GENOMICS

11,1088-1096

(1991)

Genomic Organization and Chromosomal Localization of the Human CTP Synthetase Gene (UPS) MASATAKE YAMAUCHI, *,I NORIKO YAMAUCHI, *a’ GERALDINE PHEAR,* NIGEL K. SwRR,t TOMMY MARTINSSON,* ANDREAS WEITH,~ AND MARK MEUTH*** *Cell Mutation Laboratory and tHuman Genetic Resources Laboratory, Imperial Cancer Research Fund, C/are Hall Laboratories, Blanche Lane, South Mimms, Potters Bar, Hertfordshire, EN6 3LD, United Kingdom; *Department of Clinical Genetics, University of Goteborg, 5-4 16 85 Goteborg, Sweden; and §Research institute of Molecular Pathology, Dr. Bohr-Case 7, A- 1030 Vienna, Austria Received

May 20, 1991;

INTRODUCTION

The synthesis of cytidine nucleotides in cells is achieved by the amination of UTP in the reaction UTP + ATP + glutamine + CTP + ADP + Pi + glutamate. This rate-limiting reaction, catalyzed by the enzyme CTP synthetase (CTPS, EC 6.3.4.2), is also a key regulatory step as it is subject to activation by GTP and inhibition by CTP (McPartland and Weinfeld, 1979). Mutations eliminating the inhibition by CTP produce many secondary effects in cultured mammalian cells: (i) increased intracellular pools of

0888s7543/91$3.00

Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

July 26, 1991

CTP and dCTP (Meuth et al., 1979b; Trudel et al., 1984; de Saint Vincent and Buttin, 1980); (ii) resistance to several drugs used in cancer chemotherapy (e.g., arabinosyl cytosine, &fluorouracil, 3-deazauridine, thymidine, 5-bromodeoxyuridine, and DNA alkylating agents; Meuth et al., 1982; Aronow et al., 1984; Kaufman, 1986; Chu et al., 1984); and (iii) an increased rate of spontaneous mutation (Meuth et al., 1979a; Chu et al., 1984; Aronow et al., 1984). In addition, significantly elevated activities of CTPS in rapidly growing liver (Kizaki et al., 1980), colon (Weber et al., 1980), and lung tumors (Weber et al., 1979) have been reported. Given the numerous secondary effects of alterations of CTPS activity, we are attempting to analyze the regulation of this enzyme in normal and tumor cell lines. Toward this end we have recently described the cloning of the human CTPS cDNA through chromosome-mediated complementation of CTPS-deficient CHO cells (Kelsall and Meuth, 1988; Yamauchi et al., 1990). To further facilitate these analyses, we describe here the cloning, organization, and mapping of CTPS genomic sequences.

To elucidate the organization of the human genomic sequences encoding CTP synthetase (CTPS), fragments homologous to the cDNA were isolated from genomic X libraries. The fragments cloned were overlapping and cover over 40 kb. Cotransfection of the DNAs into CTPS-deficient, cytidine-requiring CHO mutants can transform them to cytidine-independent growth, indicating that the complete structural gene has been isolated. Direct sequencing and enzymatic amplification of the cloned genomic fragments revealed that the coding sequences are distributed to 19 exons covering about 36 kb. Multiple transcriptional start sites were detected by primer extension in a G+C-rich 5’ flanking sequence that is separated from the translational start by an -3-kb intron. A panel of human-rodent somatic cell hybrids and the CTPS cDNA were used to assign the structural gene to the short arm of human chromosome 1. This assignment was further refined through the use of somatic cell hybrids bearing fragments of the short arm of the chromosome, allowing localization to lp36.11-~31, a region notable for its disruption in many types of tumors. 0 1991 Academic Press, Inc.

