Mammalian Cells Have Two Functional RCC1 Proteins Produced by Alternative Splicing

Journal of Cell Science 107, 2203-2208 (1994) 2203 Printed in Great Britain © The Company of Biologists Limited 1994

Mammalian cells have two functional RCC1 proteins produced by alternative splicing

Junko Miyabashira, Takeshi Sekiguchi and Takeharu Nishimoto* Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Maidashi, Higashi-ku, Fukuoka 812, Japan *Author for correspondence

SUMMARY

Previously we cloned two human RCC1 cDNAs that that had already been determined, and was found to be differed in their noncoding region. In this study, we have located between the 6th and 7th exons, designated as the 6′ found new human and hamster RCC1 cDNAs, which have exon. Both the 5′ and 3′ ends of the 6′ exon correspond to an even more different coding region from that of the pre- the GT-AG rules for splicing, indicating that human RCC1- viously cloned RCC1 cDNAs yet can complement the RCC1 I mRNAs are produced by alternative splicing. The ﬁnding mutation in the tsBN2 cell line. The newly found RCC1 that both humans and hamsters have the insertion at the cDNAs encode a protein (designated as RCC1-I) that has same RCC1 site suggests that the pattern of alternative an insertion of 31 (human) and 13 (hamster) amino acids splicing in the RCC1 gene has been conserved through at valine25 in the N-terminal region outside the RCC1- evolution. seven repeat. The inserted nucleotide sequence was searched for, within the human RCC1 genomic sequence Key words: RCC1, alternative splicing, tsBN2

INTRODUCTION required for coupling the completion of DNA replication with the initiation of mitosis. However, it remains unclear how the The RCC1 gene has been found to be mutated in the tsBN2 arrest in the G1 phase occurs. Interestingly, when tsBN2 cells cell line, which is a temperature-sensitive (ts) mutant of the synchronized by serum deprivation were allowed to grow by BHK21 cell line derived from golden hamsters (Uchida et al., the addition of serum at 39.5¡C, the nonpermissive tempera- 1990), and to encode a very abundant chromosomal protein ture, both RNA synthesis and protein synthesis were greatly (Ohtsubo et al., 1989). RCC1 functions as a guanine inhibited (Nishimoto et al., 1981). This previous ﬁnding nucleotide-releasing protein on the Ran, which is a nuclear suggested to us that in tsBN2 cells, chromatin was condensed small Ras-like G protein (Bischoff and Ponstingl, 1991). even in the G1 phase at the nonpermissive temperature, Homologues of RCC1 have been cloned from hamster, resulting in an inhibition of total RNA synthesis and protein Xenopus (Nishitani et al., 1990), Drosophila (BJ1) (Frasch, synthesis. However, it is also possible that decondensation of 1991), S. cerevisiae (SRM1/PRP20/MTR1) (Clark et al., 1989; chromatin, which is carried out in the G1 phase in order to start Aebi et al., 1990; Kadowaki et al., 1993) and S. pombe (pim1- the cell cycle, was prevented at the nonpermissive temperature. d1+) (Matsumoto and Beach, 1991; Sazer and Nurse, 1994). Consistent with this notion, Sazer and Nurse (1994) suggested Except for pim1-d1+, these homologues have been reported to that a ts mutant of S. pombe, pim1-d1ts, which ceases to grow be functionally exchangeable (Ohtsubo et al., 1991; Fleis- at the end of mitosis, like tsBN2 cells, has a defect in chromatin chmann et al., 1991; Clark et al., 1991), indicating that RCC1 decondensation. pim1-d1ts, however, does not prematurely has been conserved structurally and functionally through enter mitosis. On the contrary, the RCC1-homologous gene of evolution (reviewed by Dasso, 1993). S. cerevisiae has been identiﬁed as a mutant that is defective A defect in the RCC1 gene leads to various phenotypes. In in either mRNA metabolism (prp20) (Aebi et al., 1990), or in tsBN2 cells, RCC1 disappears at the nonpermissive tempera- mRNA export from the nuclei (mtr1) (Kadowaki et al., 1993), ture. Upon loss of RCC1 function, tsBN2 cells undergoing and as a mutant that restores the mating capacity to receptor- DNA replication prematurely enter mitosis and are arrested at less mutants (srm1) (Clark and Sprague, 1989). Except for the end of mitosis, showing re-formed micronuclei that contain srm1, these mutants have no relation to the cell cycle. condensed chromatin (Nishitani et al., 1991). On the other However, prp20 and pim1-d1ts, in addition to tsBN2 cells, hand, exponentially growing tsBN2 cells cease to grow in G1 accumulate mRNA in their nuclei at the nonpermissive tem- phase at the nonpermissive temperature (Nishimoto et al., perature, indicating that these mutants have a defect in mRNA 1978). Premature initiation of mitosis suggests that RCC1 is export as does mtr1 (Kadowaki et al., 1993; Amberg et al., 2204 J. Miyabashira and others

