MINIREVIEW Polymorphisms
in the Transcribed 3’ Untranslated of Eukaryotic Genes
Region
RovC. LEVITT Department
of Anesthesiology and Critical Care Medicine, Johns Hopkins Medical 600 North Wolfe Street, Baltimore, Maryland 2 1205
INTRODUCTION
approximately 1 in every 100 bp (Orkin et al., 1982; Jeffreys, 1979; Chakravarti et al., 1984, 1986; Antonarakis et al., 1982), the transcribed untranslated regions of eukaryotic genes should contain multiple nucleotide changes (depending on the length of this region) that would represent a very useful resource for genetic mapping. These sequence polymorphisms should be easily demonstrated by PCR and established electrophoretic techniques (Sheffield et al., 1989; Orita et al., 1989; Bolos et al., 1990; Traystman et aZ., 1990; Cawthon et al., 1990).
The etiology of most genetic disorders remains unknown and the vast majority of mutant genes are recognized only by the abnormal phenotype they produce. Establishing linkage with polymorphic DNA markers is often a crucial first step in the localization and eventual cloning of a given disease gene. High-resolution genetic maps will expedite the process of localizing disease genes. The construction of a high-resolution linkage map as an objective of the Human Genome Project is largely dependent on identifying DNA polymorphism for use as markers. At this time linkage maps of approximately 10 CM exist for nearly every human chromosome (Drayna and White, 1985; Donis-Keller et al., 1987; White and Lalouel, 1988; Human Gene Mapping 10, 1989, Warren et al., 1989; Petersen et aZ., 1991; Nakamura et al., 1988, O’Connell et al., 1988). Most of the markers used to construct these linkage maps are restriction fragment length polymorphisms (RFLPs) detectable by Southern blot hybridization (Southern, 1975). Although these techniques have greatly advanced our understanding of the organization of the human genome, they require nucleic acid probes, are laborious to demonstrate, and are often uninformative in linkage analyses. New classes of DNA polymorphisms, several of which can be demonstrated directly by polymerase chain reaction (PCR), have recently been described (Economou et aZ., 1990; Jeffreys et aZ., 1985; Nakamura et al., 1987; Litt and Luty, 1989; Weber and May, 1989; Semenza et al., 1984; Chebloune et al., 1988; Tautz, 1989; Saiki et al., 1985; Sinnett et al., 1990; Zuliani and Hobbs, 1990). The advantages of PCR-based methods are that they are faster and therefore less costly; they do not require nucleic acid probes; and, being directly sequence based, they are more sensitive for detecting polymorphisms at a given locus, Although these newer markers are often multiallelic and frequently heterozygous and therefore highly informative for linkage analyses, they are not generally useful for mapping cDNAs (i.e., they are not commonly found within transcribed sequences). Another potential limitation of this approach is that DNA sequence information is required to generate the oligonucleotide primers necessary for the PCR amplification. Ultimately, polymorphisms in eukaryotic genes that are efficiently demonstrated by PCR methods and can be used to map and evaluate cDNAs as candidate genes in human disorders would facilitate the scientific goals of a large number of researchers. A significant portion of the sequence information on the human genome (and that of other eukaryotes) has been derived from cloning and direct sequencing of cDNAs. Since polymorphic variation is thought to occur in noncoding sequence as frequently as GENOMICS 11,484-489 (1991) 0888-7543/91$3.00 Copyright 0 1991 by Academic Press, All rights of reproduction in any form
POLYMORPHISMS IN THE TRANSCRIBED UNTRANSLATED REGION OF EUKARYOTIC
3’ GENES
Aside from one or more putative polyadenylation, transcription termination, and/or regulatory signals, little is known about the function of the transcribed 3’untranslated region of eukaryotic genes (Birnstiel et aZ., 1985). Although the 3’ noncoding region may have a role in mRNA stability (Brawerman, 1987), it does not appear essential for mRNA translation (Kronenberg et al., 1979). Furthermore, size heterogeneity (Setzer et aZ., 1980) and a lack of evolutionary conservation (Perryman et al., 1986; Cleveland et al., 1980) support the notion that only limited discrete sequences serve a regulatory function in this region of eukaryotic transcripts. Most importantly, if the structure of the transcribed 3’ untranslated region of eukaryotic genes is largely not conserved, in contrast to coding regions, then sequence divergence within this region of the genome may represent readily accessible and highly useful markers for genetic mapping. As an initial test of the hypothesis that the transcribed 3’ untranslated region of eukaryotic genes is polymorphic, we reviewed the literature or GenBank on 27 genes (or cDNAs) that were sequenced two or more times. Twenty-four of twenty-seven of these sequences were found to contain one or more deletions and/or substitutions in their transcribed 3’ untranslated region (Table 1); three were not found to be polymorphic when the sequences from two or more determinations were compared (Nakajima et al., 1987; Sharma et al., 1989; Beauchemin et al., 1987; Oikawa et al., 1987; Barnett et al., 1988, Breslow et al., 1982; Paik et al., 1985; McLean et al., 1984). Evidence for this new class of polymorphism was found in human, chicken, bovine, and murine genes. Repeated sequence analyses of multiple cDNAs or genes from several sources have revealed that transcribed 3’ untranslated sequence variations represent true polymorphisms (Lang and Spritz, 1985; Yamano et al., 1990; Martiniuk et aZ., 1990; Lee et aZ., 1989; Sasavage et al., 1982; Bock et al., 1983; Henthorn et al., 1986). Furthermore, direct confirmation of transcribed 3’ untranslated polymor484
Inc. reserved.
Institutions,
TRANSCRIBED
3’ UNTRANSLATED
TABLE Transcribed 3’ untranslated sequence
Deletion
Myoglobin Insulin receptor Cytochrome P4502A Cytochrome P4502B M Creatine kinase Alkaline phosphatase (placental) Alkaline phosphatase (germ cell) Apolipoprotein B Apolipoprotein CII Apolipoprotein CIII C8 (complement) fl subunit C-Reactive protein y-Globin Acid a-glucosidase Argininosuccinate synthetase Prolactin (bovine) Topoisomerase I HLA-B7 al-Acid glycoprotein 1 (mouse) Lipoprotein lipase Dopamine D, receptor Pleckstrin Glucagon Calbindin Dsu (chicken)
+
? + + + + + + + + + ? + + + + f + + + + + + ? +
+ + +
+
INFORMATION 3’ UNTRANSLATED
CONTENT OF MARKERS
Assuming that transcribed 3’ untranslated sequence is no more polymorphic than introns or other noncoding sequences (i.e., approximately 1 in 100 bp is likely to be polymorphic), and the average transcribed 3’ untranslated region in our review was greater than 600 bp in length, we might anticipate multiple polymorphisms to occur within each transcribed 3’ untranslated region. Consistent with this notion, we found evidence for multiple alleles (the maximum number of alleles observed was 5, including both deletions and substitutions) in several of the examples listed in Table 1. Since multiple polymorphisms within a single transcribed 3’ untranslated region (in linkage disequilibrium) may be used collectively as a single marker, this class of polymorphism may be highly informative in linkage analyses. THE FREQUENCY UNTRANSLATED
OF TRANSCRIBED POLYMORPHISMS
3’
If this class of polymorphism occurs as frequently in the mammalian genome as in this initial review, then tran-
485
1 Substitution
phisms is available. Karanthanasis et al. (1983) demonstrated that a nucleotide substitution in the 3’ noncoding region of the apolipoprotein CIII gene alters a Sac1 restriction endonuclease site. Moreover, Bolos et al. (1990) directly demonstrated a frequent polymorphism in a portion of the transcribed 3’ untranslated region of the dopamine D, receptor by PCR amplification. POLYMORPHISM TRANSCRIBED
REGIONS
Minimum alleles
(2) (2) (2) (5)
(2) (3)
(2) (2) (2) (2) (2) (2) (2) (2) (3)
(2) (2) (2) (2) (3)
c-9
Refs. (1,851 (23,w (43,90,92) (48,W (62976) (28,33,49)
(81) (12,35,40,83) (20,25,84) (34) (27,29)
(42,89) (13,39,64,
72)
(45) (7) (50966)
(19,W (73) (41) (579%)
(3)
(8,261 (78)
(2) (2)
(5,87) (51)
scribed 3’ untranslated polymorphisms would be anticipated in as many as 90% or more of eukaryotic genes. This small sampling may not be representative of the true frequency of transcribed 3’untranslated polymorphisms. However, transcribed 3’ untranslated polymorphisms in several examples appear to be more frequent than coding sequence variation (Yamano et al., 1990, Martiniuk et aZ., 1990, Lee et aZ., 1989; Henthorn et aZ., 1986)‘ We might anticipate that certain of the examples listed in Table 1 represent sequencing errors, and not real polymorphisms. If we assume the human has between 50,000 and 100,000 genes, then if only 1 in 10 transcribed 3’ untranslated regions is in fact found to be highly polymorphic when characterized experimentally, as many as 5000 to 10,000 useful new markers may still be anticipated by studying this class of polymorphism. Nevertheless, the frequency with which sequence variations have been reported, or have been observed in our review of the literature, suggests that these polymorphisms may represent one of the more abundant classes of DNA markers for gene mapping in .eukaryotes. DETECTING
TRANSCRIBED 3’ UNTRANSLATED POLYMORPHISMS
Evidence suggests that transcribed 3’ untranslated polymorphisms can be efficiently demonstrated as a PCR product (Bolos et aZ., 1990). Polymorphisms in the PCR products produced from the amplification of transcribed 3’ untranslated regions can be rapidly analyzed for deletions by denaturing sequencing gels and for substitutions by denaturing gradient gel electrophoresis (Sheffield et al., 1989; Traystman et aZ.,1990) or nondenaturing gel single-strand
ROY C. LEVITT
486 conformational 1990).
