BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 243, 531–537 (1998)ARTICLE NO. RC988125

Triplet Repeat-Containing Ribosomal Protein L14 Genein Immortalized Human Endothelial Cell Line (t-HUE4)

Manami Tanaka,*,1 Tomoo Tanaka,*,† Masamitsu Harata,‡ Tohru Suzuki,*,‡ and Youji Mitsui**The National Institute of Bioscience and Human-Technology, 1-1 Higashi, Tsukuba Science City, Ibaraki 305, Japan;†Division of Host Defense Mechanism, Tokai University School of Medicine, Isehara, Kanagawa 259-11, Japan; and‡Department of Biotechnology, Gifu University, Yanagito, Gifu 501-1193, Japan

Received December 24, 1997

and approximately 80 different ribosomal proteins (1).A cDNA encoding human 60S ribosomal subunit pro- Ribosomal protein is abundant, low molecular weight

tein L14 (hRL14) was isolated from a human immortal (Mr õ 35,000), mostly basic proteins intrinsic to struc-endothelial cell line, t-HUE4. This cell line was estab- ture and function of the ribosome. Eukaryote ribosomelished via a series of cell lines cultured in a serum-free contains over 75 ribosomal proteins, whereas prokary-and a protein-free medium, and a directional cDNA li- otic ribosome contains only up to 52 (2, 3). Many ribo-brary has been constructed and screened in search for

somal proteins bind directly to rRNA backbone at thethe genes modulating protein synthesis machinery insites with specific primary and/or secondary structures,cell proliferation. A putative full-length clone with anand to a number of substrates for specific protein ki-open reading frame of 220 amino acids; predicted molec-nases and protein phosphatase, which may serve forular weight of 23.6 kDa. A significant identity for hRL14the fine tuning of protein synthesis.was observed with rat RL14 (85% identity), with excep-

The most ribosomal protein genes are present 7 totion of COOH-terminal region, but not with any eukary-20 copies in the genome (4), although some, such as ratote amino acid sequences so far deposited to database.ribosomal protein S5 (5), rat ribosomal protein L14 (6),The typical features of ribosomal proteins were ob-human ribosomal protein L37a (7), and human ribo-served in hRL14, as seen in nuclear targeting sequences

necessary for the transport from cytoplasm to nucleolus, somal protein L29 (8), are present as a single or twoa bZIP like (basic region-leucine zipper) element for the copies per haploid genome. Their expression is coordi-binding to rRNA, and the internal repeat sequences; the nately regulated by various means to produce equimo-pentapeptide QKA(A/S)X. The COOH-terminal region of lar amounts of protein that show a rough agreementthe transcripts contained fifteen triplet repeats (GCT; with appropriate rRNA concentrations.alanine) at nucleotide 465 to 509, which is significantly We have initiated biochemical and molecular biologicalexpanded compared to the rat RL14. However, the re- approaches to identify and characterize key moleculespeat number was all the same among the normal human which control specific functions coordinated by humanendothelial cell line and the cell lines established in the endothelial cells. Endothelial cells in the inner-most partcourse of t-HUE4 establishment. A single band with of blood vessels contribute to important roles such as;about 800 bases was identified by Northern blot analysis 1) controlling substance permeability between blood andwithout tissue specificity. This GCT repeat was found to tissues, 2) controlling blood pressure through productionbe one of the longest uninterrupted repeats in a coding or decomposition of vasoactive substances, 3) main-sequence, which were associated with the highest de-

taining antithrombus feature in the vascular endothe-gree of polymorphism. q 1998 Academic Presslium, and 4) angiogenesis. A development of immortalendothelial cell lines is valuable for these studies (9-11).

