G C h in e s e i n C h in e s e s u b j e c t s i n T a iw a n ,V o x .S an g , 2 3 ; 3 6 3 -3 6 8 ,

B l a c k w e l l , R . Q . , H . J .T a n g , C .S .L iu and C.C.Wang1970 S t r u c t u r a l i d e n t i f i c a t i o n o f h e m o g lo b in E i n F i l i p i n o s ,

T r o p . G e o g r ,M e d . , 2 2 ; 1 1 2 -1 1 4 .B r i m h a l l , B , , S . H o l l a n , R . T , J o n e s , R .D .K o lc r and J . G . S z e l e n y i

1970 M u l t i p l e a l p h a c h a i n l o c i f o r human h e m o g lo b in s ,C l i n . R e s . , Î 8 ; 1 8 4 .

C a r r e l l , R .W ., H.Lehmann a n d H ,a .Hu t c h i s o n1966 H em oglob in K oln ( b e t a 98 v a l - m e t ) ; an u n s t a b l e p r o

t e i n c a u s i n g i n c l u s i o n body a n e m ia . N a t u r e , 21 0 ; 9 1 5 - 9 1 7 .

C a r r e l l , R .W ., H.Lehmann a n d B .A .L o rk in1967 H em oglob in S y d n e y ; ( b e t a 67 ( E I I ) v a l - a l a ) ; an e m e rg -

% i n g p a t t e r n o f u n s t a b l e h e m o g lo b in s . N a tu re 215»6 2 6 -6 2 8 ,

C a r r e l l , R .W ,, and M.C.Owen1971 A new a p p r o a c h to h e m o g lo b in v a r i a n t i d e n t i f i c a t i o n ;

Hb C h r i s t c h u r c h b e t a 71 (E 15 ) p h e - s e r . B io c h im . B io p h y s .A c ta , 236 ; 5 0 7 -5 1 1 .

C h a ra c h e , S . , D . J , W e a t h e r a l l and J .B .C l e g g1966 P o ly c y th e m ia a s s o c i a t e d w i t h a h e m o g lo b in o p a th y , J ,

C l i n , I n v e s t , , 4 5 ; 8 1 3 - 8 2 2 ,C h a r a c h e , S . , a n d A.M.Mondzak

1967 H em oglobin S i n a i ; a l p h a 47 h i s ; an u n s t a b l e h e m o g lo b in c a u s i n g a c c u t e h e m o l y s i s . B lood J , H e m a t o l . , 3 0 ; 8 7 9 .

80

C h a r a c h e , S, ,1969 H em o g lob in H a s h a ro n ( a l (ha 2 47 h i s (CD5) b e t a 2 ) ;

a h e m o g lo b in f o u n d i n lo w c o n c e n t r a t i o n s . J . G l i n . I n v e s t , , 4 8 ; 8 3 4 -84 7*

C h e r n o f f , A , I . , and J . C . L i u1961 The am ino a c i d c o m p o s i t i o n o f h e m o g lo b in I I , i i n a l y t -

i c t e c h n i q u e s . B lo o d , 17 ; 5 4 .C h e r n o f f , A . I . , a n d N . h . P e t t i t

1964 The am ino a c i d c o m p o s i t i o n o f h e m o g lo b in I I I . Aq u a l i t a t i v e m etho d f o r i d e n t i f y i n g a b n o r m a l i t i e s o f t h e p o l y p e p t i d e c h a i n s o f h e m o g lo b in . B lo o d , 1 7 ; 54*

1965 The am ino a c i d c o m p o s i t i o n o f h e m o g lo b in V i . B io c h im , B io p h y s .A c t a , 9 7 ; 4 7 -6 0 ,

Chon, 5 . U . , an d B .S .S h im1969 o t u d i e s on t h e a b n o rm a l h e m o g lo b in s i n K o re a ; Hb

S o e u l I I I a n d Hb G S o e u l f o u n d i n K o re a . J . G a t h .M e d .C o l l . , 1 6 ; 1 - 1 3 .

C le g g , J . B . , M .A .H a u g h to n , ,a a d D . J . W e a t h e r a l l1965 iui im p ro v e d m eth o d f o r t h e c h a r a c t e r i z a t i o n o f human

h e m o g lo b in m u t a n t s ; i d e n t i f i c a t i o n o f a l p h a 2 b e t a2 95 g l u , h e m o g lo b in M B a l t i m o r e , N a t u r e , 2 0 7 ; 9 4 5 -947 *

1966 A bnorm al human h e m o g lo b in s ; s e p a r a t i o n a n d c h a r a c t e r i s a t i o n o f t h e a l p h a an d b e t a c h a i n s b y c h ro m a to g ra p h y a n d d e t e r m i n a t i o n o f two new v a r i a n t s , Hb C h e sa p e a k e a n d Hb J B angkok. J , M o l . B i o l * , 19 ; 9 1 - 1 0 8 ,

G le g g , J . B . , D . J . W e a t h e r a l l , -v.H.Boon an d D . i ' iu s ta fa1969 Two new h e m o g lo b in v a r i a n t s i n v o l v i n g p r o l i n e s u b

s t i t u t i o n s . N a t u r e , 2 2 2 ; 3 7 9 -3 8 0 ,C le g g , J . B . , D . J , W e a t h e r a l l a n d i . J . M i l n e r

1971 H em oglob in C o n s t a n t S p r i n g ; a c h a i n t e r m i n a t i o n m u t a n t . N a t u r e , 2 3 4 ; 3 3 7 -3 4 0 ,

C ohen , P .T .W ., A .Y a te s , A . J .B e l l i n g h a m , an d E .H .H u eh n s1973 Amino a c i d s u b s t i t u t i o n i n t h e a l p h a b e t a i n t e r s u b

u n i t c n t a c t o f h e m o g lo b in Camden B e ta 131 (H9) g l n - g l u . N a t u r e , 243; 4 6 7 -4 6 8 ,

C r o o k s to n , J . H . , D .B e a le , D , I r v i n e a n d H.Lehmann1965 A n .w h e m o g lo b in J T o ro n to ( a l p h a 5 a l a - a s p ) . N a t u r e ,

20 8 ; 1 0 5 9 -1 0 6 1 .C r o o k s t o n , J . G . , H .A .P a r q u h a r s o n , J . L . K i n d e r l e r e r and H,Lehmann

1970 H em oglob in ^ i a n i t o b a ; a l p h a 102 (G9) s e r i n e r e p l a c e d by a r g i n i n e . G a n . J .B i o c h e m . , 4 8 ; 911-914*

D e Jo n g , v.’.W ,, L .N .W e n t, a n d L . P . B e r n i n i1968 H em oglob in L e id e n ; d e l e t i o n o f b e t a 6 ac 7 lu *

N a t u r e , 2 2 0 ; 7 8 8 - 7 9 0 ,d e V r e i s , A . , H . J o s h u a , H .Lehm ann, R . L . H i l l , and R , a . F e l lo w s

1963 The f i r s t o b s e r v a t i o n o f an a b n o rm a l h e m o g lo b in i n a J e w i s h f a m i l y ; h e m o g lo b in B e i l i n s o n . B r i t . J , H a e m a to l . , 7 ; 4 8 4 * 4 8 6 ,

D h e r t e , 1 , , J . V a n d e p i t t e , J .A .M .A g e r a n d H.Lehmann1959 S t a n l e y v i l l e I an d I I ; two nc-w v a r i a n t s o f a d u l t hemo

g l o b i n . B r i t . M e d , J . , 2 , 2 8 2 -2 8 4 .E frem o v , G .D . , T .H .J .H u is m a n , L .L .S m i t n , u .B .W i l s o n , J . L .K i t c h e n s , an u R .N .W r ig h ts to n e

1969 H em oglob in R ichm ond; a h e m o g lo b in w h ic h fo rm s asymé t r i e h y b r i d s w i t h o t h e r h e m o g lo b in s . J . B i o l . C h e m , , 2 4 4 ; 6 1 0 5 -6 1 1 6 ,

E l i o n , J . , 0 ,B e l k h o d j a , H.vvajcman, and D .L a b ie1973 Two v a r i a n t s o f h e m o g lo b in D i n t h e A l g e r i a n pop

u l a t i o n . Hb D U u led R abah a n d Hb D I r a n . B io ch im . B io p j^ y s .A c ta , 3 1 0 ; 360-564*

81

E n g , A ,G , , F , V e l l a , a n d 0 . 0 . M e rry1970 Two y o s o i b l e i n s t a n c e s o f h em o g lo b in E S a sk a to o n i n

M a n i to b a . C a n , J , B i o c h e m . , 48 ; 45- 4 6 ,E n g , L . I , , and G .D u ra isa m y

1972 The s lo w m o v in g h e m o g lo b in X xom ponent i n M a lay s . H u m .H e re d . , 2 2 ; 1 1 8 - 1 2 5 .

E ng , L . I . , W . ia - ib a d i , F . 'W e s te n d o rp , 0 . 0 . E frem ov , J .B . .ve lson ,C ,A .R e y n o l d s , a n d T .H .J .H u is m a n

1974 h e m o g lo b in a 2 I n d o n e s i a o r a l p h a 2 b e t a 2 69 (E13) g l y - a r g , B i o c h i m ,B io p h y s ,A c ta , 229; 3 5 5 -3 4 2 .