analyses
cDNA MAPPING UNTRANSLATED
(Orita
et al., 1989; Bolos
WITH TRANSCRIBED POLYMORPHISMS
et al.,
3’
In the Human Genome Project, several investigators have urged the usefulness of mapping cDNAs created from mRNAs of tissue-specific and developmental stage-specific types. Even though the function of most such cDNAs may not yet be known, this map will provide candidate genes for scrutiny in connection with genetic disorders that have been mapped to the same region and they will be useful starting points for sequencing. The existence of abundant polymorphisms in the transcribed 3’ untranslated portion of these genes will further enhance the value of a saturated cDNA map of the human genome; a majority of the cDNAs mapped should be usable as DNA markers in linkage studies. Indeed, the existence of polymorphism in the cDNA should provide a method for mapping the cDNA to specific chromosomal sites, namely, linkage against the CEPH panel, in addition to in situ hybridization. Since the DNA sequence surrounding these transcribed 3’ untranslated markers will be known, this class of polymorphism can also serve as sequence tagged sites (Olson et al., 1989) in the construction of a physical map of the chromosomes (e.g., contig mapping of cosmid and YAC clones (Burke et aZ., 1987; McCormick et al., 1989) ) Generating sequence information around anonymous DNA markers for the construction of a physical map of the chromosomes is particularly laborious and expensive, making this characteristic associated with transcribed 3’ untranslated markers particularly useful. SUMMARY In this review we present preliminary evidence for a new class of polymorphism that may be used in a systematic way to map cDNAs efficiently and to expedite the construction of a high-resolution genetic map of the human genome. Ultimately, transcribed 3’ untranslated polymorphisms will warrant further study because they should be widely distributed throughout the genome within transcribed sequences, and they can be readily identified as a result of cDNA cloning and sequencing. Furthermore, these markers should be universally available on the basis of the sequence data and highly useful in linkage analyses. ACKNOWLEDGMENTS Many thanks to Dr. V. A. McKusick for his contributions and critique of this manuscript and to Dr. Mark Rogers for his suggestions and enthusiastic support. Note added in proof.A strategy for identifying polymorphisms in specific genes similar to that described herein was published by S. E. PodusuIo, M. Dean, U. Kolch, and S. J. O’Brien (1991, J. Hum. Genet. 49: 106-111).
REFERENCES 1. AKABOSHI, E. (1985). Cloning of the human myoglobin gene. Gene 33: 241-249. 2. ANTONARAKIS, S. E., BOEHM, C. D., GIARDINA, P. J., AND KAZAZIAN, H. H., JR. (1982). Nonrandom association of polymorphic restriction sites in the beta-globin gene cluster. Proc. N&l. Acad. Sci. USA 79: 137-141. 3. BARNEW, T., GOEBEL, S. J., NOTHDURFT, M. A., AND ELTING, J. J. (1988). Carcinoembryonic antigen family: Characterization of cDNAs coding for NCA and CEA and suggestion of nonrandom sequence variation in their conserved loop-domains. Genomics 3: 59-66. 4. BEAUCHEMIN, N., BENCHIMOL, S., COURNOYER,D., FUKS, A., AND STANNERS, C. P. (1987). Isolation and characterization of full-length functional cDNA clones for human carcinoembryonic antigen. Mol. Cell Biol. 7: 3221-3230. 5. BELL, G. I., SANCHEZ-PESCADOR, R., LAYBOURN, P. J., AND NAJARIAN, R. C. (1983). Exon duplication and divergence in the human preproglucagon gene. Nature 304: 368-371. 6. BIRNSTIEL, M. L., BUSSLINGER, M., AND STRUB, K. (1985). Transcription termination and 3’ processing: The end is in site! Cell 41: 349-359. 7. BOCK, H.-G. O., Su, T. S., O’BRIEN, W. E., AND BEAUDET, A. L. (1983). Sequence for human argininosuccinate synthetase cDNA. Nucbic Acids Res. 11: 6505-6512. 8. BOLOS, A. M., DEAN, M., LUCAS-DERSE, S., RAMSBURG, M., BROWN, G. L., AND GOLDMAN, D. (1990). Population and pedigree studies reveal a lack of association between the dopamine D, receptor gene and alcoholism. JAMA 264: 31563160. 9. BRAWERMAN, G. (1987). Determinants of messenger RNA stability. Cell 48: 5-6. 10. BRESLOW, J. L., MCPHERSON, J., NUSSBAUM, A. L., WILLIAMS, H. W., LOFQUIST-KAHL, F., KARATHANASIS, S. K., AND ZANNIS, V. I. (1982). Identification andDNA sequence of a human apolipoprotein E cDNA clone. J Biol. Chem. 267: 14639-14641. 11. BURKE, D. T., CARLE, G. F., AND OLSON, M. Y. (1987). Cloning of large segments of exogenous DNA into yeast by means of artificial chromosome vectors. Science 236: 806-812. 12. CARLSSON, P., OLOFSSON, S. O., DARNFORS, C., AND BJURSELL, G. (1986). Analysis of the human apolipoprotein B gene: Complete structure of the B-74 region. Gene 49: 29-51. 13. CAVALLESCO, C., FORGET, B. G., DERIEL, J. K., WILSON, L. B., WILSON, J. T., AND WEISSMA.N, S. M. (1980). Nucleotide sequence of human Oy globin messenger RNA. Gene 12: 215-221. 14. CAWTHON, R. M., WEISS, R., GANGFENG, X., VISKCJCHIL, D., CULVER, M., STEVENX, J., ROBERTSON, M., DUNN, D., GxsTELAN, R., O’CONNELL, P., AND WHITE, R. (1990). A major segment of the neurofibromatosis type I gene: cDNA sequence, genomic structure, and point mutations. Cell 62: 193201. 15. CHAKRAVARTI, A., BUETOW, K. H., ANTONARAKIS, S. E., WABER, P. G., BOEHM, C. D., AND KAZAZUN, H. H., JR. (1984). Nonuniform recombination within the human betaglobin gene cluster. Am. J. Hum. Genet. 36: 1239-1258. 16. CHAKRAVARTI, A., ELBEIN, S. C., AND PER-, M. A. (1986). Evidence for increased recombination near the human insulin gene: Implication for disease association studies. Proc. Natl. Acad. Sci. USA 83: 1045-1049. 17. CHEBLOUNE, Y. U., PAGNIER, J., TRABUCHET, G., FAURE, C., VERDIER, G., LAFIIE, D., AND NIGON, V. (1988). Structural analysis of the 5’ flanking region of the beta-globin gene in
TRANSCRIBED
18.
19.
20.
21.
22. 23.
24.
25.
26.
27.
3’ UNTRANSLATED
African sickle cell anemia patients: Further evidence for three origins of the sickle cell mutation in Africa. Pmt. Nutl. Ad. Sci. USA 86: 4431-4435. CLEWLAND, D. W., LOPATA, M. A., MACDONALD, R. J., CoWAN, N. J., R~R, W. J., AND KIRSCHNER, M. W. (1980). Number and evolutionary conservation of a- and @-tubulin and cytoplasmic @- and y-actin genes using specific cloned cDNA probes. Cell 20: 95-105. D’ARPA, P., MACHLIN, P. S., RATIRE, H., ROTHFIELD, N. F., CLEVELAND, D. W., AND EARNSI-IAW, W. C. (1988). cDNA cloning of human DNA topoisomerase I: Catalytic activity of a 67.7-kDa carboxyl-terminal fragment. Proc. Natl. Acad. Sci. USA 86: 2543-2547. DAS, H. K., JACKSON, C. L., MILLER, D. A., LEFF, T., AND BRFSLOW, J. L. (1987). The human apolipoprotein C-II gene sequence contains a novel chromosome 19-specific minisatellite in its third intron. J. Biol. Chem. 262: 4787-4793. DONIS-KELLER, H., GREEN, P., HELMS, C., CARTINHOUR, S., WEIFFENBACH, B., STEPHENS, K., KEITH, T. P., BOWDEN, D. W., SMITH, D. R., LANDER, E. S., BOTSTEIN, D., AKOTS, G., REDIKER, K. S., Gmvrus, T., BROWN, V. A., RISING, M. B., PARKER, C., POWERS, J. A., WAIT, D. E., KAUFFMAN, E. R., BRICKER, A., PHIPPS, P., MULLER-KAHLE, H., FIJLTON, T. R., NC, S., SCHUMM, J. W., BRAMAN, J. C., KNOWLTON, R. G., BARKER, D. F., CROOKS, S. M., LINCOLN, S. E., DALY, M. J., AND ABRAHAMSON, J. (1987). A genetic linkage map of the human genome. Cell 51: 319-337. DRAYNA, D., AND WHITE, R. (1985). The genetic linkage map of the human X chromosome. Science 230: 753-758. EBINA, Y., ELLIS, L., JARNAGIN, K., EDERY, M., GRAF, L., CLAUSER, E., Ou, J., ~IARZ, F., KAN, Y. W., GOLDFINE, I. D., ROTH, R. A., AND RU~TER, W. J. (1985). The human insulin receptor cDNA: The structural basis for hormone-activated transmembrane signalling. Cell 40: 747-758. ECONOMOU, E. P., BERGEN, A. W., WARREN, A. C., AND ANTONARAKIS, S. E. (1990). The polydeoxyadenylate tract of Ah repetitive elements is polymorphic in the human genome. Proc. Natl. Ad. Sci. USA 87: 2951-2954. Fo~o, S. S., LAW, S. W., AND BREWER, H. B., JR. (1987). The human preproapolipoprotein C-II gene: Complete nucleic acid sequence and genomic organization. FEBS Lett. 213: 221-226. GUSELLA, J. F., WEXLER, N. S., CONNEALLY, P. M., NAYLOR, S. L., ANDERSON, M. A., TANZI, R. E., WATKINS, P. C., OrTINA, K., WALLACE, M. R., SAKAGUCHI, A. Y., YOUNG, A. B., SHOULDSON, I., BONILLA, E., AND MARTIN, J. B. (1983). A polymorphic DNA marker genetically linked to Huntington’s disease. Nature 306: 234-238. HAEFLIGER, J.-A., TSCHOPP, J., NARDELLI, D., WAHLI, W., KOCHER, H.-P., TOSI, M., AND STANLEY, K. K. (1987). Complementary DNA cloning of complement C8@ and its sequence homology to C9. Biochemistry 26: 3551-3556.
