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Two Nonallelic Trnamet Genes Are Located in the P23-*Ql2 Region of Human Chromosome 6 (Transfer RNA/Gene Mapping/Southern Blot Analysis) S

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Two Nonallelic Trnamet Genes Are Located in the P23-*Ql2 Region of Human Chromosome 6 (Transfer RNA/Gene Mapping/Southern Blot Analysis) S Proc. Natl Acad. Sci. USA Vol. 80, pp. 5027-5031, August 1983 Genetics Two nonallelic tRNAMet genes are located in the p23-*ql2 region of human chromosome 6 (transfer RNA/gene mapping/Southern blot analysis) S. L. NAYLOR*, A. Y. SAKAGUCHI*, T. B. SHOWS*, K.-H. GRZESCHIKt, M. HOLMESt, AND M. ZASLOFF§ *Department of Human Genetics, Roswell Park Memorial Institute, New York State Department of Health, Buffalo, New York 14263; tInstitut fur Humangenetik, Westfilische Wilhelms-Universitat, Monster, Federal Republic of Germany; tDepartment of Microbiology, Medical College of Virginia, Richmond, Virginia 23298; and §Human Genetics Branch, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20205 Communicated by Elizabeth F. Neufeld, May 6, 1983 ABSTRACT Two nonallelic human tRNAlet genes were as- MATERIALS AND METHODS signed to chromosome 6 by filter hybridization of DNA from hu- Human cell lines used included W138 man-rodent somatic cell hybrids by using probes containing unique (a diploid fibroblast), sequences from the regions flanking each tRNA¶et gene. These GM2859 [46,X,t(X;11)(qll;pll)] (12), GM3322 [46,X,t(X; unique sequence probes thus allowed each tRNA~et gene to be l1)(q22;q13)] (12), CaVa [46,X,t(X;22)(q22;ql3)] (13), GM194 analyzed individually in cell hybrids. Both tRNAiet genes seg- [46,X,t(X;3)(q28;p21)] (14), GM2808 [46,XX,t(3;17)(p21;p13)] regated in the hybrid cells with the chromosome 6 enzyme mark- (14), DUV [46,X,5(X.;15)(p11;q11)] (15), GM1006 (from a pa- ers, soluble malic enzyme and the mitochondrial form of super- tient with mucolipidosis II) (16), and GM654 (from a patient oxide dismutase, and also with a karyotypically normal chromosome with mannosidosis) (16). The GM lines were obtained from the 6. By using hybrid clones containing translocations that divide Human Genetic Mutant Cell Repository (Camden, NJ). chromosome 6 into five segments, both tRNAiet genes were as- Somatic cell hybrids were made by the fusion (17) of human signed to the p23--ql2 region. These results raise the possibility fibroblasts (or leukocytes) to mouse cell lines containing se- that other tRNAlet genes may be syntenic with the two described lectable markers. The hybrids were maintained on hypoxan- in this study and illustrate the utility of using unique flanking se- thine/aminopterin/thymidine (HAT) medium (18). Hybrid lines quences to identify members of a multigene family. were designated WIL (WI38 and LTP mouse cells) (19), REW (WI38 and RAG mouse cells) (19), XER (GM2859 and RAG) It has been estimated that the human haploid genome contains (12), EXR (GM3322 and RAG) (12), REX (CaVa and RAG) (13), 1,000-2,000 tRNA genes and that there are 50-60 chromato- XTR (GM194 and RAG) (14), TSL (GM2808 and LMTK-) (14), graphically distinct tRNA species (1). These observations imply DUA (DUV and A9) (15), ICL (GM1006 and LMTK-) (16), and that some tRNA genes are redundant. Recently, this has been MAR (GM654 and RAG) (16). shown to be the case for human tRNAM" genes, which occur To regionally localize the tRNAet genes on chromosome 6, in 12-15 nonallelic loci within the human genome (2). In other hybrids made with human cells containing translocations of organisms, genes for tRNA isoaccepting species can be scat- chromosome 6 were analyzed. The hybrids were constructed tered throughout the genome or arranged in tandem repeats by the fusion of (i) IT fibroblasts [46,XX,t(6;7)(ql2;pl4)] to mouse (3). For example, in Saccharomyces eight tyrosine tRNA genes A9 cells (ITA9); (ii) cell fibroblasts [46,XY,t(2;6)(p23;p23)] to are dispersed over six chromosomes (4), whereas in Drosophila mouse A9 cells (Call A9); and (iii) GM610 fibroblasts [46,XX, eight tRNA genes are located in 10 kilobases (kb) of DNA (5, t(6;18)(q21;pll)] to mouse RAG cells (GM610 RAG) (20, 21). 