E n g ,1 , 1 . , a n d E .X .E ad o n o1 9 5 8 H em o g lo b in 0 ( B u g in e s e X) i n S u la w e s i . B r i t , M e d . J . ,

1 ; 1461- 1462,F a i r b a n d s , V .F . , J . E .M a ld o n a d o , L ,C h a r a c h e , >,nd S .H .B o y er

1971 F a m i l i a l e r y t h r o c y t o s i s due t o e l e c t r o p h o r e t i c a l l y u n d e t e c t a b l e h e m o g lo b in w i t h im p a i re d d i s s o c i a L i o n .Hb Malmo a l p h a 2 b e t a 2 9 7 ; h i s - g i n . t e y o C l in ,F r o c , , 4 6 ; 7 2 1 - 7 2 7 .

F e s s a s , C . , A K a r a k l i s , K .L o u k o p o u lo s , G .b ta m a to y a n n o p o u lo s , a n d P . F e s s a s

19 65 H eo m g lo b in N i c o s i a ; an a l p h a c h a i n v a r i a n t a n a i t sc o m b i n a t i o n w i t h b e t a t h a l a s s e m i a , p r i t . J . H a e m a t o l . ,1 1 ; 323 - 3 3 0 .

F e s s a s , P . , A . K a l t s o y a , D .L o u k o p o u lo s , and L .o .N i l s o n1969 On t h e c h e m ic a l s t r u c t u r e o f h em o g lo b in U p p s a la .

H u m .H e re d . , 1 9 ; 1 5 2 - 1 5 8 ,F e s s a s , P . , A . K a r a k l i s , an d N .G n a f a k is

1961 A f u r t h e r a b n o r m a l i t y o f f o e t a l h e m o g lo b in . A c ta H a e m a t o l , , 2 5 ; 6 2 - 7 0 ,

F e s s a s , P . , N . M a s t r o k a l o s , a n d G .F o s t e r o p o u lo s1959 New v a r i a n t o f human f e t a l h e m o g lo b in . N a tu r e ,

1 8 3 ; 30- 32 /F l a t s , G.

1971

F r e n c h , E, 1971

H em o g lo b in T a k ; a v a r i a n t w i th a d d i t i o n a l r e s i d u e s a t t h e e n d s o f t h e b e t a c h a i n s . L a n c e t , 1 ; 7 3 2 -7 3 3 . A . , a n d H.LehmannI s h e m o g lo b in G P h i l a d e l p h i a l i n k e d to a lp h a t h a l a s s e m ia ? ACta H a e m a to l . , 4 6 ; 1 4 9 -1 5 6 .

F u j i t a , 8 . ,1972 Oxygen e q u i l i b r i u m c h a r a c t e r i s t i c s o f abno rm al hemo

g l o b i n s ; H i r o s e ( b e t a 37 s e r ) . L F e r r a r a ( a lp h a 47 g l y ) , B r o u s s a i s ( a l p h a 90 a s n ) , and Bhofcir ( b e t a 58 a r g ) , J . C l i n . I n v e s t , , 51 ; 2520 -252 9 .N.S tu d y on t h e am ino a c i d s u b s t i t u t i o n i n h em o g lo b in At ago ; an a b n o rm a l human h e m o g lo b in . S e ik a g a k u ,4 2 ; 3 4 1 -3 4 9 .K . , a n d K .K atoH e m o ly t ic a n e m ia due to a new abnorm al hemof l o b i n , i r o c . 8 t h C o n g r , I n t e r n . S o c , H e m a t o l . , 2 ; 1220 -122 5 .

G a jd u s e k j B .C . , J . G u i a r t , R . L . K i r k , R . Q , C a r r e l l , D . L . I r v i n e , P’.A .K y n b c h , an d H.Lehmann

1966 The g e n e t i c s o f human h e m o g lo b in v a r i a n t s ; t h e gen e t i c c o d e . A lso a d e s c r i p t i o n o f th e f i r s t new hemog l o b i n o b s e r v e d i n a P a c i f i c p o p u l a t i o n ; hem o g lo b in J T o n g a r i k i , New Z e a la n d M e d . J , , 65 ; 8 4 9 -8 5 6 , H em og lob in J T o n g a x i k i ; t h e f i r t new h em o g lo b in v a r i a n t f o u n d i n a P a c i f i c (M e la n e s ia n ) p o p u l a t i o n .J . M e d , G e n e t . , 4 ; 1-6%

F u j i w a r a ,1970

I d ik u ta k e ,1961

1967

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G a l l o , E , , L . i U g l i a t t i , G .R ic c o , P . G . l i n c h , G ,P in n a an d U, Mazza

1972 A c a s e o f h e m o g lo b in J S a r d e g n a / b e t a t h a l a s s e m i ad o u b l e h e t e r o z y g o s i s , A c ta H a e m a t o l . , 4 5 ; 3 1 1 - 3 2 0 ,

G e r a l d , 1 , S , , a n d W .L .E f ro n1961 C h e m ic a l s t u d i e s o f s e v e r a l v a r i a n t s o f h e m o g lo b in

M. f r o c . N a t , A c a d . S c i . U. 8 . , 4 7 ; 1 7 5 8 -1 7 6 7 .G i l l e s p i e , J . E . Ü , , G . J , W h i t e , M . J . E i l i s , G .H .H e a v e n , w.B, G r a t z e r , E .H , b h o o t e r , a n d R .M .m .P ar lch o u se

1959 A h e m o g lo b in w i t h u n u s u a l a l k a l i n e d é n a t u r a t i o n p r o p e r t i e s i n a T u r k i s h - C y p r i o t woman. N a t u r e , 1 8 4 ; 1 8 7 6 -1 8 7 7 .

G lynn , K . i , , J . A . f e n n e r , a n d J . R . a m i t h1968 F a m i l i a l e r y t h r o c y t o s i s . a d e s c r i p t i o n o f t h r e e

f a m i l i e s , one w i t h h e m o r , lo b in Y p s i l a n t i . i i n n , I n t e r n . W e d . , 6 9 ; 7 6 9 - 7 7 6 ,

G o t t l i e b , A. J , , A . i . e s t r e p o , a n d H .A . l t a n o1964 H em o g lo b in J M e d e l l i n ; c h e m ic a l an d g e n e t i c s t u d i e s .

F e d e r a t i o n P r o o . , 2 3 ; 1 7 2 - 1 7 5 .G r e e r , J .

1971a T h r e e d i m e n s i o n a l s t r u c t u r e o f a b n o rm a l m u ta n t human h e m o g l o b i n s . C o ld S p r i n g H a rb o r Symposium on QUcUi- t i t a t i v e B i o l o g y , 3 6 ; 3 1 5 - 3 2 3 .

1971b T h r e e d i m e n s i o n a l s t r u c t u r e o f a b n o rm a l h e m o g lo b in sK a n sa s an d R ich m o n d . J . ü d l a . S t a t e D e n t . A s s o c . , 5 9 ; 9 9 - 1 0 5 .

H anada , M ,, a n d D .L .K u c icn ag e l1963 The c h a r a c t e r i z a t i o n o f h e m o g lo b in S h im o n o s e k i .

B lo o d , 2 4 ; 6 2 4 - 6 3 5 .H e l l e r , P .

1969 H em o g lo b in M; a n e a r l y c h a p t e r i n t h e s a g a o f m o le c u l a r p a t h o l o g y . A n n . I n t e r n . M e d . , 7 0 ; 1 0 3 8 -1 0 4 1 .

H e l l e r , t . , R .D .C o le m a n , a n d V . J . Y a k u l i s1966 H em o g lo b in M Hyde P a r k ; a new v a r i a n t o f a b n o rm a l

m e t l ie m o g lo b in , J . G l i n . I n v e s t . , 4 5 ; 1 0 2 1 ,H i l l , R . L . , R .W .S w en so n , ano H ,C .Schw ^iTtz

1960 C h a r a c t e r i z a t i o n o f a c h e m ic a l a b n o r m a l i t y o,n hemog l o b i n G, J . B i o . C h e m . , 2 3 5 ; 3 1 8 2 -3 1 o 7 .

H uism an, T . H . J , , t n a V . P . a y d e n s t u y c k e r1962 D i f f e r e n c e i n g r o s s s t r u c t u r e o f two e l e c t r o h o r -

e t i c a l l y i d e n t i c a l m in o r h a e m o g lo b in c o m p o n e n ts . N a t u r e , 1 9 3 ; 4 8 9 - 4 9 1 .

H o l l a n , S . R . , a n d J . S z e l e n y i1965 Some new d a t a on h e m o g lo b in K i s k u n h a l a s . I r o c . 9 t h

C o n g , E a r , S o c , H a e m a t o l , , S . K a r g e r , N .Y . , 5 3 8 - 5 4 2 ,H o l l a n , S . R . , J . S z e l e n y i , B . B r i m h a l l , M .^ x ie r s t , R . T . J n e s and Z . S t o c k l e n

1972 M u l t i p l e c h a i n l o c i f o r human h e m o g lo b in s ; Hb J - Buda a n d G - P e s t . N a t u r e , 2 3 5 ; 4 7 - 5 0 ,

H o l l a n , S . R . , J . S z e l e n y i , M .M i l t a n y i , D .C h a r l e s w o r t h , P .A . L o r k in , a n d H ,Lehm ann

1970 U n s t a b l e h e m o g lo b in d i s e a s e c a u s e d h e m o g lo b in S a n t a ^tnna; b e t a 08 l e u - p r o . i i e m a t o l o g i c a , 4 ; 1 4 1 - 1 5 5 .

