Isolation of a human X chromosome-linked gene essential for progression from G1 to S phase of the cell cycle

Experimental

Cell Research 169 (1987) 395-407

Isolation of a Human X Chromosome-Linked Gene Essential Progression from Gl to S Phase of the Cell Cycle

for

TAKESHI SEKIGUCHI,’ MICHIHIRO C. YOSHIDA,’ MUTSUO SEKIGUCHI’ and TAKEHARU NISHIMOTO’~* ‘Department

of Biology, Faculty of Science, Kyushu University 33, Fukuoka 812, and 2Chromosome Research Unit, Faculty of Science, Hokkaido University, Sapporo 060, Japan

The tsBN462 cell line, a temperature-sensitive (ts) mutant isolated from the hamster cell line, BHK21113, cannot progress into S phase at 39S”C, following the release from isoleucine deprivation. The mutant cells were transfected with high molecular weight (HMW) DNA from human KB cells, and several human DNA bands were found to be conserved through three cycles of ts+ transformation. Conserved human DNA was isolated from the cosmid library of the secondary ts+ transformant (K-l-l), using 32Plabelled total human DNA as a probe. The isolated human DNA covers about 70 kb of human DNA flanked with hamster DNA, and originates from the human X chromosome. The middle part (56 kb) of the isolated human DNA was conserved through the primary, secondary and tertiary ts+ transformation, without gross rearrangement. 0 1987 Academic Press, Inc.

Animal cells undergo highly organized cell-cycle events, namely Gl , S, G2 and M phases. In the Gl phase, the cells may continue to grow, or they may remain in a state of arrest, depending on cell density, concentration of growth factors and other elements [l, 21. Once a cell has passed out of the Gl phase, it is committed to completing the S, G2 and M phases. Some c-oncogenes are assumed to be involved in the regulation of early Gl phase [3]. Thus, information on the molecular basis of Gl progression is vital to elucidate the regulation of cell reproduction. We isolated several temperature-sensitive (ts) mutants defective in cell growth, from the BHK21113 cell line [4]. One of these mutants, the tsBN462 cell line, has a ts defect in the progression of Gl phase. The mutation site of this cell line is located on the X chromosome [4]. tsBN462 cells were transfected with high molecular weight (HMW) DNA from human KB cells, and human DNA retained through tsf transformation was isolated from the cosmid library of a ts+ transformant, by probing with the human specific ‘A/u’ sequence. A 56 kb DNA sequence, originating from the human X chromosome was found to be conserved through three cycles of tsf transformation. * To whom offprint requests should be addressed. Copyright @ 1987 by Academic Press, Inc. All rights of reproduction in any form reserved 0014.4827/87 $03.00

396 Sekiguchi et al. MATERIALS

AND METHODS

Cell Lines The KB cell line, derived from human laryngeal carcinoma, was used as a donor for gene transfer. The Bl cell line, thymidine kinase-negative (TK-) cell line derived from a BHK21 cell line [5], was used as a control of the homologous gene transfer. The tsBN462 cell line, ts mutant of the BHK21 cell line 141,was used as a recipient of gene transfer. A primary human tibroblast cell line from a normal male, and from an abnormal female with the 48XXXX karyotype, were kindly provided from Dr K. q Ohno (Division of Child Neurology, Institute of Neurological Sciences, Tottori University School of Medicine). The human-mouse hybrid clones, H/F2p5 cell line, contain human chromosomes 14, 21 and X, while the C13-lD3 cell line contains human chromosomes 14 and 21. The C2B5 cell line, a hybrid between mouse L cell-derived A9 cell line and human diploid fibroblast, GM1696 carrying an X/7 chromosome translocation (X;7) (q21:p22) [6], was a gift from Dr Nobuyoshi Shimizu (Department of Molecular Biology, Keio University School of Medicine).