1 Present address: National Institute of Radiological 4-9-l Anagawa, Chiba, Japan. ’ To whom correspondence should be addressed.

revised

MATERIALS

AND

METHODS

Cell Culture CHO cell strains were routinely grown on a-MEM medium (Gibco) supplemented with 5% fetal calf serum, 5% horse serum, and thymidine (10 PM) as described previously (Kelsall and Meuth, 1988, Yamauchi et al., 1990). Cytidine (20 PM) was added to cultures of the cytidine auxotroph CR-2 (Kelsall and Meuth, 1988). All the somatic cell hybrids used in this work have been described previously and are appropriately referenced in the text, figures, or tables.

Sciences,

1088

GENOMIC

DNA Extraction, Hybridization

Southern

Blotting,

ORGANIZATION

and

Genomic DNA was extracted using standard methods, and lo-15 gg was digested with EcoRI. The digested DNA was fractionated on 0.8% agarose gels and transferred to either Hybond N (Amersham International) or GeneScreen Plus membranes (NEN) following the manufacturer’s instructions. The ctps probes specific for 5’ (nucleotides 1-714) and 3’ (nucleotides 714-2758) portions of the human CTP synthetase cDNA (Yamauchi et aZ., 1990; EMBL database accession number X52142) were radiolabeled with 32P by random priming (Feinberg and Vogelstein, 1983) and hybridized to the membranes under standard conditions. The filters were washed to a final stringency of 0.1 X SSC, 0.1% SDS at 65’ for 20 min. Preparation

of Genomic Libraries

Genomic DNA libraries were prepared in the X DASH vector using Sau3A partially digested DNA purified from cultured HeLa cells essentially as described by Little (1987). HeLa cells (10’) growing in suspension were spun down and resuspended in cold Tris (10 mM), EDTA (1 mM), NaCl(400 mM). Proteinase K was added to a concentration of 250 pg/ml, followed by Sarkosyl (1.0% final concentration). After 1 h the digest was gently extracted with phenol-chloroform-isoamyl alcohol (49:49:1) and the aqueous phase was dialyzed against three changes of 10 mM Tris (pH 7.6), 1 mJ4 EDTA (1 liter) at 4°C. Test digests using 40 ~1 of this DNA solution were then performed utilizing decreasing concentrations of Sau3A followed by fractionation on 0.2% agarose gels. The enzyme concentration giving fragments of 20-30 kb was used in a scaled-up digestion followed by phenol-chloroform extraction. This product was fractionated on a lo-40% sucrose gradient and the DNA in the fractions collected was sized on 0.2% agarose gels. Those fractions giving fragments of 20-30 kb were ethanol-precipitated and the DNA was resuspended in Tris-EDTA to a concentration of -0.5 mg/ml. This DNA was used in ligation reactions with BamHI-digested X DASH (Stratagene) with a 3:l excess of insert to vector at 15°C overnight. Ligations were packaged using Gigapack gold (Stratagene) before plating on the selective host P2392. Packaging efficiency was - 106/pg insert DNA. Recombinant libraries plated onto megaplates (NUNC) were transferred to nitrocellulose filters and were probed with 5’ (nucleotides 1-714) and 3’ (nucleotides 714-2758) portions of the human CTP synthetase cDNA. Positives obtained were mapped for EcoRI restriction endonuclease sites and the overlaps

OF

THE

HUMAN

CTPS

between the cloned these maps. Transfection

1089

GENE

DNAs

were

determined

of CTP Synthetase-Deficient

from

Cells

Exponentially growing CHO CR-2 cells (Kelsall and Meuth, 1988) were plated at a density of 500,000/ lo-cm dish on a-MEM medium supplemented with 5% fetal calf serum, 10 PM thymidine, and 20 PM cytidine. The next day this medium was changed and further supplemented with 0.01 mg/ml gentamycin, 0.001 mg/ml amphotericin B, 0.1% polyethylene glyco1 1540, and 15 n&f Hepes(Na), pH 7.1. These cells were then exposed to the DNA-calcium phosphate co-precipitate (Yamauchi et al., 1990) for 16 h with DMSO (10%) added for the last 30 min. This medium was then exchanged for fresh growth medium containing 7.5% fetal calf serum, 10 PM thymidine, 20 PM cytidine, 0.01 mg/mI gentamycin, and 0.001 mg/ ml amphotericin B. After a 24-h recovery the cells were plated in selective medium containing 7.5% dialyzed fetal calf serum, thymidine, gentamycin, and amphotericin B. Colonies forming after 2-4 weeks were picked. DNAs purified from cultures of the survivors were analyzed for the presence of human CTP synthetase sequences by Southern blot analysis using the human cDNA fragments as probes. Amplification of Intron Chain Reaction

