MOLECULAR AND CELLULAR BIOLOGY, Feb. 1990, p. 577-584 Vol. 10, No. 2 0270-7306/90/020577-08$02.00/0 Copyright © 1990, American Society for Microbiology

Premature Chromosome Condensation Is Induced by a Point Mutation in the Hamster RCC1 Gene SANAE UCHIDA,t TAKESHI SEKIGUCHI, HIDEO NISHITANI, KUMI MIYAUCHIt MOTOAKI OHTSUBO, AND TAKEHARU NISHIMOTO* Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Higashi-ku, Maidashi, Fukuoka 812, Japan Received 1 August 1989/Accepted 21 October 1989

At the nonpermissive temperature, premature chromosome condensation (PCC) occurs in tsBN2 cells derived from the BHK cell line, which can be converted to the Ts' phenotype by the human RCC1 gene. To prove that the RCC1 gene is the mutant gene in tsBN2 cells, which have RCC1 mRNA and protein of the same sizes as those of BHK cells, RCC1 cDNAs were isolated from BHK and tsBN2 cells and sequenced to search for mutations. The hamster (BHK) RCC1 cDNA encodes a protein of 421 amino acids homologous to the human RCC1 protein. In a comparison of the base sequences of BHK and BN2 RCC1 cDNAs, a single base change, cytosine to thymine (serine to phenylalanine), was found in the 256th codon of BN2 RCC1 cDNA. The same transition was verified in the RCC1 genomic DNA by the polymerase chain reaction method. BHK RCC1 cDNA, but not tsBN2 RCC1 cDNA, complemented the tsBN2 mutation, although both have the same amino acid sequence except for one amino acid at the 256th codon. This amino acid change, seine to phenylalanine, was estimated to cause a profound structural change in the RCC1 protein.