Cell Culture and Transformation All cell lines except H/F2p5 and C2B5 cells were cultured in Dulbecco’s modified Eagle medium (DME). The hybrid, H/F2p5 and C2B5 cell line, containing either whole or part (q21-qter) of the X chromosome was cultured in DME containing 1x10m4M hypoxanthine, 4x10-‘M aminopterin, 1.6~ 10e5M thymidine and 1x 10m4Mglycine (HAT) [7]. All culture media contained 10% fetal calf serum (FCS), penicillin (100 U/ml) and streptomycin (100 l&ml). Cultivation of cells was performed in plastic dishes (Nunc), in a humidified atmosphere of 10% CO,-90% air. Except for the ts mutants, all cell lines were incubated at 37°C. Cultures of tsBN462 cells were maintained at 33.5”C, permissive temperature. Ts+ cells were selected at 39.5”C, non-permissive temperature. ‘Bansformation of the tsBN462 cell line was performed by the calcium phosphate precipitation technique 181,using the system of fluctuation test [9]. Cultures were washed with TD buffer; Tris-buffered saline without Ca’+ and Mg2+ (TD) containing 136.8 mM NaCl, 5 mM KCI, 0.7 mM NaZHP04 and 25 mM Tris-Cl (pH 7.4).

Isolation

of Nucleic Acid and Filter Hybridization

HMW DNA was isolated from cultured cells by phenol extraction of SDS-disrupted cells [8]. Plasmid DNAs were isolated using the procedure of Maniatis et al. [lo]. Restriction endonucleases were purchased from Takara Shuzo (Kyoto, Japan) and digestion was carried out under the conditions recommended by the supplier. DNA samples were subjected to electrophoresis in horizontal slab gels of 1% agarose (Nakarai Co., Kyoto, Japan) in 0.04 M T&-acetate, 2 mM EDTA (pH8) at 50 V (C/V) overnight. After treatment with alkaline buffer and then with neutralization buffer, gels were transferred to nitrocellulose filters (Schleicher L Schiill), according to Southern [ll]. DNA probes with a specific activity of about 2x10” dpm/ug were prepared by nick translation, using [a-32P] dCTP (Amersham) [lo]. DNA-DNA hybridization was performed as follows: DNA blotted on nitrocellulose filter was incubated at 70°C for 1 h in solution containing 4x SET (lx SET consisted of 0.15 M NaCl, 0.03 M B-is-Cl, pH8.0 and 2 mM EDTA), 10x Denhardt solution (0.02% BSA, 0.02% Ficoll 400, 0.02% Polyvinyl Pyrrolidone) and 0.1% SDS, and then prehybridized with salmon testis DNA at 70°C for 1 h in prehybridization solution containing 4x SET, 10x Denhardt solution, 0.1% SDS, 0.1% sodium pyrophosphate and JO @ml of salmon testis DNA. Hybridization with nick-translated DNA was carried out at 42°C for 40 h in the condition of 50% formamide, 1X Denhardt solution, 100 I&ml of salmon testis DNA, 50 mM sodium phosphate and 2x SSC as the stringent condition. When BLUR8 and total human DNA was used as a probe, 5x SSC was used instead of 2x SSC in order to detect human specific repeated sequence. Plasmid BLUR8 contains the human-specific ubiquitous ‘Ah’ sequence [12]. After hybridization, the 32P-labelled probe was washed as follows; filters were incubated in 2~ SET and 0.1% SDS at room temperature for 10 mitt, then in fresh 2x SET and 0.1% SDS at 70°C for 30 min, and washed twice in 1x SET and 0.1% SDS at 70°C for 30 min. When BLUR8 and total human DNA was used as a probe, the filters were exposed to X-ray film at -70°C. Otherwise. filters were further washed twice in 0.2~ SET and 0.1% SDS at 70°C for 30 min. As a Exp Cd Res 169 (1987)

X chromosomal

gene of cell cycle

397

standard probe of human X and autosomal chromosomes, we used plasmid pcD-hprt containing cDNA of the HGPRT gene [13], and plasmid pNP5 containing the 5’ single copy flanking region of the N-ras genomic DNA which locate on chromosome 1 1211 (a gift from Dr K. Shimizu of our laboratory).

Construction

and Screening of DNA Libraries

To prepare a genomic DNA library, HMW DNA was isolated from the tsC secondary transformant; K-l-l cell line and then partially digested with EcoRI, BamH I or MboI. Fragments ranging from 35 to 45 kb were isolated by preparative sucrose density grandient (lO?&tO% centrifugation), mixed with the cosmid vector, pHC79(19), and then ligated with T4 DNA ligase (Takara Shuzo). Ligated DNA was packaged in vitro, using the packaging kit (Amersham). The packaged cosmids were introduced into E. coli 49OA(r-, m-, met, rhr, leu, recA) [14]. Cosmid libraries were screened with 32P-labelled total human DNA according to Benton & Davis [15]. Strongly hybridizing colonies were purified and rescreened. To isolate DNA fragments ranging from 24 to 35 kb, we constructed the cosmid vector pTAK1 (pHC79 containing 8-kb BamH I-Hind III fragment in alkA gene of E. coli [ 161as a spacer). From the cloned human DNA insert, several Alu-free human DNA fragments were subcloned into plasmid pBR322 or pBR325, as described by Maniatis et al. [lo]. All cloning experiments were carried out according to the guidelines for Recombinant DNA Research issued by the Ministry of Education, Science and Culture of Japan.