28. HENTHORN,
P. S., KNOLL, B. J., RADUCHA, M., ROTHBLUM, K. N., SLAUGHTER, C., WEISS, M., LAFFERTY, M. A., FISCHER, T., AND HARRIS, H. (1986). Products of two com-
mon alleles at the locus for human placental alkaline phosphatase differ by seven amino acids. Proc. Natl. Acad. Sci. USA 83: 5597-5601. 29. HOWARD, 0. M. Z., RAO, A. G., AND SODETZ, J. M. (1987). Complementary DNA and derived amino acid sequence of the /3subunit of human complement protein C8: Identification of a close structural and ancestral relationship to the LYsubunit and C9. Biochemistry 26: 3565-3570. 30. Human Gene Mapping 10: New Haven Conference (1989). Cytogerzet. Cell Genet. 51: 1-1147.
REGIONS
487
31. JEFFXEYS, A. J. (1979). DNA sequence variants in the G gamma-, A gamma-, delta-, and beta-globin genes in man. Cell 18: l-10. 32. +hFFREYS, A. J., WnSON, V., AND THEIN, S. L. (1985). Hypervariable ‘minisatellite’ regions in human DNA. Nature 314: 67-73. 33. KAM, W., CLAUSSER, E., KIM, Y. S., KAN, Y. W., AND RUTTER, W. J. (1985). Cloning, sequencing, and chromosomal localization of human term placental alkaline phosphatase cDNA. Proc. Natl. Acad. Sci. USA 82: 8715-8719. 34. KARATHANASIS, S. K., MCPHERSON, J., ZANNIS, V. I., AND BRESLOW, J. L. (1983). Linkage of human apolipoproteine A-I and C-III genes. Nature 304: 371-372. 35. KNO?T, T. J., WALLIS, S. C., POWELL, L. M., PEASE, R. J., LUSIS, A. J., BLACKHART, B., MCCARTHY, B. J., ~~LQ%EY, R. W., LEW-WILSON, B., ANII SCO?T, J. (1986). Complete cDNA and derived protein sequence of human apolipoprotein B-100. Nucleic Acids Res. 14: 7501-7503. 36. KORNBLIH?T, A. R., VIBE-PEDERSEN, K., AND BARALLE, F. E. (1983). Isolation and characterization of cDNA clones for human and bovine fibronectins. Proc. Natl. Acad. Sci. USA 80: 3218-3222.
37. KORNBLIHTT, A. R., VIBE-PEDERSEN, K., AND BARALLE, F. E. (1984). Human fibronectin: Cell specific alternative mRNA splicing generates polypeptide chains differing in the number of internal repeats. Nucleic Acids Res. 12: 5853-5868. 38. KRONENBERG, H. M., ROBERTS, B. E., AND EFSTRATIADIS, A. (1979). The 3’ noncoding region of @globin mRNA is not essential for in vitro translation. Nucleic Acids Res. 6: 153-166. 39. LANG, K. M., AND SPRPIZ, R. A. (1985). Cloning specific complete polyadenylated 3’-terminal cDNA segments. Gene 33: 191-196.
40. LAW, S. W., GRANT, LACKNER,
S. M., HIGUCHI, K., HOSPA?TANKAR, K., LEE, N., AND BREWER, H. B., JR. (1986).
A., Hu-
man liver apolipoprotein B-100 cDNA: Complete nucleic acid and derived amino acid sequence. Proc. Natl. Acad. Sci. USA 83: 8142-8146. 41. LEE, S.-C., CHANG, C.-J., LEE,Y.-M., LEJ, H.-Y., LAI,M.-Y., AND CHEN, D.-S. (1989). Molecular cloning of cDNAs corresponding to two genes of al-acid glycoprotein and characterization of two alleles of AGP-1 in the mouse. DNA 8: 245251.
42.
Lnr, T., ZON, G., SORAVIA, E., LIP, T.-Y., AND N. D. (1985). Genomic DNA sequence for human C-reactive protein. J. Biol. Chm. 260: 13377-13383. 43. LEVITT, R. C., SIENKIEWICZ, M. L., AND ZASLOFF, M. (1985). Molecular analysis of a human cytochrome Pm likely to be involved in anesthetic metabolism. Anesthesiology 63: A549. 44. LITT, M., AND LUTY, J. A. (1989). A hypervariable microsatellite revealed by in vitro amplification of a dinucleotide repeat within the cardiac muscle actin gene. Am. J. Hum. Genet. 44: 397-401. 45. ~TINIUK, F., MEHLER, M., TZALL, S., MJ%XEDITH, G., AND HIRSCHHORN, R. (1990). Sequence of the cDNA and 5’-flanking region for human acid cy-glucosidase, detection of an intron in the 5’ untranslated leader sequence, definition of 18bp polymorphisms, and differences with previous cDNA and amino acid sequences. DNA Cell Bid 9: 85-94. 46. MCCORMICK, M. K., SHERO, J. H., CHEUNG, M. C., KAN, LEI, K.-J., GOLDMAN,
Y. W., HIETER,
P. A., AND ANTONARAKIS,
S. E. (1989).
Con-
struction of human chromosome al-specific yeast artificial chromosomes. Proc. Natl. Acad. Sci. USA 86: 9991-9995. J. W., ELSHOURBAGY, N. A., CHANG, D. J., MAH47. MCLEAN, LEY, R. W., AND TAYLOR,
J. M.
(1984).
Human
tein E mRNA. J. Bid. C&m. 269: 6498-6504.
apolipopro-
ROY C. LEVITT
488
48. MILES, J. S., SPURR, N. K., GOUGH, A. C., JO-, T., MCLAREN, A. W., BROOK, J. D., AND WOLF, C. R. (1988). A novel human cytochrome P450 gene (P450IIB): Chromosomal localization and evidence for alternative splicing. Nucleic Acids Res. 16: 5783-5795. 49. MILLAN, J. L. (1986). Molecular cloning and sequence analysis of human placental alkaline phosphatase. J. Biol. Chem. 261: 31123115. 50. MILLER, W. L., THIRION, J. P., AND MARTIAL, J. A. (1980). Cloning of DNA complementary to bovine prolactin mRNA. Endocrinology 107: 851-854. 51. MING~I, P. P., CANCELA, L., FUJISAWA, Y., THEOFAN, G., AND NORMAN, A. W. (1988). Molecular structure of chicken vitamin D-induced calbindin-D,sx gene reveals eleven exons, six Car+-binding domains, and numerous promoter regulatory elements. Mol. Endocrinol. 2: 355-365. 52. NAKAJIMA, H., NOGUCHI, T., YAMASAKI, ‘I’., KONO, N., TANAKA, T., AND TARUI, S. (1987). Cloning of human muscle phosphofructokinase cDNA. FEBS Lett. 223: 113-116. 53. NAKAMURA, Y., LEPPERT, M., O’CONNELL, P., WOLFF, R., HOLM, T., CULVER, M., MARTIN, C., FUJMOTO, E., HOFF, M., KUMLIN, E., AND WHITE, R. L. (1987). Variable number of tandem repeat (VNTR) markers for human gene mapping. Science 235: 1616-1622. 54. NAKAMURA, Y., O’CONNELL, P., SONE, C., LALOUEL, J. M., AND WHITE, R. (1988). An extended genetic linkage map of markers for human chromosome 10. Genomics 3: 389-392. 55. O’CONNELL, P., LATHROP, G. M., LEPPERT, M., NAKAMURA, Y., MULLER, V., LALOUEL, J. M., AND WHITE, R. (1988). Twelve loci form a continuous linkage map for human chromosome 18. Genomics 3: 367-372. 56. OIKAWA, S., NAKAZATO, H., AND KOSAKI, G. (1987). Primary structure of human carcinoembryonic antigen (CEA) deduced from cDNA sequence. Biochem. Biophys. Res. Commun. 142: 511-518. 57. OKA, K., TKALCEVIC, G. T., NAKANO, T., TUCKER, H., ISHIMURA-OKA, K., AND BROWN, W. V. (1990). Structure andpolymorphic map of human lipoprotein lipase gene. B&him. Biaphys. Acta 1049: 21-26. 58. OLSON, M., HOOD, L., CANTOR, C., AND BOTSTEIN, D. (1989). A common language for physical mapping of the human genome. Science 245: 1434-1435. 59. ORITA, M., SUZUKI, Y., SEKIYA, T., AND HAYASKI, K. (1989). Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics 5: 874-879.
65.
Science 66.