1993). It is therefore logical that phenomena caused by a defect methods (Maniatis et al., 1989), and was then amplified using the in the RCC1 gene should reflect some aspects of RCC1 following synthetic nucleotides as primers: 5′ primer, GCCGACGT- function. GCACCAAGGACAGGAAG, and 3′ primer, AGCCAGGCCCTCA- pim1-d1ts can be suppressed by overexpression of the G GAGGCTTCATCA for KB cells; and 5′ primer, TCCCCGCAACG ′ protein spi1/fyt1, the S. pombe gene homologous to Ran AGGACAGGAAGATG, and 3 primer, ACTACTTCAGAAACAC- (Matsumoto and Beach, 1991; Sazer and Nurse, 1994). In CCGGACCGA for tsBN2 cells. The PCR products were subcloned into the pCRII vector (Invitrogen). addition, both prp20 and mtr1 can be suppressed by overexpression of the G protein GSP/CNR, the S. cerevisiae gene Transformation of tsBN2 cells homologous to Ran (Belhumeur et al., 1993; Kadowaki et al., Amplified RCC1 cDNA was integrated into a mammalian expression − 1993). Suppression of these rcc1 mutations by the overex- vector, pcDEB∆ containing the hygromycin-resistant gene hygr pression of Ran-homologues indicates that Ran functions (kindly provided by Y. Nakabeppu, Kyushu University), and co-trans- downstream of RCC1 (reviewed by Dasso, 1993). The other fected into tsBN2 cells (2×105 cells/100 mm dish) with pSV2 neo mutant, srm1, however, cannot be suppressed by the overex- (200 ng/dish) by the calcium phosphate precipitation method pression of Ran homologues (Kadowaki et al., 1993). We described by Sekiguchi et al. (1988). After incubation at 33.5¡C for two days, transfected cells were cultured either in the presence of reason that srm1 may have a defect in accepting a signal from r a position upstream of RCC1. Geneticin (Sigma) (0.8 mg/ml) at 33.5¡C for selection of neo colonies, or in a normal medium at 39.5¡C for selection of ts+ trans- To investigate the upstream and downstream pathways of formants. About two weeks later, surviving cells were fixed with the RCC1-Ran system, we began by isolating suppressors of formaldehyde and were stained with crystal violet. tsBN2 cells that overcome their temperature sensitivity by a In the case of double transfection with RCC1 and RCC1-I, the mutation outside the RCC1 gene. In this way, we found that RCC1 cDNA was first integrated into another mammalian expression animal cells have two functional RCC1 proteins produced by vector, pcDL-SRα296 (Takebe et al., 1988) and then transfected into alternative splicing. Previously, the human RCC1 gene was tsBN2 cells to isolate primary ts+ transformants. The RCC1-I cDNA found to have two promoters, resulting in two RCC1 mRNAs carried on the pcDEBD vector was then transfected into primary ts+ that have the same coding region (Ohtsubo et al., 1987; Furuno transformants, with the hygr colonies being isolated at 39.5¡C. et al., 1991). Furthermore, our present results indicate that the expression of the RCC1 gene is subject to alternative splicing in the coding region. The pattern of alternative splicing of the RCC1 gene is the same for both human and hamster, suggest- RESULTS ing that the alternative splicing pattern of RCC1 genes is conserved through evolution. Identification of two RCC1 mRNAs differing in their coding regions Isolation of ts+ revertants from cultures of