H o l l e n d e r , A . , P . a .L o r k i n , H .Lehm ann e n d B .S v e n s o n1969 New u n s t a b l e h e m o g lo b in ; B o r a s ; b e t a 86 (F 4 ) l e u -

a r g . N a t u r e , 2 2 2 ; 9 5 3 - 9 5 5 .

83

H o n i g , G.H,1973 H em oglobin Abraham L i n c o l n ; 32 (B 14) l e u - p r o * An

u n s t a b l e v a r i a n t p r o d u c in g s e v t r e hemol, t i c a n e m ia .J . C l i n , I n v e s t . , 2 2 ; 1 7 4 6 -1 7 5 5 .

H u e h n s , E , R , , a n d E . M . S h o o t e r1961 P o ly I j e o t i d e c h a i n s o f h e m o g lo b in A 2 . N a t u r e , 189 ;

918.1965 Human h e m o g lo b in s , J .M e d .G e n . , 2 ; 4 8 - 9 0 .1966 F u r t h e r s t u d i e s on t h e i s o l a t i o n a n d p r o p e r t i e s o f

a l o h a c h a i n s u b u n i t s o f h e m o g lo b in . B i o c h e m .J , ,1 0 Î ; 8 4 3 - 8 5 1 .

H u ism an , T . H . J , , A ,K .B row n, G .L .E fre m o v , J .B .V / i l e o u , G . a .R e y n o l d s , H.Uy a n d L . L . S m i t h

1971 H em oglobin S a v a n n a h (B 6 24 b e t a g l y - v a l ) ; a n u n s t a b l e v a r i a n t c a u s i n g a n e m i a w i t h i n c l u s i o n b o d i e s .J . C l i n . I n v e s t . , 5 0 ; 6 5 0 -6 5 9 .

H u is m a n , T . H . J . , a . M . D o z y , B , F . M o r t o n a n a J . B . W i l s o n1965 A f e t a l h e m o g lo b in w i t h a b n o rm a l gamma p o l y p e p t i d e

c h a i n s ; H em oglobin W arren . B lo o d ; 2 6 ; 6 6 8 -6 7 6 .H uism an , T . H . J . , C .A .R e y n o ld s , a .m .D o zy and. J . B . W ilso n

1969 H em oglobin L a b i n g a o r a l p h a 2 d e l t a 2 136 g l y - a s p o b s e r v e d i n t h e A m e r i c a n N eg ro , B i o c h i m . B i o p h y s .ACta, 1 7 5 ; 2 2 3 -2 2 5 .

H u is m a n , T . H . J , , a . N . v v r i g h t s t o n e a n d J . B , W i l s o n1972 H e m o g lo b in K o i y a , t h e p r o d u c t o f f u s i o n a n d o f p o l y

p e p t i d e c h a i n s . A r c h . B i o c h e m . B i o p h y s . , 15 3 ; 6 5 0 - 8 5 3 .H u n t , J . A . , a n d V . m . I n g r t m

1961 J abnorm al human h e m o g lo b in s V I. The c h e m ic a l d i f f e r e n c e b e tw e e n h e m o g lo b in s A :_nd w, , B io c h im . L j.ophys. A c ta , 4 9 ; 5 2 0 -5 3 6 ,

H untsm an , R . G . , M .H a l l , H,Lehmann and P .k ,S u k u m a ra n1963 A s e c o n d end a t h i r d a b n o rm a l h e m o g lo b in i n N o r f o l k ,

Hb G N o r f o lk a n d Hb D N o r f o l k . B r i t . ^ e d . J . , 1 ; 7 2 0 - 7 2 2 ,

Hyde, R .D .1972 H em oglob in S o u th a m to n , a l p h a 106 (G8) l e u - p r o ; an

u n s t a b l e v a r i a n t p r o d u c in g s e v e r e h e m o l y s i s . L . .n c e t ,2 ; 1 1 7 0 -1 1 7 2 ,

H yde, n . D . , J . L . K i n d e r l e r e r , H .Lehm ann, a n d M . r . H a l l1971 H em oglobin J R a ja ^ p e n ; a l p h a 90(FG 2) l y s - t h r . B io c h im .

B io p h y s .A c t a , 2 4 3 ; 5 1 5 -5 1 9 .I m a i , K , , H .M cr im o to , M .K o ta n i , b . B h i b a t a , T . i ' î i y a j i , .n u K,M atsutom o

1970 S t u d i e s on t h e f u n c t i o n o f a b n o rm a l h e m o g lo b in s .Oxygen e q u i l i b r i u m o f a b n o rm a l h e m o g lo b in s .

Im a m u ra , T . , S . F u j i t a , Y . O h t a , M.H a n a d a , and T . Y a n a s e1969 H em oglobin Y o s h iz u k a (G 10 (1 0 8 ) b e t a a s n - a s p ) ; a

new v a r i e n t w i t h r e d u c e d oxygen a f f i n i t y f ro m a J a p a n e s e f a m i l y . J . G l i n . I n v e s t . , 4 8 ; 2 3 4 1 -2 3 4 8 ,

I n g , R .Y .K . , J .H .G r o o k s t o n , J . A .D w o ra tz e k , anc, K .L .B u r n i e 1969 Alpha t h a l a s s e m i a ; f i v e c a s t s o f h e m o g lo b in M d i s

e a s e i n t a r e e o r i e n t a l C a n a d ia n f a m i l i e s . C an.M ed. A s s o c . J . , 9 9 ; 4 9 - 5 6 .

I n g r a m , V.M.1957 Gene m u t a t i o n s i n human h e m o g lo b in ; t h e c h e m ic a l

d i f f e r e n c e i^et w e e n n o r m a l and s i c k l e c e l l h e m o g l o b i n . N a t u r e , 18U; 3 2 6 - 3 2 8 ,

84

I n g r a m , V.M,1963 The h e m o g lo b in s i n g e n e t i c s eind e v o l u t i o n . N .Y . ,

C o lu m b ia U n i v e r s i t y i r e s s .I t t n o , H .a .

1957 The human h e m o g lo b in s ; t h e i r p r o p e r t i e s ano , . e n e t i c c o n t r o l . A d v . l r o t e i n d h h e m . , 12 ; 2 1 5 .

I t a n o , H .A , , a n d J . V . N e e l1950 A new i n h e r i t e d a b n o r m a l i t y o f human h e m o g lo b in ,

i r o c , N a t . n o a d . B e e , , 5 6 ; 6 1 3 - 6 1 7 .I t a n o , H .A . , iuid E .A ,R o b in s o n

I 9 6 0 G e n e t i c c o n t r o l o f t h e a l p h a an d b e t a c h a i n s o f hemog l o b i n , I r o c . N a t . A c a d . B e i . U . ü . , 4 6 ; 1 4 9 2 ,

J a v i d , J ,19 75 M e d ic a l g r a n d r o u n d s ; H em o g lo b in S O - n r a b i a i n a

b l a c k l im e r i c a n . A m .J .M e d .B c i . , 2 6 5 ; 2 6 6 - 2 7 4 .J o h n s t o n , F .b ,

1 9 7 0 G e n e t i c E in th r o p o lo g y ; some c o n s i d e r a t i o n s . C u r r e n t D i r e c t i o n s i n A n th r o p o lo g y , 3 ; 9 9 - 1 0 4 .

J o n e s , R .T . , B, x a r i ro h a l l , luid K .R .H uehns1968 S t r u c t u r a l c n a r ^ c t e r i z a t i o n o f h e m o g lo b in N-S. a t t l e ,

B i o c h i m .B i o p h y s .A c t a , 1 5 4 ; 2 7 B -2 8 4 .J n e s , R . T . , B . B r i m h a l l , and T .H .J .H u is m a n

19 66 H e m o g lo b in F r e i b u r g ; a b n o rm a l h e m o g lo b in due t o th e d e l e t i o n o f a s i n g l e am ino a c i d r e s i d u e . B c i e n c e , 1 5 4 ; 1 0 2 4 -1 0 2 7 .

1967 B t r u c t u r a l c h a r a c t e r i z a t i o n o f tv/o d e l t a c h a i n v a r i a n t s . H em o g lo b in A '2 a n d h e m o g lo b in F l a t b u s h , J . B i o l .C h e m . , 2 4 2 ; 5 1 4 1 -5 4 5 .

J o n e s , R . T , , B . B r i m h a l l , a n d R . L i s k e r1968 C h e m ic a l c h a r a c t e r i z a t i o n o f h e m o g lo b in m ex ico and

h e m o d o b i n C h i a p i s . B i o c h i m .B i o p h y s ,A c t a , 1 5 4 ; 48 8 - 4 9 5 . .