Sequences

by Polymerase

Sense and antisense oligonucleotides (20-mers) spaced at intervals along the CTP synthetase cDNA were used as primers and the genomic X DASH clones as templates in polymerase chain reactions. In this manner the number of exons and distances separating most of them were defined. Polymerase chain reactions (Saiki et al., 1988) were performed as described previously (Phear et al., 1989). The EcoRI fragments of the genomic X DASH clones were also subcloned into pUC19 and, in some cases, exon sequences were mapped relative to these restriction endonuclease sites by polymerase chain reaction using appropriate exon and vector sequences as primers. When these sites were near the exons, the intervening portions were simply sequenced. DNA

Sequence Analysis

of Exonllntron

Junctions

Sequence determination by the dideoxy chain termination technique (Sanger et al., 1977) was carried out using 2 pg of alkaline denatured EcoRI genomic DNA fragments cloned into pUC19 as templates and sense or antisense exon sequences as primers. Reactions were performed using Sequenase (United States Biochemical Corp.) under the conditions recommended by the manufacturer.

1090

YAMAUCHI

probe:

ET

AL.

Cloning and Mapping of the CTPS Gene 5’ CTPS 123

3’ CTPS 123

-12 7.2

3.3

I .3

FIG. 1. Southern blot analysis of human genomic sequences encoding CTPS. DNA prepared from human cell lines 293 (track l), HeLa (track 2), and SW620 (3) was digested with EcoRI, fractionated on agarose gels, and transferred to filters. These blots were then probed with 5’ and 3’ portions of the CTPS cDNA. The size (kb) of each fragment is indicated.

Primer Extension Ten nanograms of an oligonucleotide (20-mer) extending upstream from nucleotide -24 (Yamauchi et aZ., 1990) was end-labeled with [y3’P]ATP to a specific activity of - lo6 cpm/ng. This was annealed to 20 pg of total RNA in 400 n&f KCl, 10 miVPipes, pH 6.4, by heating at 85°C for 10 min, followed by incubation at 65°C for 15 min. The reverse transcription was performed at 42°C for 1 h as recommended by the supplier (Anglian Biotechnology). The product was analyzed on a 6% polyacrylamide urea gel and compared with an appropriate sequencing ladder.

RESULTS

Southern Blot Analysis of Genomic Sequences Encoding CTP Synthetase To approximate the size of the region encoding the human CTPS, EcoRI digests of human DNA were fractionated by electrophoresis on agarose gels and transferred to membrane filters. The resulting blots were probed with 5’ and 3’ portions of the CTPS cDNA. Figure 1 shows that the structural gene is large, since the cDNA probes hybridize to seven distinct EcoRI fragments covering almost 40 kb.