Eucaryotic cells grow by going through the four phases of (30a). Futhermore, we cloned the Xenopus RCC1 cDNA and the cell cycle: G1, S, G2, and M. The G1 and G2 phases are found its product in Xenopus oocytes (H. Nishitani, H. the regulatory periods for entering into the S and M phases, Kobayashi, M. Ohtsubo, and T. Nishimoto, J. Biochem., in respectively (32). In the G2 phase, several proteins, such as press) in which mRNA of cyclin, an activator of MPF, is cyclin (31, 42), are newly synthesized and promote the present but not translated until oocyte maturation (41). cascade of events leading to activation of maturation or Thus, the presence of RCC1 protein in oocytes indicates M-phase-promoting factor (MPF), which is present as a another aspect of this protein. latent form in the interphase (7, 20). Mitotic cells containing Although these findings suggested an interesting function the activated MPF condense the interphase chromatin by of RCC1 protein, it is important for further analysis of RCC1 cell fusion (13, 33), resulting in so-called premature chromo- function relevant to the tsBN2 mutation to determine some condensation (PCC). whether the RCC1 gene is indeed a mutant gene in tsBN2 We isolated a temperature-sensitive mutant cell line, cells or is a suppressor gene of the tsBN2 mutation. In the called tsBN2, from the BHK-21 cell line; the mutant cells case of lower eucaryotes such as yeasts, this question can be show PCC at the nonpermissive temperature, depending on answered by gene disruption (35, 37). However, in mamma- new protein synthesis (26, 28). DNA replication in the lian cells homologous recombination is rare (19); therefore, mutant cells was shown to cease upon chromosome conden- that sequences of both the parental and the mutant gene sation (27, 28). Therefore, eucaryotic cells possess a regula- must be determined to verify that the complementing gene is tory mechanism that recognizes the completion of DNA the mutant gene. In this study, we isolated and sequenced synthesis and switches on the cascade of events leading to both mutant and wild-type RCC1 cDNAs and found that chromosome condensation (43). Since tsBN2 cells showing RCC1 cDNA of tsBN2 cells did not complement the tsBN2 PCC condense the chromatin of interphase cells by cell mutation and had a point mutation. We further identified this fusion (10), MPF was activated in this cell line at the mutation site in the genomic DNA. nonpermissive temperature. This finding suggested that ac- tivation of MPF was halted by a mechanism encoded by the MATERIALS AND METHODS gene(s) that may serve as a negative regulator for the onset of chromosome condensation. Cell lines. The tsBN2 cell line is a temperature-sensitive To investigate the molecular mechanism of PCC induction mutant derived from the BHK-21 cell line (26). The tsBN2- in tsBN2 cells, we isolated the human gene, RCC1, that N9 cell line is a thymidine kinase-negative derivative of the complements the tsBN2 mutation (14). This gene encodes a tsBN2 cell line (14). protein of 45 kilodaltons that consists of 421 amino acids and Cell culture and transformation. All cell lines were cul- has seven internal homologous repeated domains of about 60 tured in Dulbecco modified Eagle medium supplemented amino acids (30). Recently, this putative RCC1 protein was with 10% calf serum, penicillin (100 U/ml), and streptomycin detected in nuclei and found (100 jg/ml) in a humidified atmosphere containing 10% Co2. to have DNA-binding activity Cultures of tsBN2 cells were maintained at 33.5°C, the permissive temperature, and those of BHK-21 cells were * Corresponding author. maintained at 37°C. Ts' transformants were selected at t Present address: Hoechst Japan Ltd., Pharma Research Labo- 39.5°C, the nonpermissive temperature. Neomycin-resistant ratories, 1-3-2 Minamidai, Kawagoe, Saitama 350, Japan. (Neo') cells were selected by the addition of G418 (geneticin; t Present address: Department of Biophysics, Faculty of Science, 400 jig/ml; Sigma Chemical Co., St. Louis, Mo.). Kyoto University, Sakyo-ku, Kyoto 606, Japan. Transformation of tsBN2 cells was performed by the 577 578 UCHIDA ET AL. MOL. CELL. BIOL. calcium phosphate precipitation technique (44), using B pSV2neo (40) as a control for calculating the efficiency of A 1 2 1 2 3 4 transfection. kDa Isolation of nucleic acid and filter hybridization. Exponen- 97 tially growing cells were washed twice with ice-cold TD 28S- * buffer (Tris-buffered saline without Ca2' and Mg2' and 5 - 6 containing 136.8 mM NaCl, 5 mM KCl, 0.7 mM Na2HPO4, and 25 mM Tris chloride [pH 7.4]) and harvested with a NS p45 1. Teflon scraper. High-molecular-weight DNA was isolated 18S .1111_ -: 43 from cultured cells by phenol extraction of the sodium dodecyl sulfate-disrupted cells (38). From the total RNA isolated, the poly(A)+ RNA was purified by oligo(dT) chro- matography (30). Southern and Northern (RNA) blot hybrid- ization were performed as described previously (30, 38). 