Proc. Natl Acad. Sci. USA Vol. 80, pp. 5027-5031, August 1983 Genetics

Two nonallelic tRNAMet are located in the p23-*ql2 region of human 6 (transfer RNA/ 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 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 (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 (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. To assign the tRNA!Iet genes detected by the 1.15- and 2X NaCI/Cit/0. 1% NaDodSO4 at room temperature then washed 1.45-kb fragments to chromosomes, DNA samples were iso- in 0.1 X NaCI/Cit/O. 1% NaDodSO4 at 50'C for 45 min. Suc- lated from several human-rodent somatic cell hybrids, each with cessive washes were used to reduce background radioactivity, a unique set of human chromosomes, and analyzed by Southern if necessary. The filters were then exposed to Kodak XAR film blot analysis using a BamHI digest of the cell hybrid DNAs and with DuPont Cronex Lightning Plus intensifying screens at hybridization with the tRNA~et probes. Because these probes -70'C. Autoradiograms were developed after 1-10 days of ex- detect only human sequences, only DNA from those cell hy- posure. A r RESULTS 1.15kb fragment tRNA0et Gene Arrangement in Human DNA. Santos and 1.45 kb fragment Zasloff (2) have shown that 2 of the approximate 13 tRNAiet genes of the human genome are nonallelic as judged by re- 9 4- O kb striction endonuclease digestion, sequence analysis, and het- eroduplex analysis of sequences identified in A Charon 4A phage 6.0 kb libraries of human fetal DNA (2). Restriction endonuclease maps of these two loci derived in a previous study (2) are shown in Fig. 1. EcoRI restriction fragments bearing tRNAM" genes were 3.1 kb- isolated from Charon phage Ahtml (1. 15-kb fragment) and Ahtm2 (1.45-kb fragment), labeled by nick-translation (26), then used H M - + + + + - M H to probe mouse and human genomic DNA cleaved with BamHI, using the filter hybridization method of Southern (24). As shown FIG. 2. Hybridization of the tRNA¶et probes to human, mouse, restriction and hybrid DNAs. (A) Hybridization of the 1.45-kb fragment to DNA in Fig. 2, both fragments detect specific BamHI from a human fibroblast line (WI38) (H), a mouse cell line (LM/TK-) fragments in human but not mouse genomic DNA. With these (M), and hybrids resulting fromnthe fusion ofthese two cells that either conditions of hybridization and time of film exposure, mouse hybridize to the 1.45-kb fragment (+) or, like the mouse parental cells, tRNA ~et gene sequences are not detected. Because of its short do not hybridize (-). (B) Hybridization of the 1.15-kb fragment to the length and secondary structure, the 72-base-pair tRNAet se- same DNAs. Downloaded by guest on September 26, 2021 Genetics: Naylor et aL Proc. Natl Acad. Sci. USA 80 (1983) 5029 Table 1. Segregation of two nonallelic genes of tRNAlet with human chromosomes in somatic cell hybrids Reaction with Cell tRNAMet probe Chromosomes hybrid 1.45 kb 1.15 kb 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X 11/X X/11 15/X 3/17 17/3 X/3 3/X XER-7* + + + + + + + + + + + + - + + + + -- + -- -- + + - XER-9* - - - + - - -+ + - +- + -- + + - - + -- + - XER11* + + + - + + -+ + + + - + - -+ + + + - + - + - + + EXR-3t + + + + + + + + + + + + + + + + + + ++ + + + - + + EXR-9t + + + + + + + + + + - + + + + + + + + + + + + + - + + DUA-1At -----+ TSL-2§ + + -+ --+ + ---+ -+ + + -+ + + XTR-3BSAgBI - -+.+ - - + ICL15 - + + + + MAR-2 - - + + + + + + + + + + + + + WHLL14 -- -- + + Southern blot analysis and karyotyping ofhybrid clones were performed on cells in the same passage. At least 30 metaphases were analyzed for each hybrid line. A hybrid was considered positive for a given human chromosome if one copy was present in at least 10% of the metaphases. *The 11/X translocation chromosome is Xqter--Xq11::11p11-11qter and the X/11 chromosome is Xpter--+Xqll::llpll--dlpter. tThe 11/X translocation chromosome is Xqter-)Xq22::11ql3--)llpter and the X/11 chromosome is Xpter- .Xq22::11ql3-4llqter. tThe 15/X translocation chromosome is 15pter-+15ql1::Xp1--Xpter. §The 3/17 translocation chromosome is 3qter-,3p21::17p13-d17pter and the 17/3 chromosome is 3pter-33p21::17p13- 17qter. 1The X/3 translocation chromosome is 3qter--3q21::Xq28--Xpter and the 3/X chromosome is 3pter-*3q21::Xq28--*Xqter.