67.
Natl.
Acad.
Sci. USA
70.
71.
72.
73.
74.
SETZER, D. R., MCGROGAN, M., NUSNBER, J. H., AND SCHIMKE, R. T. (1980). Size heterogeneity in the 3’ end of dihydrofolate reductase messenger RNAs in mouse cells. Cell SHARMA, P. M., REDDY, G. R., VORA, S., BABIOR, B. M., AND MCLACHLAN, A. (1989). Cloning and expression of a human muscle phosphofrutokinase cDNA. Gene 77: 177-183. SHEFFIELD, V. C., Cox, D. R., LEXMAN, L. S., AND MYERS, R. M. (1989). Attachment of a 40-base-pair G + C-rich sequence (GC-clamp) to genomic DNA fragmenta by the polymerase chain reaction results in improved detection of singlebase changes. Proc. Natl. Acad. Sci. USA 86: 232-236. SINNE’IT, D., DERAGON, J-M., SIMARD, L. R., AND LABUDA, D. (1990). Alumorphs-Human DNA polymorphisms detected by polymerase chain reaction using Ah-specific primers. Genomics 7: 331-334. SLIGHTOM, J. L., BLECHL, A. E., ANIJ SMITHIES, 0. (1989). Human fetal ‘y and *y-globin genes: Complete nucleotide sequences suggest that DNA can be exchanged between these duplicated genes. CeU 21: 627-638. SOOD, A. K., PAN, J., BIRO, P. A., PEREIRA, D., SRNASTAVA, R., REDDY, V. B., DUCEMAN, B. W., AND WEISSMAN, S. M. (1985). Structure and polymorphism of class I MHC antigen mRNA. Immunogenetics 22: 101-121. SOUTHERN, E. M. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol.
75.
77.
78.
82: 3445-3449.
PERRYMAN, M. B., KERNER, S. A., BOHLMEYER, T. J., AND ROBERTS, R. (1986). Isolation and sequence analysis of a fulllength cDNA for human M creatine kinase. Biachem. Biaphys. Res. Commun. 140: 981-989. 63. PETERSEN, M. B., SKUGENHAUPT, S. A., CHAKRAVARTI, A., LEWIS, J. G., WARREN, A. C., AND ANTONARAKIS, S. E. (1991). A linkage map of 27 markers on human chromosome 21. Genomics 9: 407-419. 64. POON, R., KAN, Y. W., AND BOYJZR,H. W. (1978). Sequence of
1350-1354.
22:361-370. 69.
60.
Proc.
230:
SASAVAGE, N. L., NILSON, J. H., HOROWITZ, S., AND ROTTMAN, F. M. (1982). Nucleotide sequence of bovine prolactin messenger RNA-Evidence for sequence polymorphism. J. Biol. Chem. 257: 678-681. SEMENZA, G. L., MALLXX, P., SURREY, S., DEMROSSO, K., PONCZ, M., AND SCHWARTZ, E. (1984). Detection of a novel DNA polymorphism in the beta-globin gene cluster. J. Bial. Chem.259:6045-6048.
68.
76.
ORKIN, S. H., KAZAZIAN, H. H., JR., ANTON-IS, S. E., GOFF, S. C., BOEHM, C. D., SEXTON, J. P., WABER, P. G., AND GIARDINA, P. J. (1982). Linkage of beta-tbalassaemia mutations and beta-globin gene polymorphisms with DNA polymorphisms in human beta-globin gene cluster. Nature 296: 627-631. 61. PAIK, Y. K., CHANG, D. J., REARDON, C. A., DAVIES, G. E., MAHLEY, R. W., AND TAYLOR, J. M. (1985). Nucleotide sequence and structure of the human apolipoprotein E gene.
the 3’-noncoding and adjacent coding regions of human y-globin mRNA. Nucleic Acids Res. 6: 4625-4630. SAIKI, R. K., SCHARF, S., FALLOONA, F., MULLIS, K. B., HORN, G. T., ERLICH, H. A., AND ARNHEIM, N. (1985). Enzymatic amplification of beta-globm genomic sequences and restriction site analysis for diagnosis of sickle cell anemia.
62.
Biol.
98: 503-517.
TAUTZ, D. (1989). Hypervariability of simple sequences as a general source for polymorphic DNA markers. Nucleic Acids Res. 17: 6463-6471. TRASK, R. V., STRAUSS, A. W., AND BILLADELLO, J. J. (1988). Developmental regulation and tissue-specific expression of the human muscle creatine kinase gene. J. Bial. Chem. 263: 17142-17149. TRAYSTMAN, M. D., HIGUCHI, M., KASPER, C. K., ANTONARAKIS, S. E., ti KAZAZIAN, H. H. (1990). Use of denaturing gradient gel electrophoresis to detect point mutations in the factor VIII gene. Genomics 6: 293-301. TYERS, M., HASLAM, R. J., RACHUBINSKI, R. A., AND HARLEY, C. B. (1989). Molecular analysis ofpleckstrin: The major protein kinase C substrate of platelets. J. Cell. Biochem. 40: 133-145.
79.
80.
ULLRICH, A., BELL, J. R., CHEN, E. Y., HERRERA, R., PETRUZZELLI, L. M., DULL, T. J., GRAY, A., COUSSEN, L., LIAO, Y.-C., TSUEOKAWA, M., MASON, A., SEXFIURG,P. H., GRUNFELD, C., ROSEN, 0. M., AND RAMACHANDRAN, J. (1985). Human insulin receptor and its relationship to the tyrosine kinase family of oncogenes. Nature 313: 756-761. WARREN, A. C., SLAUGENHAUPT, S. A., LEWIS, J. G., CHAKRAVARTI, A., AND ANTONARAKIS, S. E. (1989). A genetic
TRANSCRIBED
3’ UNTRANSLATED
linkage map of 17 markers on human chromosome 21. Gerwmics 4: 579-591. 81. WATANABE, S., WATANABE, T., LI, W. B., SOONG, B.-W., AND CHOU, J. Y. (1989). Expression of the germ cell alkaline phosphatase gene in human choriocarcinoma cells. J. Bid. Chem. 264: 12611-12619. 82. WEBER, J. L., AND MAY, P. E. (1989). Abundant class of human DNA polymorphisms which can be typed using the polymerase chain reaction. Am. J. Hum. Genet. 44: 366-396. 83. WEI, C. F., CHEN, S. H., YANG, C. Y., MARCEL, Y. L., MILNE, R. W., LI, W. H., SPARROW, J. T., GOTTO, A. M., JR., AND C&IAN, L. (1985a). Molecular cloning and expression of partial cDNAs and deduced amino acid sequence of a carboxylterminal fragment of human apolipoprotein B-100. Proc. Natl. Ad. Sci. USA 82: 7265-7269. 84. WEI, C. F., TSAO, Y. K., ROBBERSON, D. L., Gorro, A. M., JR., BROWN, K., AND CHAN, L. (198513). The structure of the human apolipoprotein C-II gene: Electron microscopic analysis of RNA:DNA hybrids, complete nucleotide sequence, and identification of 5’ homologous sequences among apolipoprotein genes. J. Biol. C&m. 260: 15211-15221. 85. WELLER, P., JEFFREYS, A. J., WILSON, V., AND BLANCHETOT, A. (1984). Organization of the human myoglobin gene. EMBO J. 3: 439-446. 86. WHITE, R., AND LALOUEL, J. M. (1988). Sets of linked genetic markers for human chromosomes. Annu. Rev. Genet. 22: 259-279. 81. WHITE, J. W., AND SAUNDERS, G. F. (1986). Structure of the human glucagon gene. Nucleic Acids Res. 12: 4719-4730.
REGIONS
489
88. WION, K. L., KIRCHGESSNER, T. G., LUSIS, A. J., SCHOTZ, M. C., AND LAWN, R. M. (1987). Human lipoprotein iipase complementary DNA sequence. Science 236: 1638-1641. 89. Woo, P., KORENBERG, J. R., AND WHITEHEAD, A. S. (1985). Characterization of genomic and complementary DNA sequence of human C-reactive protein, and comparison with the complementary DNA sequence of serum amyloid P component. J. Biol. Chem. 260: 13384-13388. 90. YAMANO, S., NAGATA, K., YAMAU)E, Y., KATO, R., GELBOIN, H. V., AND GONZALEII, F. J. (1989a). cDNA and deduced amino acid sequences of human P450 IIA3 (CYP2A3). Nucleic Acids Res. 17: 4888. 91. YAMANO, S., NHAMBURO, P. T., AOYAMA, T., MEYER, U. A., INABA, T., KALOW, W., GELBOIN, H. V., MCBRIDE, 0. W., AND GONZALEZ, F. J. (1989b). cDNA cloning and sequence and cDNA-directed expression of human P450 IIBl: Identification of a normal and two variant cDNAs derived from the CYPBB locus on chromosome 19 and differential expression of the IIB mRNAs in human liver. Biochemistry 28: 73407348.
92.