Macromolecular

Synthesis in Synchronized

Cultures

tsBN462 cells were seeded at a concentration of 1x lo4 tells/35-mm dish and synchronized at Gl(0) phase by incubating at 33.5”C in low serum medium (DME containing 0.25 % calf serum) for 24 h, and then in isoleucine-depleted (ileu-) medium for 24 h, as described [17]. Following the addition of isoleucine, half of the culture was incubated at 39.5”C. The synthesis of DNA, RNA and protein at both 33.5 and 39.5”C was measured by the cumulative incorporation of radioactive precursors into TCA (trichloroacetic acid)-insoluble material [18]. at the GZIS boundary. Cultures synchronized at Gl(0) phase were then incubated in the growth medium containing 2.5 mM of hydroxyurea (HU) for 17 h at 33.5”C. Following removal of HU, one series of the cultures was cumulatively labelled with [3H]thymidine at 33.5”C and the other at 39.5”C. Synchronizafion

Chromosome Staining Chromosome prepared from hybrid cells, H/F2p5 and C13-lD3 cells, were stained with quinacrine mustard (50 &ml) and Hoechst 33258 (0.5 @ml) dissolved in a McIlvane’s buffered solution (pH 7.0) according to Yoshida et al. [25].

RESULTS S Phase of tsBN462 Cells Is Not Initiated

at a Non-Permissive

Temperature

The tsBN462 cell line was isolated from the BHK21 cell line by FUdR selection at 37.5-38°C after mutagenesis with MNNG [4]. This line belongs to the B group, mutants of which cannot progress from Gl to S phase. For confirmation, cultures of tsBN462 cells were synchronized at Gl(0) phase by low serum and isoleucine deprivation at 33.5”C. Following the addition of isoleucine, half of the culture was incubated at 39.X, and the other half at 33S”C. To estimate the ability of cells to synthesize DNA, RNA and protein, cells were labelled with L3Hlthymidine, [3H]uridine or [3H]amino acid mixture, and the radioactivity incorporated into the acid-insoluble materials was determined. Exp Cell Res 169 (1987)

398

Sekiguchi et al. 20

(b) t

2

4

6

6

10

12

2 hours

4 after

6

6 ileu

lo

12

2

4

6

8

10

12

addition

Fig. 1. Macromolecular synthesis in synchronized cultures of tsBN462 cell line. tsBN462 cells were

plated at 1~10~ per 35-mm dish and allowed to grow for 18 h, followed by low serum, ileusynchronization at 33.X. After the addition of isoleucine, half of the cultures were incubated at 39.X. Cultures were cumulatively labelled with (a) [3H]thymidine (1.0 @i/ml, 5~ 10W6Mthymidine); (b) 3H-amino acid mixture (2 @i/ml); (c) [3H]uridine (2 @/ml, uridine 2.2 t&ml). Every 1 or 2 h, duplicate cultures were processed to count the radioactivity incorporated into acid-insoluble materials of cells. O-0, incubation at 335°C; O-0, at 395°C.

After the addition of isoleucine, DNA synthesis began at the 4th h at 335°C but at 395°C the DNA synthesis observed even at the 12th h was scanty (fig. 1a). Total protein synthesis continued almost normally at 39.K for 8 h, whereafter the rate decreased (fig. 1b). Total RNA synthesis continued rather normally at 39.X until the 5th h after addition of isoleucine and then decreased (fig. 1 c). These observations suggested that both RNA and protein synthesis in tsBN462 cells are normal at both permissive and non-permissive temperature during the progression of Gl phase. To determine whether the absence of DNA synthesis at 39.5”C is due to a defect in the initiation of S phase or to a defect in DNA synthesis, progression of the S phase was then examined by cumulative labelling with [3H]thymidine after synchronization at the Gl/S boundary with HU treatment. The amount of [‘Hlthymidine incorporated into the acid-insoluble materials was always greater at 39.5” than at 33.5”C. The ratio of [3H]thymidine incorporation at high and low temperatures was 1.3-1.7, thereby suggesting that the progression of S phase is normal in tsBN462 cells, at 39.5”C. From these data we considered that tsBN462 cells may have a ts defect with regard to initiation of the S phase, or at some point in the Gl phase where these cells cannot pass at 39.5”C. Transformation

of tsBN462 Cell Line to ts+ Phenotype

with Human DNA

To determine whether the tsBN462 mutation can be complemented by normal human DNA, these cells were plated at a concentration of 100 ceils/35-mm dish into 38 dishes and incubated at 33.5”C for 2 weeks. Cells from each 35-mm dish Exp Cell Res 169 (1987)

