Hum Genet (1991) 88:119-121
9 Springer-Veflag1991
Chromosome mapping of the human cytidine-5'-triphosphate synthetase (CTPS) gene to band lp34.1-p34.3 by fluorescence in situ hybridization Ei-ichi Takahashi I, Masatake Yamauchi 1, Hideo Tsuji 1, Akitsu I-litomi x, Mark Meuth 2, and Tada-aki Hori I 1Division of Genetics, National Institute of Radiological Sciences, 4-9-I Anagawa, Chiba, 260 Japan 2Imperial Cancer Research Fund, Clare Hall Laboratories, Blanche Lane, South Mimms, Herts EN6 3LD, UK Received April 16, 1991
Summary. The human cytidine-5'-triphosphate synthetase (CTPS) gene was mapped by a direct mapping system combined with fluorescence in situ hybridization and replicated prometaphase R-bands. By high-resolution banding analysis, the signals were localized to band 34.1-34.3 of the short arm of c h r o m o s o m e 1; l p 3 4 . 1 p34.3. Simple procedures for the detection of R-bands are described.
Introduction Cytidine-5'-triphosphate synthetase (CTPS, U T P : L-glutamine ligase, E C 6.3.4.2) is one of the key regulatory enzymes in pyrimidine biosynthesis. CTPS activity has been isolated and purified from both prokaryotic (Lieberman 1956; Long and Pardee 1967) and mammalian (Hurlbert and Kammen 1960; Savage and Weinfeld 1970) sources. CTPS is subject to feedback inhibition by its product, CTP. The loss of this regulation has numerous effects on cells, including an increased rate of spontaneous mutation and resistance to cancer chemotherapeutic agents (Trudel et al. 1984). Recently, Yamauchi et al. (1990) have successfully cloned the human CTPS gene using chromosome-mediated gene transfer. Sequence analysis o f c D N A revealed that the CTPS gene has been strikingly conserved from E. coli to human (Yamauchi et al. 1990). This suggests that CTPS activity and regulation are crucial for cell proliferation. A n o t h e r key regulatory e n z y m e gene in pyrimidine biosynthesis, thymidylate synthase (TS), has been isolated (Ayusawa et al. 1984) and m a p p e d to 18p11.32 by fluorescence in situ hybridization (Hori et al. 1990). T o elucidate the organization of pyrimidine biosynthetic genes in the human g e n o m e , it is important to map the CTPS gene. Fluorescence in situ hybridization (FISH) techniques have made it possible to localize D N A sequences on chromosomes (Langer et al. 1981; Pinkel et al. 1986; Lawrence et al. 1988). A direct mapping system has
Offprint requests to: E. Takahashi
been recently developed, which is based on F I S H combined with replicated prometaphase R-bands (Takahashi et al. 1990a, 1991). For genomic clones containing repetitive sequences, a suppression hybridization procedure has been established with total human D N A (Landegent et al. 1987; Pinkel et al. 1988; Lichter et al. 1990; Hori et al. 1990). These two systems facilitate a more rapid and efficient method not only for gene mapping but also for genome mapping by the construction of cytogenetic maps using chromosome-specific genomic libraries (Hori et al. 1990; Yamakawa et al. 1991; Tokino et al. 1991). In the present communication, we report the chromosome mapping of the human CTPS gene by these systems.
Materials and methods Preparation of R-banded chromosomes The R-banding method for direct mapping with FISH has been described elsewhere (Takahashi et al. 1990a, 1991). This method is based on cell-synchronization with excess thymidine prior to incorporation of bromodeoxyuridine (BrdU) into replicating DNA. The procedures of lymphocyte-culture, thymidine- and BrdUtreatment, and chromosome preparation were carried out in a routine manner (Takahashi et al. 1990a, 1991). In the previous reports (Takahashi et al. 1990a, 1991) a mercury vapor lamp was used for the detection of R-bands. This has been modified in the present study by the use of a black light and a hot plate. The BrdUincorporated chromosome preparations were stained with I ttg/ml Hoechst 33258 in Serensen's phosphate buffer (pH 6.8) for 5 rain, rinsed and then mounted in 2 x SSC (standard saline citrate, pH 7.8) under coverslips. The chromosome slides were heated at 75~ for 3 min on a hot plate (Hirasawa), and then exposed for 6 rain at 75"C under a black light (Toshiba, 20W, FL20SBLB) at a distance of 1.5 cm. The exposed slides were rinsed in distilled water, dried and stored at -80~ until use.