The mammalian ribosome is a macromolecule assem- As a long course of our studies, we have developed humanbly which consists of four different molecules of RNA and porcine immortal endothelial cell lines, which could

grow in serum- or protein-free medium, for easy purifica-tion of novel bioactive substances (9). In fact, these cell1 To whom correspondence should be addressed. The National In-

stitute of Bioscience and Human-Technology, 1-1 Higashi, Tsukuba lines have been used to isolate and analyze highly im-Science City, Ibaraki 305, Japan. Fax: /81-298-54-6503. E-mail: portant physiological substances such as growth factorstmanami@nibh.go.jp. and endothelin(s) (10-12).Nucleotide sequence data reported in this paper have been submit-

A cDNA library was thus constructed from one of theted to the DDBJ/EMBL/GenBank DNA databases with the AccessionNo. D87735. immortal human endothelial cell lines, t-HUE4 (9), and

0006-291X/98 $25.00Copyright q 1998 by Academic PressAll rights of reproduction in any form reserved.

531

AID BBRC 8125 / 6948$$$581 01-26-98 10:37:31 bbrcg AP: BBRC

Vol. 243, No. 2, 1998 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

partial amino acid sequences were obtained by peptide sequencingthe library was further screened for the characteristicof the proteins secreted abundantly in the culture media of t-HUE4genes which are responsible for their immortality, pro-cells. The oligonucleotide 5*-CA(G/A)CT(G/A/T/C)TA(C/T)TA(C/T)(C/liferative capacity, and vigorous protein synthesis in a A)G(G/A/T/C)CA(T/C)CA(T/C)-3 * was thus synthesized and further

protein-free culture media. Herein we report the clon- used for screening of t-HUE4 cDNA library using the GIBCO BRLGeneTrapper cDNA Positive Selection System (16, 17) according toing and the deduced amino acid from the nucleotidethe manufacturer’s instructions (Life Technologies, Inc., USA). Thesequences of human ribosomal protein L14 (hRL14)captured single strand cDNAs were converted to double strand DNAcloned from the t-HUE4 cDNA library. The character-with a DNA polymerase, and transformed into ElectroMAX DH10B

ized hRL14 showed a strong identity with rat ribosomal cells (Life Technologies, Inc., USA). Colonies on the plates containingprotein L14 recently reported (6), with exception at 100 mg/ml ampicillin were screened by colony hybridization as de-

scribed previously (18, 19). The positive clones were further screenedCOOH-terminal region. Fifteen triplet repeats (GCT:by sequencing the inserted cDNA.alanine) were identified at amino acid 150-164. Al-

DNA sequencing. Plasmid DNA from the positive colonies ob-though the triplet repeat in hRL14 showed a significanttained as above was prepared using QIAprep Spin Plasmid Kit (QIA-expansion compared with rat RL14 gene (6), the repeatGEN Inc.). Nucleotide sequencing of the inserted DNA was per-number was all the same among the nomral human formed with the DNA Sequencer 373S (Applied Biosystems) by Sang-

endothelial cell line (HUVEC), and the different stages er’s method (20). Sequencing of PCR product was performed usingof cell lines obtained from human umbilical vein en- TA cloning Kit (Invitrogen, Corp.) as previously described (18). The

DNA sequences were analyzed with the basic local alignment searchdothlial cells (HUVEC) through the course of t-HUE4tool program for nucleotides (BLASTN) and amino acids (BLASTX)establishment. Like minisatellites, triplet repeats have(21) in DNA Data Bank of Japan (DDBJ).been found to be widely dispersed in human genomic

Northern blot analysis. Multiple tissue northern blots membraneDNA, and the length of the triplet repeats of several(CLONTECH Laboratories, Inc.) containing human RNA samplesgenes in the database was found to be highly polymor- was used for the study of expression level of hRL14 gene in various

phic (13, 14). The obtained results were discussed in human tissues. A whole region of hRL14 gene was used as a probethe light of genetic instability, repeat stability, and the to hybridize with tissues 2 mg of poly (A)/ RNA per lane. The condi-

tion for hybridization and washing were as previously described (18,characteristics of hRL14 gene.19). The size of the mRNAs was estimated from RNA standards (LifeTechnologies, Inc.).

