Human Chromatid Cohesin Component hRad21 Is Phosphorylated in M Phase and Associated with Metaphase Centromeres

Sister chromatids duplicated in S phase are connected with each other during G2 and M phase until the onset of anaphase. This chromatid cohesion is essential for correct segregation of genetic material to daughter cells. Recently, understanding of the molecular mechanisms governing chromatid cohesion in yeast has been greatly advanced, whereas these processes in mammalian cells remain unclear. We report here biochemical and cytological analyses of human Rad21, a homologue of the yeast cohesin subunit, Scc1p/Mcd1p. hRad21 is a nuclear phosphorylated protein. Its abundance does not change during the cell cycle, and it becomes hyperyphosphorylated in M phase. Most hRad21 is not associated with chromatin when the nuclear envelope breakdown takes place in prophase. However, a detailed analysis of the spread chromosomes indicated that hRad21 remains associated with prometaphase-like chromosomes along their entire lengths. The mitotic chromatin-bound hRad21 becomes dissociated in a highly regulated manner because hRad21 remains specifically at the centromeres but disappears from the arm regions on metaphase-like chromosomes. Interestingly, hRad21 at the metaphase centromeres appears to be present at the inner pairing domain where the two sister chromatids are supposed to be in intimate contact. These results suggest that hRad21 has a critical role in chromatid cohesion in human mitotic cells. Sister chromatids duplicated in S phase are connected with each other during G2 and M phase until the onset of anaphase. This chromatid cohesion is essential for correct segregation of genetic material to daughter cells. Recently, understanding of the molecular mechanisms governing chromatid cohesion in yeast has been greatly advanced, whereas these processes in mammalian cells remain unclear. We report here biochemical and cytological analyses of human Rad21, a homologue of the yeast cohesin subunit, Scc1p/Mcd1p. hRad21 is a nuclear phosphorylated protein. Its abundance does not change during the cell cycle, and it becomes hyperyphosphorylated in M phase. Most hRad21 is not associated with chromatin when the nuclear envelope breakdown takes place in prophase. However, a detailed analysis of the spread chromosomes indicated that hRad21 remains associated with prometaphase-like chromosomes along their entire lengths. The mitotic chromatin-bound hRad21 becomes dissociated in a highly regulated manner because hRad21 remains specifically at the centromeres but disappears from the arm regions on metaphase-like chromosomes. Interestingly, hRad21 at the metaphase centromeres appears to be present at the inner pairing domain where the two sister chromatids are supposed to be in intimate contact. These results suggest that hRad21 has a critical role in chromatid cohesion in human mitotic cells. immunofluorescence human Rad21 polyacrylamide gel electrophoresis phosphate-buffered saline propidium iodide Accurate chromosome segregation during mitosis into two daughter cells is one of the requirements for stable genome maintenance. Until recently, the molecular mechanisms regulating chromatid cohesion had not been well understood. However, recent studies, mostly conducted in budding yeast, have outlined the process generally (reviewed in Ref.1Nasmyth K. Peters J.-M. Uhlmann F. Science. 2000; 288: 1379-1384Crossref PubMed Scopus (379) Google Scholar). Concurrent with DNA replication, the sister chromatids become connected along their entire length. Several distinct groups of proteins are involved in establishing and maintaining chromatid cohesion. The most intensively investigated protein complex, budding yeast cohesin, consists of four subunit proteins, Scc1p/Mcd1p, Scc3p, Smc1p, and Smc3p (2Michaelis C. Ciosk R. Nasmyth K. Cell. 1997; 91: 35-45Abstract Full Text Full Text PDF PubMed Scopus (1168) Google Scholar, 3Guacci V. Koshland D. Strunnikov A. Cell. 1997; 91: 47-57Abstract Full Text Full Text PDF PubMed Scopus (686) Google Scholar, 4Toth A. Ciosk R. Uhlmann F. Galova M. Schleiffer A. Nasmyth K. Genes Dev. 1999; 13: 320-333Crossref PubMed Scopus (500) Google Scholar), and serves as a physical glue between sister chromatids. Cohesin is phylogenetically conserved. Rad21p in fission yeast and human Rad21 (hRad21) are homologues of Scc1p/Mcd1p (5Birkenbihl R.P. Subramani S. Nucleic Acids Res. 1992; 20: 6605-6611Crossref PubMed Scopus (212) Google Scholar, 6McKay M.J. Troelstra C. van der Spek P. Kanaar R. Smit B. Hagemeijer A. Bootsma D. Hoeijmakers J.H. Genomics. 