D N A probes Two overlapping human genomic clones of the CTPS gene, H21 and II7221 in LDASHII (Yamauchi et al. 1991), were used as DNA probes in the present study. H21 (a 20kb insert) covers the Y-end of the gene and II7221 (an 18kb insert) covers the T-side with a 2 kb overlap.
120
Fig. la-d. R-banded pro-metaphases (a whole, b--fl partial) after in situ hybridization with the biotinylated CTPS gene. Arrows indicate signals on lp34.1-p34.3
Fluorescence in situ hybridization The procedures of FISH have been described elsewhere (Lawrence et al. 1988; Takahashi et al. 1989, 1990b, 1991; Hori et al. 1990). Some modifications were made for the labelling and DNA purification techniques as well as in the hybridization solution. We labeled probe DNA by a nick translation system (Boehringer, Germany) using biotin 16-dUTP (Boehringer) for 1.5 h at 15~ The deoxynucleoside triphosphates were two times more concentrated (40 IxM each) than those of Boehrigner's method. Labeling was stopped by heating (65~ 10min), and the labeled DNA fragments were purified by ethanol precipitation; 4M CH3COONI"L, 2.5 ~tl + 100% ethanol, 56~tl + sonicated herring sperm DNA (10 mg/ml) containing tRNA (E. coli, 10mg/ml, Boehringer), 2 I.d/ 20 I~1labeling solution ( - 800C, 15 rain). The pellets were dissolved in 100% formamide, after centrifugation (15000 rpm, 5 rain, room temperature). For the elimination of repetitive sequences, a suppression hybridization procedure with total human DNA, reported by Hori et al. 1990, was applied with a slight modification. Preincubation (37~ 2 h) was not performed in the present study. For the CTPS clones, 50 times excess amounts of total human placenta DNA were added to the above probe DNA in formamide, and then denatured at 75~ for 10 rain. The hybridization solution consisted of bovine serum albumin (BSA, 20mg/ml, Boehringer), 10 x SSC and 50% dextran sulfate (1 : 2: 2). The hybridization solution (10 g.l), with an equal volume of the mixture of probe DNA and total human DNA denatured in formamide, was pipetted onto the denatured chromosomes. The concentration of probe DNA was 500 ng/20 p.l hybridization mixture per slide. The procedures of hybridization, rinsing and detection were performed in a routine manner (Takahashi et al. 1989, 1990b, 1991; Hori et al. 1990). The chromosomes treated by the present R-banding system were counterstained with 0.5 ~tg/ml propidium iodide in antibleach mounting medium. Ektachrome film (Kodak, ASA100) was used for microphotography (filter combination, Nikon B-2A); exciter, 450-490 nm, barrier, 520nm. Results and discussion We have examined 100 typical R-banded pro-metaphase plates for both the H21 and II7221 probes. The hybridization efficiency of both probes were similar. Of the
plates examined, 53% exhibited complete double spots on both homologs, 41% were incomplete single and/or spots on either or both homologs, and in the others (6%) no spots were detectable. By high-resolution banding analysis, the signals of the H21 probe were localized to the p34 region of the short ann of c h r o m o s o m e 1. The location of the II7221 signal was the same as that of H21. No signals were observed on the other chromosomes. The CTPS gene could, therefore, be assigned to the subband lp34.1-p34.3 (Fig. 1a-d). There are difficulties in identifying the precise