X chromosomal

gene of cell cycle

399

were then replated into one lOO-mm dish. Two days later, HMW human DNA (20 ug/dish) was calcium-precipitated and added to the cultures of 20 dishes (2~ lo5 cells/dish) for transfection. As a control, cultures of five dishes were transfected with DNA of Bl (BHK21 Tk-) cell line and the remaining cultures were transfected with DNA of tsBN462 cell line itself (table 1). Following the removal of medium containing DNA precipitates, cultures were washed with TD, fed fresh medium, incubated at 335°C for 24 h and then at 39S”C for 2 weeks to select ts+ transformants. The medium was replaced every 3 or 4 days. Similar DNA transfection experiments were performed twice more. Total 10 tsf cell lines were obtained following transfection with human DNA (table 1). From these tsf cell lines, DNAs were extracted, digested with EcoR I and analysed by Southern blot hybridization, using 32P-labelled total human DNA as a probe. Three cell lines out of 10 tsf cell lines contained human DNA. The representative results was shown in fig. 2. The content of human DNA in tsf cell lines was varied from extremely low (K-6) to high (K-l). Since the ts+ K-8 cell line does not have any other DNA band than that of the recipient, tsBN462 cell line, we considered that ts+ K-8 cells is a spontaneous revertant of tsBN462 cells which used to occur during cultivation of ts mutants [9]. Using the A/u-free human specific probe (B fragment in fig. 4) the human specific 17 kb band was detected in DNAs from K1, K-6 and K-7, but not in DNAs from other ts+ cell lines (data not shown). From these results, the frequency for transforming tsBN462 cells to ts+ phenotype by total human DNA was estimated to be 2.7~10~‘. Since the primary DNA transformants may contain DNA unrelated to the putative human gene which complements tsBN462 mutation, the secondary transfection was then performed using DNA from the primary ts+ transformant cell line, K-l, which contains a lot of human DNA. Two ts+ cell lines containing human DNA were obtained from cultures of the tsBN462 cell line transfected with DNA of K-l cells (table 1, expt 4). Through the primary and secondary tsf transformation, several DNA bands hybridizing to ‘*P-labelled BLUR8 DNA were conserved (indicated by arrowTable 1. Transformation gene transfer

of tsBN462 cells to ts+ phenotype

by DNA-mediated

No. of ts+ colonies/number of culture dishes exposed to DNAsb DNA source

Expt 1

Expt 2

Expt 3

Expt 4

Expt 5

KB (Human) Bl (BHK21 tk-) K-l (ts’ primary transformant) K-l-l (ts+ secondary transformant) tsBN462

5(1)“/20 215 o/13

3(2)/20 4120 -

2(0)/15 4115

l/15 3(2)/U

-

2120

Y/15

l/15

3(3)/20 O/20

a Numbers in parentheses show numbers of ts+ colonies containing human DNA. b The mean number of tsBN462 cells per dish is about 2x lo5 cells. Exp Cell Res 169 11987)

400

Sekiguchi et al.

-1.7

a 1234567

b 1234567

Fig. 2. Presence of human DNA in ts+transformed cells. HMW DNA (20 ug) from ts+ transformants and tsBN462 cells were digested by EcoR I, electrophoresed in 1% agarose gel. After treatment with alkaline and neutralization buffers, DNA in the gel was transferred to nitrocellulose filters, as described by Southern [ll]. The filter was hybridized with (a) “P-labelled total human DNA, (b) BLUR8. (a) Each lane contains DNA of the following cell lines: 1, tsBN462 cells; 2, secondary ts+ transfectant (K-l-2); 3, secondary ts+ transfectant (K-l-l); 4, primary ts+ transfectant (K-l); 5, primary ts+ transfectant (K-7); 6, primary ts+ transfectant (K-6); 7, primary ts+ transfectant (K-8) (revertant). (b) Each lane conatains DNA of following cell lines: 1, tsBN462 cells; 2, primary ts+ transfectant (K-6); 3, primary ts+ transfectant (K-l); 4, secondary ts+ transfectant (K-l-l); 5, secondary ts+ transfectant (K-l-2); 6, tertiary ts+ transfectant (K-l-2-l); 7, tertiary ts+ transfectant (K-l-2-3). Arrowheads indicate the conserved DNA band and its MW estimated by co-electrophoresed Hind III-digested lc1857 DNA.