1996; 36: 305-315Crossref PubMed Scopus (72) Google Scholar). Smc1p and Smc3p are found in budding yeast, Xenopus, and mammals (7Strunnikov A.V. Larionov V.L. Koshland D. J. Cell Biol. 1993; 123: 1635-1648Crossref PubMed Scopus (259) Google Scholar, 8Losada A. Hirano M. Hirano T. Genes Dev. 1998; 12: 1986-1997Crossref PubMed Scopus (519) Google Scholar, 9Schmiesing J.A. Ball J., A.R. Gregson H.C. Alderton J.M. Zhou S. Yokomori K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12906-12911Crossref PubMed Scopus (92) Google Scholar). They are members of the SMC (structuralmaintenance of chromosome) protein family that is characterized by the presence of coiled-coil domains and ATPase domains (reviewed in Ref. 10Hirano T. Genes Dev. 1999; 13: 11-19Crossref PubMed Scopus (198) Google Scholar). Very recently, two Scc3p homologues, SA1 and SA2, have been found in Xenopus and human cells (11Losada A. Yokochi T. Kobayashi R. Hirano T. J. Cell Biol. 2000; 150: 405-416Crossref PubMed Scopus (258) Google Scholar). Interestingly, in Xenopus, two distinct classes of cohesin, termed x-cohesinSA1 and x-cohesinSA2, are present. These two complexes share Xenopus (X)SCC1, XSCC2, and XRAD21 and differ via containing either XSA1 or XSA2 (11Losada A. Yokochi T. Kobayashi R. Hirano T. J. Cell Biol. 2000; 150: 405-416Crossref PubMed Scopus (258) Google Scholar). Immunodepletion of Xenopus cohesin from egg extracts led to a failure of chromatid cohesion (8Losada A. Hirano M. Hirano T. Genes Dev. 1998; 12: 1986-1997Crossref PubMed Scopus (519) Google Scholar). Therefore, the cohesin complex is likely to be conserved in all eukaryotes, including humans. However, the precise roles of cohesin may be different between budding yeast and other eukaryotes. In budding yeast, Scc1p/Mcd1p abruptly dissociates from chromatin at the onset of anaphase (2Michaelis C. Ciosk R. Nasmyth K. Cell. 1997; 91: 35-45Abstract Full Text Full Text PDF PubMed Scopus (1168) Google Scholar, 4Toth A. Ciosk R. Uhlmann F. Galova M. Schleiffer A. Nasmyth K. Genes Dev. 1999; 13: 320-333Crossref PubMed Scopus (500) Google Scholar). In contrast, ∼95% of Xenopus cohesion molecules (XSMC1, XSMC3, and XRAD21) dissociate from chromatin at the entry of mitosis, much earlier than the metaphase-anaphase transition (8Losada A. Hirano M. Hirano T. Genes Dev. 1998; 12: 1986-1997Crossref PubMed Scopus (519) Google Scholar). Similarly, in indirect immunofluorescence (IF)1 experiments, it has been found that human Smc1p and mouse Rad21 (called PW29) are mostly excluded from mitotic chromosomes (9Schmiesing J.A. Ball J., A.R. Gregson H.C. Alderton J.M. Zhou S. Yokomori K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12906-12911Crossref PubMed Scopus (92) Google Scholar, 12Darwiche N. Freeman L.A. Strunnikov A. Gene (Amst.). 1999; 233: 39-47Crossref PubMed Scopus (104) Google Scholar). Therefore, most cohesins are apparently absent on metaphase chromatids in higher eukaryotes. It is not known what molecules or conditions are responsible for the chromatid cohesion immediately before anaphase. Two models have been proposed to explain the apparent inconsistency between the timings of the cohesin-chromatin dissociation and the mitotic chromatid separation (8Losada A. Hirano M. Hirano T. Genes Dev. 1998; 12: 1986-1997Crossref PubMed Scopus (519) Google Scholar). The first model proposes that cohesin molecules are responsible for interphase-specific chromatid cohesion and that some yet unidentified mitosis-specific cohesion machinery is responsible for the chromatid cohesion from prophase until the onset of anaphase. The second model hypothesizes that the same cohesin complex is required for both interphase- and mitosis-specific chromatid cohesions. However, in this model, the complex dissociates from chromatin in two steps, whereby most cohesin is released from chromatin at the entry into mitosis. The remaining cohesin connects chromatids in metaphase and dissociates from chromatin at the onset of anaphase. The recent discovery of Xenopus (X)SA proteins revealed that a small population of XSA1 is associated with the metaphase chromosomes formed in Xenopus cell-free extracts, thus supporting the second model (11Losada A. Yokochi T. Kobayashi R. Hirano T. J. Cell Biol. 2000; 150: 