J o n e s , R .T , . , R »D .G olem an , a n d l . H e l l e r f1 9 6 6 The s t r u c t u r a l a b n o r m a l i t y o f h e m o g lo b in M K a n k ak e e ,

J . B i o l . C h e m , , 241 ; 2 1 3 7 - 2 1 4 3 .J o i i e s , R . T . , il, J . G r im e s , R. W. C a r r e l l , a n d H.Lehmann

1967 K o ln h e m o g lo b in o p a th y ; f u r t h e r d . . t a a n a a c o m p a r is o n w i t h o t h e r H e in z bod y a n e m ia s . B r i t . J . H a e m a t o l . ,13; 3 9 4 ,

[.mes, R . T , , H .r , O sg o o d , B . B r i m h a l l , and R . D . n o l e r1967 H em o g lo b in Y akim a; I , C l i n i c a l a n d b i o c h e m i c a l ,

s t u d i e s . J . C l i n , I n v e s t . , 4 6 ; 1 8 4 0 .J o n e s , n . T . , R . D .K o l e r , M .D u e rs t^ a n d Z .B to c k l e n

1 9 7 2 H e m o g lo b in C a s p e r ; f u r t h e r e v i d e n c e t h a t h e m o g lo b in m u t a t i o n s a r e n o t ran d o m . A d v .E x p .M e d .B i o l . , 2 8 ; 7 9 - 9 8 .

K am el, K . , K .C .H o erm an a n d A.Y.Awny1967 H e m o g lo b in a l p h a 2 b e t a 2 l y s ; c h e m ic a l i d e n t i f i c

a t i o n i n an L g y p t i o n f a m i l y . S c i e n c e , 1 5 6 ; 3 9 7 -3 9 8 .K e e l i n g , M .M ., L .L .O g d e n , R .N . W r i g h t s t o n e , J . B , W i l s o n , D.A.R e y n o ld s , J . L . K i t c h e n s a n d T .H .J .H u is m a n

1971 h e m o g lo b in L o u i s v i l l e ( b e t a 42 (GDI) p h e - l e u ) ; a n u n s t a b l e v a r i a n t c a u s i n g m i l d h e m o l y t i c a n e m ia . J , C l i n . I n v e s t . , 5 0 ; 2 3 9 5 - 2 4 0 2 ,

K e n d a l l , A .G , , R .D . B a r r , A ,L a n d , H ,Lehm ann1973 H em o g lo b in J N y an za ; 21 (B2) a l a - a l p , B :o c h e m .B io

p h y s . A c t a , 3 1 0 ; 357- 3 5 9 .

85

Kim, C . b . , and B .S .Shim1 9 6 9 Hem oglobin v a r i a n t s i n t h e K o re a n p o p u l a t i o n , Hb

Soeu l I and Hb S o e u l I I , J , G a t h . i ' i . d , C o l l . , 16 ; 7 3 - 8 3 ,K in g , M,A.

1972 An u n s t a b l e h e m o g lo b in w i t h r e d u c e d o x y g e n a f f i n i t y ;Hb P e t e r b o r o u g h , 5 (G 13 ) v a l - p h e , i t s i n t e r a c t i o n w i th no rm al h e m o g lo b in c'uid w i t h h e m o g lo b in Be c o r e . B r i t . J . H a e m a t o l , , 2 2 ; 1 2 5 - 1 3 4 ,

K l e i n h a u e r , E . F , , G ,A ,R e y n o ld s , a .M.Do z y , u . ^ . N e l s o n , H .R.M o o re s , M .P .B e re n so n , C .S , W r i g h t , a n d T .H .J .H u is m a n

1968 Hem oglobin Bibb a a lp h a 2 136 oro b e t a 2 , an u n x t a b l e a l p h a c h a in a b n o rm a l h e m o g lo b in , b io c h im . B io h y s . A c ta . 154; 2 2 0 -2 2 2 .

K l e i n h a u e r , B .P .1971 Hemoglobin T u b in g e n ; a new b e t a c h a i n v a r i a n t (BTP

10-21 ) w i th i n c r e a s e d s ijon t e n e c u s o x i d a t i o n , K l i n , r to c h e n s c h r . , 4 9 ; 6 5 1 .

K o n o te y - . ih u lu , F . I . D . , E, G a l l o , H.Lehmann ;.nd B . i i i r i f e lh a n n1969 Hemo[-lobin K o rle -B u ( b e t a 73 a s p - a s n ) i n c o m b i n a t i o n

w i th Hb S. J ,W e d ,G e n . , 5 ; 1 0 7 - 1 1 1 ,K o n o te y -A b u lu , F . I . D . , J . L . K i n a e r l e r e r , H .n eh m an n , onu B.R in g e lh a n n

1971 Hem oglobin ü s u - G h r i s t i o n s b o r g ; a n w b e t a c h a i n v a r i a n t o f h e m o g lo b in ( b e t a 52 (D5) a s p - a s n ) i n comb i n a t i o n w i t h Hb Ü, J ,M e d .G e n . , 8 ; 3 0 2 - 3 0 5 .

K r a u s , L .M ,, T .M iy a j i , I . I u c h i an d a .P .K r a u s1966 C h a r a c t e r i z a t i o n o f a l p h a 22 gul-NH2 i n h e m o g lo b in

Memphis, h em o g lo b in M e m p h is /8 , a new v a r i a n t o f m o le c u l a r d i s e a s e . B i o c h e m i s t r y , 5 ; 3 7 0 1 - 3 7 0 8 .

K u r a c h i , b . , M.Hermodson, H .H ornung a n d G ,S ta m a to y a n n o u o u lo s1975 S t r u c t u r e o f h e m o g lo b in S e a i t l e , N a t u r e , 2 4 3 ; 2 7 5 -

276.K usarno to , Y,

1967 Hemoglobin Kumamoto. Kumamoto h e u . J , , 2 0 ; 1 - 1 0 .L a b i e , D . , and G .S c h a p i ra

1966 New v a r i a n t o f h e m o g lo b in g . H em o g lo b in G - P a r i s ,N ah u re , 209; 1 0 3 3 -1 0 3 4 .

L a b i e , D. , W .A .S ch roeder and T .H .J .H u is m a n1966 The amino ; ;c id s e q u e n c e o f t h e d e l t a - b e t a c h a i n s o f

h em o g lo b in L e g o re A u g u s ta , H em o g lo b in L e p o re Washi n g t o n . B i o c h im .B io p h y s .A c ta , 1 2 7 ; 4 2 8 - 4 3 7 .

L a b i e , D , , J .R o s a , u .B e lk h o d j a , and R .R i e r n e1971 Hem oglobin T o u lo u se a l p h a 2 b e t a 2 (E 1 0 ) l y s - g l u .

S t r u c t u r e and c o n s e q u e n c e s i n m o l e c u l a r p a t h o l o g y . B io c h im .B io p h y s .A c ta , 2 3 6 ; 2 0 1 - 2 0 7 ,

L a b o s s i e r e , a . , J . R . H i l l , and F . V e l l a »1971 A nw h em o g lob in v a r i a n t ; Hb E dm on ton . C l i n , B i o c h e m . ,

4 ; 11 4 -117 .L a b o s s i e i - e , A , , F . V e l l a , J . H i e b e r t , no P . G a l b r a i t h

1972 Hei-.oglobin Deer Lodge a l p h a 2 b e t a 2 2 h i s - a r g , O l i n . B iochem ., 5 ; 4 6 - 5 0 ,

L a b o s s i e . e , a , , F .V e l l a £uid J .J . H i l l1971 Hem oglobin I i n a w h i t e f a m i l y i n S a s k a t o o n , C l i n .

B io ch e m ,, 4 ; 1 0 4 -1 1 3 ,L a n g , A . , h .Lehm ann, P.R.KlcCurdy a n d L . l i e r c t ,

1972 I d e n t i f i c a t i o n o f h e m o g lo b in G G e o rg e to w n , B io c h e m , , B io p h y s .A c ta , 278 ; 5 7 - 6 1 ,

8 6

L ehm ann, H.1962 h e m o g lo b in s a n d h e m o g l o b i n o p a t h i e s . I n H.Lehmann

a n d D .B e tk e ( E d s . ) H em o g lo b in C o l lo q u iu m . G eorg Thiem e V e r l a g , S t u t g a r t , 1 - 1 4 .

L ehm ann, H , , D ,B e a le , a n d P .S .B o i-D o k u1964 H em o g lob in G A c c a r a . N a t u r e , 2 0 3 ; 3 6 3 -3 6 5 ,

Lehm ann, H , , a n d R .W ,C a r r e l l1968 D i f f e r e n c e s b e tw e e n a l p h a an d b e t a c h a i n m u ta n t s o f Hb.

P o s s i b l e d u p l i c a t i o n o f t h e a l p h a g e n e , B r i t , M e d . J . 4 ; 748,Lehm ann, H , , a n d D .C h a r l e s w o r th

1970 O b s e r v a t i o n s on h e m o g lo b in P (Congo t . p e ) . B iochem ,J . , 118 ; 1 2 - 1 3 .

Lehm ann, H . , a n d R .G . l t in t s m a n1966 M a n 's h e m o g lo b in s , J . B . L i p p i n c o t t . P h i l a d e l p h i a ,

L i v i n g s t o n e , P .B ,1958 A n t h r o p o l o g i c a l i m p l i c a t i o n s o f t h e s i c k l e c e l l g e n e

d i s t r i b u t i o n i n West A f r i c a , Am.Anth., 6 0 ; 5 3 3 ,1967 A bnorm al h e m o g lo b in s i n human p o p u la i o n s . A l d i n e .