6). Interestingly, the tRNAmet genes of Xenopus along with other Also included was a human fibroblast-RAG hybrid clone (RAG tRNA gene species are tightly clustered and located within a SU 3-1-2-3) that had undergone a spontaneous deletion of 6q 3.18-kb region of DNA tandemly repeated several hundred times [del(6ql4--6qter)] . (7-10). However, it appears from the studies of Santos and Zas- Analyses of Somatic CellIHybrids. The human chromosome loff (2, 11) that the 15 or so human tRNAmet genes are not tightly composition of the hybrid cells was determined in two ways. clustered as judged by Southern analysis of human genomic First, human chromosomes were identified by karyotypic anal- DNA and a detailed analysis of four cloned segments of human ysis of hybrid cellsTrypsin-Giemsa banding of chromosomes DNA bearing individual tRNAIet genes. DNA sequence and was performed as described (13). Second, a large number of cell heteroduplex analyses of these four recombinant A Charon 4A hybrids were analyzed for human enzyme markers whose genes clones established that two tRNAMet loci were nonallelic be- have been assigned to specific human chromosomes. A profile cause there was little homology in flanking sequences (2). Be- of 29 human enzyme markers thus reflected the chromosome cause the tRNAmet genes were each found on a separate re- composition of a given hybrid. Enzyme markers are listed in combinant clone, these genes must be located greater than 20 Table 2, and original references.for details of the electropho- kb apart in the human genome (2). resis and enzyme detection are given by Shows et al (22). We have utilized the unique sequences flanking these two DNA was isolated from hybrids (23), and, at the same pas- tRNAMet genes to probe DNA from human-mouse cell hybrids sage, cell homogenates for isozyme analysis and metaphase to determine their chromosomal location. The results of this spreads for karyotyping were made as described (22). Ten mi- study localizing both genes in the same region of chromosome crograms of DNA was digested with a restriction endonuclease 6 suggest that other tRNA genes may be in this region and raise in the buffers recommended by the supplier. The resulting DNA questions about the arrangement of the tRNA gene family in fragments were separated by electrophoresis in 1% agarose gels man. in Tris/acetate buffer, pH 8.2. Molecular weights were esti- The publication costs of this article were defrayed in part by page charge Abbreviations: kb, kilobase(s); NaCl/Cit, 0.15 M NaCI/0.015 M sodium payment. This article must therefore be hereby marked "advertise- citrate; ME1, malic enzyme (soluble); SOD2, superoxide dismutase ment" in accordance with 18 U.S.C. §1734 solely to indicate this fact. (mitochondrial). 5027 Downloaded by guest on September 26, 2021 5028 Genetics: Naylor et aL Proc. Natl. Acad. Sci. USA 80 (1983) E 5Hjp Hi Hi XHa HI Ha TI EI 1.15kb fragment Hh Ho Hh Hp H A Ha Hh Hi Hh A Hh T* El § >Hi Hi H B Hp E tff- NIK( )I/ I I 1.45kb fragment FIG. 1. Restriction maps of tRNAMet and flanking sequences. (Upper) The 1.15-kb EcoRI fragment from Ahtml (2). (Lower) The 1.45-kb EcoRI fragment from Ahtm2 (2). The cleavage sites are abbreviated as A, Alu I; B, BamHI; E, EcoRI; H, HinduI; Ha, Hae III; Hh, Hha I; Hi, Hinfi; Hp, Hpa II; T, Taq I; X, Xba I. mated by comparing the mobilities of DNA fragments to the quence, probably identical in man and mouse (2), generates a mobilities of those produced by a HindIII digestion of bacte- considerably weaker hybridization signal than the 15- to 20-fold riophage A. larger human-specific sequences contained on the probes. Thus, Detection of tRNAMet Sequences. DNA from agarose gels whereas the unique flanking regions are detected after an over- was transferred onto nitrocellulose filters (Schleicher & Schuell) night exposure of a Southern blot of human DNA, tRNA4et- by the method of Southern (24). Blots in heat-sealable bags were specific sequences are visualized with comparable intensity only hydrated and incubated at 420C in a solution containing 50% after a 15- to 20-day exposure (unpublished data). In particular, (vol/vol) formamide, 5X NaCI/Cit (lx NaCl/Cit = 0.15 M it can be seen in Fig. 2 that the 1.15-kb probe detects a 10-kb NaCI/0.015 M sodium citrate), 5X Denhardt's solution (lx restriction fragment in human genomic DNA cut with BamHI, Denhardt's solution = 0.02% bovine serum albumin/0.02% Fi- whereas the 1.45-kb probe reveals two fragments 6.0 and 3.1 coll/0.02% polyvinylpyrrolidone), 50 mM sodium phosphate at kb in size. These results are consistent with BamHI restriction pH 6.5, denatured sonicated salmon sperm DNA at 500 ,ug/ml, maps previously determined for these gene loci in recombinant and 0.1% NaDodSO4 (25). After 24 hr, this mixture was re- phage (ref. 2 and Fig. 1) and support previous studies which placed by a hybridization mixture containing 50% formamide, suggest that these genes are nonallelic. Furthermore, a similar 10% dextran sulfate, 5X NaCl/Cit, lx Denhardt's solution, 20 analysis of other human DNA preparations has not detected mM sodium phosphate at pH 6.5, and 0.1% NaDodSO4 along polymorphisms (unpublished data), which supports our con- with 5 x 106 cpm of labeled probe that had been denatured by tention that two distinct loci have been detected. boiling with sonicated salmon sperm DNA at 200 ,4g/ml. After Segregation of Two tRNAket Genes with Human Chromo- 24-48 hr of hybridization at 420C, blots were rinsed briefly in somes.
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