405-416Crossref PubMed Scopus (258) Google Scholar). In this paper, we describe the biochemical and cytological behaviors of hRad21. We aimed to better understand the roles of hRad21 particularly in metaphase. We observed a small but significant population of hRad21 associated with colcemid-induced mitotic chromosomes. These results suggest that the mitotic cohesion is mediated by cohesin, further underscoring the conserved mechanisms regulating chromatid cohesion and separation in eukaryotes. Full-length hRad21 cDNA was isolated from a HeLa cell cDNA library by reference to the published hRad21 cDNA sequence (6McKay M.J. Troelstra C. van der Spek P. Kanaar R. Smit B. Hagemeijer A. Bootsma D. Hoeijmakers J.H. Genomics. 1996; 36: 305-315Crossref PubMed Scopus (72) Google Scholar). The cloned cDNA was completely sequenced. The resulting sequence was identical to the published clone. The full-length hRad21 cDNA was then subcloned into pGEX5X-1 (Amersham Pharmacia Biotech). glutathione S-transferase-fused hRad21 was expressed in Escherichia coli. The recombinant protein contained in the inclusion body was denatured and purified using Prep Cell Model 491 (Bio-Rad). The purified protein was mixed with Freund's complete adjuvant and injected into rabbits to obtain the anti-hRad21 antisera. To generate the anti-C-hRad21 antibodies, a sequence of hRad21 was with and was to the The resulting purified by was from and from was a from was to Ref. D. first for in all and in for with at by for with (Amersham Pharmacia by using (Amersham Pharmacia Biotech). In and using a of DNA was in each proteins by of at Pharmacia to the the was during of the protein. on Two In the first cells with for at In the second cells with at for and then with for cells with containing and and then with for at by and with for at as in containing propidium iodide or and by HeLa cells at S phase by a and as in K. B. B. P. F. C. M. J. 1998; PubMed Scopus Google Scholar). In experiments, cells in a with to a of for to by the Cell with the cell with antibodies, and the complex was by protein (Amersham Pharmacia Biotech). proteins on and to a The was first to the same was for hRad21 by using The by a HeLa cells with and Cell in of and cell in cell of and with a using the with a to that most cells had been and nuclear by at from the at with the and by the in containing either or for on was from by of the was to Ref. S. Cell. Full Text PDF PubMed Scopus Google HeLa cells at S phase a and To obtain metaphase-like cells released from the and for by for in the presence of To obtain prometaphase-like cells released from the and for or by for in the presence of The cells with a for and to a by The cells using for with for by and in and with anti-hRad21 and mouse These by and (Amersham Pharmacia DNA was with by a yeast Scc1p/Mcd1p is a nuclear protein that with chromatin from the metaphase-anaphase transition (2Michaelis C. Ciosk R. Nasmyth K. Cell. 1997; 91: 35-45Abstract Full Text Full Text PDF PubMed Scopus (1168) Google Scholar, 3Guacci V. Koshland D. Strunnikov A. Cell. 1997; 91: 47-57Abstract Full Text Full Text PDF PubMed Scopus (686) Google Scholar). Scc1p/Mcd1p abundance is regulated in a cell The protein is absent in in and and in anaphase (2Michaelis C. Ciosk R. Nasmyth K. Cell. 1997; 91: 35-45Abstract Full Text Full Text PDF PubMed Scopus (1168) Google Scholar, 3Guacci V. Koshland D. Strunnikov A. Cell. 1997; 91: 47-57Abstract Full Text Full Text PDF PubMed Scopus (686) Google Scholar). In is absent in and most in (2Michaelis C. Ciosk R. Nasmyth K. Cell. 1997; 91: 35-45Abstract Full Text Full Text PDF PubMed Scopus (1168) Google Scholar, 3Guacci V. Koshland D. Strunnikov A. Cell. 1997; 91: 47-57Abstract Full Text Full Text PDF PubMed Scopus (686) Google Scholar). hRad21 is most in S G2 (6McKay M.J. Troelstra C. van der Spek P. Kanaar R. Smit B. Hagemeijer A. Bootsma D. Hoeijmakers J.H. Genomics. 1996; 36: 305-315Crossref PubMed Scopus (72) Google Scholar). Therefore, we first investigated hRad21 protein the cell Full-length human Rad21 (hRad21) cDNA was isolated from a HeLa cell cDNA of the cDNA a sequence of complete to the published one (6McKay M.J. Troelstra C. van der Spek P. Kanaar R. Smit B. Hagemeijer A. Bootsma D. Hoeijmakers J.H. Genomics. 