C h ic a g o .1969 Gene f r e q u e n c y d i n e s o f t h e b e t a h e m o g lo b in l o c u s

i n v a r i o u s human p o p u l a t i o n s a n d t h e i r s i m u l a t i o n by m o d e ls i n v o l v i n g d i f f e r e n t ! 1 s e l e c t i o n . Hum,B i o l . , 41 ; 2 2 3 - 2 3 6 ,

L o k ic h , J . J .1973 H em oglob in B righam ( a l p h a 2 b e t a 2 100 p r o - l e u ) , A

h e m o g lo b in v a r i a n t a s s i c i a t e d w i t h f a m i l i a l e r y t h r o c y t o s i s . J . C l i n . I n v e s t . , 5 2 ; 2 0 6 0 -2 0 6 7 ,

L o r k i n , P . A . , H ,Lehm ann, V . P . F a i r b a n k s , G .B e rg lu n d a n d T.L e o n h a r d t

1970 Two new p a t h o l o g i c a l h e m o g lo b in s ; O lm sted ( b e t a 141 l e u - a r g ) ana Malmo ( b e t a 97 h i s - g l n ) . B i o c h e m . J . ,1 1 9 ; 6 8 ,

L o r k i n , P . A . , D .C h a r l e s w o r t h , H .Lehm ann, S .R a h b a r , G .T u c h in a d a a n d L . I .E n g

1970 Two h e m o g lo b in s w a l p h a 74 (E P3) a n d a l p h a 75 (E F4) a s p a r t i c a c i d - h i s t i d i n e . B r i t . J , H a e m a t o l . , 1 9 ; 1 1 7 - 128,

M e k a w a , M.1 9 70 An am ino .^c id s u b s t i t u t i o n i n an a b n o rm a l human hemo

g l o b i n , Hb N a g a s a k i . N a g a s a k i I g a k k a i E a s s i , 44 ;5 3 9 - 5 4 8 ,

Kiaekawa, M ,, T . Maekawa, N . I h j i w a r a , K .T a b a r a , a n d G. 11a t su d a1970 H em oglob in N a g a s a k i ( a l p h a 2 b e t a 2 17 g l u ) ; A new

a b n o rm a l human h e m o g lo b in fo u n d i n one f a m i l y i n N a g a s a k i ; B io c h e m ic a l s t u d i e s o n h e m o g lo b in s a n d myog l o b i n s . I n t . J . P r o t e i n R e s . , 2 ; 1 4 7 - 1 5 5 ,

M arengo-R ow e, A . J . , D .B e a le and H.Lehmann1968 New h e m o g lo b in v a r i a n t f rom S o u th e r n A r a b i a ; G-

A u d h a l i a l p h a 23 (B4) g l u t a m i c a c i d r e p l a c e s v a l i n e a n d t h e v a r i a b i l i t y o f B4 i n human h e m o g lo b in .N a t u r e , 2 1 9 ; 1 1 6 4 -1 1 6 9 .

M a r t i , H .R . , D .B e a le a n d H.Lehmann1967 H em oglob in K o l l i k e r ; a new a c q u i r e d h e m o g lo b in a p

p e a r i n g a f t e r s e v e r e h e m o l y s i s ; a l u h a 2 m in u s 141 a r g b e t a 2 , A c ta H a e m a to l . , 3 7 ; 174.

M a r t i , H .R . , C . P i k , a n d P .M osim ann19 64 L in e n e u e h a m o g lo b in I v a r i a n t e ; Hb I I n t e r l a k e n ,

A c ta H a e m a to l , , 31 ; 9 - 1 6 ,

8 7

K iatsum oto , K.1971 H em oglobin G ifu ( b e t a 80 a s n - l y s ) , a new s lo w m oving

h e m o g lo b in d e t e c t e d from two f a m i l i e s o f J a p a n e s e ,AC t a H a e m a to l , J a p a n , 3 4 ; 4 7 9 -4 8 3 ,

M i l n e r , P . P . , G , M i l l e r , K .G re y , M .S e a k in s , W.W.DeJong a n d L,N. W e n t

1970 H em oglobin Q Arab i n f o u r Negro f a m i l i e s and i t s i n t e r a c t i o n w i t h h e m o g lo b in s S and 0 , New E n g . J .M ed ., 2 8 3 ; 1 4 1 7 -1 4 2 5 .

M in n ic h , V . , J , K , C o r d o n n i e r , W .J .W i l l i a m s , eind G.V.M oore1962 A lp h a , b e t a and gamma h e m o g lo b in p o l y p e p t i d e c h a i n s

d u r i n g t h e n e o n a t a l p e r i o d w i t h d e s c r i p t i o n o f a f e t a l fo rm o f h e m o g lo b in D S t , L o u i s . B lo o d , 19 ; 137 ,

M in n ic h , V , , R . J . H i l l , P .D .K h u re , and M .E .A nderson1965 H em oglobin Hope; a b e t a c h a i n v a r i a n t . B lo o d , 25 ;

8 3 0 - 8 3 8 .M i y a j i , T . , I . I u c h i , K.Yamamoto, Y.Ohba and S . S h i b a t a

1967 Amino a c i d s u b s t i t u t i o n o f h e m o g lo b in Ube I I s u c c e s s f u l a p p l i c a t i o n o f p a r t i a l h y d r o l y s i s o f a pept i d e w i t h 5/0 a c e t i c a c i d , C l i n , G h i m . a c t a , 16; 3 4 7 -3 5 2 .

M i y a j i , T , , Y .U hba , K.Yamamoto, S . S h i b a t a , I . I u c h i , and H.B,H a m il to n

1968 H em oglob in H i j iy a m a ; a f a s t m oving h e m o g lo b in i n a J a p a n e s e f a m i l y . S c i e n c e , 159; 2 0 4 ,

M i t a j i , T . , Y .O hba , K.Yamamoto, S . S h i b a t a , I . I u c h i , and M.T ak e n ak a

1968 A J a p a n e s e h e m o g lo b in v a r i e t y . N a t u r e , 2 1 7 ; 8 9 .M y a j i , T . , H .S u z u k e , Y .O hba, and S . S h i b a t a

1966 H em oglobin A genogi ( a l p h a 2 b e t a 2 90 l y s ) , a new s low m oving h e m o g lo b in o f a J a p a n e s e f a m i l y ie s e m - b l i n g h e m o g lo b in E, O l in .C h im .A c ta , 14; 6 2 4 -6 2 9 ,

M o tu ls k y , A.Ü,1960 M e ta b o l ic p o ly m o rp h ism s and t h e r o l e o f i n f e c t i o u s

d i s e a s e i n human e v o l u t i o n . H u m ,B io l . , 32 ; 2 8 - 6 2 ,M eK usick , V.

1971 M e n d e l l i a n I n h e r i t e n c e i n Man. The J o h n s H opk ins 1 r e s 8 , B a l t i m o r e .

I - iu l l e r , C . J . , a n d S.K ingm a1961 H em oglobin Z u r i c h ; a lp h a 2 b e t a 2 63 a r g . B io ch im ,

B io p h y s .A c ta , 5 0 ; 5 9 5 ,N a g a i , M ,, Y . S u g i t a , a n d Y.Yoneyama

1972 Oxygen e q u i l i b r i u m and c u r c u l a r d e c i i r o i s m o f Hb R a n ie r ( a l p h a 2 b e t a 2 145 t y r - c y s ) , J .B i o l . C h e m . , 2 4 1 , 2 8 5 -2 9 0 ,

N a g e l , R . , H .M .Ranney, A .S . J a c o b s , H .M .A nderson and R . A . K r i t z l e r1964 H em oglobin C o lu m b ia ; an a l p h a c h a i n v a r i a n t fo u n d

i n a R u s s i a n J e w i s h f a m i l y ; i t s a s s o c i a t i o n w i t h a h e m o l y t i c s t a t e . A b s t r a c t s o f t h e A nnual M e e t in g o f Lhe A m erican S o c i e t y o f numan G e n e t i c s , 2 .

N ance , W .E ., M .C o n n e a l ly , K.W.Kang, T .R e td , J , S c h r o d e r , andS .R o sa

1970 G e n e t ic l i n k a g e a n a l y s i s o f human h e m o g lo b in v a r i a n t s . /A n .J .H u m .G en e t. ; 2 2 ; 4 5 3 -4 5 9 .

N o w ic k i , L . , and N .M a r t in1971 H em oglobin F r a n k f u r t ; a new u n s t a b l e h e m o g lo b in a s

t h e c a u s e o f H e inz body anem ia i n a German f a m i l y , K l i n .W o c h e n s c h r . , 4 9 ; 1 1 1 5 -1 1 2 0 ,

- V " : / / 8 8

O h ta , Y . , K .T am aeda, I .S t i m i d a , and T .Y an ase1971 H em oglobin M iy a d a , a b e t a - d e l t a f t i e io n p e p t i d e ( a n t i -

L e p o re ) t y p e d i s c o v e r e d i n a J a p a n e s e f a m i l y , N a t , New B i o l , , 2 3 4 ; 2 1 8 -2 2 0 ,

O lso n , J . S . , and Q .d ,G ib s o n1972 The f u n c t i o n a l p r o p e r t i e s o f h e m o g lo b in B e t h e s d a

( a l p h a 2 b e t a 2 145 h i s ) , J . B i o l . C h e m . , 2 4 7 ; 3 6 6 2 - 3670 .

O n o d e ra , S ,1973 H em oglobin S e n d a i ; an u n s t a b l e h em o g lo b in d i s c o v e r e d

i n h e m o l y t i c a n e m ia acc o m p a n ie d by i n t e r s t i t i a l n e p h r i t i s , A c ta H e m o l , J a p a n , , 36; 3 2 - 4 5 ,

Osgood, E , E , , R . T . J o n e s , B .B r im h a l l and R .D .K o le r1967 H em oglob in Y akim a ; c l i n i c a l and b io c h e m i c a l s t u d i e s .