heads in fig. 2). These DNA bands are 17, 7.2, 5.4, 3.2, 2.55, 2 and 1.7 kb in size (as estimated by cleaved d phage DNA marker). These human specific DNA bands were conserved even after the tertiary transfection of tsBN462 cells with DNA of the secondary ts+ transformant, K-l-2 (fig. 2). Isolation

of Human DNA retained in ts+ Transformants

DNA from the secondary ts+ transformant cell line, K-l-l, derived from the primary ts+ transformant; K-l, was partially digested with restriction enzyme BamH I, EcoR I or Mbo I, and used to construct a genomic library in a cosmid vector, pHC79. The library was screened by colony hybridization with nicktranslated 32P-labelled total human DNA. Seven cosmid clones containing human DNA insert were isolated. Restriction maps of these recombinant cosmid clones were constructed with the use of EcoR I and BamH I (fig. 3). To examine whether the isolated human DNA is present in ts+ transformants through three cycles of DNA transfection, four fragments (fig. 4A-D), were subcloned into pBR322 or pBR325. Using these subcloned DNAs as probes, DNAs from KB cells and from ts+ transformants digested with BamH I or EcoR I Exp Cell Res 169 (1987)

X chromosomal ECORI ,

,:,

BamHl

E-26

401

Fig. 3. A composite partial restriction map of DNA in cosmid clones. Seven recombinant cosmid clones containing human DNA were isolated from a genomic library of secondary ts+ transformant, K-l-l. Restriction enzymes used for isolating cosmid clones were EcoR 1 (E21, E-24, E-28), Mbo I (M-63, M-66) and BamH I (B- 1, T-2). The EcoR I sites are shown over the line and the BamH I sites under the line.

I

L “‘,“““,’

gene of cell cycle

T-2 M-66 E-21 B-1 E-24 M-63

1Okb

were analysed with Southern blot hybridization. Probe A hybridized to the 28.5 kb BamH I fragment (cosmid T-2) and to the 8.7-kb &OR I fragment, as shown in fig. 4. Using this probe, the 28.5kb BamHI fragment proved to be conserved from the original KB cells to a final tertiary tsf transformant through three cycles of ts+ transformation. However, the size of the region corresponding to the 8.7-kb EcoRI fragment in the secondary ts+ transformant changed through transformation, thereby suggesting that a part of this region may not be essential and is probably located at the end of the putative human gene (fig. 4a).

1

probe:

EcoRl

A m

BarnHI

(a)1 26.5k-

23456

B

6.7kb

78910

CD 17kb

I

7.2kb 1Okb

I

28.5kb (b)

16.2kb

---~-ska (C)l

12345676910

2345676910

-

12kb cd)1

2345676

-17k 9k-

12k-7.2k

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Fig. 4. Conservation of original human DNA through ts+ transformation. DNAs (2Opg) from original KB cells or human cells with a karyotype 48XxXx, from the primary transformant (K-l), from the secondary transformant (K-l-l), from the tertiary transformant (K-l-2-l) and from tsBN462 cells were digested with BamHI or EcoRI, electrophoresed in 1% agarose gel and analysed according to Southern [II], using the following DNA fragments as probes, A(a), B(b), C(c) and D(d), whose locations are shown in the restriction map drawn on the top. Each lane contain cellular DNA as following. (a) Lanes: 1, human 48XXXX; 2, 8, K-l; 3, 7, K-l-l; 4, 6, K-l-2-l; 5, 10, tsBN462; 9, KB (human). Since the filter for lanes l-5 had been used for hybridization with probe B, the lower band corresponds to the 16.2-kb BamH I fragment which hybridized to the probe B. (b) Lanes: I, Human cells with a karyotype (48XxXx); 2, 7, K-l; 3, 8, K-l-l; 4, 9, K-l-2-l; 5, 10, tsBN462; 6, KB. (c) Lanes 1, 6, KB; 2, 7, K-l; 3,8, K-l-l; 4,9, K-l-2-l; 5, 10, tsBN462. (d) Lanes: 1,5, K-l; 2,6, K-l-l; 3, 7, K-l-2-l; 4, 8, tsBN462. (u-c) Lanes: l-5 and (6) 14 were digested with EarnHI. (u-c) Lanes 6-10 and (6) 5-8 were digested with EcoRI. The hamster DNA were confirmed to exist within the 8.7 kb of EcoRI fragment and the 12 kb of BamH I fragment (fragment with oblique lines), by hamster repeat sequences (data not shown). Exp Cell Res 169 (1987)

402

Sekiguchi et al. 3.5k 1

00,.