MATERIALS AND METHODSPCR amplification of genomic DNA and RNA. Primers used for

the amplification of genomic DNA and RNA were as follows: forwardCell lines and the culture conditions. In a series of our studies to primer 5*-GCA CCT GGT ACT AAG GGT ACT GC-3 *; reverse primer

isolate bioactive substances from the culture medium of mammalian 5*-GGA GCA GGC GCT GCT TTC TGG C-3* (indicated by arrowscells, Kobayashi et al. (9) have established t-HUE2 clone which could in Figure 1). PCR was performed using 30 cycles of 95 7C (30 s), 62grow in a serum-free medium and then t-HUE4 clone in protein-free 7C (30 s), and 72 7C (1 min) using Premix Taq (Takara Shuzo, Japan)culture from a human transformed endothelial cell line, ECV304. according to the manufacturer’s instructions. The PCR product wereNormal human endothelical cells (HUVEC) were isolated from an subject to the gel-electrophoresis in a 12.5 % GeneGel (Pharmaciaumbilical cord vein and subcultivated in MCDB 151 medium (Sigma, Biotech) by GenePhor system (Pharmacia Biotech).St. Louis, USA) supplemented with 15% fetal bovine serum (FBS)up to 70 population-doubling levels (PDL) of in vitro lifespan as pre-

RESULTS AND DISCUSSIONviously described (9, 10). t-HUE2 was established as a subclonewhich can grow in serum-free medium, ASF301 (Ajinomoto, Tokyo,Japan) that contained albumin (100 mg/L), transferrin (10 mg/L), The Nucleotide Sequence of Recombinant cDNAinsulin (1 mg/L), and epidermal growth factor (EGF, 0.01 mg/L) as Encoding hRL14protein components (9-11). t-HUE4 was selected from t-HUE2 cellsby related cloning of cells that could grow in a lower concentration The positive selection of cDNA using GeneTrapperof protein components, and finally established as a stable cell line system and the following colony hybridization yieldedwhich can be subcultured in completely protein-free media (9). They

many colonies which contained different new se-were maintained in vitro with a basal medium such as Ham’s F-12without any supplement. quences. One of the clones, t-HUE4-4, had an open

reading frame which showed a strong identity with theConstruction of cDNA library. A cDNA library was constructedsequence of amino acids encoded rat ribosomal proteinusing SuperScript Plasmid system (Life Technologies, Inc.) according

to the manufacturer’s instructions (15, 16). Total RNA from 51106 of L14 (6). A complete sequencing of the t-HUE4-4 cDNAgrowing phase t-HUE4 cells was prepared with GIBCO/BRL TRIzol insert revealed that it contained a whole region of thereagent, and the poly(A)/ RNA was isolated using oligo(dT) cellulose human ribosomal protein L14 (hRL14), excluding the(MESSAGE MAKER Reagent Assembly, Life Technologies, Inc.).

poly(A)/ tail, is 722 bp in length (Fig. 1). The cDNASynthesized cDNA was directionally cloned into the NotI-SalI regionof the eukaryotic expression vector pCMV-SPORT (Life Technologies, clone contained the whole region of hRL14, and theInc.). After transformation into ELECTROMAX DH10B cells (Life insert had a 5* noncoding sequence of 17 bases, a singleTechnologies, Inc.), 5.61106 independent clones were amplified once open reading frame of 663, a 3 * noncoding sequence ofusing a semi-solid culture procedure to help preservation of the dis-

42, and a poly(A)/ tail. In the other two reading framestribution of clones in the original library. This library was furtherthe sequence are interrupted by termination codons.screened for its quality by random sequencing of the insert DNA.The open reading frame begins at an ATG codon atScreening of t-HUE4 cDNA library. Degenerate oligonucleotidesnucleotide 18 and ends with a stop codon (TAA) at posi-were used to capture the specific t-HUE4 cDNA which encodes the

amino acid sequences derived from preliminary examinations. The tion 680; it spans 220 amino acids (Fig. 1). The hexamer

532

AID BBRC 8125 / 6948$$$581 01-26-98 10:37:31 bbrcg AP: BBRC

Vol. 243, No. 2, 1998 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

FIG. 1. The nucleotide sequence and the predicted amino acid sequence of human ribosomal protein L14 (hRL14). Bold letters showthe repetitive region of ‘‘GCT (amino acid: alanine)’’ at nucleotide 465 to 509 . Both forward and reverse primer to amplify the alaninerepeat region are shown by arrows, and the nucleotides covered with a box are polyadenylation site.