C l i n . R e s . , 1 5 ; 134 .O s t e r t a g , W., a n d E .w .S m i uh

1969 H em oglobin L e p o re B a l t im o r e ; a t h i r d t y p e o f b e t a - a l p h a c r o s s o v e r . D u r . J .B i o c h e m . , 10; 3 7 1 - 3 7 6 ,

O v e r ly , W .L., A .R o s e n b e rg a n d J .W ,H a r r i s .1966 H em oglobin M r e s e r v e ; s t u d i e s on i d e n t i f i c a t i o n a n d

c h a r a c t e r i z a t i o n . J . L a b . C l i n . M e d . , 6 9 ; 6 2 - 8 7 ,OzBoylu, S,

1972 C o n g e n i t a l m e th em o g lo b in e m ia due t o h e m o g lo b in M I w a te . A c ta H a e m a to l . , 4 7 ; 2 2 5 -2 3 2 .

i a u l i n g , L,1949 S i c k l e c e l l a n e m ia ; a m o le c u la r d i s e a s e . S c i e n c e ,

110; 5 4 3 - 5 4 8 .l e i u t z , M. P . , a n d H,Lehmann

1968 M o le c u la r p a t h o l o g y o f human h e m o g lo b in . N a t u r e ,219 ; 9 0 2 - 9 0 9 .

l i e r c e , L . E . , K.McCoy, a n d G .E .R a th1963 A new hemoglobin var ian t with s ic k l in g p r o p e r t ie s .

New E n g , J .M e d , , 2 6 8 ; 8 6 2 -8 6 6 ,l i e t s c h m a n n , H,

1971 G ty o c h e in ic a l s t u d i e s o f a new h e m o g lo b in o p a th y (Hb V i e n n a ) , Wien K l i n . W o r s c h r . , 8 2 ; 3 6 2 -3 6 7 ,

i l s c i o t t a , A .V , , S . N . E b r e , and J . E .H i n z1963 C l i n i c a l a n d l a b o r a t o r y f e a t u r e s o f two v a r i a n t s of

m e th e m o g lo b in d i s e a s e , J . L a b . C l i n , M e d , , 5 4 ; 7 3 - 8 7 ,P o o t r a k u l , S . , a n d G .H .D ix o n

1970 H em oglobin M a h id o l ; a new h em o g lo b in a l p h a c h a i n m u ta n t . C a n , J .B i o c h e m . , 48 ; 1 0 6 6 -1 0 7 8 .

P r a t o , V ,, E , G a l l o , G .R ic c o , V .Piazza, G .E ia n c o , and H ,Lehm ann1970 H e m o ly tic a n e m ia due t o h em o g lo b in T o r in o . B r i t ,

J . H a e m a t o l . , 1 9 ; 1 0 5 -1 1 5 ,Rah b a r . S . , D, B e a l e , W. A, I s a a c s , . n d H.Lehmann

1967 Abnormal h e m o g lo b in s i n I r a n . O b s e r v a t i o n s o f a new v a r i a n t h e m o g lo b in J I r a n ( a l p h a 2 b e t a 2 77 h i 8 - a s p ) . B r i t . M e d . J . , 1 ; 6 7 4 -6 7 7 ,

R a h b a r^ s . , J . L . K i n d e r l e r e r , an c H.Lehmann1969 H em oglobin L Pej s i an G u l f ; a lp h a 57 (B6) g l y - a r g .

A c ta H a e m a to l , , 4 2 ; 1 69 -1 7 5 .R a h b a r , S . , H .S o o u d i , an d P .Daneshm and

1972 H em oglobin J P a r l e ( a lp h a 12 a l a - a s p ) I n two I r a n i a n f a m i l i e s , A c ta H a e m a to l . , 4 7 ; 4 3 - 4 8 ,

R anney , H.M. ' ^ *1954 O b s e r v a t i o n s on t h e i n h e r i t en ce o f s i c k l e c e l l hemo

g l o b i n and h e m o g lo b in C , . j , 0 1 i n . I n v e s t . , 3 3 ; 1 6 3 4 .

8 9

R a n n e y , H .M ., A . S . J a c o b s , e n d R .G .N a g e l1967 H em o g lo b in New Y o rk . N a t u r e , . 2 1 3 ; 8 7 6 - 8 7 8 .

R a n n e y , H .M ., a . S . J a c o b s , L .Uraden, a n d R .Z a l u s k y1968 H em og lo b in R i v e r d a l e - B r o n x ; a n u n s t a b l e h e m o g lo b in

r e s u l t i n g from t h e s u b s t i t u t i o n o f a r g i n i n e f o r g l y c i n e a t h e l i c a l r e s i d u e B6 o f t h e b e t a p o l y p e p t i d e c h a i n , B i o c h i m .B i o p h y s .R e s ,Commun., 35 ; 1 0 0 4 -1 0 0 5 .

R a n n ey , H .M ., A . S . J a c o b s , B .R am ot, and T .B .B r a d l e y1969 H em o g lo b in NYU; a d e l t a c h a i n v a r i a n t , a l o h a 2 d e l t a

2 12 l y s . J . C l i n , I n v e s t . , 4 8 ; 2 0 5 7 -2 0 6 2 ,A a p e r , a . B . , J .A .M .A g e r , and H.Lehmann

I 9 6 0 H em og lo b in S i n g a p o r e - B r i s t o l . A f a s t h e m o g lo b in f o u n d i n i n f a n t s , B r i t . M e d . J , , 1 ; 1 5 3 7 -1 5 3 9 ,

R a p e r , a . B . , L .B .Gam m ack, B .R .H u e h n s , an d A .M .^ h o o te rI9 6 0 Fou r h e m o g lo b in s i n one i n d i v i d u a l . A o tu d y o f t h e

g e n e t i c i n t e r a c t i o n o f Hb G and Hb C, s r i t . M e d . J , ,1 1 ; 1 2 5 7 ,

R e e d , C . S . , R .H am pson , S ,G o rd o n , R .T . J .m e s , M .J .N o v e , B .B r im h a l l M .J ,E d w a r d s , and R .D .K o le r

1 9 68 E r y t h r o c y t o s i s s e c o n d a r y t o i n c r e a s e d oxygen s i f f i n i t y o f a m u ta n t h e m o g lo b in . Hb Dempsey, B lo o d , 3 1 ; 6 2 3 ,

R e y n o ld s , C . a . , and T .H .J .H u is m a n19 66 H em o g lo b in R u ss o r a l p h a 2 51 a r g b e t a 2 . B io c h im ,

B i o p h y s .A c t a , 13 0 ; 5 4 1 -5 4 3 , n h i n e s m i t h , H , S . , W .A .S c h ro e d e r , and L . P a u l i n g

1957 A q u a n t i t a t i v e s t u d y o f t h e h y d r o l y s i s o f human d i - n i t r o p h e n y l (DNP) g l o b i n ; t h e num ber a n d k i n d o f p o l y p e p t i d e c h a i n s i n n o rm a l a d u l t human h e m o g lo b in ,J ,A m .C h e m .S o c . , 7 9 ; 4 6 8 2 ,

R i e d e r , R . F . , F .A .O s k i , ano J . B , C l e g g1968 PIb P h i l l y ; b e t a 35 ( 0 1 ) t y r - p h e . I n c r e a s e d s u b u n i t

d i s s o c i a t i o n due to l o s s o f a h y d ro g e n bon d a t t h e a l p h a - 1 - b e t a - 1 c n t a c t . C . i n . R e s , . 16 ; 5 4 0 ,

1969 H em o g lo b in P h i l l y ( b e t a 35 t y r - p h e ) ; s t u d i e s i n t h e m o l e c u l a r p a ih o lo g y o f h e m o g lo b in s , J . C l i n . I n v e s t . , 4 8 ; 1 6 2 7 -1 6 4 2 ,

R ohe , R .A ,1973 H em og lo b in D I r a n a l p h a 2 b e t a 2 22 g l u - g i n i n a s

s o c i a t i o n w i t h t h a l a s s e m i a . B lo o d , 4 2 ; 4 5 5 -4 6 2 ,R o s , G .V an, D .B e a l e , and H.LePanann

1968 Hemo l o b i n S t a n l e y v i l l e I I ( a l p h a 78 a s n - l y s ) , B r i t . M e d . J . , 4 ; 9 2 - 9 3 ,

R o s a , J , , G . P . B r i z a r d , A .G ib a n d , I . B e u z a r d , J . C . C o u r v a l i n , M. C o h e n - S o l a l , M .G .G a re l , a n d J . T h i l l e t

19 72 H em o g lob in S t . E t i e n n e ; a l p h a A b e t a 92 h i s - g l n (F 8 ) a new u n s t a b l e h e m o g lo b in w i t h no home on t h e b e t a c h a i n s . N o u v .R e v .F T .H e m a to l . , 12; 6 9 1 -6 9 9 .