localization of the CTPS signal to the p34 region, because the R-positive (p34.1 and p34.3) and R-negative (p34.2) band cannot be clearly separated. By detailed analysis, however, the CTPS signal could be localized distal to the Rnegative p33 band and very close to it. Thus, the localization of the signal must be to the R-positive p34.1 band. In mammals, early replicating R-positive bands contain constitutive or active (house-keeping) genes, which are GC-rich in their D N A sequences (Holmquist et al. 1982; Goldman et al. 1984). The CTPS gene is a typical housekeeping gene functioning in cell proliferation. Another pyrimidine biosynthesis gene, thymidylate synthase (TS), has been mapped by F I S H and its precise localization was to the R-positive band 18p11.32 (Hofi et al. 1990). These facts support the localization of the CTPS gene to the R-positive band lp34.1. The following cytogenefic assignments have been made around the region of lp34: Epstein Barr virus insertion site 1 (EBVS1, lp35), and the MYCL1 and JUN oncogenes (lp32), mapped by non-isotopic in situ hybridization, linkage and somatic cell hybrid analyses (for review see Bruns and Sherman 1989); two c o m m o n fragile sites, fra(1)(p36), F R A 1 A and fra(1)(p32), F R A 1 B (for review see Sutherland and Ledbetter 1989). Breakpoints involved in chromosomal rearrangements (deletions and translocations) in several tumor types have also been mapped to this region ( l p 3 2 - p 3 6 , for review see Trent et al. 1989): neuroblastoma (Fong et al. 1989; Weith et al. 1989), malignant melanoma (Dracopoli et al. 1989), colorectal carcinoma (Leister et al. 1990), multiple endocrine neoplasia type IIA (Mathew et al. 1987; Yang et al. 1990), glioma, malignant lymphoma, and T cell acute
121
lymphoblastic l e u k e m i a (Begley et al. 1989). H o w e v e r , the region a r o u n d l p 3 4 c a n n o t be clearly s e p a r a t e d by banding so that such descriptions might by a m b i g u o u s . It is of i m p o r t a n c e to e x a m i n e the precise m a p p i n g and ordering o f C T P S , E B V S 1 , M Y C L 1 and J U N by F I S H in relation to t u m o r cell breakpoints and fragile sites. G i v e n the wide r a n g e o f cellular effects i n d u c e d b y the loss o f regulation of C T P S activity and the altered level o f the e n z y m e in certain t u m o r s ( W e b e r et al. 1980), it is intriguing that C T P S m a p s to this putative h o t spot o f c h r o m o s o m e instability. F u r t h e r w o r k is being directed at examining the structure of g e n o m i c C T P S s e q u e n c e s in these various t u m o r cell types. Acknowledgements. Our thanks are due to Ms. S. Tsuji for technical assistance. This work was supported in part by the Special Coordination Funds for Promoting Science and Technology, Science and Technology Agency, Japan.