YAMANO, S., TATSUNO, J., AND GONZALEZ, F. J. (1990). The CYP2A3 gene product catalyzes coumarin 7-hydroxylation in human liver microsomes. Biochemistry 29: 1322-1329. 93. ZHOU, B.-S., BASTOW, K. F., AND CHENG, Y. C. (1989). Characterization of the 3’ region of the human DNA topoisomerase I gene. Cancer Res. 49: 3922-3927. 94. ZULIANI, G., AND HOBBS, H. H. (1990). A high frequency of length polymorphisms in repeated sequences adjacent to Alu sequences. Am. J. Hum. Genet. 46: 963-969.
in the Transcribed 3’ Untranslated of Eukaryotic Genes
Region
RovC. LEVITT Department
of Anesthesiology and Critical Care Medicine, Johns Hopkins Medical 600 North Wolfe Street, Baltimore, Maryland 2 1205
INTRODUCTION
approximately 1 in every 100 bp (Orkin et al., 1982; Jeffreys, 1979; Chakravarti et al., 1984, 1986; Antonarakis et al., 1982), the transcribed untranslated regions of eukaryotic genes should contain multiple nucleotide changes (depending on the length of this region) that would represent a very useful resource for genetic mapping. These sequence polymorphisms should be easily demonstrated by PCR and established electrophoretic techniques (Sheffield et al., 1989; Orita et al., 1989; Bolos et al., 1990; Traystman et aZ., 1990; Cawthon et al., 1990).
The etiology of most genetic disorders remains unknown and the vast majority of mutant genes are recognized only by the abnormal phenotype they produce. Establishing linkage with polymorphic DNA markers is often a crucial first step in the localization and eventual cloning of a given disease gene. High-resolution genetic maps will expedite the process of localizing disease genes. The construction of a high-resolution linkage map as an objective of the Human Genome Project is largely dependent on identifying DNA polymorphism for use as markers. At this time linkage maps of approximately 10 CM exist for nearly every human chromosome (Drayna and White, 1985; Donis-Keller et al., 1987; White and Lalouel, 1988; Human Gene Mapping 10, 1989, Warren et al., 1989; Petersen et aZ., 1991; Nakamura et al., 1988, O’Connell et al., 1988). Most of the markers used to construct these linkage maps are restriction fragment length polymorphisms (RFLPs) detectable by Southern blot hybridization (Southern, 1975). Although these techniques have greatly advanced our understanding of the organization of the human genome, they require nucleic acid probes, are laborious to demonstrate, and are often uninformative in linkage analyses. New classes of DNA polymorphisms, several of which can be demonstrated directly by polymerase chain reaction (PCR), have recently been described (Economou et aZ., 1990; Jeffreys et aZ., 1985; Nakamura et al., 1987; Litt and Luty, 1989; Weber and May, 1989; Semenza et al., 1984; Chebloune et al., 1988; Tautz, 1989; Saiki et al., 1985; Sinnett et al., 1990; Zuliani and Hobbs, 1990). The advantages of PCR-based methods are that they are faster and therefore less costly; they do not require nucleic acid probes; and, being directly sequence based, they are more sensitive for detecting polymorphisms at a given locus, Although these newer markers are often multiallelic and frequently heterozygous and therefore highly informative for linkage analyses, they are not generally useful for mapping cDNAs (i.e., they are not commonly found within transcribed sequences). Another potential limitation of this approach is that DNA sequence information is required to generate the oligonucleotide primers necessary for the PCR amplification. Ultimately, polymorphisms in eukaryotic genes that are efficiently demonstrated by PCR methods and can be used to map and evaluate cDNAs as candidate genes in human disorders would facilitate the scientific goals of a large number of researchers. A significant portion of the sequence information on the human genome (and that of other eukaryotes) has been derived from cloning and direct sequencing of cDNAs. Since polymorphic variation is thought to occur in noncoding sequence as frequently as GENOMICS 11,484-489 (1991) 0888-7543/91$3.00 Copyright 0 1991 by Academic Press, All rights of reproduction in any form
POLYMORPHISMS IN THE TRANSCRIBED UNTRANSLATED REGION OF EUKARYOTIC
3’ GENES
Aside from one or more putative polyadenylation, transcription termination, and/or regulatory signals, little is known about the function of the transcribed 3’untranslated region of eukaryotic genes (Birnstiel et aZ., 1985). Although the 3’ noncoding region may have a role in mRNA stability (Brawerman, 1987), it does not appear essential for mRNA translation (Kronenberg et al., 1979). Furthermore, size heterogeneity (Setzer et aZ., 1980) and a lack of evolutionary conservation (Perryman et al., 1986; Cleveland et al., 1980) support the notion that only limited discrete sequences serve a regulatory function in this region of eukaryotic transcripts. Most importantly, if the structure of the transcribed 3’ untranslated region of eukaryotic genes is largely not conserved, in contrast to coding regions, then sequence divergence within this region of the genome may represent readily accessible and highly useful markers for genetic mapping. As an initial test of the hypothesis that the transcribed 3’ untranslated region of eukaryotic genes is polymorphic, we reviewed the literature or GenBank on 27 genes (or cDNAs) that were sequenced two or more times. Twenty-four of twenty-seven of these sequences were found to contain one or more deletions and/or substitutions in their transcribed 3’ untranslated region (Table 1); three were not found to be polymorphic when the sequences from two or more determinations were compared (Nakajima et al., 1987; Sharma et al., 1989; Beauchemin et al., 1987; Oikawa et al., 1987; Barnett et al., 1988, Breslow et al., 1982; Paik et al., 1985; McLean et al., 1984). Evidence for this new class of polymorphism was found in human, chicken, bovine, and murine genes. Repeated sequence analyses of multiple cDNAs or genes from several sources have revealed that transcribed 3’ untranslated sequence variations represent true polymorphisms (Lang and Spritz, 1985; Yamano et al., 1990; Martiniuk et aZ., 1990; Lee et aZ., 1989; Sasavage et al., 1982; Bock et al., 1983; Henthorn et al., 1986). Furthermore, direct confirmation of transcribed 3’ untranslated polymor484
Inc. reserved.
Institutions,
TRANSCRIBED
3’ UNTRANSLATED
TABLE Transcribed 3’ untranslated sequence
Deletion
Myoglobin Insulin receptor Cytochrome P4502A Cytochrome P4502B M Creatine kinase Alkaline phosphatase (placental) Alkaline phosphatase (germ cell) Apolipoprotein B Apolipoprotein CII Apolipoprotein CIII C8 (complement) fl subunit C-Reactive protein y-Globin Acid a-glucosidase Argininosuccinate synthetase Prolactin (bovine) Topoisomerase I HLA-B7 al-Acid glycoprotein 1 (mouse) Lipoprotein lipase Dopamine D, receptor Pleckstrin Glucagon Calbindin Dsu (chicken)
+
? + + + + + + + + + ? + + + + f + + + + + + ? +
+ + +
+
INFORMATION 3’ UNTRANSLATED
CONTENT OF MARKERS
Assuming that transcribed 3’ untranslated sequence is no more polymorphic than introns or other noncoding sequences (i.e., approximately 1 in 100 bp is likely to be polymorphic), and the average transcribed 3’ untranslated region in our review was greater than 600 bp in length, we might anticipate multiple polymorphisms to occur within each transcribed 3’ untranslated region. Consistent with this notion, we found evidence for multiple alleles (the maximum number of alleles observed was 5, including both deletions and substitutions) in several of the examples listed in Table 1. Since multiple polymorphisms within a single transcribed 3’ untranslated region (in linkage disequilibrium) may be used collectively as a single marker, this class of polymorphism may be highly informative in linkage analyses. THE FREQUENCY UNTRANSLATED
OF TRANSCRIBED POLYMORPHISMS
3’
If this class of polymorphism occurs as frequently in the mammalian genome as in this initial review, then tran-
485
1 Substitution
phisms is available. Karanthanasis et al. (1983) demonstrated that a nucleotide substitution in the 3’ noncoding region of the apolipoprotein CIII gene alters a Sac1 restriction endonuclease site. Moreover, Bolos et al. (1990) directly demonstrated a frequent polymorphism in a portion of the transcribed 3’ untranslated region of the dopamine D, receptor by PCR amplification. POLYMORPHISM TRANSCRIBED
REGIONS
Minimum alleles
(2) (2) (2) (5)
(2) (3)
(2) (2) (2) (2) (2) (2) (2) (2) (3)
(2) (2) (2) (2) (3)
c-9
Refs. (1,851 (23,w (43,90,92) (48,W (62976) (28,33,49)
(81) (12,35,40,83) (20,25,84) (34) (27,29)
(42,89) (13,39,64,
72)
(45) (7) (50966)
(19,W (73) (41) (579%)
(3)
(8,261 (78)
(2) (2)
(5,87) (51)
scribed 3’ untranslated polymorphisms would be anticipated in as many as 90% or more of eukaryotic genes. This small sampling may not be representative of the true frequency of transcribed 3’untranslated polymorphisms. However, transcribed 3’ untranslated polymorphisms in several examples appear to be more frequent than coding sequence variation (Yamano et al., 1990, Martiniuk et aZ., 1990, Lee et aZ., 1989; Henthorn et aZ., 1986)‘ We might anticipate that certain of the examples listed in Table 1 represent sequencing errors, and not real polymorphisms. If we assume the human has between 50,000 and 100,000 genes, then if only 1 in 10 transcribed 3’ untranslated regions is in fact found to be highly polymorphic when characterized experimentally, as many as 5000 to 10,000 useful new markers may still be anticipated by studying this class of polymorphism. Nevertheless, the frequency with which sequence variations have been reported, or have been observed in our review of the literature, suggests that these polymorphisms may represent one of the more abundant classes of DNA markers for gene mapping in .eukaryotes. DETECTING
TRANSCRIBED 3’ UNTRANSLATED POLYMORPHISMS
Evidence suggests that transcribed 3’ untranslated polymorphisms can be efficiently demonstrated as a PCR product (Bolos et aZ., 1990). Polymorphisms in the PCR products produced from the amplification of transcribed 3’ untranslated regions can be rapidly analyzed for deletions by denaturing sequencing gels and for substitutions by denaturing gradient gel electrophoresis (Sheffield et al., 1989; Traystman et aZ.,1990) or nondenaturing gel single-strand
ROY C. LEVITT
486 conformational 1990).