brids containing the complementary human sequences yield signment of the tRNA el genes to a specific chromosome was hybridizing restriction fragments. By comparing the human made. Table 1 summarizes the results obtained from a set of chromosomal content of hybrid cells as determined by the anal- 11 karyotyped hybrid cells screened for the presence or ab- ysis of marker enzymes assigned to each chromosome and by sence of the BamHI fragments complementary to the 1.15- and karyotyping with the results of the Southern blot analyses, as- 1.45-kb probes bearing nonallelic tRNA"et gene loci. Both

Table 2. Segregation of two nonallelic genes of tRNAMet with enzyme markers tRNA genes/enzyme marker Chromosome Enzyme marker +/+ +/- -/+ -/- % discordant 1 PEPC 2 5 2 10 37 2 IDH1, MDH1 8 2 3 12 20 3 ACY1 9 1 8 7 36 4 PEPS 5 3 5 7 40 5 HEXB 6 4 5 10 36 6 ME1, SOD2 9 0 1* 15 4 7 GUSB 7 4 3 12 28 8 GSR 7 2 7 7 39 9 AK1, ACO1 1 9 0 15 36 10 GOT1 8 2 7 8 36 11 LDHA 9 1 6 9 28 12 LDHB, PEPB 6 5 8 7 48 13 ESD 5 5 4 11 36 14 NP 10 0 8 6 33 15 MPI,PKM2 8 2 5 10 28 16 APRT 3 7 3 12 40 17 GALK 10 0 13 2 52 18 PEPA 8 2 9 6 44 19 GPI 7 3 4 11 28 20 ADA 7 3 4 11 28 21 SOD1 9 1 12 3 52 22 ACO2 3 7 4 10 46 X G6PD 8 2 10 5 48 Electrophoretic and detection procedures forenzyme markers are in references listed in ref. 22. Enzyme markers are peptidase C (PEPC), isocitrate dehydrogenase (soluble) (IDH1), malate dehydrogenase (sol- uble) (MDH1), aminoacylase 1 (ACYM), peptidase S (PEPS), hexosaminidase B (HEXB), malic enzyme (soluble) (ME1), superoxide dismutase (mitochondrial) (SOD2), P-glucuronidase (GUSB), phosphoserine phosphatase (PSP), glutathione reductase (GSR), adenylate kinase 1 (AK1), aconitase (soluble) (ACO1), glutamate oxaloacetate transaminase (soluble) (GOT1), lactate dehydrogenase A (LDHA), lactate de- hydrogenase B (LDHB), peptidase B (PEPB), esterase D (ESD), nucleoside phosphorylase (NP), man- nosephosphate isomerase (MPI), pyruvate kinase muscle form (PKM2), adenine phosphoribosyltransfer- ase (APRT), galactokinase (GALK), peptidase A (PEPA), phosphoglucose isomerase (GPI), adenosine deaminase (ADA), superoxide dismutase (soluble) (SOD1), aconitase (mitochondrial) (ACO2), and glu- cose-6-phosphate dehydrogenase (G6PD). *This hybrid contained the enzyme SOD2 but not ME1, suggesting that it has a broken chromosome 6. Downloaded by guest on September 26, 2021 5030 Genetics: Naylor et aL Proc. Natl. Acad. Sci. USA 80 (1983) Table 3. Regional assignment of TRM1 and TRM2 Reaction with tRNAie probe Hybrid Region present 1.15 kb 1.45 kb - RAG SU 3-1-2-3 6pter--ql4 + + 12 J TRMITRM2 ITA9-1-2 6q12-*qter - - Call A9-1-13 6p23-.qter + + >2 Call A9-1-9-3 6pter-*p23 GM610 RAG-4-5-1 6pter-.q21 + + GM610 RAG-5-23 6q21-+qter The presence of translocation chromosomes was determined karyo- 23 typically on an aliquot ofcells, the remainder ofwhich was used in the 5 isolation of DNA for Southern blot analyses. sSh, probes reacted with cell hybrid DNA only when human chro- mosome 6 was present. No other chromosome segregated con- 6 cordantly with these genes. We conclude from these data that 7 both loci are found on chromosome 6. All positive DNA prep- FIG. 3. Localization ofthe two tRNAMet genes to the p23-*ql2 re- arations exhibited BamHI patterns consistent with the pattern gion of human chromosome 6. Arrows indicate the location of breaks seen in human genomic DNA, as illustrated in Fig. 2. in the human translocated cells used for hybrid formation. We have characterized an additional 25 cell hybrids for the presence or absence of marker enzymes that have previously ternatively, related genes have been found at widely dispersed been assiged to each human chromosome (22) as well as for the sites, as seen with prolactin and CSH (30, 32). The organization two tRNA et genes (Table 2). Sequences complementary to both of individual gene families may not be the same in all species the 1.15- and 1.45-kb probes were detected in hybrid cell lines of organisms; 5S RNA genes may be either clustered or dis- containing human malic enzyme (ME1) and the mitochondrial persed (33), depending on the species studied. In the case of form of superoxide dismutase (SOD2), whose genes have been tRNA genes in eukaryotic systems, no general picture of overall assigned to chromosome 6 (27, 28). All other markers showed structural and functional organization has emerged except that :20% discordancy. These data support our contention that both tRNA genes can be tandemly repeated and embedded in ho- tRNAMet gene loci are on human chromosome 6. However, one