References Ayusawa D, Takeishi K, Kaneda S, Shimizu K, Koyama H, Seno T (1984) Isolation of functional cDNA clones for human thymidylate synthase. J Biol Chem 259:14361-14364 Begiey CG, Aplan PD, Davey MP, Nakahara K, Tchorz K, Kurtzberg J, Hershfield MS, Haynes BF, Cohen DI, Waldmann TA, Kirsch IR (1989) Chromosomal translocation in a human leukemic stem-cell line disrupts the T-cell antigen receptor 8chain diversity region and results in a previously unreported fusion transcript. Proc Natl Acad Sci USA 86:2031-2035 Bruns GAP, Sherman SL (1989) Report of the committee on the genetic constitution of chromosome 1. (10th International Workshop on Human Gene Mapping) Cytogenet Cell Genet 51 : 67-90 Dracopoli NC, Harnett P, Bale SJ, Stanger BZ, Tucker MA, Housman DE, Kefford RF (1989) Loss of alleles from the distal short arm of chromosome 1 occurs late in melanoma tumor progression. Proc Natl Acad Sci USA 86: 4616-4618 Fong CT, Dracopoli NC, White PS, Merrill PT, Griffith RC, Housman DE, Brodeur GM (1989) Loss of heterozygosity for the short arm of chromosome I in human neuroblastomas: correlation with N-myc amplification. Proc Natl Acad Sci USA 86: 3753-3757 Goldman MA, Holmquist GP, Gray MC, Caston LA, Nag A (1984) Replication timing of genes and middle repetitve sequences. Science 224: 686-692 Holmquist G, Gray M, Porter T, Jordan J (1982) Characterization of Giemsa dark- and light-band DNA. Cell 31 : 121-129 Hori T, Takahashi E, Ayusawa D, Takeishi K, Kaneda S, Seno T (1990) Regional assignment of the human thymidylate synthase (TS) gene to chromosome band 18pl 1.32 by nonisotopic in situ hybridization. Hum Genet 85: 576-580 Hurlbert RB, Kammen HO (1960) Formation of cytidine nucleotides from uridine nucleotides by soluble mammalian enzymes: requirements for glutamine and guanosine nucleotides. J Biol Chem 235 : 443--449 Landegent JE, Jansen in de Wal N, Dirks RW, Baas F, Ploeg M van der (1987) Use of whole cosmid cloned genomic sequences for chromosomal localization by non-radioactive in situ hybridization. Hum Genet 77: 366-370 Langer PR, Waldrop AA, Ward DC (1981) Enzymatic synthesis of biotin-labeled polynucleotides: novel nucleic acid affinity probes. Proc Natl Acad Sci USA 78: 6633-6637 Lawrence JB, Villnave CA, Singer RFI (1988) Sensitive, high-resolution chromatin and chromosome mapping in situ: presence and orientation of two closely integrated copies of EBV in a lymphoma line. Cell 52: 51-61 Leister I, Weith A, Briiderlein S, Cziepluch C, Kangwanpong D, Schlag P, Schwab M (1990) Human colorectal cancer: high
frequency of deletions at chromosome lp35. Cancer Res 50: 7232-7235 Lichter P, Chang CJ, Call K, Hermanson G, Evans GA, Housman D, Ward D (1990) High-resolution mapping of human chromosome 11 by in situ hybridization with cosmid clones. Science 247 : 64-69 Lieberman I (1956) Enzymatic amination of uridine triphosphate to cytidine triphosphate. J Biol Chem 222: 765-775 Long CW, Pardee AB (1967) Cytidine triphosphate synthetase of Escherichia coli B. J Biol Chem 242: 4715-4721 Mathew CGP, Smith BA, Thorpe K, Wong Z, Royle NJ, Jeifreys AJ, Ponder BAJ (1987) Deletion of genes on chromosome 1 in endocrine neoplasia. Nature 328: 524-526 Pinkel D, Straume T, Gray JW (1986) Cytogenetic analysis using quantitative, high-sensitivity, fluorescence hybridization. Proc Natl Acad Sci USA 83 : 2934-2938 Pinkel D, Landegent J, Collins C, Fuscoe J, Segraves R, Lucas J, Gray J (1988) Fluorescence in situ hybridization with human chromosome-specific libraries: detection of trisomy 21 and translocations of chromosome 4. Proc Nail Acad Sci USA 85: 9138-9142 Savage CR, Weinfeld H (1970) Purification and properties of mammalian liver cytidine triphosphate synthetase. J Biol Chem 245 : 2529-2535 Sutherland GR, Ledbetter DH (1989) Report of the committee on cytogenetic markers. (10th