analyses
cDNA MAPPING UNTRANSLATED
(Orita
et al., 1989; Bolos
WITH TRANSCRIBED POLYMORPHISMS
et al.,
3’
In the Human Genome Project, several investigators have urged the usefulness of mapping cDNAs created from mRNAs of tissue-specific and developmental stage-specific types. Even though the function of most such cDNAs may not yet be known, this map will provide candidate genes for scrutiny in connection with genetic disorders that have been mapped to the same region and they will be useful starting points for sequencing. The existence of abundant polymorphisms in the transcribed 3’ untranslated portion of these genes will further enhance the value of a saturated cDNA map of the human genome; a majority of the cDNAs mapped should be usable as DNA markers in linkage studies. Indeed, the existence of polymorphism in the cDNA should provide a method for mapping the cDNA to specific chromosomal sites, namely, linkage against the CEPH panel, in addition to in situ hybridization. Since the DNA sequence surrounding these transcribed 3’ untranslated markers will be known, this class of polymorphism can also serve as sequence tagged sites (Olson et al., 1989) in the construction of a physical map of the chromosomes (e.g., contig mapping of cosmid and YAC clones (Burke et aZ., 1987; McCormick et al., 1989) ) Generating sequence information around anonymous DNA markers for the construction of a physical map of the chromosomes is particularly laborious and expensive, making this characteristic associated with transcribed 3’ untranslated markers particularly useful. SUMMARY In this review we present preliminary evidence for a new class of polymorphism that may be used in a systematic way to map cDNAs efficiently and to expedite the construction of a high-resolution genetic map of the human genome. Ultimately, transcribed 3’ untranslated polymorphisms will warrant further study because they should be widely distributed throughout the genome within transcribed sequences, and they can be readily identified as a result of cDNA cloning and sequencing. Furthermore, these markers should be universally available on the basis of the sequence data and highly useful in linkage analyses. ACKNOWLEDGMENTS Many thanks to Dr. V. A. McKusick for his contributions and critique of this manuscript and to Dr. Mark Rogers for his suggestions and enthusiastic support. Note added in proof.A strategy for identifying polymorphisms in specific genes similar to that described herein was published by S. E. PodusuIo, M. Dean, U. Kolch, and S. J. O’Brien (1991, J. Hum. Genet. 49: 106-111).
REFERENCES 1. AKABOSHI, E. (1985). Cloning of the human myoglobin gene. Gene 33: 241-249. 2. ANTONARAKIS, S. E., BOEHM, C. D., GIARDINA, P. J., AND KAZAZIAN, H. H., JR. (1982). Nonrandom association of polymorphic restriction sites in the beta-globin gene cluster. Proc. N&l. Acad. Sci. USA 79: 137-141. 3. BARNEW, T., GOEBEL, S. J., NOTHDURFT, M. A., AND ELTING, J. J. (1988). Carcinoembryonic antigen family: Characterization of cDNAs coding for NCA and CEA and suggestion of nonrandom sequence variation in their conserved loop-domains. Genomics 3: 59-66. 4. BEAUCHEMIN, N., BENCHIMOL, S., COURNOYER,D., FUKS, A., AND STANNERS, C. P. (1987). Isolation and characterization of full-length functional cDNA clones for human carcinoembryonic antigen. Mol. Cell Biol. 7: 3221-3230. 5. BELL, G. I., SANCHEZ-PESCADOR, R., LAYBOURN, P. J., AND NAJARIAN, R. C. (1983). Exon duplication and divergence in the human preproglucagon gene. Nature 304: 368-371. 6. BIRNSTIEL, M. L., BUSSLINGER, M., AND STRUB, K. (1985). Transcription termination and 3’ processing: The end is in site! Cell 41: 349-359. 7. BOCK, H.-G. O., Su, T. S., O’BRIEN, W. E., AND BEAUDET, A. L. (1983). Sequence for human argininosuccinate synthetase cDNA. Nucbic Acids Res. 11: 6505-6512. 8. BOLOS, A. M., DEAN, M., LUCAS-DERSE, S., RAMSBURG, M., BROWN, G. L., AND GOLDMAN, D. (1990). Population and pedigree studies reveal a lack of association between the dopamine D, receptor gene and alcoholism. JAMA 264: 31563160. 9. BRAWERMAN, G. (1987). Determinants of messenger RNA stability. Cell 48: 5-6. 10. BRESLOW, J. L., MCPHERSON, J., NUSSBAUM, A. L., WILLIAMS, H. W., LOFQUIST-KAHL, F., KARATHANASIS, S. K., AND ZANNIS, V. I. (1982). Identification andDNA sequence of a human apolipoprotein E cDNA clone. J Biol. Chem. 267: 14639-14641. 11. BURKE, D. T., CARLE, G. F., AND OLSON, M. Y. (1987). Cloning of large segments of exogenous DNA into yeast by means of artificial chromosome vectors. Science 236: 806-812. 12. CARLSSON, P., OLOFSSON, S. O., DARNFORS, C., AND BJURSELL, G. (1986). Analysis of the human apolipoprotein B gene: Complete structure of the B-74 region. Gene 49: 29-51. 13. CAVALLESCO, C., FORGET, B. G., DERIEL, J. K., WILSON, L. B., WILSON, J. T., AND WEISSMA.N, S. M. (1980). Nucleotide sequence of human Oy globin messenger RNA. Gene 12: 215-221. 14. CAWTHON, R. M., WEISS, R., GANGFENG, X., VISKCJCHIL, D., CULVER, M., STEVENX, J., ROBERTSON, M., DUNN, D., GxsTELAN, R., O’CONNELL, P., AND WHITE, R. (1990). A major segment of the neurofibromatosis type I gene: cDNA sequence, genomic structure, and point mutations. Cell 62: 193201. 15. CHAKRAVARTI, A., BUETOW, K. H., ANTONARAKIS, S. E., WABER, P. G., BOEHM, C. D., AND KAZAZUN, H. H., JR. (1984). Nonuniform recombination within the human betaglobin gene cluster. Am. J. Hum. Genet. 36: 1239-1258. 16. CHAKRAVARTI, A., ELBEIN, S. C., AND PER-, M. A. (1986). Evidence for increased recombination near the human insulin gene: Implication for disease association studies. Proc. Natl. Acad. Sci. USA 83: 1045-1049. 17. CHEBLOUNE, Y. U., PAGNIER, J., TRABUCHET, G., FAURE, C., VERDIER, G., LAFIIE, D., AND NIGON, V. (1988). Structural analysis of the 5’ flanking region of the beta-globin gene in
TRANSCRIBED
18.
19.
20.
21.
22. 23.
24.
25.
26.
27.
3’ UNTRANSLATED
African sickle cell anemia patients: Further evidence for three origins of the sickle cell mutation in Africa. Pmt. Nutl. Ad. Sci. USA 86: 4431-4435. CLEWLAND, D. W., LOPATA, M. A., MACDONALD, R. J., CoWAN, N. J., R~R, W. J., AND KIRSCHNER, M. W. (1980). Number and evolutionary conservation of a- and @-tubulin and cytoplasmic @- and y-actin genes using specific cloned cDNA probes. Cell 20: 95-105. D’ARPA, P., MACHLIN, P. S., RATIRE, H., ROTHFIELD, N. F., CLEVELAND, D. W., AND EARNSI-IAW, W. C. (1988). cDNA cloning of human DNA topoisomerase I: Catalytic activity of a 67.7-kDa carboxyl-terminal fragment. Proc. Natl. Acad. Sci. USA 86: 2543-2547. DAS, H. K., JACKSON, C. L., MILLER, D. A., LEFF, T., AND BRFSLOW, J. L. (1987). The human apolipoprotein C-II gene sequence contains a novel chromosome 19-specific minisatellite in its third intron. J. Biol. Chem. 262: 4787-4793. DONIS-KELLER, H., GREEN, P., HELMS, C., CARTINHOUR, S., WEIFFENBACH, B., STEPHENS, K., KEITH, T. P., BOWDEN, D. W., SMITH, D. R., LANDER, E. S., BOTSTEIN, D., AKOTS, G., REDIKER, K. S., Gmvrus, T., BROWN, V. A., RISING, M. B., PARKER, C., POWERS, J. A., WAIT, D. E., KAUFFMAN, E. R., BRICKER, A., PHIPPS, P., MULLER-KAHLE, H., FIJLTON, T. R., NC, S., SCHUMM, J. W., BRAMAN, J. C., KNOWLTON, R. G., BARKER, D. F., CROOKS, S. M., LINCOLN, S. E., DALY, M. J., AND ABRAHAMSON, J. (1987). A genetic linkage map of the human genome. Cell 51: 319-337. DRAYNA, D., AND WHITE, R. (1985). The genetic linkage map of the human X chromosome. Science 230: 753-758. EBINA, Y., ELLIS, L., JARNAGIN, K., EDERY, M., GRAF, L., CLAUSER, E., Ou, J., ~IARZ, F., KAN, Y. W., GOLDFINE, I. D., ROTH, R. A., AND RU~TER, W. J. (1985). The human insulin receptor cDNA: The structural basis for hormone-activated transmembrane signalling. Cell 40: 747-758. ECONOMOU, E. P., BERGEN, A. W., WARREN, A. C., AND ANTONARAKIS, S. E. (1990). The polydeoxyadenylate tract of Ah repetitive elements is polymorphic in the human genome. Proc. Natl. Ad. Sci. USA 87: 2951-2954. Fo~o, S. S., LAW, S. W., AND BREWER, H. B., JR. (1987). The human preproapolipoprotein C-II gene: Complete nucleic acid sequence and genomic organization. FEBS Lett. 213: 221-226. GUSELLA, J. F., WEXLER, N. S., CONNEALLY, P. M., NAYLOR, S. L., ANDERSON, M. A., TANZI, R. E., WATKINS, P. C., OrTINA, K., WALLACE, M. R., SAKAGUCHI, A. Y., YOUNG, A. B., SHOULDSON, I., BONILLA, E., AND MARTIN, J. B. (1983). A polymorphic DNA marker genetically linked to Huntington’s disease. Nature 306: 234-238. HAEFLIGER, J.-A., TSCHOPP, J., NARDELLI, D., WAHLI, W., KOCHER, H.-P., TOSI, M., AND STANLEY, K. K. (1987). Complementary DNA cloning of complement C8@ and its sequence homology to C9. Biochemistry 26: 3551-3556.