tsBN2 cells that can grow at the nonpermissive temperature was simply the starting MATERIALS AND METHODS point of this study. Our original purpose was to investigate the RCC1-Ran system by isolating ts+ revertants of tsBN2 cells Cell lines and culture conditions that have a mutation outside the RCC1 gene. Thus, in order to The tsBN2 cell line is a ts mutant of the BHK21 cell line derived from determine whether the original mutation of the RCC1 gene golden hamsters. The ts+ transformants of tsBN2 cells were con- exists in these ts+ revertants, we amplified the coding region of structed by transfecting the RCC1 and RCC1-I cDNAs as described + (Sekiguchi et al., 1988). All cell lines were cultured in Dulbecco’s the RCC1 mRNA expressed in ts revertants of tsBN2 cells. modified Eagle’s medium (DMEM) containing 10% calf serum and Amplified RCC1 cDNAs were subcloned into the pcRII vector maintained at 33.5¡C (tsBN2), at 37.5¡C (BHK21, KB), and at 39.5¡C and then digested with EcoRI. Since the RCC1 cDNA has only (ts+ transformants of tsBN2 cells) in a humidified atmosphere con- one EcoRI site in the coding region, two EcoRI fragments, 0.4 taining 10% CO2. kb and 0.9 kb were expected to be produced by digestion with EcoRI. Immunoblotting analysis After complete digestion, about one fifth of the cDNA Cellular proteins electrophoresed on a 12.5% SDS-polyacrylamide gel clones was found to have EcoRI fragments longer than 0.9 kb, were transferred to a PVDF membrane (Immobilon-P, Millipore) in along with the 0.4 kb fragment. A representative result is transfer buffer (50 mM Tris, 40 mM glycine, 15% methanol and 0.02% SDS). Subsequently, the filter was blocked in blocking buffer shown in Fig. 1B, lanes 3 and 4 (hamster). Newly found (20 mM Tris, pH 7.5, 150 mM NaCl and 4% skim milk), and was hamster RCC1 cDNAs with an EcoRI fragment longer than 0.9 then incubated in the same buffer containing the immune serum. The kb were sequenced and found to have an additional nucleotide membrane was incubated in reaction buffer (20 mM Tris, pH 7.5, 150 of thirty-nine base pairs between the guanine and the thymidine mM NaCl and 0.25% Tween-20) containing horseradish peroxidase- of the valine25 codon in the N-terminal region outside the linked anti-rabbit IgG. The protein that reacted with the antibody RCC1-seven repeat, resulting in the replacement of valine25 was detected with the enhanced chemiluminescence (ECL) kit with a peptide of 14 amino acids (Fig. 2, hamster). Based on (Amersham). this result, the protein encoded by the newly found RCC1 The anti-Xenopus RCC1 antibody was prepared against Xenopus cDNA was calculated to have a molecular mass of 46.5 kDa. RCC1 (Nishitani et al., 1990) and the Nishi-4 antibody was prepared Previously, we found that some of the Xenopus RCC1 has an using the synthetic human RCC1 N-terminal peptide 24 (KSKKVKVSHRSHSTE) as the antigen (Ohtsubo et al., 1989). additional amino acid at the same site, that is, between lysine and valine25 (Nishitani et al., 1990). Taking into account all RT-PCR these findings, we made the assumption that an insertion at Total cellular RNA of KB and tsBN2 cells was prepared by guanidine valine25 probably holds some important biological meaning. In Alternative splicing of the RCC1 gene 2205