. . -.-.‘:

E-20

l7k

7.2k

’

ECORI I

I

-_ /,

r

II, : probe

BiWlHl

T-2 -

B-l

1Okb

E-21

:a)

1234

(b)

I 2 3

Cc)

I 2 3

(d)

1 2

3

-1 7k

Fig. 5. Detection of human DNA in ts+ transformants of tsBN462 cells transfected with cosmid DNA. (a) DNAs (20 ug) from ts+ transformants and from tsBN462 cells were digested with EcoRI and hybridized with total human DNA as a probe. Each lane contains DNA of the following cell lines. Lanes: I, tsBN462; 2, the secondary ts+ transformant, K-l-l; 5, ts+ transfectant, 3-1-ll[transfected with BamH I digested and religated two cosmid DNAs (T-2 and B-l) and intact cosmid DNA of E-281 (table 2, line 1); 4, ts+ transfectant, 3-6-2(transfected with two cosmid DNAs, T-2 and E-21) (table 2, line 4). (b-d) Twenty ug of DNA from tsBN462 cells, the ts+ transfectant, 3-I-11and the secondary ts+ transformant, K-l-l, were digested with EcoRI and electrophoresed in 1% horizontqal agarose gel and analysed according to Southern [ 111under the stringent hybridization condition, using as a probe I(b), probe II (c), probe III (rf). Probes I, II and III originated from the region of 3.5, 17 and 7.2 kb EcoR I fragment (top). (b. c) Lanes I; (d) 2 contain DNA of tsBN462 cells. (b, c) Lanes: 2 and (d) I, DNA of the secondary ts+ transformant, K-l-l. (&-&) Lane 3, DNA of the ts+ transfectant, 3-l-11.

Consistently, this 8.7-kb fragment contained the hamster DNA sequence (data not shown). In the same manner, using probe B, the 17-kb EcoRI fragment and the 16.2-kb BamHI fragments were conserved through ts+ transformation (fig. 4 b). Using probe C, the EcoR I fragment (7.2 kb) and the BamH I fragment (9 kb) located on the right end of the cloned human DNA proved to be conserved through ts+ transformation (fig. 4~). Probe D can hybridize to 7.2-kb EcoRI fragment and to the 12-kb BamHI fragment which contains both human and flanking hamster DNA (data not shown). Using probe D, the length of the BamH I fragment (12 kb) in the secondary ts+ transformant (K-I-I) changed considerably through ts+ transformation, thereby suggesting that this part may not be essential for complementing tsBN462 mutation (fig. 46). Probe C also hybridized with extra DNA bands which are well conserved in both hamster and human genes. We considered that these extra DNA bands originated from a gene different from the presently isolated human gene, since these DNAs did not hybridize with the probe D. Thus, these data show that cloned human DNA covers about 70 kb of human DNA flanked with hamster DNA and that the middle part (about 56 kb) of the isolated human DNA is conserved through three Exp Cell Res 169 (1987)

X chromosomal

gene of cell cycle

403

cycles of ts+ transformation, without gross rearrangement. This region may be essential for complementing the tsBN462 mutation. To determine whether the cloned human DNA carries the activity transforming tsBN462 cells to the tsf phenotype, a single cosmid clone or a mixture of two or three clones was transfected into tsBN462 cells. No cosmid clone could, by itself, transform tsBN462 cells to the ts+ phenotype (data not shown). Two ts+ transformants containing input human DNA were obtained after transfection with a mixture of cosmid clones (table 2, fig. 5a). One of ts+ transformants, 3-1-11 cells, appeared after transfection with a mixture of three cosmid DNAs, two of which were digested completely with BamH I and then ligated by T4 DNA ligase before transfection (table 2, line 1). The three EcoR I fragments with BamH I site and size of 7.2, 17 and 3.5 kb, which were digested with BamHI and religated with T4 ligase before transfection, were suggested to be recovered in the ts+ 3-111cell line following ts+ transformation, using probes I, II and III which contain, or are close to, BamH I site (fig. 5 6, c, 6). Dark repetitive bands in fig. 5 b, 3 and fig. 5 c may be caused by a high copy number of the putative EcoR I fragment integrated. The other ts+ transformant, 3-6-2, appeared after transfection with a mixture of two cosmid DNAs; T-2 and E-21. These two cosmids shared the 3.5 kb of EcoR I fragment and covered the middle 56 kb region of isolated human DNA. The findings support the observation that the middle 56 kb region of isolated human DNA was conserved through three cycles of ts+ transformation. Assignment