R o s a , J , , N .M a le k in a , D .V e rg o z , and R .D u n e t1965 Une n o u v e l l e h é m o g lo b in e a b n o rm a le ; Hb J a l p h a P a r i s

12 a l a - a s p , N o u v .R e v ,F r .H e m a t o l . , 6 ; 4 2 3 -4 2 6 , S a d i k a r i o , A . , H.Duma, G .E frem o v , V .M la d e n o n s k i , a n d M .A ndreeva

1968 Hb J ; a n a l p h a c h a i n v a r i a n t f o u n d i n a I-iacedonian f a m i l y . A c ta B i o l , J u g o s l a v . S e r . G . l u g o s l a v . P h y s i o l , P h a rm a c o l .A C t a , 4 ; 1 9 1 -1 9 6 ,

S a lo m o n , H . , I . T a t a r s k i , N .D a n c e , E .R .H u e h n s , a n d a . M . s h o o t e r 1965 A new h e m o g lo b in v a r i a n t fo u n d ixi a B ed u in t r i b e ;

h e m o g lo b in Kambam. I s r a i l i J .M e d .S o i . , 1 ; 8 3 6 - 8 4 0 ,

90

S a n s o n e ,1965

ü a n s o n e ,1967

G , , and 0 . t i c k F a m i l i a l h e m o l y t i c a n e m ia w i t h e r y t h r o c y t e i n c l u s i o n b o d i e s , b i l i f u s c i n u r i a and a b n o rm a l h e m o g lo b in (Hb G a l l i e r a G e n o v a ) , B r i t , J , H e m a t o l . , 1 1 ; 5 1 1 -5 1 7 .

G , , R . W ,G a r r e l l , ;.nd H,Lehmann H em oglob in Genova b e t a 28 (BIOJ l e u - p r o . N a t u r e ,2 1 4 ; 8 7 7 - 8 7 9 ,

S a t h i a p a l a n , R . , an d M ,G .R ob in son1968 H e r e d i t a r y h e m o l y t i c a n em ia due t o an a b n o rm a l hemo

g l o b i n (Hb K i n g 's C o u n ty ) , B r i t , J , H a e m a t o l , , 1 5 ; 5 7 9 -5 8 7 .

S c h n e i d e r , R .G , , J . B . A l p e r i n , B . B r i m h a l l , an d R .T . J o n e s1969 H em o g lob in P ( a l p h a 2 b e t a 2 117 a r g ) ; s t r u c t u r e and

p r o p e r t i e s , J , L a b . C l i n . M e d . , 7 3 ; 6 1 6 -6 2 2 .S c h n e i d e r , R .G , , B . B r i m h a l l , R . T , J o n e s , n . B r y a n t , G .B ,M i t c h e l l fuid A. I . G o ld b e rg

1971 H em oglob in F t ,W o r th ; a l p h a 27 g l u - g l y (B 8 j ; a v a r i a n t p r e s e n t i n u n u s u a l ly ' lo w c o n c e n t r a t i o n s , ^ io o h lm , B i o p h y s ,A c t a , 2 4 3 ; 1 6 4 -1 6 9 .

S c h n e i d e r , R . G . , and R .T , J o n e s1965 H em og lob in F T e x a s ; a gamma c h a i n v a r i a n t . S c i e n c e ,

1 4 8 ; 2 4 0 .S c h n e i d e r , R . G . , S .V e d a , J . P . A l p o r i n , B .B r i m h a l l , a n d R . i . J o n e s

1969 H em o g lob in S a b in e b e t a 91 ( F ? ) l e u - p r o ; a n u n s t a b l e v a t i a n t c a u s i n g s e v e r e an em ia w i t h i n c l u s i o n b o d i e s . New E n g . J ,M e d . , 2 8 0 ; 7 3 9 - 7 4 5 ,

S c h r o e d e r , W.a . , and R .T , J o n e s1965 Some a s p e c t s o f t h e c h e m i s t r y and f u n c t i o n o f human

a n d a n im a l h e m o g lo b in s , F o r t c h r . d e r Chemie u r g a n - i s c h e r N a t u r s t o f f e . , 2 3 ; 1 1 3 -1 9 4 ,

S c h r o e d e r , W .A ,, J . R . S h e l t o n , T .H ,J .H u is m a n , J . B , S h e l t o n , l , F , K l e i n h a u e r , A .M ,B ozy , an d B .K o b b e rso n

1968 E v id e n c e f o r m u l t i p l e s t r u c t u r a l g e n e s f o r t h e gamma c h a i n o f human f e t a l h e m o g lo b in , P r o c . N a t ' l , A cad , S o i . ( W a s h . ) , 5 3 7 - 5 4 4 .

S c o t t , J . L . , A H a u t , G .E .C a r tv n ? ig h t , an d W.M.vVintrobeI 9 6 0 C o n g e n i t a l h e m o l y t i c d i s e a s e a s s o c i a t e d w i t h r e d

c e l l i n c l u s i o n b o d i e s , a b n o rm a l p ig m e n t m e ta b o l i s m an d an e l e c t r o p h o r e t i c h e m o g lo b in a b n o r m a l i t y .B lo o d , 16; 1 2 3 9 -1 2 5 2 ,

8 ,Amino a c i d s u b s t i t u t i o n i n h e m o g lo b in M a k i ta , J , G io c h e m ,, 6 3 ; 1 9 3 -1 9 o ,

5 . , I . I u c h i , T .M a z a g i , a n d I .T a k e d a H e m o g lo b in o p a th y i n J a p a n , B u l l .Y a m a g u c h i M e d .S c h o o l , 10 ; 1 - 9 ,

5 . , I . I u c h i , T . M i y a j i , S .U e d a , an d I . T a k e d a H e m o ly t ic d i s e a s e a s s o c i a t e d w i t h t h e p r o d u c t i o n o f a b n o rm a l h e m o g lo b in cOid i n t r a e r y t h r o c y t o c y t i c H e in z b o d i e s , N ippon K e t s u c k i Gsikkai Z a s s h i , 2 6 ; 1 6 4 -1 7 3 ,

5 . , T .M i y a j 1 , I . l u c h i , and A ,Tam ura S u b s t i t u t i o n o f t y r o s i n e f o r h i s t i d i n e (87 i n t h e a l p h a c h a i n o f M I w a t e ) , N ippon K e t s u c k i G akkai Z a s s h i , 2 7 ; 1 3 - 1 8 .

5 . , T . M i y a j i , S .U e d a , I . I u c h i , a n a I .T a d e d a H em oglob in H i k a r i ; a f a s t m oving homo l o b i n fo u n d i n two u n r e l a t e d J a p a n e s e f a m i l i e s , C l in ,C h e m ,A c ta , 1 0 ; 1 0 1 -1 0 5 ,

- h i b a t a ,1968

S h i b a t a ,1963

S h i b a t a ,1963

S h i b a t a ,19o4

S h i b a t a ,1964

91

Shim , B . S . , ü .U .C h on , Ï . H . L e e , Y .S ,K ang , K .J .H o n g » and O .S .Kim1 9 6 9 F o u r K orean hem o g lo b in m u t a n t s , i iu m .H e r e d , , 19;

1 7 0 -1 7 3 .S h i m i z u , A , , A .T s u g i t a , A .H a y a sh i , an d Y.Yamamura

1 9 65 The p r im a ry s t r u c t u r e o f h e m o g lo b in M i w a t e . B io - c h im ,B io p h y s ,A c ta , 107 ; 2 7 0 - 2 7 7 .

S i c k , K , , B ,B e a le , B . I r v i n e , H.Lehm ann, P . T . G o o d a l l , and S.Mac Kou g a l l

1967 H em oglobin G Copenhagen a n d h e m o g lo b in J C am bridge; two new b e t a v a r i a n t s o f h e m o g lo b in a , B ioch im . B io p h y s .A c ta , H O ; 2 3 1 -2 4 2 .

S i l v e s t r i o n i , E . , and I .B ia n c o1961 H em ato logy a s s o c i a t i o n o f h e m o g lo b in N and m ic r o -

c y te a e m ia i n a S a r d i n i a n f a m i l y , N a t u r e , 1 9 1 ;1 2 0 8 - 12 09 .

1 9 6 3 A new v a r i a n t o f human f e t a l h e m o g lo b in ; F Roma,B > o d , 2 2 ; 5 4 5 -5 5 3 .

S i l v e s t r i o n i , E . , 1 . B ia n c o , and C .B r a n c a t i1 9 6 3 h e m o g lo b in s N ;-.nd 1- i n I t a l i a n f a m i l i e s . N a tu r e ,

2 0 0 ; 6 o 8 -6 5 9 .S m i th , E .V i,, j i d J . V ,T o r b t r t

1 9 5 8 o tu d y o f two abnorm l h e m o g lo b in s w i t h e v id e n c e f o r a new g e n e t i c l o c u s f o r h e m o g lo b in f o r m a t i o n . B u l l , J o h n s Hopkins H o s p . , 102; 3 8 - 4 5 ,

S t a m a to y a n n o p o u lo s , G . , a . J.A daiuSon, A .Y o s h id a , an d S. H e in en b e rg1967 H em oglobin R a n i e r ; an a d u l t a l d a l a i r e s i s t a n t hemo

g l o b i n a s s o c i a t e d w i th e r y t h r o c y t o s i s . B lood J . H e m a to l . 30 ; 8 7 9 -8 8 0 ,

S ta m a to y a n n o p o u lo s , G , , x. E .N u te , J .W , Aoam son, A, J .B e l l i n g h a m , a n d B ,F d n k

1 9 7 3 H em oglobin O lym pia; b e t a 20 v a l - m e t ; a n e l e c t r o p h o r e t i c a l l y s i l e n t v a r i a n t a s s o c i a t e d w i t h h ig h oxygen a f f i n i t y and e r y t h r o c y t o s i s , J . C l i n , I n v e s , ,5 2 ; 3 4 2 -3 4 9 ,

S ta m a y o y a iu io p o u lo s , G . , and A .Y o sh id a19 69 S i c k l e c e l l a l k a l i r e s i s t a n c e i n h e m o g lo b in R a n ie r

( b e t a 145 t y r - h i s ; . S c i e n c e , 166 ; 1 0 0 5 -1 0 0 6 ,S t a n v l o v i c , M.