AATAAA for polyadenylation is at position 699-704, 24 ber of basic residues (13 arginyl, 37 lysyl, and 3 histi-dyl) over acidic ones (7 aspartyl and 4 glutamyl). Clus-nucleotides upstream of the start of the poly (A)/

ters of basic residues in ribosomal proteins are commonstretch (22). Pyrimidine sequences are found at the im-and in hRL14 there are 18 in a sequence of 40 residuesmediate 5* end of the most, if not all, eukaryotic ribo-at positions 93-132.somal protein mRNAs; the presence of the polypyrimid-

The alanine content of hRL14, 22.8 mol %, is unusu-ine stretch can be taken to confirm that the mRNAally high; the average for mammalian ribosomal pro-includes a region where transcription started (1). Theteins is 8 mol % (1, 6). Thirty one of the 49 alanines5* end pyrimidine sequences, which have been sug-are clustered in the 72 carboxyl-terminal residues (Fig.gested to play a role in the regulation of ribosomal1) including a sequence of 15 consecutive alanines. Theprotein mRNA translation (23) were absent from thisother mammalian ribosomal proteins with a high ala-transcript.nine content are P0 (12 mol %), P1 (20 mol %), and P2

The Primary Structure of hRL14 (17 mol %) (24), whereas a typical mammalian proteinThis cDNA sequence predicts a molecular weight of has about 7 mol% alanine (25). In these three proteins

the alanines are also clustered at their carboxyl termi-23.6 kDa with pI 11.6. This pI is due to an excess num-

533

AID BBRC 8125 / 6948$$$581 01-26-98 10:37:31 bbrcg AP: BBRC

Vol. 243, No. 2, 1998 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

FIG. 2. Multiple sequence alignment of hRL14 and rat RL14. Identical amino acids are shown as boxes and lack of amino acids in ratRL14 as ‘‘-’’.

nus. There are exceptions that include: myristoylated common in ribosomal proteins with little functionalinterpretation (6).alanine-rich C-kinase substrate (26 mol %) (26); a hu-

man signal recognition particle protein 14 (18 mol %) Between residues 100 and 119 there are overlappingputative nuclear targeting sequences RKAKMTDFD-(27); and histone H1 (25 mol %) (28, 29). The latter two

are nucleic acid binding proteins. In these proteins, RFKVMKAKKMR that confirm to the bipartite consen-sus sequence (30). Nuclear targeting sequences are be-alanines are observed in clusters. However, their func-

tion is yet unknown. In histone H1, alanines and ly- lieved to be necessary for the transport of eukaryoticribosomal proteins from the cytoplasm where they aresines form a highly charged random coil at the carboxyl

terminus (29). Of these alanine rich proteins only synthesized to the nucleolus where they are assembledon nascent rRNA.hRL14 and histone H1 have additional significant

amino acid sequence similarities. There is a bZIP like (basic region-leucine zipper) ele-ment (31, 32), RFKVMKAKKMRNRIIKNEVKK-LQK-There are internal repeat sequences in hRL14; the

pentapeptide QKA(A/S)X occurs in tandem four AALLKASPKKA, between residues 109 and 143. Thebasic region is supposed for to be DNA recognition andtimes at position 181 to 212. Internal repeats are

FIG. 3. Multiple sequence alignment of human homologue genes; hRL14, CAG-isl7, and CTG-B33. Identical amino acids are shown asboxes and lack of amino acids as ‘‘-’’.

534

AID BBRC 8125 / 6948$$$581 01-26-98 10:37:31 bbrcg AP: BBRC

Vol. 243, No. 2, 1998 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the leucine zipper is for dimerization through coiled-coil formation. Other ribosomal proteins have bZIP-like motifs (33, 34) that may bind to rRNA, while theirfunction has not confirmed (34).