International Workshop on Human Gene Mapping) Cytogenet Cell Genet 51:452-458 Takahashi E, Hori T, Lawrence JB, McNeil J, Singer RH, O'Connell P, Leppert M, White R (1989) Human type II collagen gene (COL2A1) assigned to chromosome 12q13.1--q13.2 by in situ hybridization with biotinylated DNA probe. Jpn J Hum Genet 34: 307-311 Takahashi E, Hori T, O'Connell P, Leppert M, White R (1990a) R-banding and nonisotopic in situ hybridization: precise localization of the human type II collagen gene (COL2A1). Hum Genet 86:14-16 Takahashi E, Hori T, Sutherland GR (1990b) Mapping of the human type II collagen gene (COL2A1) proximal to fra(12)(q13.1) by nonisotopic in situ hybridization. Cytogenet Cell Genet 54:84--85 Takahashi E, Hori T, O'Connell P, Leppert M, White R (1991) Mapping of the MYC gene to band 8q24.12-q24.13 by R-banding and distal to fra(8)(q24.11), FRA8E, by fluorescence in situ hybridization. Cytogenet Cell Genet 57:109-111 Tokino T, Takahashi E, Mori M, Tanigami A, Glaser T, Park JW, /ones C, Hori T, Nakamura Y (1991) Isolation and mapping of 62 new RFLP markers on human chromosome 11. Am J Hum Genet 48: 258-268 Trent JM, Kaneko Y, Mitelman F (1989) Report of the committee on structural chromosome changes in neoplasia. (10th International Workshop on Human Gene Mapping) Cytogenet Cell Genet 51 : 533-562 Trudel M, Genechten TV, Meuth M (1984) Biochemical characterization of the hamster thy mutator gene and its revertants. J Biol Chem 259: 2355-2359 Weber G, Lui MS, Takeda E, Denton JE (1980) Enzymology of human colon turnouts. Life Sci 27: 793-799 Weith A, Martinsson T, Cziepluch C, Bruderlein S, Amler LC, Berthold F, Schwab M (1989) Neuroblastoma concensus deletion maps to lp36.1-2. Genes Chrom Cane 1 : 159-166 Yamakawa K, Takahashi E, Saito H, Sato T, Oshimura M, Hori T, Nakamura Y (1991) Isolation and mapping of 75 new DNA markers on human chromosome 3. Genomics 9: 536-543 Yamauchi M, Yamauchi N, Meuth M (1990) Molecular cloning of the human CTP synthetase gene by functional complementation with purified human metaphase chromosomes. EMBO J 9: 2095-2099 Yamauchi M, Yamauchi N, Phear G, Spurt NK, Matinsson T, Weith A, Meuth M (1991) Genomic organization and chromosomal localization of the human CTP synthetase gene. Genomics (in press) Yang KP, Nguyen CV, Castillo SG, Samaan NA (1990) Deletion mapping on the distal third of chromosome lp in multiple endocrine neoplasia type IIA. Anticancer Res 10: 527-534
9 Springer-Veflag1991
Chromosome mapping of the human cytidine-5'-triphosphate synthetase (CTPS) gene to band lp34.1-p34.3 by fluorescence in situ hybridization Ei-ichi Takahashi I, Masatake Yamauchi 1, Hideo Tsuji 1, Akitsu I-litomi x, Mark Meuth 2, and Tada-aki Hori I 1Division of Genetics, National Institute of Radiological Sciences, 4-9-I Anagawa, Chiba, 260 Japan 2Imperial Cancer Research Fund, Clare Hall Laboratories, Blanche Lane, South Mimms, Herts EN6 3LD, UK Received April 16, 1991
Summary. The human cytidine-5'-triphosphate synthetase (CTPS) gene was mapped by a direct mapping system combined with fluorescence in situ hybridization and replicated prometaphase R-bands. By high-resolution banding analysis, the signals were localized to band 34.1-34.3 of the short arm of c h r o m o s o m e 1; l p 3 4 . 1 p34.3. Simple procedures for the detection of R-bands are described.