Fig. 1. Amplification of human and hamster RCC1 cDNAs. (A) Amplified cDNA subcloned into the vector. RCC1 cDNA was amplified from total RNA of KB and ts+ revertants of tsBN2 cells as described in Materials and Methods. PCR products were subcloned into the pCRII vector (Invitrogen). The rectangle of cDNA indicates the protein-coding region. Dotted lines indicate the region of the pCRII vector. Dotted arrows indicate the positions of the primer sequences used for RT-PCR. E indicates the EcoRI site. (B) Gel-electrophoresis of amplified RCC1 cDNAs digested with EcoRI. Human and hamster RCC1 cDNAs subcloned into the pCRII vector were digested with EcoRI and then electrophoresed on a 1.5% agarose gel. M, 100 bp ladder (Pharmacia). a and b indicate the positions of the EcoRI fragments, 0.9 kb and 0.4 kb, respectively. order to confirm this notion, we amplified human RCC1 inserted into the newly found human RCC1 cDNA. It turned mRNA as described in Materials and Methods. out to be the sequence located between the 6th and 7th exons The human RCC1 cDNA has a single EcoRI site at the same (designated as the 6′ exon) (Fig. 3). The nucleotide sequence position as the hamster RCC1 cDNA, so that two fragments of of the 6′ exon precisely fitted the coding frame of RCC1 (Fig. 0.4 kb and 0.9 kb should be produced by digestion with EcoRI 2). Furthermore, the base-sequences outside the 6′ exon were as is the case with hamster RCC1 cDNA (Fig. 1). Upon found to be AG (5′ end) and GT (3′ end) (Furuno et al., 1991), digestion, a similar result was also obtained in the case of which correspond to the GT-AG rules for splicing (Mount, humans, that is, one tenth of the amplified human RCC1 cDNA 1982). The newly found human RCC1 cDNA, therefore, was clones was found to have an EcoRI fragment longer than 0.9 verified to have been produced from the original RCC1 mRNA kb (Fig. 1B, lanes 1 and 2). Sequence analysis indicated that by alternative splicing. newly found human RCC1 cDNAs have an additional We designated the newly found RCC1 cDNA as RCC1-I nucleotide sequence of 93 base pairs, which is within the frame (insertion). of human RCC1 protein and which is located at the same site as in the hamster protein, replacing valine25 with a peptide of Complementation of tsBN2-mutation with RCC1-I 32 amino acid residues (Fig. 2A, human). This means that it cDNA encodes a protein of molecular mass, 48 kDa. To examine the biological meaning of alternative splicing in The inserted nucleotides and amino acids seem to be well the RCC1 gene, the RCC1-I cDNA was integrated into a conserved between human and hamster (Fig. 2C), suggesting mammalian expression vector, pcDEB∆ (Nakabeppu et al., that this may bear some significance, either functionally or 1993) and was then transfected into tsBN2 cells. As a control, structurally, for the RCC1 protein. RCC1 cDNA carried on the same vector was also transfected into tsBN2 cells. In order to estimate the efficiency of the trans- Insertion produced by alternative splicing of RCC1 fection, the pSV2 neo cDNA was co-transfected with RCC1 mRNA and RCC1-I cDNAs, and the number of neor colonies was Previously, we determined the whole nucleotide sequence of counted. the human RCC1 gene (Furuno et al., 1991). In this genomic RCC1-I cDNA complements tsBN2 cells efficiently (Fig. 4, nucleotide sequence, we searched for the nucleotide sequence closed circle). At low doses of transfected DNA, RCC1-I 2206 J. Miyabashira and others