31 Construction and screening of cDNA libraries. The cDNA FIG. 1. Identification of RCC1 mRNA and its product in BHK- library of the poly(A)+ RNA of BHK21/13 and tsBN2 cells 21 and tsBN2 cells. (A) Total RNAs extracted from BHK-21 and was constructed by using the Amersham cDNA synthesis tsBN2 cells were electrophoresed in a 1.2% agarose gel containing system (Amersham Corp., Arlington Heights, and the formaldehyde and transferred to a nylon filter. The filter was Ill.) hybridized with an c-32P-labeled wild-type RCC1 cDNA probe. Amersham XgtlO cDNA cloning system and then screened as Each lane contained 40 ,ug of total RNA. The size of the RNA was described previously (38). estimated by using 28S and 18S rRNAs. The arrowhead indicate the Subcloning of DNA fragments into vector DNAs. By using position of RCC1 mRNA. (B) Total protein extracted from BHK-21 the procedure of Maniatis et al. (18), DNA fragments were and tsBN2 cells was electrophoresed and immunoblotted with the subcloned into bacteriophage (M13mp18) or plasmid anti-Xenopus RCC1 protein (lanes 1 and 2) or stained with (pUC118) vectors to determine the base sequence or into the Coomassie brilliant blue (lanes 3 and 4). Lanes 1 and 3, BHK-21 pKCRS eucaryotic expression vector derived from pKCRH cells; lanes 2 and 4, tsBN2 cells. (22) to express the cloned cDNA in hamster cells. Restric- tion endonucleases were purchased from TAKARA Shuzo (Kyoto, Japan), and digestion was carried out under the RESULTS conditions recommended by the supplier. Identification of RCC1 mRNA and protein in tsBN2 cells. Sequencing. After the inserts of phage X clones were RCC1 mRNA detected in tsBN2 cells by Northern blot subcloned into pUC118, a series of overlapping deletions analysis, using as a probe human RCC1 cDNA (30), indi- was created, using a TAKARA deletion kit as recommended cated that the hamster RCC1 gene is present and is tran- by the supplier. The nucleotide sequence was determined by scribed in tsBN2 cells. Since a temperature-sensitive muta- the chain termination method (36). In some cases, single- tion is usually caused by a point mutation (2), BHK and stranded M13 phage templates were used. tsBN2 cells would have RCC1 mRNAs and proteins of the PCR. The polymerase chain reaction (PCR) was carried same-sizes if the RCC1 gene is a mutant gene in tsBN2 cells. out as described by Randall et al. (34), using Taq polymerase Before investigating the mutation site of the RCC1 gene in (Cetus Corp., Emeryville, Calif.). A 1-,ug sample of genomic tsBN2 cells, we verified the presence of RCC1 mRNA and DNA extracted from BHK-21 or tsBN2 cells was subjected its product in tsBN2 cells. to 30 cycles of amplification in 50 mM KCl-10 mM Tris For quantitative comparison ofthe concentrations of RNA chloride (pH 8.3)-1.5 mM MgCl2-0.01% (wt/vol) gelatin-200 specific to the RCC1 gene in BHK and tsBN2 cells, in ,uM each deoxynucleotide triphosphate-50 pmol of primers- addition to the comparison of size, total RNAs were ex- 2 U of polymerase in a final volume of 100 ,ul. The primers tracted from BHK and tsBN2 cells growing exponentially at had the sequences 5'-ACTCCTGCTCCCCAAGTGTG-3' 33.5°C and analyzed by Northern blot hybridization. By and 5'-AAGCTGGTGATAGTTCGAGA-3', respectively. using BHK RCC1 cDNA as a probe, RCC1 mRNAs of the The DNA was denatured by heating at 94°C for 1 min and same size were detected in BHK and tsBN2 cells, which annealed with the primers at 54°C for 2 min; the polymeriz- indicated no difference in size of the RCC1 mRNAs in the ing reaction was carried out for 2 min except for the last two cell lines (Fig. 1A, lanes 1 and 2). The amounts of cycle, when the reaction was carried out for 7 min for RCC1-specific RNAs in BHK and tsBN2 cells seemed to be complete elongation. similar. The lower band at 18S was caused by cross-hybrid- The PCR product was purified from a 2% agarose gel, ization with rRNA, which accounts for about 90% of total using DEAE-paper. The fragments was blunt ended with a RNA. In the poly(A)+ RNA fraction, only a single band was large fragment of polymerase I, subcloned into the pUC8 detected with BHK RCC1 cDNA used as a probe (data not vector, and then sequenced. shown). Analysis of protein structure. The amino acid sequence of Total proteins were extracted from exponentially growing the RCC1 protein was analyzed by using the algorithm of BHK and tsBN2 cells, electrophoresed in the presence of Chou and Fasman (5) programmed in DNASIS (Hitachi sodium dodecyl sulfate (16), and then analyzed by immuno- Software Engineering Co., Ltd., Japan). blotting, using the antibody against the Xenopus RCC1 Immunoblot analysis. Cells were lysed in buffer containing protein, which recognizes hamster and human RCC1 protein 62.5 mM Tris hydrochloride (pH 6.8), 10 mM 2-mercapto- in addition to the Xenopus protein (Nishitani et al., in press). ethanol, 3% (wt/vol) sodium dodecyl sulfate, and 20% glyc- The antibody recognized two closely located proteins, with erol. Cellular proteins were electrophoresed in a 10.5% molecular masses to 45 to 46 kilodaltons, in extracts of both sodium dodecyl sulfate-polyacrylamide slab gel (16) and BHK and tsBN2 cells (Fig. 1B, lanes 1 and 2). Hence, the analyzed by immunoblotting as described previously (3). two cell lines apparently produce RCC1 proteins of the same The antibody used was directed against the Xenopus RCC1 size. protein (Nishitani et al., in press). In addition, the amount of RCC1 protein was lower in VOL. 10, 1990 RCC1 GENE AND tsBN2 GENE 579