of the Cloned DNA on Human X Chromosome

Since the mutation of the tsBN462 cell line is linked to the HGPRT gene [4], the isolated human DNA most likely originated from the human X chromosome. To confirm this, Southern blotting experiments were carried out using genomic DNAs obtained from cultured fibroblasts of a normal male (46XY) and of an abnormal female with a 48XXXX karyotype. As a probe, we used human Alu-free

Table 2. Transformation

of tsBN462 cells to ts+ phenotype

Cosmid DNA (2 ugkosmid/dish)”

Number of ts+colonies/number of dishes (Ah+ colonies)b

1. 2. 3. 4.

l(l)/5 O/5 3(0)/5 2(1)/5

T-2+B-I+E-28’ E-28+M-66+B-1 E-28+M-66 T-2+E-21

with cosmid clones

a As a carrier DNA for transfection, 20 ug of DNA from tsBN462 cells were co-precipitated with cosmid DNA. b The number in parentheses shows number of ts+ colonies containing human DNA. ’ DNAs of cosmid T-2 and B-l were completely digested with BamH I and religated with T4 DNA ligase. Before transfection, ligated DNAs were mixed with DNA of cosmid E-28 and co-precipitated with calcium phosphate. Exp Cell Res 169(1987)

404

Sekiguchi et al.

Fig. 6. Assignment of the cloned DNA on human X chromosome (I). HMW DNAs (20 pg) from a 46XY man and a 48XXXX woman were digested with EcoR I, electrophoresed in 1% agarose gel, and transferred to nitrocellulose filters according to Southern [I I]. The DNA blotted filter was hybridized with the probe B in fig. 4 a. After washing, the same filter was hybridized with (b) pcD-hprt DNA; (c) with pNP5 DNA. (a-c) Lanes: 1 contains DNA of 46XY; 2, DNA of 48XXXX.

fragment B, which is located on the 17-kb EcoRI fragment of the cloned human DNA. To confirm the validity of dosage effect of X-chromosome we also used, as a probe, DNA of the HGPRT gene located on the X chromosome, and the single copy 5’ flanking region DNA of N-ras gene located on the chromosome 1 and which has no homology with the H-ras gene [21]. As shown in fig. 6, the fragment B hybridized with a 17-kb fragment in human EcoRI-digested DNA. The hybridization intensity of DNA from an abnormal female (4X) is greater than that of DNA from a normal male (XY). The same DNA-blotted filter was washed and rehybridized to cDNA of HGPRT gene, and then to the DNA of the plasmid, pNP5 containing the 5’ region DNA of the N-t-as gene [21]. The cDNA of HGPRT gene hybridized strongly to a 8-kb fragment in EcoR I-digested human DNA. The difference in hybridization intensity between DNA from cells with one X and DNA from cells with 4X is similar to the case of the human Alu-free fragment B. On the other hand, the DNA of the N-ras gene hybridized, with equal intensity, to a 7-kb fragment in both human EcoRIdigested DNA. The 8 and 7 kb are the size of the expected EcoRI fragment of the HGPRT gene and the N-ras gene, respectively, for the probes we used [20, 211. These results suggest that the cloned human DNA is derived from the human X chromosome. This proposal was confirmed by the finding that the 17 kb human DNA band was found in the DNA from a human-mouse hybrid H/F2p5, carrying human chromosomes 14, 21 and X, but not in the DNA from a human-mouse hybrid, C13-lD3, which carries only No. 14 and 21 human chromosome (fig. 7a). The karyotype of these hybrids was shown in fig. 7 b, c. This 17-kb human DNA band was not found in DNA from the human-mouse hybrid C2B5 cell line, Exp Cell Res 169 (1987)