Fig. 2. Analysis of the nucleotides inserted into human and hamster RCC1 cDNAs. (A) Sites of the insertion in human and hamster RCC1 cDNAs. The numbers indicate the positions of amino acids belonging to the RCC1 protein. Amino acids are shown by a single capital letter while nucleotides are shown by a small letter. The insertion occurred between guanine (g) and thymidine (t) in the valine25 codon of the human and hamster RCC1 proteins. (B) Nucleotide sequence of insertions found in human and hamster RCC1 cDNAs. Underlined nucleotides indicate sequences flanking the insertion. Asterisks (*) indicate nucleotides that are identical between the human and hamster sequences. (C) An amino acid sequence deduced from the inserted nucleotide sequence. Amino acids that are identical between human and hamster RCC1-I are shown by asterisks (*). cDNA conferred the ts+ phenotype on tsBN2 cells with a Total cellular proteins extracted from KB cells were elec- frequency relatively higher than the original RCC1 cDNA. At trophoresed and subjected to immunoblotting analysis. As a all doses of transfected RCC1 and RCC1-I cDNAs, the number control, total cellular proteins extracted from ts+ transformants of neor colonies was similar (about 70 neor colonies/200 ng of of tsBN2 cells that were transfected with human RCC1-I or pSV2 neo per 2×105 cells) indicating that the efficiency of RCC1 cDNAs were also assayed for the presence of RCC1 and transfection was constant. This result shows that not only RCC1-I protein by immunoblotting. Since ts+ transformants of RCC1 cDNA but also RCC1-I cDNA has the ability to com- tsBN2 cells were cultured at 39.5¡C, the endogenous hamster plement the tsBN2-mutation. RCC1 protein became lost in these transformants due to the tsBN2-mutation (Nishitani et al., 1991). Thus, only the Identification of the RCC1-I protein in human cell lines intron To confirm the existence of human RCC1-1 protein in the cells, 1.8kb total cellular proteins extracted from human KB cells were assayed by immunoblotting. As antibodies, we used the anti-