Sau3A partial digests of human genomic DNA purified from HeLa cells were ligated to BamHI-cut X DASH. The resultant libraries were screened for recombinants bearing 5’ and/or 3’ portions of the human CTPS cDNA. Several independent recombinant phage were obtained in these screens and EcoRI sites in the inserts were mapped. Maps of four inserts delineated fragments similar to those identified on Southern blots of genomic DNA digests probed with the cDNA fragments (Figs. 1 and 2). The sequences covered by these inserts were overlapping and stretched nearly 40 kb (Fig. 2). The short 2-kb overlap of the inserts of XH21 and XII7221 was confirmed by the hybridization of the 5’ 6-kb EcoRI fragment of XII’221 with the 3.2-kb 3’ EcoRI fragment of H21. This was more precisely determined by the positioning of exon 9 in the overlapping portions of the two fragments (see below). Sites for the infrequently cutting restriction endonucleases A&I, NarI, and BssHII were detected in the most 5’ portion of the insert in the recombinant phage H21, indicating the presence of a CpG-rich region characteristic of the 5 portion of an active gene. Clones extending further downstream than IIRl were also obtained in the screens (e.g., XI131, Fig. 2). However, these sequences did not appear to be essential for the activity of CTPS, as they were modified in some of the tertiary cellular transformants (Yamauchi et al., 1990) from which the CTPS sequences were originally cloned (data not presented). Thus it appeared that the functional CTP synthetase may be included in the three overlapping X DASH clones. Complementation of the Cytidine Growth Requirement of CTP Synthetose-Deficient Mutants by Transfection of the Cloned Overlapping Fragments To directly test this conclusion, combinations of the cloned genomic DNAs were transfected into cytidine-requiring, CTPS-deficient mutants to determine their ability to transform these cells to cytidineindependent growth. Table 1 shows that cotransfection of phage DNAs XH21 and XII7221 produced colonies in cytidine-deficient medium at a frequency of -5 X 10W6.Cotransfection of three overlapping recombinant phage (XH21, XII’221, and XIIR1, Fig. 2) did not further enhance the frequency of colony formation. Transfection of the noncontiguous clones H21 and RR1 resulted in a single colony, although transfection of the individual X DNAs alone failed to produce colonies. Several of the colonies growing in the absence of cytidine after cotransfection of XH21 and XII7221

GENOMIC

ORGANIZATION

OF

THE

HUMAN

CTPS

1091

GENE

1

40

kb

Nr Na Nr Nr 5 H21

b II7

221

R 111

1

kI1

31

FIG. 2. Restriction endonuclease maps of CTPS cDNA (top) and genomic DNAs (bottom). The coding portion of the cDNA is indicated by the filled region; numbers below the line indicate size (in kb). Restriction endonuclease sites (E, EcoRI; Bg, BglII) in the cDNA and their equivalent positions within the genomic sequence are indicated (dotted lines). Exons in the genomic map are numbered and represented by vertical lines. All EcoRI sites in the region are indicated (E) as are rare cutting sites (Bs, BssHII; Na, NaeI; Nr, N~FI). The map positions of the overlapping X phage inserts covering the region are also presented.

were picked, and DNA purified from these strains was analyzed for the presence of human CTPS sequences. Figure 3 shows that these colonies contained human CTPS genomic sequences specific for both 5’ and 3’ cDNA probes, as the distinctive patterns of human

TABLE Complementation Cells by Genomic Complete cDNA DNA source” None HZ1 II7 221 11a 1 H21+ II’ 221 H21 + IIR 1 H21 + II’ 221 + IF 1

1

of CTP Synthetase-Deficient X Phage Clones Homologous to the

Amount added (Pi?)

cells transfected (X106)

0

0.84 1.0

20 20 20 20+ 20 20 + 20 20 + 20 f20

n See Fig. 2 for map positions

1.0 1.2 1.7 1.8 2.1

of X inserts.

Colonies formed

Frequency (X10”)

370 CGGAGGULGA

500

380 GarnCGCTGG

510 CAAAULGCCT

CCTCCCCTTC

570 GGGGCAGAWL

580 CGCCACCACC

CGCCCGG!xT

640

650 GCCTGCG

400 CCAGGAGCCC

410 ACAGT-G

I 420 P.CcATCGGAT

530

540 GCTTGGGCCA

550 CAGGCTGGG?.

+ 560 CGGAAGCAUL

600 CWULCTACGG

CCGC~TC~CC

ET

AL.