28. HENTHORN,
P. S., KNOLL, B. J., RADUCHA, M., ROTHBLUM, K. N., SLAUGHTER, C., WEISS, M., LAFFERTY, M. A., FISCHER, T., AND HARRIS, H. (1986). Products of two com-
mon alleles at the locus for human placental alkaline phosphatase differ by seven amino acids. Proc. Natl. Acad. Sci. USA 83: 5597-5601. 29. HOWARD, 0. M. Z., RAO, A. G., AND SODETZ, J. M. (1987). Complementary DNA and derived amino acid sequence of the /3subunit of human complement protein C8: Identification of a close structural and ancestral relationship to the LYsubunit and C9. Biochemistry 26: 3565-3570. 30. Human Gene Mapping 10: New Haven Conference (1989). Cytogerzet. Cell Genet. 51: 1-1147.
REGIONS
487
31. JEFFXEYS, A. J. (1979). DNA sequence variants in the G gamma-, A gamma-, delta-, and beta-globin genes in man. Cell 18: l-10. 32. +hFFREYS, A. J., WnSON, V., AND THEIN, S. L. (1985). Hypervariable ‘minisatellite’ regions in human DNA. Nature 314: 67-73. 33. KAM, W., CLAUSSER, E., KIM, Y. S., KAN, Y. W., AND RUTTER, W. J. (1985). Cloning, sequencing, and chromosomal localization of human term placental alkaline phosphatase cDNA. Proc. Natl. Acad. Sci. USA 82: 8715-8719. 34. KARATHANASIS, S. K., MCPHERSON, J., ZANNIS, V. I., AND BRESLOW, J. L. (1983). Linkage of human apolipoproteine A-I and C-III genes. Nature 304: 371-372. 35. KNO?T, T. J., WALLIS, S. C., POWELL, L. M., PEASE, R. J., LUSIS, A. J., BLACKHART, B., MCCARTHY, B. J., ~~LQ%EY, R. W., LEW-WILSON, B., ANII SCO?T, J. (1986). Complete cDNA and derived protein sequence of human apolipoprotein B-100. Nucleic Acids Res. 14: 7501-7503. 36. KORNBLIH?T, A. R., VIBE-PEDERSEN, K., AND BARALLE, F. E. (1983). Isolation and characterization of cDNA clones for human and bovine fibronectins. Proc. Natl. Acad. Sci. USA 80: 3218-3222.
37. KORNBLIHTT, A. R., VIBE-PEDERSEN, K., AND BARALLE, F. E. (1984). Human fibronectin: Cell specific alternative mRNA splicing generates polypeptide chains differing in the number of internal repeats. Nucleic Acids Res. 12: 5853-5868. 38. KRONENBERG, H. M., ROBERTS, B. E., AND EFSTRATIADIS, A. (1979). The 3’ noncoding region of @globin mRNA is not essential for in vitro translation. Nucleic Acids Res. 6: 153-166. 39. LANG, K. M., AND SPRPIZ, R. A. (1985). Cloning specific complete polyadenylated 3’-terminal cDNA segments. Gene 33: 191-196.
40. LAW, S. W., GRANT, LACKNER,
S. M., HIGUCHI, K., HOSPA?TANKAR, K., LEE, N., AND BREWER, H. B., JR. (1986).
A., Hu-
man liver apolipoprotein B-100 cDNA: Complete nucleic acid and derived amino acid sequence. Proc. Natl. Acad. Sci. USA 83: 8142-8146. 41. LEE, S.-C., CHANG, C.-J., LEE,Y.-M., LEJ, H.-Y., LAI,M.-Y., AND CHEN, D.-S. (1989). Molecular cloning of cDNAs corresponding to two genes of al-acid glycoprotein and characterization of two alleles of AGP-1 in the mouse. DNA 8: 245251.
42.
Lnr, T., ZON, G., SORAVIA, E., LIP, T.-Y., AND N. D. (1985). Genomic DNA sequence for human C-reactive protein. J. Biol. Chm. 260: 13377-13383. 43. LEVITT, R. C., SIENKIEWICZ, M. L., AND ZASLOFF, M. (1985). Molecular analysis of a human cytochrome Pm likely to be involved in anesthetic metabolism. Anesthesiology 63: A549. 44. LITT, M., AND LUTY, J. A. (1989). A hypervariable microsatellite revealed by in vitro amplification of a dinucleotide repeat within the cardiac muscle actin gene. Am. J. Hum. Genet. 44: 397-401. 45. ~TINIUK, F., MEHLER, M., TZALL, S., MJ%XEDITH, G., AND HIRSCHHORN, R. (1990). Sequence of the cDNA and 5’-flanking region for human acid cy-glucosidase, detection of an intron in the 5’ untranslated leader sequence, definition of 18bp polymorphisms, and differences with previous cDNA and amino acid sequences. DNA Cell Bid 9: 85-94. 46. MCCORMICK, M. K., SHERO, J. H., CHEUNG, M. C., KAN, LEI, K.-J., GOLDMAN,
Y. W., HIETER,
P. A., AND ANTONARAKIS,
S. E. (1989).
Con-
struction of human chromosome al-specific yeast artificial chromosomes. Proc. Natl. Acad. Sci. USA 86: 9991-9995. J. W., ELSHOURBAGY, N. A., CHANG, D. J., MAH47. MCLEAN, LEY, R. W., AND TAYLOR,
J. M.
(1984).
Human
tein E mRNA. J. Bid. C&m. 269: 6498-6504.
apolipopro-
ROY C. LEVITT
488
48. MILES, J. S., SPURR, N. K., GOUGH, A. C., JO-, T., MCLAREN, A. W., BROOK, J. D., AND WOLF, C. R. (1988). A novel human cytochrome P450 gene (P450IIB): Chromosomal localization and evidence for alternative splicing. Nucleic Acids Res. 16: 5783-5795. 49. MILLAN, J. L. (1986). Molecular cloning and sequence analysis of human placental alkaline phosphatase. J. Biol. Chem. 261: 31123115. 50. MILLER, W. L., THIRION, J. P., AND MARTIAL, J. A. (1980). Cloning of DNA complementary to bovine prolactin mRNA. Endocrinology 107: 851-854. 51. MING~I, P. P., CANCELA, L., FUJISAWA, Y., THEOFAN, G., AND NORMAN, A. W. (1988). Molecular structure of chicken vitamin D-induced calbindin-D,sx gene reveals eleven exons, six Car+-binding domains, and numerous promoter regulatory elements. Mol. Endocrinol. 2: 355-365. 52. NAKAJIMA, H., NOGUCHI, T., YAMASAKI, ‘I’., KONO, N., TANAKA, T., AND TARUI, S. (1987). Cloning of human muscle phosphofructokinase cDNA. FEBS Lett. 223: 113-116. 53. NAKAMURA, Y., LEPPERT, M., O’CONNELL, P., WOLFF, R., HOLM, T., CULVER, M., MARTIN, C., FUJMOTO, E., HOFF, M., KUMLIN, E., AND WHITE, R. L. (1987). Variable number of tandem repeat (VNTR) markers for human gene mapping. Science 235: 1616-1622. 54. NAKAMURA, Y., O’CONNELL, P., SONE, C., LALOUEL, J. M., AND WHITE, R. (1988). An extended genetic linkage map of markers for human chromosome 10. Genomics 3: 389-392. 55. O’CONNELL, P., LATHROP, G. M., LEPPERT, M., NAKAMURA, Y., MULLER, V., LALOUEL, J. M., AND WHITE, R. (1988). Twelve loci form a continuous linkage map for human chromosome 18. Genomics 3: 367-372. 56. OIKAWA, S., NAKAZATO, H., AND KOSAKI, G. (1987). Primary structure of human carcinoembryonic antigen (CEA) deduced from cDNA sequence. Biochem. Biophys. Res. Commun. 142: 511-518. 57. OKA, K., TKALCEVIC, G. T., NAKANO, T., TUCKER, H., ISHIMURA-OKA, K., AND BROWN, W. V. (1990). Structure andpolymorphic map of human lipoprotein lipase gene. B&him. Biaphys. Acta 1049: 21-26. 58. OLSON, M., HOOD, L., CANTOR, C., AND BOTSTEIN, D. (1989). A common language for physical mapping of the human genome. Science 245: 1434-1435. 59. ORITA, M., SUZUKI, Y., SEKIYA, T., AND HAYASKI, K. (1989). Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics 5: 874-879.
65.
Science 66.
67.
Natl.