1996; 36: 305-315Crossref PubMed Scopus (72) Google Scholar), thus that a protein of with a molecular of Two different one the glutathione S-transferase-fused recombinant full-length hRad21 protein expressed in and the other a These purified by using the hRad21 cDNA both the recombinant hRad21 protein that was expressed in the cells cells with protein in cells that the same in with that of the hRad21 These because the with the to of hRad21 The apparent molecular of the hRad21 protein as using was than that from supposed of budding yeast (2Michaelis C. Ciosk R. Nasmyth K. Cell. 1997; 91: 35-45Abstract Full Text Full Text PDF PubMed Scopus (1168) Google Scholar), fission yeast protein R. Subramani S. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar), and Xenopus Rad21 XRAD21 (8Losada A. Hirano M. Hirano T. Genes Dev. 1998; 12: 1986-1997Crossref PubMed Scopus (519) Google Scholar). To that the hRad21 we and hRad21 cDNA in using a proteins in the presence of either or and then by proteins by whereas proteins to with anti-hRad21 and anti-C-hRad21 in the hRad21 protein in was as a protein this protein was specifically by both anti-hRad21 and anti-C-hRad21 we that it is hRad21 protein. HeLa cells using a and The cells at from the and first by by The These using as the protein and was to the that proteins with the It is known that is during S phase R. D. B. Biol. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar). was in most at and to a at that most of the cells in S phase at In contrast, was most at and and at and These results that the cells in phase at to and from M phase at analysis the of the cell from the proteins In these the abundance of as by anti-C-hRad21 during the cell It is known that Scc1p/Mcd1p is at the onset of anaphase F. F. Nasmyth K. 1999; PubMed Scopus Google Scholar). We observed some proteins in hRad21 this we not these from hRad21 or had However, we not of these small in M that not to the chromatid separation We found that hRad21 was in but not in at all of the cell We found that hRad21 was not present in not hRad21 was not by not These results that hRad21 is not a protein or a protein to hRad21 may be associated with nuclear at all cell including M phase. It has been that fission yeast Rad21p and budding yeast are phosphorylated from S phase to anaphase R. Subramani S. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar). We hRad21 is phosphorylated in a cell this HeLa cells a and The cells at released from the to each the cells for in the presence of was for the cell by hRad21 was using anti-hRad21 from cell extracts containing the same of cells. The in and the proteins a the was to the same was for hRad21 using a analysis with anti-C-hRad21 these experiments, it was found that hRad21 is phosphorylated most at and from the the analysis revealed that these mostly from M phase these results that hRad21 becomes in M phase. To the that hRad21 is in M phase we the in a HeLa cells at a and of the cells released from the and for and then at the metaphase by the cells with for These and along with the with for to each analysis of indicated that the two of cells at and hRad21 was from the and cell using anti-hRad21 containing different protein and where and contained and protein than for each of using and a The was to by with anti-hRad21 the in the analysis the and in their whereas the This apparently observed in the for and that the protein abundance in this of protein In contrast, of the than a to the and to a when we by the by the we cell in hRad21 both the and the In both the cells in the of hRad21 with the cells. Therefore, we that hRad21 is in M phase. In the of we observed and to hRad21 and The apparent molecular of the as from and these not with anti-hRad21 in the we that these are not hRad21. We as phosphorylated proteins and have and Interestingly, the of hRad21 and associated proteins and in the cells higher than found with the cells and phosphorylated proteins, with apparent molecular of and specifically found in the hRad21 from the cells. these proteins as proteins and and we not the of these the results suggest that cohesin may be regulated by during M phase. We the of hRad21 in the HeLa cells by using anti-hRad21 HeLa cells with with or in The with to the DNA and by using anti-hRad21 and hRad21 was in and was not present in the cells with to most of the was to whereas hRad21 These results the biochemical results and that hRad21 is a nuclear protein associated with nuclear hRad21 is involved in sister chromatid as has been one the of hRad21 to change in M phase. However, it has been that Rad21 is dissociated