Comparison of the Amino Acid Sequencesof hRL14-Related Genes

The amino acids sequence of hRL14 was comparedusing the computer programs BLASTN and BLASTX(21), with the amino acid sequences of more than 1,400ribosomal proteins contained in a library that we havecompiled, and to nucleotide sequences in the GenBankdata base. Our sequence is related to a number of pre-viously unidentified partial amino acid sequences fromhuman, rat, mouse, plants and yeast. The predictedamino acid sequence of hRL14 cDNA exhibits 85%identity to rat ribosomal protein RL14 (Fig. 2) (6). Theprimary structure of rat ribosomal protein and thehRL14 amino acid sequence is 92% identical over thefirst 147 residues, and is 75% similar in 56 residuesafter the repetitive region in the hRL14, suggesting animportant function of N-terminal domain. This regionof relatively low homology is observed to be rich inpositively charged amino acids and contain similarnumbers of basic residues in both rat and human se-quences (Fig. 2).

The hRL14 also showed a significant identity withFIG. 4. The molecular length of alanine repeat region in differentthe human protein sequence CTG-B33 (accession num- stages of human cell lines. PCR amplification of genomic DNA from

ber L10376) (13) and CAG-isl7 (14). CTG-B33 shares HUVEC (lane 1), t-HUE2 (lane 2), and t-HUE4 (lane 3) was per-91 % amino acid identity with hRL14, and CAG-isl7 formed using the primers indicated in Materials and Methods sec-

tion. 100 bp ladder (Life Technologies, Inc.) was used as a marker.shares 91 % (Fig. 3). CTG-B33 was identified from hu-man brain cDNA library as an element responsible forneuropsychiatric illness (13), and CAG-isl7 was identi-fied from human pancreatic islets in search for the morphic CAG-isl7 in human population, suggests that

this repeat sequence was expanded through the evolu-genes responsible for diabetes (14). The significant dif-ferences among these three amino acid sequences were tional process with a certain amount of instability

among the populations in the same species. The chro-the number of the GCT (alanine) repeat sequence(boxed in bald letters in Fig. 4), and the entire differ- mosome location of hRL14 gene was assigned on 3p,

within 10.9 cM gap between polymorphic markersences in COOH-terminal region. The repeat unit ‘‘GCT’’at nucleotide 465 to 509, observed in open reading D3S1260 and D3S1289, as reported previously using

CTG-B33 (13). The diversity found at COOH terminalframe, was expanded in t-HUE4 compared with CTG-B33 and CAG-isl7. However, there is no differences in region still remains unknown.

A variety of tumors, including cultivated immortal-alanine repeat numbers among normal and differentstages of transformed human endothelial cell lines, ized cells, have been found to harbor alterations in re-

petitive sequences. Such alterations very often reflectHUVEC, t-HUE2, and t-HUE4, as detected by PCRamplification using both RNA and genomic DNA fol- a malfunction in the replication or repair of DNA which

persists throughout the lifespan of the cell. Therefore,lowed by the sequencing (Fig. 4). This GCT repeat wasfound to be one of the longest uninterrupted repeats we examined the possibility of genomic mutation in

our cell lines using human mutL-related homologuein a coding sequence among the clones, which wereassociated with the highest degree of polymorphism hMLH1 sequence (35), the gene responsible for multi-

ple advanced cancer (MMAC1 or PTEN) (36, 37); micro-among the patients and normal samples (13, 14). Trip-let repeats tend to expand with pathogenic conse- satellite markers on chromosome 2, 7, and 11 (38-40).

However, no alterations were detected so far amongquences, however, the defined function of these repeatswith polymorphisms is only partially understood. The the cell lines HUVEC, t-HUE2, and t-HUE4, used in

the present study (data not shown).present data, taken together with the findings ofshorter alanine repeat of rat RL14 gene and the poly- Although the mechanism of triplet repeat expansion

535

AID BBRC 8125 / 6948$$$581 01-26-98 10:37:31 bbrcg AP: BBRC

Vol. 243, No. 2, 1998 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

FIG. 5. Northern blot analysis of hRL14 expression. Each lane contained 2mg of polyadenylated RNA from adult human tissues, andthe probe consisted of the whole region of t-HUE4 RL14 gene.