Xenopus RCC1 antibody (Nishitani et al., 1990) and the anti- EXON6 GTAA GCAG EXON6' GTGG TCAG EXON7 human RCC1 peptide antibody, Nishi-4, which was prepared 93bp against peptide 1 from lysine19 to glutamic acid33 of RCC1, as 0.6kb 1.1kb described in Materials and Methods (Ohtsubo et al., 1989). 25 Fig. 3. Position of the newly found exon in the human RCC1 gene. Since RCC1-I was estimated to have an insertion at valine , The nucleotide sequence inserted into the human RCC1 cDNA was it could not be recognized by the antibody Nishi-4. On the searched for within the human RCC1 genomic sequence (Furuno et other hand, the anti-Xenopus RCC1 antibody prepared for the al., 1991). The length of the introns is shown for each case of whole Xenopus RCC1 protein should be able to recognize both splicing and the nucleotide sequence of introns proximal to the exon RCC1 and RCC1-I proteins. is also shown. Alternative splicing of the RCC1 gene 2207

200

s 100

Fig. 5. Immunoblotting analysis of cellular protein extracted from 0 KB cells and ts+ transformants. (A) Total cellular proteins were 1 10 100 1000 10000 electrophoresed on a 12.5% SDS-polyacrylamide gel and ng DNA/dish immunoblotted with the anti-Xenopus RCC1 antibody (lanes 1-4), or with the antibody Nishi-4 (lanes 5-8). Lanes 1 and 5, KB cells. Lanes Fig. 4. Complementation of tsBN2 cells with the RCC1-I and RCC1 2 and 6, ts+ transformants of tsBN2 cells transfected with RCC1-I cDNAs. Various doses of RCC1 and RCC1-I cDNAs that were cDNA. Lanes 3 and 7, ts+ transformants of tsBN2 cells transfected carried on the expression vector pcDEB∆, and 200 ng of pSV2 neo with RCC1 cDNA. Lanes 4 and 8, ts+ transformants of tsBN2 cells DNA were co-transfected into tsBN2 cells as described in Materials transfected with both RCC1-I and RCC1 cDNAs. (B) Presence of and Methods. Colonies were fixed and stained with crystal violet to RCC1-I protein in KB cells. Total cellular proteins of KB cells and enable them to be counted. The vertical axis indicates the number of ts+-transformants of tsBN2 cells transfected with RCC1-I cDNA ts+ transformants per dish: mean value of two dishes. Open circles, alone or with both RCC1 and RCC1-I cDNAs were electrophoresed RCC1. Solid circles, RCC1-I. on a 12.5% SDS-polyacrylamide gel using the apparatus Model AE- 6450 (ATTO INC) for 3 hours in order to obtain a good separation of RCC1 and RCC1-I proteins, and were then assayed by immunoblotting using the antibody to Xenopus RCC1. The blotting proteins encoded by the transfected human RCC1 or RCC1-I filter was overexposed in order to detect any small amount of RCC1- cDNA could be recognized by the antibody. I in the KB cells. Lane 1, KB cells. Lanes 2 and 3, ts+ transformants As expected, the antibody to the Xenopus RCC1 protein rec- transfected with RCC1 alone, and with both RCC1 and RCC1-I ognized both human RCC1 and RCC1-I proteins (Fig. 5A). On cDNAs, respectively. a indicates the position of the RCC1-I protein the contrary, the antibody, Nishi-4, recognized only RCC1 while b indicates the position of the RCC1 protein. (lower bands in Fig. 5A). The difference in molecular mass (3 kDa) between the human RCC1 and RCC1-I proteins, which was estimated based on the putative amino acid sequence, was clearly observed as a delay of band-migration between two human cells can be calculated as having four kinds of mRNAs bands in the cell lysate of ts+ transformants expressing both transcribed from the RCC1 gene. It is an interesting problem RCC1 and RCC1-I proteins (Fig. 5A, lane 4). for future study to ascertain which mRNA has a tendency to In human KB cells, the band corresponding to the RCC1-I be subject to alternative splicing: that which is transcribed from protein was observed after a longer exposure (Fig. 5B, lane 1, the distal promoter; or that which is transcribed from the shown by arrow a). Since KB cells contained a much smaller internal promoter. amount of RCC1-I compared with RCC1, we used a thin, long Since both the 5′ and 3′ ends of the 6′ exon correspond to gel to distinguish RCC1-I from RCC1. Thus, it became clear the GT-AG rules for splicing and the nucleotide sequence of that the RCC1-I protein actually exists in KB cells, although the 6′ exon precisely fits the RCC1 frame encoded by both the the amount is very low. 6th and 7th exons, the previously found RCC1 protein is appar- ently produced by skipping the 6′ exon. In this context, the RCC1-I protein should be the original form of RCC1. In both DISCUSSION humans and hamsters, the extra amino acids were found to be inserted into the same site (valine25). Furthermore, it has been The human RCC1 gene has two promoters, at the distal 5′ end noted previously that Xenopus RCC1 protein has an amino acid and in the internal region of the gene (Ohtsubo et al., 1987; insertion at the same site as in the human and hamster RCC1 Furuno et al., 1991). Only RCC1 mRNA transcribed from the proteins (Nishitani et al., 1990). It is therefore apparent that the internal promoter has been detected in HeLa cells (Furuno et alternative splicing pattern of the RCC1 gene has been well al., 1991). The