Cell Cycle Control in the Fission Yeast Schizosaccharomyces pombe

Transcription

1 Journal of Generul Microbiology ( 1989, 131, Printed in Great Britain 2123 Cell Cycle Control in the Fission Yeast Schizosaccharomyces pombe The Tenth Fleming Lecture ByPAUL NURSE Imperial Cancer Research Fund, 44 Lincoln s Inn Fields, London WCZA 3PX, UK (Delivered at the 103rd Ordinary Meeting of the Society for General Microbiology, I April 1985) My interest in the mitotic cell cycle was first stimulated by two major sources. The first of these was Murdoch Mitchison s book published in 1971, The Biology of the Cell Cycle (Mitchison, 1971), which introduced me to the cell cycle and the problem of its control. The second was Lee Hartwell s work, also published in the early ~OS, describing the isolation and characterization of cell division cycle mutants in the budding yeast Saccharomyces cereuisiae (Hartwell, 1974). This work elegantly demonstrated the power of genetic analysis for investigating problems of the cell cycle. I decided to initiate a similar study using the fission yeast Schizosaccharomyces pombe. My reason for choosing this organism was because its cell cycle had been extensively studied in the past and was rather more typically eukaryotic than that of budding yeast (Mitchison, 1970). Saccharomyces cerevisiae divides by budding and has an extended mitotic phase which occupies much of the cycle (Nurse, 1985b). A mitotic spindle forms early, usually in the middle of S-phase, and so there does not appear to be a normal G2 period. In contrast, Schizosaccharomycespombe divides by medial fission and has discrete S and mitotic phases with an intervening G2 period. Its chromosomes also become condensed during mitosis, although there is no breakdown of the nuclear membrane. I joined Murdoch Mitchison s Edinburgh laboratory in 1974 and began isolating cell division cycle mutants in collaboration with Pierre Thuriaux and Kim Nasmyth. About 100 of these cdc mutants were isolated as temperature sensitive lethals unable to complete the cell cycle when incubated at their restrictive temperature (Nurse et al., 1976; Nasmyth & Nurse, 1981). These mutants could be easily identified because the cells failed to divide, but continued to grow and therefore became highly elongated. The mutants defined a total of 27 cdc genes which are necessary for successful completion of the mitotic cell cycle. The genes were shown to be required at the different stages during the cell cycle such as G 1, S-phase, G2 or the mitotic phase. However, it was not clear which of these genes were important for cell cycle control. One important control is the point in the mitotic cycle where cells become committed to that cycle and are then unable to undergo any of the other alternative developmental fates which are open to the cell. These fates include conjugation, sporulation and entry into stationary phase. The functions of two cdc genes, cdcd and cdcl0, were shown to be required before a cell can become committed to the mitotic cycle (Nurse & Bissett, 1981). Mutants in these two genes can undergo conjugation if challenged to do so but mutants in other genes which block later in the cell cycle are committed to that cycle and are unable to conjugate if challenged. Both of these gene functions are required during the G 1 period although cdcd is unique amongst all cdc genes in that it is also required late in G2, just before the mitotic phase. It is at the G1 block point of cdc2 where cells are uncommitted to the mitotic cycle. This point of commitment defined by the cdc2 and cdcl0 genes has been called start after the analogous control identified in the budding yeast by Hartwell, Pringle and their co-workers (Hartwell, 1974). A second control point has also been identified in the G2 period which determines the time of initiation of mitosis. This was revealed by the isolation of wee mutants which undergo mitosis and cell division prematurely at a reduced cell size (Nurse, 1975 ; Thuriaux et al., 1978). Study of temperature sensitive wee mutants showed that they were altered in functions which are rate limiting for the traverse of G2 and thus determine the timing of mitosis. The wee mutants were found to map in two genes, wee1 and cdc SGM