1 9 7 1 Hemog l o b i n M Novi S ad , h e d . R r i g l , , 2 4 ; 2 7 5 -2 7 8 , S te a d m a n , J . G . , A .Y a te s , and i^ .K .huehns

1970 I d i o p a t h i c Heinz body a n e m ia ; Hb B r i s t o l ( b e t a 67 (E 11) v a l - a s p ) , B r i t . J . H a e m a t o l . , 1 8 ; 4 3 5 -4 4 6 ,

S u k u m a ra n , T ,1 9 7 2 H em oglobin w I n d i a a; e o c i a t e d w i t h t h a l a s s e m i a ob

s e r v e d i n t h r e e Sinidhi f a m i l i e s , J .M ed. G e n . , 9; 4 3 6 -4 4 2 .

S u m id a , I .1973 Hem oglobin S aw ara ; a lp h a 6 (A 4 ) a l a - a s p . B ioch im .

B io p h y s .A c ta , 322 ; 2 3 -2 6 ,S w en so n , R . T , , R . L . H i l l , H.Lehmann, an d R . T . 3 , J im

19 62 A c h e m ic a l abnorm ality^ i n h e m o g lo b in G from C h in ese i n d i v i d u a l s , J .B i o l .C h e m . , 2 3 7 ; 1 5 1 7 -1 5 2 0 .

S y d e n s t r y c k e r , V . P . , B .H o r to n , R. A .P a y n e , .u idT .H , J .H u ism an1961 S t u d i e s on a f a s t h e m o g lo b in v a r i a n t f o u n d i n a

Negro f a m i ly i n a s s o c i a t i o n w i t h t h a l a s s e m i a , C l i n . O h e m .^ c ta , 6 ; 6 7 7 -6 8 5 ,

T a k a o k i , M ,, Y .O hba, K.Yamamoto, S . S h i o a t a , I . I u c h i , and H.B, H a m i l to n

1 9 68 Hem oglobin H if iy a m a ; a new f a s t m o v in g h e m o g lo b in i n a J a p a n e s e f a m i l y . S c i e n c e , 1 5 9 : 2 0 4 -2 0 6 .

9 2 *

T a n a k a , K .R , , Y ,o,W ong, and L ,H ,G r e e n b e r g1969 H em oglob in G H ar lem ; a new h e m o g lo b in c o m b i n a t i o n

a s s o c i a t e d w i t h h e m o ly t i c an em ia and s i c k l i n g and c o m p l i c a t e d b y h e m a tu r e a , J . C l i n . I n v e s . , 4 8 ; 8 3 a .

T e n t e r i , L , , M .C .S o r c i n i , and O .E u c e l l a1972 H em oglobin A bruzzo b e t a 143 (H 2 1 ) h i s - a r g , C l i n ,

C h im .A c ta , 3 8 ; 2 5 8 - 2 6 2 ,T u c h in a d a , S . , D ,B e a le , i-md H.Lehmann

1965 A n^w h a e m o g lo b in i n a T h a i f a m i l y , A c a s e o f hemog l o b i n S i r i r a j b e t a t h a l a s s e m i a , B r i t . M e d , J , , 1; 1 5 8 3 -1 5 8 5 .

V e l l a , P ,1973 H em oglobin W in n ip eg ; a l p h a 2 75 a s p - t y r , C l i n ,

B io c h e m , , 6 ; 6 6 - 7 0 ,V e l l a , P . , J .A .A g e r , a ao H,Lehmann

1958 An a b n o rm a l h e m o g lo b in i n C h in e s e ; h e m o g lo b in 0 ,N a t u r e , 182 ; 4 6 0 - 4 6 1 ,

V e l l a , P . , B .C h a r l e s w o r t h , l . A . L o r k i n , an a H,Lehmann1970 H em oglobin Bro u s a i s ; a l p h a 90 l y s - a s n , G a n . J .B i o

chem, , 4 3 ; 9 O8 - 91O.V e l l a , P . , and B ,Graham

1969 A v a r i a n t o f h e m o g lo b in A2 i n J Î l b e r t a I n d i a n s .C l i n ,B i o c h e m , , 2 ; 4 5 5 -4 6 0 ,

V e l l a , P . , J . n . H i l l , B , W i l t s h i r e , an d H,Lehmann1971 H em oglob in J T o r o n to ; a l p h a 5 a l a - a s p , C l i n , B i o

chem , , 4 ; 137- 1 4 0 ,V e l l a , P . , R .H ,W e l l s , J .A . A g e r , and H.Lehmann

1958 A h e m o g lo b in o p a th y i n v o l v i n g h e m o g lo b in G . n d a new (Q) h e m o g lo b in . B r i t . M c a . J . , 1 ; 7 5 2 -7 5 5 .

V e l l a , P . , S .C .W ong, J . B .W i l s o n , a n a T .H ,J .H u is m a n1972 H em o glob ins A - 2 -S p h a k ia and A-2-Nyu i n C a n a d a , Can.

J ,B i o c h e m , , 5 0 ; 8 4 1 - 8 4 5 .V o g e l , P. ^

1969 l o i n t m u t a t i o n s and humxm h e m o g lo b in v a r i a n t s . Hu- m a n g e n e t i k , 8 ; 1 - 2 7 ,

Wade, R .T . , T , J e n k i n s , a n a L . i i . i .u e h n s1967 H em oglob in v a r i a n t i n Bushmen; h e m o g lo b in B b e t a

Bushman, a l p h a 2 b e t a 2 16 g l y - a r g . N a t u r e , 2 1 6 ; 68 8 ,Wajcraan, i i . , 0 , B e lk h o d ja , cUid B .L a b ie

1970 H em oglobin N i o r t , a new u n s t a b l e h e m o g lo b in . B u l l . S o c .G h im .B io , , 5 2 ; 1 2 2 3 -1 2 3 1 .

W ajcman, H . , J .M .B o ig n e , B .L a b i e , and l . B e r i n g e1969 H em oglobin D Los A n g e le s , E v id e n c e o f uhe s t r u c t u r a l

a n om aly o b t a i n e d by v a t i o u s m e th o d s . B io c h im ,B io p h y s .A c t a , 1 88 ; 5 5 - 5 8 ,

ivajcraan, H . , B .L a b i e , a n d G .E c h a p i r a1 9 7 ^ Two h e m o g lo b in v a r i a n t s w i t h d e l e t i o n s ; T o u r s ; t h r

87 (P 3 ) d e l e t e d and S t . A n t o i n e g l y - l e u 7 4 -7 5 (E 18- 19) . d e l e t e d , ^^G oüsequences f o r oxygen a f f i n i t y an d p r o t e i n s t a b i l i t y , B i o c h i m ,B io p h y s .A c ta , 2 9 5 ; 4 9 5 -5 0 4 .

w ajcm an , H . , a L e ro u x , a n d B . L a b i e '1973b ^ n o t i o n a l p r o p e r t i e s o f h e m o g lo b in Hammersmith.

B io c h im ie , 5 5 ; 1 1 9 -1 3 5 .Wajcman, H . , e t . a l ,

1973c H em oglob in S e t i f ; G 1 94 a s p - t y r , A new c h a i n hemog l o b i n v a r i a n t w i t h s u b s t i t u t i o n o f t h e r e s i d u e i n v o lv e d i n h y d ro g e n b o n d in g b e tw e e n u n l i k e s u b u n i t s . PEBS L e t t . , 2 7 ; 299- 3 0 0 ,

' 5 - , 9 . ' ^

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W a l l e t , L . H . B , , a n d J ,B ,R o b i n s o n1^68 Human h e m o g lo b in v a r i a n t s and t h e i r i d e n t i f i c a t i o n ,

B u t t e r s v / o r t h , L ondon ,W a s i , P ,

1972 I n c i d e n c e o f h e m o g lo b in T h a i ; a r e - e x a m i n a t i o n o fa l p h a t h a l a s s e m i a d i s e a s e s , Ann,Hum,Gen, , 35 ; 4 6 7 - 47Ü,

W a t s o n - v j i l l i a m s , E. J . , D, B e a l e , B . i r v i n e , and H,Lehmann1965 A new h e m o g lo b in , D I b i d a n ( b e t a 87 t h r - l y s ) p r o

d u c in g no s i c k l e c e l l Hb 1) d i s e a s e s w i t h Hb 3 , N a t u r e , 2 0 5 ; 1 2 7 3 -1 2 7 5 ,

W e a t h e r a l l , D . J .1 9 6 4 H em og lob in J B a l t i m o r e c o e x i s t i n g i n a f a m i l y w i t h

h e m o g lo b in 8 - 1 . B u l l , J o h n s H o p k in s H o s p . , 1 1 4 ; 1 - 1 2 , W e a t h e r a l l , D , J . , a n d C .B a g l i o n i

1962 A f e t a l h e m o g lo b in o f u n u s u a l g e n e t i c i n t e r e s t .B lo o d , 2 0 ; 6 7 5 .

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