200 TABLE 1. Ability of various RCC1 cDNA clones to transform tsBN2 cells to the Ts' phenotypea Avg no. of Ts' and G418 colonies/dish with Sac! Accl Smal EcoRI Xbal given mode of selection DNA source G418 resistance pBH231 - 39.5°C Expt 1 Expt 2 Expt 1 Expt 2 None 0 0 0 0 pKCRS-BH21, 200.5 110 266.5 315 Xbal pSV2neo Accl Smal EcoRI pKCRS-BH21', 240.5 116 0 0.5 I I pSV2neo pBN112 CODING REGION pKCRH-BN112, 66.5 212 0.5 0.5 pSV2neo FIG. 2. Restriction maps of isolated wild-type and mutant RCC1 pKCRH-BN112', 73 176 69.5 260 cDNAs. The restriction maps of isolated hamster wild-type (pBH21) pSV2neo and mutant (pBN112) RCC1 cDNA clones were determined by using the restriction enzymes AccI, EcoRI, SacI, SmaI, and XbaI. The a tsBN2 cells (2.0 x 105/dish) were plated onto 100-mm-diameter dishes. putative coding regions are indicated, as is the size scale (in base After incubation for 24 h at 33.5TC, four dishes were transfected with 200 ng pairs). The hatched bar in pBN112 indicates the region in which the of pSV2neo and 250 ng of RCC1 cDNA; as a carrier, 20 F.g of tsBN2 cellular base sequence differed from that of DNA was used. Transfected cells were incubated for 2 days at 33.5TC, and pBH21. then two of the dishes were incubated either at 39.5TC or in the presence of G418 at 33.5aC. After incubation for 10 to 14 days, the number of colonies was counted. tsBN2 cells than in BHK cells. Since both lanes (Fig. 1B, lanes 1 and 2) contained almost the same amount of total proteins (Fig. 1B, lanes 3 and 4), the lower content seemed (pBN112); there was no other base change in the region to be specific to the RCC1 protein of tsBN2 cells, probably encoding the RCC1 protein. With this base transition, serine because of a tsBN2 mutation. at the 256th codon is converted to phenylalanine (Fig. 5). These data suggested that tsBN2 cells may have a point The same base change was identified in all seven of the mutation in the RCC1 gene. To prove this idea, we cloned independently isolated tsBN2 cDNA clones that covered the both the BHK RCC1 and the tsBN2 RCC1 cDNA. 256th codon of the RCC1 open reading frame (data not Cloning of hamster RCC1 cDNA from BHK-21 cells. A shown). hamster wild-type XgtlO cDNA library was constructed by Base substitution in the tsBN2 genomic DNA. To verify the using poly(A)+ RNA extracted from BHK-21 cells and was presence of the single base change found in the tsBN2 RCC1 screened with human RCC1 cDNA, pCD40 (30), as a probe. cDNA in the chromosome of tsBN2 cells, the nucleotide After screening of 5 x 105 plaques, 12 positive clones were sequence of RCC1 genomic DNA was determined by the isolated. The inserts of positive clones were subcloned into PCR method (34) (Fig. 6). the pUC118 vector, and restriction maps were determined. Chromosomal DNA was extracted from BHK-21 and According to these maps, the putative full-length cDNA was tsBN2 cells, and the region of the RCC1 genome covering constructed (Fig. 2). This cDNA was recloned into pKCRH, the 256th codon of the RCC1 protein was amplified, using a eucaryotic expression vector (22), and cotransfected into synthetic nucleotides as primers. The nucleotide sequence of tsBN2 cells with pSV2neo. The putative full-length cDNA two synthetic primers and the region to be amplified is clone, pBH21, complemented the tsBN2 mutation with high shown in Fig. 6A. These primers were selected on the basis frequency (Table 1), indicating that this cDNA contains the of the fact that the region to be amplified was located in a intact full-length hamster (BHK) RCC1-coding region. single exon (with reference to the nucleotide sequence of the The nucleotide sequence of BHK RCC1 cDNA, deter- human RCC1 genomic DNA [N. Furuno et al., manuscript in mined as described in Materials and Methods, showed that preparation], since the nucleotide sequence of the hamster the cloned cDNA was about 2.6 kilobase pairs long and had RCC1 genomic DNA has not been determined). The ampli- an open reading frame of 1,263 base pairs, encoding a protein fied fragments were purified from agarose gel, blunt ended with a molecular mass of 45 kilodaltons (Fig. 3), the same with the large fragment ofEscherichia coli polymerase I, and size as that of human RCC1 protein (30). The estimated ligated into the pUC8 vector. Seven DNA fragments were BHK RCC1 protein, which was highly homologous (94%) cloned independently from the BHK-21 and from the tsBN2 with the human RCC1 protein through the entire portion, cell line, and their base sequences were determined. Since had the seven homologous repeated domains present in the our data suggested an autosomal location of the RCC1 gene human protein (Fig. 4). in the hamster cell line (29, 30), more than two clones were Cloning of BN2 RCC1 cDNA. A cDNA library was con- analyzed for each cell line to determine the mutation site. structed from the poly(A)+ RNA of tsBN2 cells and The mutation site was found in an area where the C-to-T screened, using the BHK RCC1 cDNA as a probe. After transition occurs (Fig. 6A). Representative results of assays screening of 7 x 105 plaques, 18 positive clones were to determine the base sequence of the DNA fragments isolated and sequenced to precisely determine location of the amplified by PCR are shown in Fig. 6B and C. The same open reading frame. According to the base sequence, the transition was detected in all clones examined. Thus, the full-length tsBN2 RCC1 cDNA, pBN112, was constructed base transition in the cDNA clones is present in the corre- (Fig. 2). The nucleotide sequences of pBN112 was compared sponding RCC1 genomic DNA region. with that of pBH21 (Fig. 5). Southern blot analysis of EcoRI-digested DNA of both According to these data, the cytosine of the second base at tsBN2 and BHK cells, using as a probe the BHK RCC1 the 256th codon of the BHK RCC1 cDNA (pBH21) was cDNA, showed that the RCC1 genes of the two cell lines had replaced with a thymine in the tsBN2 RCC1 cDNA the same genomic structure (data not shown). 580 UCHIDA ET AL. MOL. CELL. BIOL.