genomic region encoding CTPS: (i) the coding sequences are highly similar to those of Escherichia coli (48% amino acid identity, 74% similarity); (ii) the overlapping genomic clones bearing this sequence are able to transform cytidine-requiring CTPS-deficient CHO mutants to cytidine-independent growth; and (iii) a hamster cell strain deficient in CTPS activity (CR-2; Kelsall and Meuth, 1988) also has a partial deletion of hamster CTPS coding sequences detectable on Southern blots. The chromosomal localization of CTPS to human chromosome lp36.11-p31 is also notable considering the rearrangement of this region in many types of tumors. Frequent deletions of this region have been reported for malignant melanoma (Dracopoli et al., 1989), colorectal carcinoma (Leister et al., 1990), and multiple endocrine neoplasia type IIA (Mathew et al,

FIG. 4. Sequence of 5’ flanking region of CTPS. Primer extension from an oligonucleotide complementary to nucleotides 812832 indicates multiple start sites with a major start at nucleotide 556 (vertical arrow). Extensions from an oligonucleotide complimentary to nucleotides downstream from the translational start (MET) start also indicate multiple transcription starts. Intron sequences are indicated by lowercase letters and short inverted repeats by dashed arrows. The short direct repeat in the G+C-rich region is underlined. Sequence numbering commences from the first EcoRI site on the restriction endonuclease map in Fig. 2.

DHB, a chromosome 1 with a deletion of the lp31pter region; and AE2 only the long arm of chromosome 1. Since all three human CTPS EcoRI fragments were detected in hybrids BE1 and EA3, but were not observed in DHB and AE2 (Fig. 5), it is evident that CTPS maps in the region deleted in DHB but not EA3, i.e., lp36.11-~31.

- 8.5

DISCUSSION Here we have presented the organization and properties of the human genomic sequences encoding CTP synthetase. The structural gene, covering about 35 kb in 19 exons, is somewhat larger than those encoding other enzymes involved in pyrimidine synthesis or salvage. The region encoding human thymidylate synthetase spans 16 kb (Kaneda et al., 1990); thymidine kinase covers 14-16 kb (Bradshaw, 1983; Lin et aZ., 1983); while the CAD gene, encoding the trifunctional enzyme responsible for the first three steps of pyrimidine synthesis, is spread over about 25 kb (Wahl et al,, 1982). In comparison, the gene encoding the purine salvage enzyme hypoxanthine guanine phosphoribosyltransferase covers 44 kb in 9 exons (Pate1 et al., 1986). Several lines of evidence now support our conclusion that the clones that we isolated represent the

FIG. 5. Southern blot analysis of human-mouse hybrid cell lines bearing fragments of human chromosome 1. DNA purified from the indicated hybrid, mouse, and human cells was digested by EcoRI. The fragment sizes indicated are those homologous to the human 5’ CTPS probe. Strain BE1 contains an entire human chromosome 1; EA3, a chromosome 1 with a deletion spanning lp36.12-pter; DH8, a chromosome 1 with a deletion of the lp31pter region; AE2, only the long arm of the chromosome [Ref. (ZO)].

GENOMIC

ORGANIZATION

OF

THE

TABLE Assignment

Hybrid

name

of the Human

Mog34A4 LSR34S49 HORLIllBGP SIR74ii F4Sc13CI12 3W4CL5 DT1.2.4 FGlO DUR4R3 FSTS/lO CTP41A2 o +, chromosome ’ Only short arm

1

2

3

+

+

-

++++++-+++++++-++-+

+ + + + -

-++---+--++++-+++++ + + +-+-------+-++---+---++ + +b ------

4

5

6

7

8

9

3.

chromosome 11

12

Gene retained”

13

14

15

16

17

18

19

20

21

22

X

Ref.

+

+ +

--+

t

+

-

+ -

+ + + + +

(35) (36) (1) (45) (5) (27) (37) (15) (34) (15) (34)

+--+++-++-+---+ -+------+++--++++-++ +--+--+-+----+--+--+ + + -++-+-+-+-++-+--+-+-+ +---++------+--+--~--

retained; -, chromosome of chromosome 1 retained.