mologous sequences such as in amphibia (7-10); however, this hybrid that expressed human ME1 did not contain sequences may be related to gene amplification required for embryologi- that hybridize to either the 1.15- or 1.45-kb probe. This hybrid cal development in these organisms. clone did not contain SOD2 activity, which suggests that a dele- We have shown here that two nonallelic human tRNAM" genes tion or other rearrangement of chromosome 6 may have oc- are both positioned within about 2% of the human genome even curred in this hybrid. though sequence analysis of these loci showed little homology The combined data from chromosome and marker enzyme of flanking sequences (2). These results raise the interesting studies indicate that both tRNAMet loci are located on human possibility that others of the 15 or so tRNA4et loci may reside chromosome 6. For convenience, we designate the gene de- on chromosome 6; it is necessary to isolate other tRNAmet loci tected by the 1.15-kb probe TRMI and the gene hybridizing in order to address this question. However, if one calculates with the 1.45-kb probe, TRM2. the probability of any 2 of 15 tRNA4et genes being found by Two tRNA~4t Genes Are Within the p23-+q12 Region of chance within any 2% segment of the human genome, a value Chromosome 6. Hybrids segregating portions of chromosome of 8.4% is obtained. Hence it is premature to conclude that all 6 were analyzed to further define the chromosomal location for tRNAMet genes will be found clustered on chromosome 6, par- the two tRNAMet loci studied here. Cultured cells from three ticularly because the factors determining whether a multigene individuals having reciprocal translocations involving chromo- family will be clustered or found at various chromosomal lo- some 6 were used in the formation of human-rodent somatic cations are essentially unknown. Roy et al. (34) have recently cell hybrids (20, 21). One additional hybrid (RAG SU 3-1-2-3) isolated a 1.65-kb segment of human DNA encoding three dif- had a spontaneous deletion of the long arm of chromosome 6 ferent amino acid accepting species of tRNA, those for lysine, [del(6ql4-->qter)] (20, 21). Table 3 summarizes the chromo- glutamine, and leucine. The 5' flanking sequences of each gene somal constitution of the hybrids used. Note that the key hy- shared only limited homology. The cluster of three tRNAs was brids RAG SU 3-1-2-3 (6pter-*q14) and Call A9 1-13 (6p23-3 not part of a tandem repeat because no other tRNA genes were qter) contain both tRNA etloci, whereas IT A9 1-2 (6q12-* detected within the additional 11 kb of human DNA flanking qter) lacks the sequences, indicating that these genes must be the genes. Whether the cluster forms a dispersed repeating unit between 6p23-4ql2 (Fig. 3). This, taken with the results from is not known (34). The chromosome location of this group of the other translocation hybrids, supports our contention that tRNA genes is not presently known, but the close linkage of both TRM1 and TRM2 reside in the p23--ql2 region of chro- three different tRNA species adds uncertainty as to whether mosome 6. isoaccepting species such as tRNAMet will necessarily be syn- tenic. DISCUSSION This study provides the assignment for two tRNA genes in It is generally accepted that multigene families result from the the human genome, contributes to our understanding of how duplication and relocation of an ancestral precursor gene. Such the 1,000 or so human tRNA genes may have evolved and are families may remain clustered, as is the case for the ,-globin structured, and provides additional physical markers for the hu- family located on human chromosome 11 (29), growth hormone man genome. To look at individual tRNAMet genes, we have (GH) and chorionic somatomammotropin (CSH) on chromo- employed unique flanking sequences to locate members of a some 17 (30), or the histone genes on chromosome 7 (31). Al- multigene family. Finally, as recombinant clones bearing other Downloaded by guest on September 26, 2021 Genetics: Naylor et al. Proc. Natl. Acad. Sci. USA 80 (1983) 5031 human tRNA genes are isolated, studies such as these should 16. Champion, M. J. & Shows, T. B. 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L., Elliott, R. W., Brown, J. A. & Shows, T. B. (1982) 33. Fedoroff, N. (1979) Cell 16, 697-710. Am. J. Hum. Genet. 34, 235-244. 34. Roy, K. L., Cooke, H. & Buckland, R. (1982) Nucleic Acids Res. 15. Champion, M. J., Brown, J. A. & Shows, T. B. (1978) Cytogenet. 10, 7313-7322. Cell Genet. 22, 498-502. Downloaded by guest on September 26, 2021