from chromosomes (8Losada A. Hirano M. Hirano T. Genes Dev. 1998; 12: 1986-1997Crossref PubMed Scopus (519) Google Scholar). We in the of hRad21 in metaphase cells. Two one and the other We found these two to the same and the results from cells are in HeLa cells with DNA by using and anti-hRad21 some hRad21 to be associated with chromatin during prophase when the two are not yet a significant was dissociated from chromatin hRad21 was in prophase with some of it apparently to chromatin However, in metaphase and it that most hRad21 had completely dissociated from the chromatin and was associated with the The was particularly in and because of the hRad21 was at during and in two daughter cells. To understand the of the nuclear envelope breakdown and hRad21 dissociation from we cells by using anti-hRad21 and entry to mitosis, a population of hRad21 was excluded from chromatin of cells that had been nuclear envelope breakdown It was not to this of hRad21 had associated with the chromatin and then dissociated from it in prophase or the had associated with the However, some of hRad21 apparently dissociated as the chromatin further the of M a of hRad21 in the when the nuclear envelope had apparently Therefore, it is that hRad21 be to all hRad21 was the of the by the hRad21 in mitotic it was to hRad21 with chromosomes in M phase or We spread prometaphase-like and metaphase-like chromosomes to hRad21 is present on chromosomes. To this HeLa cells with and the spread chromosomes by We found that significant of hRad21 associated along the of the prometaphase-like chromosomes These not observed when the was by the and when chromosomes by that the not The hRad21 along the of chromosomes. To better understand the of hRad21 on the we centromeres using a protein that to We found that hRad21 was at both the centromeres and the arm regions on prometaphase-like chromosomes In contrast, when we further metaphase-like we found that hRad21 at centromeres a revealed that hRad21 mostly from the arm regions and specifically at centromeres of metaphase-like chromosomes Interestingly, when as two hRad21 was not at regions but at inner regions between the two sister a these suggest that hRad21 in M phase is dissociated from chromatin in two The arm hRad21 is dissociated during the prophase to metaphase whereas the hRad21 remains associated with metaphase-like chromosomes. It is that of Scc1p/Mcd1p is associated with chromatin in budding yeast during metaphase F. F. Nasmyth K. 1999; PubMed Scopus Google Scholar), whereas cohesin molecules are mostly excluded from chromatin in prophase (8Losada A. Hirano M. Hirano T. Genes Dev. 1998; 12: 1986-1997Crossref PubMed Scopus (519) Google Scholar, 9Schmiesing J.A. Ball J., A.R. Gregson H.C. Alderton J.M. Zhou S. Yokomori K. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 12906-12911Crossref PubMed Scopus (92) Google Scholar). Therefore, it has not been cohesin a role in the mitotic chromatid cohesion in higher eukaryotes. In this we have found that a small but significant of hRad21 is at centromeres of colcemid-induced metaphase-like chromosomes. hRad21 and associated proteins are in M phase. These results highly suggest that hRad21 has a critical role in the chromatid cohesion and separation in M as has been in budding In human and mouse revealed that sister chromatid arm regions are with each other to whereas the regions are connected until the onset of chromatid segregation in anaphase PubMed Scopus Google Scholar). This that chromatids may be in a process in mammalian cells. In the first the arm regions are during prophase to metaphase. this the remaining cohesion at centromeres serves as a two sister chromatids. In the second that at the metaphase-anaphase the cohesion is and the two chromatids and by by a has been in a of as and human (reviewed in Ref. PubMed Scopus Google Scholar). The of hRad21 that we have found with mitotic chromosomes are well with these cytological We have found that hRad21 is present along the entire of chromosomes during prophase to However, the hRad21 and the hRad21 remains on the colcemid-induced metaphase-like chromosomes. Therefore, the of hRad21 on mitotic chromosomes to the of cohesion between mitotic chromatids. These results suggest that cohesin containing hRad21 is responsible for the chromatid cohesion in M phase. this was for it was that a small population of SA1 protein is associated with metaphase chromosomes both in Xenopus egg cell-free extracts and cells A. Hirano T. Biol. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). recently, mouse protein expressed in HeLa cells was to be specifically associated with regions in metaphase chromosomes S. A. Peters J.-M. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). that the hRad21 protein as the expressed protein and further the of a highly regulated of cohesin complex on mitotic chromosomes as revealed in the Scc1p/Mcd1p has been to with regions along the chromosome at regions N. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, C. V. Koshland D. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, T. K. Nasmyth K. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). yeast cohesion becomes further at regions on metaphase-like chromosomes by a N. Cell. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). Therefore, in both yeast and cohesin is at regions to the onset of anaphase. It is to that these cohesin associated with centromeres are the of the that anaphase. The recent by S. A. Peters J.-M. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google this by the biochemical and cytological behaviors of HeLa cells cohesin and mitotic processes in Xenopus cell-free Interestingly, we found that the hRad21 on the metaphase-like chromosomes to be present between two sister in some of the on human chromosome two different classes of and M. T. PubMed Scopus Google Scholar). the to but does it has been that is with on spread metaphase-like but a that hRad21 in regions that the sister and the inner domain between the two that a of to may be involved in sister chromatid cohesion at Several of the of hRad21 are different from that of yeast Scc1p/Mcd1p. We found that hRad21 not change during the cell This is different from that of budding yeast, Scc1p/Mcd1p have been found to the cell We found that a small of hRad21 associated with prophase The remaining and population of hRad21 not to chromatin in the the nuclear envelope breakdown had We not the of hRad21 we observed had with chromatin and dissociated in prophase or it had been to chromatin the cell we that a of hRad21 to chromatid cohesion in M a different from that of yeast Scc1p/Mcd1p F. F. Nasmyth K. 1999; PubMed Scopus Google Scholar). This that hRad21 may a role to chromatid cohesion. In of this the mitotic hRad21 the of is dissociated from chromatin and was to Therefore, it is that this of hRad21 is associated with other nuclear as nuclear and We that a of hRad21 to with and in anaphase and This is of that of the chromosome proteins J. Cell Sci. PubMed Google Scholar). It has been proposed that a of chromosome segregation processes R. P. 1999; PubMed Scopus Google Scholar). It may be to the roles of hRad21 in of mitosis. It is not known the dissociation of hRad21 from mitotic chromatin is In budding yeast, Scc1p/Mcd1p is in a manner on at the onset of anaphase. Similarly, it has been that a small of mammalian is at the transition S. A. Peters J.-M. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). In this we not a that specifically to anaphase hRad21. However, we have found that hRad21 is phosphorylated in a cell hRad21 and associated proteins, and phosphorylated most in M phase. Recently, it has been that Xenopus homologues and are phosphorylated by in proteins for and thus it has been proposed that this protein proteins in M phase from with chromatin (11Losada A. Yokochi T. Kobayashi R. Hirano T. J. Cell Biol. 2000; 150: 405-416Crossref PubMed Scopus (258) Google Scholar). Interestingly, and with anti-hRad21 in this apparent molecular in to of human proteins (11Losada A. Yokochi T. Kobayashi R. Hirano T. J. Cell Biol. 2000; 150: 405-416Crossref PubMed Scopus (258) Google Scholar), that and may be human Xenopus cohesin a unidentified apparent molecular of in (8Losada A. Hirano M. Hirano T. Genes Dev. 1998; 12: 1986-1997Crossref PubMed Scopus (519) Google Scholar). that was with anti-hRad21 in this may be the human of cohesin of the other phosphorylated proteins and revealed in this are not and are particularly because these phosphorylated proteins not in the hRad21 These proteins may be phosphorylated in mitosis or associated with the cohesin complex specifically in mitosis. these results suggest that of human cohesin are by in M may the of of hRad21 be aimed at understanding and of hRad21 are We are to K. of for critical of and on the and for and The of F. K. and K. are