Acad.
Sci. USA
70.
71.
72.
73.
74.
SETZER, D. R., MCGROGAN, M., NUSNBER, J. H., AND SCHIMKE, R. T. (1980). Size heterogeneity in the 3’ end of dihydrofolate reductase messenger RNAs in mouse cells. Cell SHARMA, P. M., REDDY, G. R., VORA, S., BABIOR, B. M., AND MCLACHLAN, A. (1989). Cloning and expression of a human muscle phosphofrutokinase cDNA. Gene 77: 177-183. SHEFFIELD, V. C., Cox, D. R., LEXMAN, L. S., AND MYERS, R. M. (1989). Attachment of a 40-base-pair G + C-rich sequence (GC-clamp) to genomic DNA fragmenta by the polymerase chain reaction results in improved detection of singlebase changes. Proc. Natl. Acad. Sci. USA 86: 232-236. SINNE’IT, D., DERAGON, J-M., SIMARD, L. R., AND LABUDA, D. (1990). Alumorphs-Human DNA polymorphisms detected by polymerase chain reaction using Ah-specific primers. Genomics 7: 331-334. SLIGHTOM, J. L., BLECHL, A. E., ANIJ SMITHIES, 0. (1989). Human fetal ‘y and *y-globin genes: Complete nucleotide sequences suggest that DNA can be exchanged between these duplicated genes. CeU 21: 627-638. SOOD, A. K., PAN, J., BIRO, P. A., PEREIRA, D., SRNASTAVA, R., REDDY, V. B., DUCEMAN, B. W., AND WEISSMAN, S. M. (1985). Structure and polymorphism of class I MHC antigen mRNA. Immunogenetics 22: 101-121. SOUTHERN, E. M. (1975). Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol.
75.
77.
78.
82: 3445-3449.
PERRYMAN, M. B., KERNER, S. A., BOHLMEYER, T. J., AND ROBERTS, R. (1986). Isolation and sequence analysis of a fulllength cDNA for human M creatine kinase. Biachem. Biaphys. Res. Commun. 140: 981-989. 63. PETERSEN, M. B., SKUGENHAUPT, S. A., CHAKRAVARTI, A., LEWIS, J. G., WARREN, A. C., AND ANTONARAKIS, S. E. (1991). A linkage map of 27 markers on human chromosome 21. Genomics 9: 407-419. 64. POON, R., KAN, Y. W., AND BOYJZR,H. W. (1978). Sequence of
1350-1354.
22:361-370. 69.
60.
Proc.
230:
SASAVAGE, N. L., NILSON, J. H., HOROWITZ, S., AND ROTTMAN, F. M. (1982). Nucleotide sequence of bovine prolactin messenger RNA-Evidence for sequence polymorphism. J. Biol. Chem. 257: 678-681. SEMENZA, G. L., MALLXX, P., SURREY, S., DEMROSSO, K., PONCZ, M., AND SCHWARTZ, E. (1984). Detection of a novel DNA polymorphism in the beta-globin gene cluster. J. Bial. Chem.259:6045-6048.
68.
76.
ORKIN, S. H., KAZAZIAN, H. H., JR., ANTON-IS, S. E., GOFF, S. C., BOEHM, C. D., SEXTON, J. P., WABER, P. G., AND GIARDINA, P. J. (1982). Linkage of beta-tbalassaemia mutations and beta-globin gene polymorphisms with DNA polymorphisms in human beta-globin gene cluster. Nature 296: 627-631. 61. PAIK, Y. K., CHANG, D. J., REARDON, C. A., DAVIES, G. E., MAHLEY, R. W., AND TAYLOR, J. M. (1985). Nucleotide sequence and structure of the human apolipoprotein E gene.
the 3’-noncoding and adjacent coding regions of human y-globin mRNA. Nucleic Acids Res. 6: 4625-4630. SAIKI, R. K., SCHARF, S., FALLOONA, F., MULLIS, K. B., HORN, G. T., ERLICH, H. A., AND ARNHEIM, N. (1985). Enzymatic amplification of beta-globm genomic sequences and restriction site analysis for diagnosis of sickle cell anemia.
62.
Biol.
98: 503-517.
TAUTZ, D. (1989). Hypervariability of simple sequences as a general source for polymorphic DNA markers. Nucleic Acids Res. 17: 6463-6471. TRASK, R. V., STRAUSS, A. W., AND BILLADELLO, J. J. (1988). Developmental regulation and tissue-specific expression of the human muscle creatine kinase gene. J. Bial. Chem. 263: 17142-17149. TRAYSTMAN, M. D., HIGUCHI, M., KASPER, C. K., ANTONARAKIS, S. E., ti KAZAZIAN, H. H. (1990). Use of denaturing gradient gel electrophoresis to detect point mutations in the factor VIII gene. Genomics 6: 293-301. TYERS, M., HASLAM, R. J., RACHUBINSKI, R. A., AND HARLEY, C. B. (1989). Molecular analysis ofpleckstrin: The major protein kinase C substrate of platelets. J. Cell. Biochem. 40: 133-145.
79.
80.
ULLRICH, A., BELL, J. R., CHEN, E. Y., HERRERA, R., PETRUZZELLI, L. M., DULL, T. J., GRAY, A., COUSSEN, L., LIAO, Y.-C., TSUEOKAWA, M., MASON, A., SEXFIURG,P. H., GRUNFELD, C., ROSEN, 0. M., AND RAMACHANDRAN, J. (1985). Human insulin receptor and its relationship to the tyrosine kinase family of oncogenes. Nature 313: 756-761. WARREN, A. C., SLAUGENHAUPT, S. A., LEWIS, J. G., CHAKRAVARTI, A., AND ANTONARAKIS, S. E. (1989). A genetic
TRANSCRIBED
3’ UNTRANSLATED
linkage map of 17 markers on human chromosome 21. Gerwmics 4: 579-591. 81. WATANABE, S., WATANABE, T., LI, W. B., SOONG, B.-W., AND CHOU, J. Y. (1989). Expression of the germ cell alkaline phosphatase gene in human choriocarcinoma cells. J. Bid. Chem. 264: 12611-12619. 82. WEBER, J. L., AND MAY, P. E. (1989). Abundant class of human DNA polymorphisms which can be typed using the polymerase chain reaction. Am. J. Hum. Genet. 44: 366-396. 83. WEI, C. F., CHEN, S. H., YANG, C. Y., MARCEL, Y. L., MILNE, R. W., LI, W. H., SPARROW, J. T., GOTTO, A. M., JR., AND C&IAN, L. (1985a). Molecular cloning and expression of partial cDNAs and deduced amino acid sequence of a carboxylterminal fragment of human apolipoprotein B-100. Proc. Natl. Ad. Sci. USA 82: 7265-7269. 84. WEI, C. F., TSAO, Y. K., ROBBERSON, D. L., Gorro, A. M., JR., BROWN, K., AND CHAN, L. (198513). The structure of the human apolipoprotein C-II gene: Electron microscopic analysis of RNA:DNA hybrids, complete nucleotide sequence, and identification of 5’ homologous sequences among apolipoprotein genes. J. Biol. C&m. 260: 15211-15221. 85. WELLER, P., JEFFREYS, A. J., WILSON, V., AND BLANCHETOT, A. (1984). Organization of the human myoglobin gene. EMBO J. 3: 439-446. 86. WHITE, R., AND LALOUEL, J. M. (1988). Sets of linked genetic markers for human chromosomes. Annu. Rev. Genet. 22: 259-279. 81. WHITE, J. W., AND SAUNDERS, G. F. (1986). Structure of the human glucagon gene. Nucleic Acids Res. 12: 4719-4730.
REGIONS
489
88. WION, K. L., KIRCHGESSNER, T. G., LUSIS, A. J., SCHOTZ, M. C., AND LAWN, R. M. (1987). Human lipoprotein iipase complementary DNA sequence. Science 236: 1638-1641. 89. Woo, P., KORENBERG, J. R., AND WHITEHEAD, A. S. (1985). Characterization of genomic and complementary DNA sequence of human C-reactive protein, and comparison with the complementary DNA sequence of serum amyloid P component. J. Biol. Chem. 260: 13384-13388. 90. YAMANO, S., NAGATA, K., YAMAU)E, Y., KATO, R., GELBOIN, H. V., AND GONZALEII, F. J. (1989a). cDNA and deduced amino acid sequences of human P450 IIA3 (CYP2A3). Nucleic Acids Res. 17: 4888. 91. YAMANO, S., NHAMBURO, P. T., AOYAMA, T., MEYER, U. A., INABA, T., KALOW, W., GELBOIN, H. V., MCBRIDE, 0. W., AND GONZALEZ, F. J. (1989b). cDNA cloning and sequence and cDNA-directed expression of human P450 IIBl: Identification of a normal and two variant cDNAs derived from the CYPBB locus on chromosome 19 and differential expression of the IIB mRNAs in human liver. Biochemistry 28: 73407348.
92.
YAMANO, S., TATSUNO, J., AND GONZALEZ, F. J. (1990). The CYP2A3 gene product catalyzes coumarin 7-hydroxylation in human liver microsomes. Biochemistry 29: 1322-1329. 93. ZHOU, B.-S., BASTOW, K. F., AND CHENG, Y. C. (1989). Characterization of the 3’ region of the human DNA topoisomerase I gene. Cancer Res. 49: 3922-3927. 94. ZULIANI, G., AND HOBBS, H. H. (1990). A high frequency of length polymorphisms in repeated sequences adjacent to Alu sequences. Am. J. Hum. Genet. 46: 963-969.