GAATTCCTTGATAXTAAGCCTGCAGTTCTGTCC ATATACTCAAGTAAGCTTTGACCCAGTCCACAXTGCTTCTTATCCACATCTTTGTACTCCTTGAGGACTCGGTTTGTG III TAAAACATGGCAGC ATCAGTCATCTCTTTC ACAAAGGGGCCAGGTTTTGCAGCCAGAGCCACCCAGCCCAGGGCCTGGATGCTTTCACTGACAGCTGACAAATGATTGAAAA 22 3 ACTTGCTGCCTCGGTTCTTCTCCCGGAAGGTTATAACTTCTTGAATCTGCTCTGAGATAGGTGCCAACAAATCAGAGAGCTTATTACCG(GCCGGCTGCTGGCCACTGAGAAGC 335S TGTAACCAGGAGAGCTCCAACTTCAGGCCTGTGTGGACCATCTCTGCATGTTTCTGCACGTCTCCCCCAATCTCCTTACTCATCTTCAACTTGTGGCCTCCCCGCAACGAGG 447 ACAGGAAG 6IG CCA CCC AAG CGC ATA GCT AAG AGA AGG TCA CCC CCA GAA GAT GCC ATT CCC AAA AGC AAG AAA GTT AAA GTC TCA 533 Met Pro Pro Lys Ar I le Ala Lys Arg Arg Ser Pro Pro Glu Asp Ala Ile Pro Lys Ser Lys Lys Val Lys Val Ser 26 CAC AGG TCT CAC AAA ACA GAA CCA GGC TTG GTG CTG ACA CTG GGC CAG GGC GAC GTG GGC CAG CTG GGG CTG GGT GAG AGT GTG 617 His Arc Ser His Lys Thr Glu Pro Gly Leu Val Leu Thr Leu Gly Gin Gly Asp Val Gly Gin Leu Gly Leu Gly Glu Ser Val 54 CTG GAG AGG AAG AAG CCG GCC TTG GTG CCC CTT CTG CAA GAT GTT GTG CAG GCT GAG GCT GGG GGC ATG CAT ACC GTG TGT CTG 701 Leu Glu Arg Lys Lys Pro Ala Leu Val Pro Leu Leu Gin Asp Val Val Gin Ala Glu Ala Gly Gly Met His Thr Val Cys Leu 82 AAC CAA AGT GGC CAG GTC TAC TCC TTT GGC TGC AAT GAT GAG GGT GCC CTG GGA AGG GAC ACA TCA GTC GAG GGC TCA GAG ATG 785 Asn Gin Ser Gly Gin Val Tyr Ser Phe Gly Cys Asn Asp Glu Gly Ala Leu Gly Arg Asp Thr Ser Val Glu Gly Ser Glu Met 110 GTC CCT GGC AAA GTG GAA CTG CAA GAG AAG GTG GTA CAA GTG TCA GCA GGG GAC AGT CAC ACA GCA GCT CTT ACT GAA GAT GGC 869 Val Pro Gly Lys Val Glu Leu Gin Glu Lys Val Val Gin Val Ser Ala Gly Asp Ser His Thr Ala Ala Leu Thr Glu Asp Gly 138 CGT GTC TTC CTC TGG GGC TCT TTC CGG GAT AAT AAC GGT GTG ATC GGG TTG TTG GAG CCC ATG AAG AAG AGC ATG GTT CCT GTT 953 Arg Val Phe Leu Trp Gly Ser Phe Arg Asp Asn Asn Gly Val Ile Gly Leu Leu Glu Pro Met Lys Lys Ser Met Val Pro Val 166 CAA GTG CAG CTG GAC ATG CCT GTG GTA AAG GTA GCC TCA GGG AAT GAC CAT TTG GTG ATG CTG ACG ACT GAC GGA GAC CTC TAC 1037 Gin Val Gin Leu Asp Met Pro Val Val Lys Val Ala Ser Gly Asn Asp His Leu Val Met Leu Thr Thr Asp Gly Asp Leu Tyr 194 ACC TTG GGG TGT GGA GAG CAG GGT CAG CTG GGC CGT GTG CCT GAG TTA TTT GCC AAC CGA GGT GGC CGT CAG GGC CTT GAG CGA 1121 Thr Leu Gly Cys Gly Glu Gin Gly Gin Leu Gly Arg Val Pro Glu Leu Phe Ala Asn Arg Gly Gly Arg Gin Gly Leu Glu Arg 222 CTC CTG GTC CCC AAG TGT GTG CTG CTG AAA TCC CGG GGA AGT CGG GGC CGT GTG CGG TTC CAA GAT GCC TTC TGT GGG GCC TAT 1205 Leu Leu Val Pro Lys Cys VaP Leu Leu Lys Ser Arg Gly Ser Arg Gly Arg Val Arg Phe Gin Asp Ala Phe Cys Gly Ala Tyr 250 CTC ACT TTT GCC ATC TCC CGT GAG GGC CAT GTA TAT GGC TTT GGC CTC TCG AAC TAT CAC CAG CTT GGA ACT CCG GGC ACT GCA 1289 Leu Thr Phe Ala le Ser Arg Glu Gly His Val Tyr Gly Phe Gly Leu Ser Asn Tyr His Gin Leu Gly Thr Pro Gly Thr Ala 278 TCT TGC TTC ATT CCT CAG AAC TTA ACA TCC TTC AAG AAT TCC ACC AAG TCC TGG GTG GGC TTC TCT GGT GGC CAG CAC CAT ACA 1373 Ser Cys Phe Ile Pro Gin Asn Leu Thr Ser Phe Lys Asn Ser Thr Lys Ser Trp Val Gly Phe Ser Gly Gly Gin His His Thr- 306 ATC TGC ATG GAT TCA GAA GGA AAA GCA TAC AGC CTG GGC AGG GCT GAA TAT GGG CGG CTG GGC CTT GGG GAG GGT GCT GAG GAG 1457 Ile Cys Met Asp Ser Glu Gly Lys Ala Tyr Ser Leu Gly Arg Ala Glu Tyr Gly Arg Leu Gly Leu Gly Glu Gly Ala Glu Glu 334 AAG AGC ATA CCC ACC CTC ATT TCC CGA CTG CCC GTC GTC TCC TCT GTG GCC TGT GGG GCT TCT GTG GGA TAT GCT GTG TCC AAG 1541 Lys Ser Ile Pro Thr Leu I le Ser Arg Leu Pro Val Val Ser Ser Val Ala Cys Gly Ala Ser Val Gly Tyr Ala Val Ser Lys 362 GAT GGT CGT GTT TTT GCC TGG GGC ATG GGC ACC AAC TAC CAG CTG GGC ACA GGG CAG GAT GAA GAT GCC TGG AGC CCT GTG GAA 1625 Asp Gly Ars Val Phe Ala Trp Gly Met Gly Thr Asn Tyr Gin Leu Gly Thr Gly Gin Asp Glu Asp Ala Trp Ser Pro Val Glu 390 ATG ACC GGC AAA CAG CTG GAG AAC CGA GTG GTC TTA ACT GTA TCC AGC GGG GGC CAG CAC ACA GTC TTA CTG GTT AAG GAC AAG 1709 Met Thr Gly Lys Gin Leu Glu Asn Arg Val Val Leu Thr Val Ser Ser Gly Gly Gin His Thr Val Leu Leu Val Lys Asp Lys 418 GAA CAG AGC 18ATGA1GTCTTTGTGGGCCTGGCTTCTGGCCCCCAACCCACCTTCACAGAACAGGAAAACAGCCAGATGCAAATTCCAGAGGCCCCTCCCCAGCCCTG1818 Glu Gin Ser 421 AACAGCTGTCATCTCCTGTCTTACCCATCACCACCACAGAATCCTTTCCTTCTGTTTCGTCCTCTTT TCTAGAATCATCCTGAGAAGTACAGGATGAGGGAGGATGGGAAGG 1 93 0 GGGTCTCAGAAGGAGTTTGCTTACTTACGTCCTTACACCGTTCCCTATATTACCCTACTTCCTGTGGTCCTAGCAGGCCCTGGCTC ATTGCCTAC AAAACCCAAAGCTTGGG 2 042 GTTTGGGTGCCCATACTCTGAAAAGTTGGGAACTCCGTTCACACCCCCATTCCTATGTGCCACTTTTTCTGTTCCTAACATCAGTTTAAAAGGAGACGGATGGTACAGTCAT 215 4 CAGATCCAGTTGTCATGGACCTATGCCTAGAGAGATCTGGACAAAGGCTTCACAAGGCTGGGGTGACCCTAAGCCAAATATTTGGCTCTAAACAGGTGTCCATGGGCAAAAA 2 266 CAGCGATCCACTTGAAAGAAATCCAACTAGCTGTCTCCCAAAAGCACCAAAGCACCCACTGTCTCTTGGGCATTGCTATGCATTCCCCATCT AGTCAGCCTTTGTGCCAAAA 2 37 8 AGGACTGG^AAGCCATGCTGGGCTCTGCTGGGCAGTCAAGGCAGGGTACTGGGGAAGCGTGGAGGGGAACACTTGGAGGGACGGAGGCTGGTCCTAAGAAAAGGAAGAACAG 2 49 0 TGGTTGAGACAGT AGTTTTGTTTTTGTTTTTTTAATTAT AAAGTATTTTGGAGGGGAGAGTGAAAGTCTTTTAACACTTTGAAT AAATTTAGAGTTTTATMAAATGGAATT 2 602 C 2603 FIG. 3. Base sequence of BHK RCC1 cDNA. The insert of pBH21 was sequenced as described in Materials and Methods.