recent finding of Kiss and Filipowicz (1993), conserved through evolution, although the length of the however, indicates that transcription from the distal promoter insertion is species-dependent. of the RCC1 gene occurs in HeLa cells. Thus, two RCC1 We believe that the presently found alternative splicing mRNAs are transcribed from two promoters in HeLa cells. product of the RCC1 gene clearly holds some significance with Additionally, our results indicate that alternative splicing regard to the function of the RCC1-Ran system. If proteins happens in the coding region of the RCC1 gene. Therefore, encoded by alternatively spliced mRNAs have the opposite 2208 J. Miyabashira and others function to each other, as is the case with some transcription complementation by the human homologue RCC1, a protein involved in the factors (Foulkes and Sasson-Corsi, 1992), it would be easy to control of chromosome condensation. Mol. Gen. Genet. 227, 417-423. evaluate the meaning of alternative splicing. However, in the Foulkes, N. S. and Sassone-Corsi, P. (1992). More is better: activators and repressors from the same gene. Cell 68, 411-414. case of the RCC1 gene, both cDNAs complement tsBN2 cells Frasch, M. (1991). The maternally expressed Drosophila gene encoding the efficiently. We noticed that the amount of RCC1-I protein was chromatin-binding protein BJ1 is a homolog of the vertebrate gene regulator extremely low compared to that of RCC1 protein in KB cells of chromatin condensation, RCC1. EMBO J. 10, 1225-1236. (Fig. 5B). To investigate whether overproduction of the RCC1- Furuno, N., Nakagawa, K., Eguchi, U., Ohtsubo, M., Nishimoto, T. and + Soeda, E. (1991). Complete nucleotide sequence of the human RCC1 gene I protein has any effect on the cells, we constructed ts trans- involved in coupling between DNA replication and mitosis. Genomics 11, formants expressing either RCC1-I alone or both RCC1 and 459-461. RCC1-I (Fig. 5). Such transformants grew normally at 39.5¡C, Kiss, T. and Filipowicz, W. (1993). Small nucleolar RNAs encoded by introns suggesting that an abundance of RCC1-I has no harmful effect of the human cell cycle regulatory gene RCC1. EMBO J. 12, 2913-2920. upon tsBN2 cells. Kadowaki, T., Goldfarb, D., Spitz, L. M., Tartakoff, A. M. and Ohno, M. (1993). Regulation of RNA processing and transport by a nuclear guanine In the case of Drosophila, the RCC1 gene is expressed only nucleotide release protein and members of the Ras superfamily. EMBO J. 12, in the early phase of development (Frasch, 1991), although the 2929-2937. RCC1 protein is present in the adult fly. Moreover, in Xenopus, Matsumoto, T. and Beach, D. (1991). Premature initiation of mitosis in yeast the RCC1 mRNA is highly expressed during oogenesis (H. lacking RCC1 or an interacting GTPase. Cell 66, 347-360. Maniatis, T., Fritsch, E. F. and Sambrook, J. (1989). Molecular Cloning: A Kobayashi, personal communication), indicating that the Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring expression pattern of the RCC1 gene seems to alter during its Harbor, New York. development. Therefore, it seems possible that the alternative Mount, S. M. (1982). A catalogue of splice junction sequence. Nucl. Acids Res. splicing pattern of the RCC1 gene changes during develop- 10, 459-472. ment. Since the 6′ exon seems to be nonessential for the cells, Nakabeppu, Y., Oda, S. and Sekiguchi, M. (1993). Proliferative activation of ′ quiescent Rat-1A cells by ∆ Fos B. Mol. Cell. Biol. 13, 4157-4166. we could create experimental animals that do not have the 6 Nishimoto, T., Eilen, E. and Basilico, C. (1978). Premature chromosome exon in the RCC1 gene. By using such animals, the meaning condensation in a ts DNA− mutant of BHK cells. Cell 15, 475-483. of alternative splicing of the RCC1 gene could be investigated Nishimoto, T., Ishida, R., Ajiro, K., Yamamoto, S. and Takahashi, T. in vivo through their development. (1981). The synthesis of protein(s) for chromosome condensation may be regulated by a post-transcriptional mechanism. J. Cell. Physiol. 109, 299- 308. We thank Dr H. Seino for his kind gift of the RCC1 cDNA inte- α Nishitani, H., Kobayashi, H., Ohtsubo, M. and Nishimoto, T. (1990). grated into pcDL-SR vector, and Dr Y. Nakabeppu for his kind gift Cloning of Xenopus RCC1 cDNA, a homolog of the human RCC1 gene: of the pcDEB∆ vector. We thank T. Ohba and Dr T. Seki for preparing complementation of tsBN2 mutation and identification of the product. J. the figures. This work was supported by Grants-in-Aid for Scientific Biochem. 107, 228-235. Research and for Cancer Research from the Ministry of Education, Nishitani, H., M. Ohtsubo, K. Yamashita, H. Iida, J. Pines, H. Yasuda, Y. Science and Culture of Japan, and from the HFSP. The English used Shibata, T. Hunter and T. Nishimoto. (1991). Loss of RCC1, a nuclear in this manuscript was corrected by Miss K. Miller (Royal English DNA-binding protein, uncouples the completion of DNA replication from Language Centre, Fukuoka, Japan). the activation of cdc2 protein kinase and mitosis. EMBO J. 10, 1555-1564. 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