Eds)., pp. 203–214, American Society of Microbiology, Washing-is uncertain, our present data indicate that the tripletton, DC.repeat number in the genome of somatic cells does not

2. Otaka, E., and Ozawa, S. (1981) Mol. Gen. Genet. 181, 176–182.change during a long course of cell division even under3. Otaka, E., K. I., and Itoh, T. (1984) Mol. Gen. Genet. 195, 544–the severe culture condition such as a serum-free or

546.protein-free media. The genomic instability is more4. Monk, R. J., Meyuhas, O., and Perry, R. P. (1981) Cell 24, 301–likely to occur by genetic alterations such as ‘‘anticipa-

306.tion’’, characterized in human genetic diseases or neu-

5. Kuwano, Y., Olvera, J., and Wool, I. G. (1992) J. Biol. Chem.rological disorders (13, 14). 267, 25304–25308.6. Chan, Y., Olvera, J., and Wool, I. G. (1996) Biochem. Biophys.

The Size of the mRNA Encoding hRL14 Res. Comm. 222, 427–431.7. Saha, D. P., Tirumalai, P. S., Scala, L. A., and Howells, R. D.To determine the size of hRL14 mRNA, Northern blot

(1993) Gene 132, 285–289.analysis was performed with mRNAs extracted from8. Law, P. T. W., Tsui, S. K. W., Lam, W. Y., Luk, S. C. W., Hwang,multiple human tissues. One distinct band of predicted

D. M., Liew, C. C., Lee, C. Y., Fung, K. P., and Waye, M. M. Y.,length (722 base pairs) was detected (Fig. 5). The ex- (1996) Biochem. Biophys. Acta 1305, 105–108.pression of hRL14 gene did not show any tissue speci- 9. Kobayashi, M., Kondo, M., and Mitsui, Y. (1991) Human Cell 4,ficity as demonstrated in the figure. 296–305.

10. Kondo, M., Ishida, N., Kobayashi, M., and Mitsui, Y. (1991) J.Cardiovascular Pharmacol. 17(Suppl. 7), S52–S54.ACKNOWLEDGMENTS

11. Kondo, M., Ishida, N., Kobayashi, M., and Mitsui, Y. (1992) Bio-The authors thank Ms. Akira Togiya of Takara Shuzo, Co., Ltd., chem. Biophys. Acta, 1134, 242–246.

Japan, for sequence service; Dr. Wu-Bo Li of Life Technologies, Inc., 12. Yanagisawa, M., Kurihara, H., Kimura, S., Tomobe, Y., Kobay-USA., for valuable discussions and technical advices. This work was ashi, M., Mitsui, Y., Yazaki, Y., Goto, K., and Masaki, T. (1988)supported by grants from Agency of Industrial Science and Technol- Nature 332, 411–415.ogy (AIST) Human Science Frontier Program; Science and Technol-

13. Li, S. H., McInnis, M. G., Margolis, R. L., Asntonaraskis, S. E.,ogy Agency (STA) Center of Excellence Program (COE); Science andand Ross, C. A. (1993) Genomics 16, 572–579.Technology Agency (STA) grant for Promotion of Basic Research;

14. Aoki, M., Koranyi, L., Riggs, A. c., Wasson, J., Chiu, K. C., Vaxil-Ministry of Education, Science, and Culture, Japan (Nos. 06670253laire, M., Froguel, P., Gough, S., Liu, L., Donis-Keller, H., andand 08559002).Permutt, M. A. (1996) Diabetes 45, 157–164.

15. Simms, D., Cizdziel, P. E., and Chomczynski, P. (1993) Focus 15,REFERENCES99–100.

16. Li, W-B., Cruber, J. E., Noon, M. C., Polayees, D., Schmidt, B.,1. Wool, I. G., Endo, Y., Chan, Y. L., and Gluck, A. (1990) in TheRibosome: Structure, Function, and Evolution (Hill, W. E., et al., and Jessee, J. (1995) Focus 17, 45–49.

536

AID BBRC 8125 / 6948$$$581 01-26-98 10:37:31 bbrcg AP: BBRC

Vol. 243, No. 2, 1998 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

17. Nisson, P., Li, W.-B., Gruber, C., Macky, J., and Jesse, J. (1995) 30. Dingwall, C., and Lackey, R. A. (1991) Trends Biochem. Sci. 16,478–481.Focus 17, 104–105.