tsBN2 RCC1 cDNA does not complement the tsBN2 muta- peared with the same efficiency as did Neor colonies. How- tion. The insert of the pBN112 clone (tsBN2 RCC1 cDNA) ever, upon transfection with pKCRH-BN112, very few or no was subcloned into the pKCRH vector, and the resultant Ts' colonies appeared (Table 1). Since pSV2neo cotrans- plasmid, pKCRH-BN112, was cotransfected into tsBN2 fected with pKCRH-BN112 transformed tsBN2 cells to Neor cells with pSV2neo, a marker to estimate transfection effi- with efficiency comparable to that of pKCRS-BH21, the ciency. As a control, the pKCRS-BH21 clone, carrying pKCRH-BN112 clone should be inserted into tsBN2 cells wild-type BHK RCC1 cDNA, and pSV2neo were cotrans- with the same efficiency as is the pSV2neo plasmid. Thus, fected into the same culture oftsBN2 cells. pKCRH-BN112 seems to be unable to complement the When pKCRS-BH21 was transfected, Ts' colonies ap- tsBN2 mutation even though the amino acid sequence en-

38 HAMST ER MPPKR IAKRRSPPEDA IPKSKKV KV SHRSHKTEPGLVLTLGQGDV-GQLGLGESV L- ERKXP-- ALV PLLQDVV ------QAEAGGMHTVCLNQSGQYY- 89 HUMAN, *********************** *******N*M ***** ***SIPE*********SX ** SFGCNDE-GALGRDTSVEGSEMVP-- --- GKVELQEKVV ------QVSAGDSHTAALTEDGRVFLW 143

G- SFRDNNGYVIGLLEP-MKKSNVP-- --- VQVQLDMPVV------KVASGNDHLVMLTTWGDLY- - 19 4

TLGCG-EQGQLGRVPELFANRG ------GRQGLERLLVPXCVLLKSRGSRGRVRFQDAFCGAYLTFA ISREGHVY-- 26 2

GFGLS-NYHQLGT-PGTA-SCF IPQNLTSFKNSTKSWV ------GFSGGQHHT ICMDSEGXAY- - 316 ***** ******* ***E ********* * ******** SLGRA-EYGRLGLGEGAEEKS- I P----TLI -SRLPVVS------SVACGASVGYAVSKDGRVF-- 367

AWGMGTNY-QLGTGQDEDAWS - -PVEMTGKQ-LENRVVL------TVSSGGQHTYVLLVKDKEQS -- 42 1

0 0 0 0 00 0 0 0 0 00 0 0 0 FIG. 4. Comparison of amino acid sequences in hamster wild-type and human RCC1 proteins. In the human sequence, * indicates an tmin2RcCidnh orespnot i lment theitheN2 mutaK pqeaed wihesnami eeff agiedtothnrepaeiatdNenes.sho Htroure of RCC1 protein. 0, amino acid conserved well among the seven repeats (30). VOL. 10, 1990 RCC1 GENE AND tsBN2 GENE 581

2 5 6 Small Xbal Small Xbal

Ala Ile $ (@ G Arg Glu Wild Type GCC ATC TCC CGT GAG pKCRS-BH21 pKCRS-BH21'