(a) 123456789

a

Fig. 7. Assignment of the cloned DNA on human X chromosome (II). (a) HMW DNAs (20 pg) from various strains were digested with EcoRI, electrophoresed in 1% agarose gel, and transferred to nitrocellulose filters according to Southern [1 11.The DNA-blotted filter was hybridized with probe B in fig. 4. Lanes J-9 contain DNA of the following cell lines: I, KB; 2, Cl3-lD3; 3, C2B5; 4, H/F2p5; 5, BHK21; 6, tsBN462; 7, secondary ts+ transformant, K-l-2; 8, secondary ts+ transformant, K-l-l; 9, primary ts+ transformant, K-l. The karyotypes of (b) Cl3-lD3; and (c) H/F2pS were analysed according to Yoshida et al. [25].

carrying the q21-qter region of the human X chromosome [6]. Thus, the cloned human DNA may locate on the pter-q21 region of the human X chromosome. DISCUSSION Although numerous studies have been done on the characteristics of the Gl phase [2], the mechanisms involved in the progression of this phase are poorly understood. To investigate Gl progression at the molecular level, we used the tsBN462 cell line as a recipient of DNA-mediated gene transfer. After release from isoleucine deprivation, little DNA synthesis occurs with the tsBN462 line, at 395°C though a considerable degree of RNA and protein synthesis is apparent at 39S”C, for 5 h. Following synchronization at the Gl/S boundary, DNA synthesis of the tsBN462 cells continued at a higher level at 39.5” than at 33S”C, thereby suggesting that the progression of S phase is normal at Exp Cell Res 169 (1987)

406 Sekiguchi et al. 39.E. Thus, we assumed that the tsBN462 cell line has a ts defect either in the progression of the Gl phase or in initiation of the S phase. Hybrid cells between the tsBN462 HGPRT- cell line and the primary human fibroblast, MRC-5 cell line grow in HAT (Hypoxanthine/Aminopterin/Thymidine) [7] medium containing 1x 10m5Mouabain at 39S”C. Therefore, we assumed that the human cells possessed a gene which could complement the ts defect in the tsBN462 cell line. This is advantageous for isolating a gene, because human DNA inserted into hamster DNA can be detected by probing the human specific ‘Alu’ repetitive sequence. The frequency of ts+ transformation of tsBN462 cells with total human DNA is low, about 3x lo-’ which is close to the spontaneous ts+ reversion frequency of tsBN462 cells. This is compatible with the result that only three lines out of 10 ts+ cell lines contain human DNA. To make sure that the primary ts+ transformant contains the whole putative human gene, we chose, as the donor DNA for the secondary transfection, the K-l cell line which contains a lot of human DNA. Several human DNA bands were found to be commonly conserved through both the secondary and tertiary transformation. These human DNAs were isolated into seven cosmid clones and human DNA inserts in isolated cosmid clones covered 70 kb of the human genomic DNA flanked with hamster DNA. As fragments in the middle of isolated human DNA are well conserved through three cycles of ts+ transformation, this conserved region may contain the putative human gene complementing tsBN462 mutation. However, the frequency of ts+ transformation did not increase, even when isolated cosmid clones were used as the transfecting DNA. The size of the putative human gene required for complementing tsBN462 mutation is no doubt too large to be inserted into a single cosmid, and so prohibits the efficient ts+ transformation of tsBN462 cells. To obtain the direct evidence for the activity of isolated human DNA, we are now searching for the cDNA corresponding to the isolated human DNA. The cloned human DNA apparently originates from the human X chromosome. This is consistent with our finding that the site of tsBN462 mutation locates in the X chromosome of hamster cells [4]. Since genes of the X chromosome have been well conserved through processes of evolution [22], it seems reasonable that the human gene complementing tsBN462 mutation locates on the X chromosome. The site of cloned human DNA in the human X chromosome was suggested to be located in the pter-q21 region of the human X chromosome. Several ts mutants with mutation sites in the X chromosome have been detected [4, 23, 241. Our observations indicate that the genes of the X chromosome which are necessary for functions of eukaryote can be isolated using mutant cells of the X chromosome as a recipient of DNA-mediated gene transfer. We thank M. Ohara (Kyushu University) for comments on the manuscript. This investigation was supported in part by scientific and cancer research grants from the Ministry of Education, Science and Culture of Japan, and by a Grant-in-Aid for Cancer Research from the Ministry of Health and Welfare of Japan. Exp Cell Res 169 (1987)

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Exp Cell Res 169 (1987)