Introduction Cytidine-5'-triphosphate synthetase (CTPS, U T P : L-glutamine ligase, E C 6.3.4.2) is one of the key regulatory enzymes in pyrimidine biosynthesis. CTPS activity has been isolated and purified from both prokaryotic (Lieberman 1956; Long and Pardee 1967) and mammalian (Hurlbert and Kammen 1960; Savage and Weinfeld 1970) sources. CTPS is subject to feedback inhibition by its product, CTP. The loss of this regulation has numerous effects on cells, including an increased rate of spontaneous mutation and resistance to cancer chemotherapeutic agents (Trudel et al. 1984). Recently, Yamauchi et al. (1990) have successfully cloned the human CTPS gene using chromosome-mediated gene transfer. Sequence analysis o f c D N A revealed that the CTPS gene has been strikingly conserved from E. coli to human (Yamauchi et al. 1990). This suggests that CTPS activity and regulation are crucial for cell proliferation. A n o t h e r key regulatory e n z y m e gene in pyrimidine biosynthesis, thymidylate synthase (TS), has been isolated (Ayusawa et al. 1984) and m a p p e d to 18p11.32 by fluorescence in situ hybridization (Hori et al. 1990). T o elucidate the organization of pyrimidine biosynthetic genes in the human g e n o m e , it is important to map the CTPS gene. Fluorescence in situ hybridization (FISH) techniques have made it possible to localize D N A sequences on chromosomes (Langer et al. 1981; Pinkel et al. 1986; Lawrence et al. 1988). A direct mapping system has
Offprint requests to: E. Takahashi
been recently developed, which is based on F I S H combined with replicated prometaphase R-bands (Takahashi et al. 1990a, 1991). For genomic clones containing repetitive sequences, a suppression hybridization procedure has been established with total human D N A (Landegent et al. 1987; Pinkel et al. 1988; Lichter et al. 1990; Hori et al. 1990). These two systems facilitate a more rapid and efficient method not only for gene mapping but also for genome mapping by the construction of cytogenetic maps using chromosome-specific genomic libraries (Hori et al. 1990; Yamakawa et al. 1991; Tokino et al. 1991). In the present communication, we report the chromosome mapping of the human CTPS gene by these systems.
Materials and methods Preparation of R-banded chromosomes The R-banding method for direct mapping with FISH has been described elsewhere (Takahashi et al. 1990a, 1991). This method is based on cell-synchronization with excess thymidine prior to incorporation of bromodeoxyuridine (BrdU) into replicating DNA. The procedures of lymphocyte-culture, thymidine- and BrdUtreatment, and chromosome preparation were carried out in a routine manner (Takahashi et al. 1990a, 1991). In the previous reports (Takahashi et al. 1990a, 1991) a mercury vapor lamp was used for the detection of R-bands. This has been modified in the present study by the use of a black light and a hot plate. The BrdUincorporated chromosome preparations were stained with I ttg/ml Hoechst 33258 in Serensen's phosphate buffer (pH 6.8) for 5 rain, rinsed and then mounted in 2 x SSC (standard saline citrate, pH 7.8) under coverslips. The chromosome slides were heated at 75~ for 3 min on a hot plate (Hirasawa), and then exposed for 6 rain at 75"C under a black light (Toshiba, 20W, FL20SBLB) at a distance of 1.5 cm. The exposed slides were rinsed in distilled water, dried and stored at -80~ until use.
D N A probes Two overlapping human genomic clones of the CTPS gene, H21 and II7221 in LDASHII (Yamauchi et al. 1991), were used as DNA probes in the present study. H21 (a 20kb insert) covers the Y-end of the gene and II7221 (an 18kb insert) covers the T-side with a 2 kb overlap.