GCC ATC T¶ C CGT GAG tsBN2 Small Xbal Small Xbal Ala Ile ph & Arg Glu FIG. 5. Mutation site in BN2 RCC1 cDNA. Sequences of nucleic acids and amino acids near the 256th codon of RCC1 protein are shown. The arrow marks the point mutation site. pKCRH-BN1 12 pKCRH-BN1 12' FIG. 7. Exchange of the 3' region between wild-type and mutant cDNAs. To construct pKCRS-BH21' and pKCRH-BN112', the coded by this clone is the same as that encoded by pKCRS- DNA fragment between the SmaI site and the XbaI site of pKCRS- BH21 except for one amino acid at the 256th codon. BH21 was exchanged with that of pKCRH-BN112. The 5' noncoding leader sequences of two hamster RCC1 clones were different, and the 3' noncoding tail was shorter exchanged to construct pKCRS-BH21' and pKCRH- in pKCRH-BN112 than in the pKCRS-BH21 clone (Fig. 2). BN112', respectively (Fig. 7). It is therefore possible that the inability ofpKCRH-BN112 to pKCRS-BH21', containing the putative mutation site de- complement the tsBN2 mutation is due to differences in rived from tsBN2 cDNA and the noncoding region of BHK these noncoding regions rather than to differences in the RCC1 cDNA, did not complement the tsBN2 mutation. coding regions of the cDNA clones. To investigate this Conversely, pKCRH-BN112', containing the 5' and 3' non- possibility, the SmaI-XbaI fragments of pKCRH-BN112 and coding regions derived from tsBN2 cDNA and the coding pKCRS-BH21, which cover the putative mutation site, were region of BHK RCC1 cDNA, did complement the tsBN2 mutation (Table 1). These results showed that the 5' non- coding leader sequences did not affect the Ts' transforma- tion and that the shortness in the 3' noncoding region of pKCRH-BN112 was not responsible for the biological activ- A ity of this clone. 5'-RCTCCTGGTCICCCRRGTGTG-3' Effect of a single amino acid change on protein structure. By using the algorithm of Chou and Fasman (5), the structures 5-4-CTCCTGGTCCCCAAGTGTGIrGCTGCTGAAATCCCGGGGAA of wild-type BHK and tsBN2 RCC1 proteins were estimated and compared (Fig. 8). GTCGGGGCCGTGTGCGGTTCCAAGATGCCTTCTGTGGGGCC The seine residue, like glycine, makes a turn in the V protein structure (1), and phenylalanine has a tendency to TATCTCACTTTTGCCATCTCCCGTGAGGGCCATGTATATGGC form a hydrophobic bond with other hydrophobic amino acids (8, 24). In the vicinity of the 256th amino acid, there are three hydrophobic amino acids: phenylalanine, valine, and TTTGGCC*TCGAACTATCACCAGCTTi -3 tyrosine at the 253rd, 261st, and 262nd residues (Fig. 4). In particular, phenylalanine at the 253rd residue will form a 3' -GflCTTGRTRGTGGTCGlH-5' strong hydrophobic bond with a new amino acid at the 256th codon in the tsBN2 RCC1 protein. Hence, the turn of folding at seine, the 256th amino acid in the BHK RCC1 protein, BG A T C CG A T C would be replaced with the Pi sheet in tsBN2 RCC1 protein, and the orientation of folding will be opposite in the C- terminal portion (Fig. 8). - _ OO - *-g DISCUSSION _s4mm _ - Since BHK and tsBN2 cells expressed RCC1 mRNAs and 1_ proteins ofthe same sizes, the RCC1 gene of tsBN2 cells was = expected to have a point mutation, which usually causes a __ temperature-sensitive mutation (2), if the RCC1 gene is indeed a mutant gene in tsBN2 cells. The result obtained is consistent with this notion. This proposal is supported by the FIG. 6. Mutation site in BN2 RCC1 genomic DNA. (A) Nucleo- lines of evidence. tide sequence amplified by the PCR method. Boxed sequences are following the synthetic primers used for amplification. The arrowhead marks First, the tsBN2 cell line was isolated from the BHK-21 the mutation site. (B and C) Genomic DNAs extracted from BHK-21 cell line after mutagenesis with N-methyl-N'-nitro-N-nitro- (B) and tsBN2 (C) cells, amplified in the vicinity of the mutation site soguanidine (26), a potent alkylating agent that induces a by the PCR method. Sequence ladders near the mutation site are cytosine-to-thymine transition (G- C--A T) (6), as is found shown. in the RCC1 gene of tsBN2 cells. 582 UCHIDA ET AL. MOL. CELL. BIOL.

FIG. 8. Alteration of secondary structure by a single point mutation in the BN2 RCC1 gene. The secondary structures of BHK (A) and BN2 (B) RCC1 proteins were drawn, by using the algorithm of Chou and Fasman (5). The region of the mutation site in each clone is enlarged, and the arrowhead marks the site of mutation.