31. Ellenberger, T. E., Brandl. C. J., Struhl, K., and Harrison, S. C.18. Tanaka, T., Iida, A., Kaneda, Y., and Tanaka, M. (1994) Int. J.(1992) Cell 71, 1223–1237.Parasitol. 24, 179–188.

32. Pathak, D., and Sigler, P. B. (1992) Current Opinion in Struc-19. Sambrook, J., Fritsch, E. F., and Maniatis, T. (1989) Moleculartural Biol. 2, 116–123.Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory

33. Chan, Y. L., Olvera, J., Gluck, A., and Wool I. G. (1994) J. Biol.Press, Cold Spring Harbor, NY.Chem. 269, 5589–5594.20. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl.

34. Hemmerich, P,m con Mikecz, A., Neumann, F., Sozeri, O., Wolff-Acad. Sci. USA 74, 5463–5467.Vorberk, G., Zoebelein, R., and Krawinkel, U. (1993) Nucleic

21. Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, Acid Res. 21, 223–231.D. (1990) J. Mol. Biol. 215, 403–410.

35. Liu, B., Nicolaides, N. C., Markowits, S., Willson, J. K., Parsons,22. Proudfoot, N. J., and Brownlee, G. G. (1976) Nature 263, 211– R. E., Jen, J., Papadopolous, N., Peltomaki, P., de la Chapelle,

214. A., Hamilton, S. R., Kinzler, K. W., and Vogelstein, B. (1995)23. Perry, R. P., and Meyuhas, O. (1990) Enzyme 44, 83–92. Nature Genet. 9, 48–55.

36. Han, H.-J., Yanagisawa, A., Kato, Y., Park, J.-G., Nakamura, Y.24. Wool., I. G., Chan, Y. L., and Gluck, A. (1996) in Translation(1993) Cancer Res. 53, 5087–5089.Control (Hershey, J. W. B., Mathews, M. F., and Sonengerg, N.,

Eds.), pp. 685–732, Cold Spring Harbor Laboratory Press, Cold 37. Risinger, J. I., Berchuck, A., Kohler, M. F., Watson, P., Lynch,Spring Harbor, NY. H. T., and Boyd, J. (1993) Cancer Res. 53, 5100–5103.

38. Peltomaki, P., Lothe, R. A., Aaltonen, L. A., Pylkkanen, L., Nys-25.Wada, K., Wada, Y., Doi, H., Ishibashi, F., Gojobaori, T., andtrom-Lahti, M., Seruca, R., David, L., Hold, R., Ryberg, D.,Ikemura, T. (1991) Nucleic Acids Res. 19 supplement, 1981–Haugen, A., Brogger, A., Borresen, A.-L., and de la Chapelloe,1986.A. (1993) Cancer Res. 53, 5853–5855.26. Seykora, J. T., Ravetch, H. V., and Aderem, A. (1991) Proc. Natl.

39. Steck, P. A., Pershaouse, M. A., Jassar, S. A., Yung, W. K. A, Lin,Acad. Sci. USA 88, 2505–2509.H., Ligon, A. H., Langford, L. A., Baumgard, M. L., Hattier, T.,

27. Chang, D. Y., Nelson, B., Bilyeu, T., Hsu, K., Darlington, G. J., Davis, T., Frye, C., Hu, R., Swedlund, B., Teng, D. H. F., andand Maraia, R. J. (1994) Mol. Cell. Biol. 14, 3949–3959. Tavtingian, S. V. (1997) Nature Genet. 15, 356–362.

28. Allan, J. Hartman, P. G., Crane-Robinson, C., and Aviles, F. X. 40. Li, J., Yen, C., Liaw, D., Podsypanina, K., Bosse, S., Wang, S. I.,(1980) Nature 288, 675–679. Puc, J., Miliaresis, C., Rodgers, L., McCombie, R., Bigner, S. H.,

Giocanella, B. C., Ittmann, M., Tycko, B., hibshoosh, H., Wigler,29. Zlatanova, J., and Yanebva, J. (1991) DNA and Cell Biol. 10,239–248. M. H., and Parsons, R. (1997) Science 275, 1943–1947.

537

AID BBRC 8125 / 6948$$$581 01-26-98 10:37:31 bbrcg AP: BBRC