120
Fig. la-d. R-banded pro-metaphases (a whole, b--fl partial) after in situ hybridization with the biotinylated CTPS gene. Arrows indicate signals on lp34.1-p34.3
Fluorescence in situ hybridization The procedures of FISH have been described elsewhere (Lawrence et al. 1988; Takahashi et al. 1989, 1990b, 1991; Hori et al. 1990). Some modifications were made for the labelling and DNA purification techniques as well as in the hybridization solution. We labeled probe DNA by a nick translation system (Boehringer, Germany) using biotin 16-dUTP (Boehringer) for 1.5 h at 15~ The deoxynucleoside triphosphates were two times more concentrated (40 IxM each) than those of Boehrigner's method. Labeling was stopped by heating (65~ 10min), and the labeled DNA fragments were purified by ethanol precipitation; 4M CH3COONI"L, 2.5 ~tl + 100% ethanol, 56~tl + sonicated herring sperm DNA (10 mg/ml) containing tRNA (E. coli, 10mg/ml, Boehringer), 2 I.d/ 20 I~1labeling solution ( - 800C, 15 rain). The pellets were dissolved in 100% formamide, after centrifugation (15000 rpm, 5 rain, room temperature). For the elimination of repetitive sequences, a suppression hybridization procedure with total human DNA, reported by Hori et al. 1990, was applied with a slight modification. Preincubation (37~ 2 h) was not performed in the present study. For the CTPS clones, 50 times excess amounts of total human placenta DNA were added to the above probe DNA in formamide, and then denatured at 75~ for 10 rain. The hybridization solution consisted of bovine serum albumin (BSA, 20mg/ml, Boehringer), 10 x SSC and 50% dextran sulfate (1 : 2: 2). The hybridization solution (10 g.l), with an equal volume of the mixture of probe DNA and total human DNA denatured in formamide, was pipetted onto the denatured chromosomes. The concentration of probe DNA was 500 ng/20 p.l hybridization mixture per slide. The procedures of hybridization, rinsing and detection were performed in a routine manner (Takahashi et al. 1989, 1990b, 1991; Hori et al. 1990). The chromosomes treated by the present R-banding system were counterstained with 0.5 ~tg/ml propidium iodide in antibleach mounting medium. Ektachrome film (Kodak, ASA100) was used for microphotography (filter combination, Nikon B-2A); exciter, 450-490 nm, barrier, 520nm. Results and discussion We have examined 100 typical R-banded pro-metaphase plates for both the H21 and II7221 probes. The hybridization efficiency of both probes were similar. Of the
plates examined, 53% exhibited complete double spots on both homologs, 41% were incomplete single and/or spots on either or both homologs, and in the others (6%) no spots were detectable. By high-resolution banding analysis, the signals of the H21 probe were localized to the p34 region of the short ann of c h r o m o s o m e 1. The location of the II7221 signal was the same as that of H21. No signals were observed on the other chromosomes. The CTPS gene could, therefore, be assigned to the subband lp34.1-p34.3 (Fig. 1a-d). There are difficulties in identifying the precise localization of the CTPS signal to the p34 region, because the R-positive (p34.1 and p34.3) and R-negative (p34.2) band cannot be clearly separated. By detailed analysis, however, the CTPS signal could be localized distal to the Rnegative p33 band and very close to it. Thus, the localization of the signal must be to the R-positive p34.1 band. In mammals, early replicating R-positive bands contain constitutive or active (house-keeping) genes, which are GC-rich in their D N A sequences (Holmquist et al. 1982; Goldman et al. 1984). The CTPS gene is a typical housekeeping gene functioning in cell proliferation. Another pyrimidine biosynthesis gene, thymidylate synthase (TS), has been mapped by F I S H and its precise localization was to the R-positive band 18p11.32 (Hofi et al. 1990). These facts support the localization of the CTPS gene to the R-positive band lp34.1. The following cytogenefic assignments have been made around the region of lp34: Epstein Barr virus insertion site 1 (EBVS1, lp35), and the MYCL1 and JUN oncogenes (lp32), mapped by non-isotopic in situ hybridization, linkage and somatic cell hybrid analyses (for review see Bruns and Sherman 1989); two c o m m o n fragile sites, fra(1)(p36), F R A 1 A and fra(1)(p32), F R A 1 B (for review see Sutherland and Ledbetter 1989). Breakpoints involved in chromosomal rearrangements (deletions and translocations) in several tumor types have also been mapped to this region ( l p 3 2 - p 3 6 , for review see Trent et al. 1989): neuroblastoma (Fong et al. 1989; Weith et al. 1989), malignant melanoma (Dracopoli et al. 1989), colorectal carcinoma (Leister et al. 1990), multiple endocrine neoplasia type IIA (Mathew et al. 1987; Yang et al. 1990), glioma, malignant lymphoma, and T cell acute
121
lymphoblastic l e u k e m i a (Begley et al. 1989). H o w e v e r , the region a r o u n d l p 3 4 c a n n o t be clearly s e p a r a t e d by banding so that such descriptions might by a m b i g u o u s . It is of i m p o r t a n c e to e x a m i n e the precise m a p p i n g and ordering o f C T P S , E B V S 1 , M Y C L 1 and J U N by F I S H in relation to t u m o r cell breakpoints and fragile sites. G i v e n the wide r a n g e o f cellular effects i n d u c e d b y the loss o f regulation of C T P S activity and the altered level o f the e n z y m e in certain t u m o r s ( W e b e r et al. 1980), it is intriguing that C T P S m a p s to this putative h o t spot o f c h r o m o s o m e instability. F u r t h e r w o r k is being directed at examining the structure of g e n o m i c C T P S s e q u e n c e s in these various t u m o r cell types. Acknowledgements. Our thanks are due to Ms. S. Tsuji for technical assistance. This work was supported in part by the Special Coordination Funds for Promoting Science and Technology, Science and Technology Agency, Japan.