Second, the C-to-T transition at the 256th codon results in consisting of seven repeats of about 60 amino acids (30). an amino acid change, serine to phenylalanine. The phenyl- DNA-binding proteins usually have two domains, a DNA- alanine has a tendency to form a hydrophobic bond with a binding domain and a regulatory domain (13, 21). Our nearby hydrophobic amino acid. The hydrophobic bond is preliminary results suggested that the DNA-binding domain important for folding of the protein structure (1, 17) and is of the RCC1 protein is in the N-terminal site outside of the stabilized by increasing the temperature to up to 58 to 600C repeated region (H. Seino et al., manuscript in preparation). (8, 15, 24). In water, the phenylalanine-phenylalanine bond The amino acid of the mutated site, seine, is located in the is stabilized maximally at 420C (24). Therefore, the estimated fourth repeat of the RCC1 protein and is conserved among structure of the tsBN2 RCC1 protein will be rigidly main- human, hamster, and Xenopus RCC1 proteins (Nishitani et tained at the nonpermissive temperature, and the RCC1 al., in press); hence, this seine probably plays an important protein will become inactive. Since tsBN2 cells grow nor- role in functions of the repeated domain. To investigate the mally at 33.50C, this estimation is consistent with the tem- role of amino acids in the protein, intragenic revertants will perature-sensitive feature of the tsBN2 mutation. be useful. In E. coli, a one-base substitution in the genes for Finally, the lower content of RCC1 protein in tsBN2 cells growth, such as rpoD, sometimes results in temperature- is probably due to the tsBN2 mutation, since the tempera- sensitive cell growth (23). These temperature-sensitive mu- ture-sensitive feature is partially expressed at the permissive tants can be reverted to Ts' by an additional intragenic or temperature. As mentioned above, the structure of the intergenic suppressor mutation. Analyses ofthese revertants RCC1 protein will be changed profoundly by the tsBN2 yielded information on the relationship between protein mutation, and a structural change will either cause RCC1 structure and function. This should also be the case for protein instability or reduce production of this protein even eucaryotic cells. In lower eucaryotes such as yeasts, tem- at the nonpermissive temperature. perature-sensitive mutations were reported to be comple- We had previously isolated two human RCC1 cDNAs with mented either by a wild-type gene corresponding to the different 5' untranslated regions (30). By comparing the base mutant gene or by a suppressor gene (11). Thus, analysis of sequences of these cDNAs with that of the entire human a suppressor gene(s) for the tsBN2 mutation will be impor- RCC1 gene, the human RCC1 gene was found to have two tant for further analysis of the RCC1 protein. The intragenic promoters corresponding to the two isolated cDNAs (Fu- suppressor is expected to provide information on the func- runo et al., in preparation). In human cells, only a single tion of seine at the 256th codon, and the intergenic suppres- broad band was detected by Northern blot analysis, using as sor will aid in elucidating the relationship between RCC1 a probe the RCC1 cDNA (30). The broad band in Fig. 1A is protein and other nuclear components. consistent with these previous results, suggesting that the The RCC1 gene resides on chromosome 1 in the human hamster RCC1 gene also has two promoters. Thus, the cell (30a), and we found that the tsBN2 mutation is not difference in the 5' untranslated regions of BHK and BN2 linked to the hypoxanthine-guanosine phosphoribosyltrans- RCC1 cDNAs may be caused by different promoters. ferase gene (29). These results indicate that the hamster RCC1 protein is a DNA-binding protein (30a) with a region RCC1 gene is located on an autosome. Therefore, we VOL. 10, 1990 RCC1 GENE AND tsBN2 GENE 583 expected two sets ofPCR patterns. However, all of the BHK 202:291-293. RCC1 gene fragments isolated independently from the li- 12. Hope, I. A., S. Mahadevan, and K. Struhl. 1988. Structural and brary of DNA amplified by PCR had the same base se- functional characterization of the short acidic transcriptional quence, as was the case with the tsBN2 RCC1 gene. activation region of yeast GCN4 protein. Science 333:635-640. 13. Johnson, R. T., and P. N. Rao. 1970. Mammalian cell fusion: There are three possibilities to explain why only one set of induction of premature chromosome condensation in interphase RCC1 genomic base sequence was obtained from both BHK nuclei. Nature (London) 226:717-722. and tsBN2 cells. First, the BHK cell line may be functionally 14. Kai, R., M. Ohtsubo, M. Sekiguchi, and T. Nishimoto. 1986. hemizygous for the RCC1 gene; hence, the allelic RCC1 gene Molecular cloning of a human gene that regulates chromosome cannot hybridize with the synthetic primer in the PCR condensation and is essential for cell proliferation. Mol. Cell. analysis. Functional hemizygosity is proposed as one of Biol. 6:2027-2032. reasons why many phenotypically recessive mutations have 15. King, J., C. Haase, and M. H. Yu. 1987. Temperature-sensitive been isolated in cultured animal cells such the CHO line (39), mutations affecting kinetic steps in protein-folding pathways, p. and its presence in cultured cell lines was indicated geneti- 109-121. In D. L. Oxender and C. F. Fox (ed.), Protein engineering. Alan R. Liss, Inc., New York. cally (9). Second, the allelic gene may be deleted in the BHK 16. Laemmli, U. K. 1970. Cleavage of structural proteins during the cell line. In these two cases, the tsBN2 mutation can be assembly of the head of bacteriophage T4. Nature (London) isolated by a single step. Finally, the RCC1 gene is homozy- 227:680-685. gous in both BHK and tsBN2 cells. In this case, two steps, 17. Lin, W. A., and R. T. Sauer. 1989. Alternative packaging mutation and loss of heterozygosity, are required to isolate arrangements in the hydrophobic core of K repressor. Nature tsBN2 cells, since this mutation is phenotypically recessive (London) 339:31-36. (4, 26). In any case, it is curious that a recessive mutation 18. Maniatis, T., E. F. Fritsch, and J. Sambrook. 1982. Molecular such as tsBN2 was isolated from the autosomal genes. The cloning: a laboratory manual. 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