-

absent:

-

+

-

t. trace-less

ANDREWS, P. W., KNOWLES, B. B., PARKAR, M., PYM, B., STANLEY, K., AND GOODFELLOW, P. N. (1985). A human cellsurface antigen defined by a monoclonal antibody and controlled by a gene on human chromosome 1. Ann. Hum. Genet. 49: 31-39. ARONOW, B., WATTS, T., LASSE?TER, J., WASHTIEN, W., AND UUMAN, B. (1984). Biochemical phenotype of 5-fluorouracilresistant murine T-lymphoblasts with genetically altered CTP synthetase activity. J. Biol. Chem. 259: 9035-9043. BEGLEY, C. G., APLAN, P. D., DAVEY, M. P., NAKAHARA, K., TCHORZ, K., KURTZBERG, J., HERSHFIELD, M. S., HAYNES, B. F., COHEN, D. I., WALDMAN, T. A., AND KIRSCH, I. R. (1989). Chromosomal translocation in a human leukemic stem-cell line disrupts the T-cell antigen receptor b-chain diversity region and results in a previously unreported fusion transcript. Proc. Natl. Acad. Sci. USA 86: 2031-2035.

4.

BRADSHAW, H. D. J. (1983). Molecular cloning and cell cyclespecific regulation of a functional human thymidine kinase gene. Proc. Natl. Acad. Sci. 80: 5588-5591.

5.

BROWN, M. H., GORMAN, P. A., SEWELL, W. A., SPURR, N. K., SHEER, D., AND CRUMPTON, M. J. (1987). The gene coding for the human T-lymphocyte CD-2 antigen is located on chromosome lp. Hum. Genct. 76: 191-195. CHU, E. H. Y., MCLAREN, J. D., LI, I.-C., AND LAMB, B.

6.

CTPS

t

REFERENCES

2.

1095

GENE

+++-------+++--+ --~------~-+-~-~----+

1987) (Yang et al., 1990). Breakpoints for translocations (tl:14) in T-cell acute lymphoblastic leukemia also fall in this region (Begley et al., 1989) and it appears to be frequently rearranged in other types of leukemias and lymphomas (Olah et al., 1989). Given the wide range of cellular responses induced by the loss of regulation of CTPS activity and the altered level of the enzyme in many tumors, it is intriguing that the CTPS gene maps to this putative cluster of chromosome rearrangements. Further work is being directed at examining the structure of genomic CTPS sequences in these various tumor cell types.

1.

10

CTPS

3

Human Human C!FPS

HUMAN

-

+

than

+

+

+

10% of cells retain

?

-

+

8.

9.

+

+

chromosome.

(1984). Pleiotropic mutants tered cytidine 5’-triphosphate 22: 701-715. 7.

-

of Chinese hamster cells with alsynthetase. Biochem. Genet.

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promoter-specific sequences in the

10.

FEINBERG, A. P., AND VOGELSTEIN, B. (1983). A technique radiolabeling DNA restriction endonuclease fragments high specific activity. And. Biochem. 132: 6-13.

11.

ISHII, S., KADONAGA, J. T., TLTAN, R., BRADY, J. N., ~MERLINO, G. T., AND PASTAN, I. (1986). Binding of the Sp-1 transcription factor by the human Harvey ras 1 proto-oncogene promoter. Science 232: 1410-1413.

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13.

for to

14.

KELSALL, A., AND MEUTH, M. (1988). Direct selection nese hamster ovary cells deficient in CTP synthetase ity. Somat. Cell Mol. Genet. 14: 149-154.

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K~Lw, C. M., POVEY, S., AND HOPKINSON, D. A. (1982). Regulation of expression of liver-specific enzymes. III. Further analysis of a series of rat hepatoma x human somatic cell hybrids. Ann. Hum. Genet. 46: 307-327. K-1, H., WILLIAMS, J. C., MORRIS, H. P., AND WEBER, G. (1980). Increased cytidine 5’-triphosphate synthetase activity in rat and human tumors. Cancer Res. 40: 3921-3927. LEISTER, I., WEITH, A., BRUDERLEM, S., CZIEPLUCH, C.,

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ET AL. ‘-

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8: 423-432.

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