References Ayusawa D, Takeishi K, Kaneda S, Shimizu K, Koyama H, Seno T (1984) Isolation of functional cDNA clones for human thymidylate synthase. J Biol Chem 259:14361-14364 Begiey CG, Aplan PD, Davey MP, Nakahara K, Tchorz K, Kurtzberg J, Hershfield MS, Haynes BF, Cohen DI, Waldmann TA, Kirsch IR (1989) Chromosomal translocation in a human leukemic stem-cell line disrupts the T-cell antigen receptor 8chain diversity region and results in a previously unreported fusion transcript. Proc Natl Acad Sci USA 86:2031-2035 Bruns GAP, Sherman SL (1989) Report of the committee on the genetic constitution of chromosome 1. (10th International Workshop on Human Gene Mapping) Cytogenet Cell Genet 51 : 67-90 Dracopoli NC, Harnett P, Bale SJ, Stanger BZ, Tucker MA, Housman DE, Kefford RF (1989) Loss of alleles from the distal short arm of chromosome 1 occurs late in melanoma tumor progression. Proc Natl Acad Sci USA 86: 4616-4618 Fong CT, Dracopoli NC, White PS, Merrill PT, Griffith RC, Housman DE, Brodeur GM (1989) Loss of heterozygosity for the short arm of chromosome I in human neuroblastomas: correlation with N-myc amplification. Proc Natl Acad Sci USA 86: 3753-3757 Goldman MA, Holmquist GP, Gray MC, Caston LA, Nag A (1984) Replication timing of genes and middle repetitve sequences. Science 224: 686-692 Holmquist G, Gray M, Porter T, Jordan J (1982) Characterization of Giemsa dark- and light-band DNA. Cell 31 : 121-129 Hori T, Takahashi E, Ayusawa D, Takeishi K, Kaneda S, Seno T (1990) Regional assignment of the human thymidylate synthase (TS) gene to chromosome band 18pl 1.32 by nonisotopic in situ hybridization. Hum Genet 85: 576-580 Hurlbert RB, Kammen HO (1960) Formation of cytidine nucleotides from uridine nucleotides by soluble mammalian enzymes: requirements for glutamine and guanosine nucleotides. J Biol Chem 235 : 443--449 Landegent JE, Jansen in de Wal N, Dirks RW, Baas F, Ploeg M van der (1987) Use of whole cosmid cloned genomic sequences for chromosomal localization by non-radioactive in situ hybridization. Hum Genet 77: 366-370 Langer PR, Waldrop AA, Ward DC (1981) Enzymatic synthesis of biotin-labeled polynucleotides: novel nucleic acid affinity probes. Proc Natl Acad Sci USA 78: 6633-6637 Lawrence JB, Villnave CA, Singer RFI (1988) Sensitive, high-resolution chromatin and chromosome mapping in situ: presence and orientation of two closely integrated copies of EBV in a lymphoma line. Cell 52: 51-61 Leister I, Weith A, Briiderlein S, Cziepluch C, Kangwanpong D, Schlag P, Schwab M (1990) Human colorectal cancer: high
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