2 2124 P. NURSE (Nurse & Thuriaux, 1980). Genetic analysis indicated that weel coded for a negative element or inhibitor of mitosis. The cdc2 gene appeared to code for an activator or positive acting element in the control. Mutants in cdc2 then can be of two different sorts, the recessive cdc temperature sensitive lethals which are unable to complete start or undergo mitosis, and the dominant wee mutants which initiate mitosis more rapidly than normal. It is also striking that a single gene, cdc2, is involved at the two major control points in the cell cycle, one in G1 at commitment and a second one in G2 determining the timing of mitosis (Nurse, 1981). A series of physiological experiments carried out in collaboration with Peter Fantes revealed more about these two major control points (Fantes & Nurse, 1977, 1978). Passage of start requires growth of the cells to a critical size more or less independent of growth rate (Nurse & Thuriaux, 1977). Initiation of mitosis also requires growth to a critical size rather larger than that necessary for start but this is modulated by the growth rate of the cells. In poor nutritional conditions cells can initiate mitosis at a smaller cell size. As a consequence these smaller cells are unable to pass start in the next cell cycle. This control system leads to an extension of the G1 period in poor growth rate conditions, leading to cells accumulating before start in a state where they are not yet committed to the mitotic cycle. If growth conditions deteriorate further, then these uncommitted cells can leave the cell cycle altogether and undergo conjugation and sporulation or entry into stationary phase. Therefore, these two controls can provide a smooth transition between the proliferative and non-proliferative states. Another gene called ranllpatl also appears to have a role in this transition (Nurse, 1985a; Iino & Yamamoto, 1985). Temperature sensitive mutants in this gene undergo conjugation and sporulation at their restrictive temperature even in good growth rate conditions. These mutants are unable to remain in the uncommitted state of the cell cycle before start and as a consequence are pushed towards conjugation and sporulation. This type of abstract genetic analysis was very good for working out the underlying controls important for regulating the cell cycle and for identifying those gene functions which appear to have a role in those controls. But, for a more complete understanding, it was important to move towards a molecular description of the control mechanisms. The most direct approach towards such an analysis is to isolate physically the genes involved and then use them as probes for investigating the molecular mechanisms of the controls. A DNA transformation system would allow the genes to be isolated by complementation or rescue of mutant function. Such a transformation system was developed in collaboration with David Beach soon after I moved from Edinburgh to the University of Sussex in 1980 (Beach & Nurse, 1981). Vectors were developed which allowed high level transformation of fission yeast and which could be maintained by autonomous replication within the cell. These vectors could then be shuttled out of fission yeast into Escherichia coli for further manipulation of the cloned DNA (Beach et a/., 1982~). Using these procedures various genes such as cdc2, cdcl0 and the mating type locus were cloned and characterized. The cloned DNAs were shown to contain these genes by integrating plasmids containing the genes via homologous recombination into the chromosome and then showing that plasmid-linked markers were now closely linked to the chromosomal sites of the genes. The DNA sequences from the mating type locus were used to demonstrate that mating type interconversion involves gene rearrangements in a manner analogous to that described in budding yeast (Beach et al., 1982b). The characterization of the cdc2 gene at Sussex was carried out in collaboration with Barbara Durkacz. The gene was found to be contained on a 5kb Hind111 fragment. Two other genes contained on the same fragment encode a 5s ribosomal RNA, one of 30 copies which are dispersed throughout the Schizosaccharomyces pombe genome, and a histone-like protein which shares about 60% homology at the amino acid level to histone H2A (John Hindley, personal communication). The cdc2 gene itself was located by Bal31 resection to a 2 kb DNA fragment which encodes a 1.6 kb polyadenylated RNA transcript. Given the central role of the cdc2 gene function in cell cycle control, it was possible that regulation of entry into and through the mitotic cycle was determined by the level of cdc2 transcript. However, this was not the case. The cdc2 transcript level did not change on shift of cells from exponential growth to stationary phase or during a synchronous culture prepared by selecting small cells at the beginning of the cell cycle

3 The Tenth Fleming Lecture 2125 using an elutriator rotor. A similar result was obtained for the cdcl0 gene transcript. This has been isolated and subcloned to a 2.6 kb DNA fragment encoding a 2.7 kb polyadenylated RNA transcript (Aves et al., 1985). The level of this RNA is also unchanged during the shift between exponential growth and stationary phase and during synchronous culture. A number of other cdc genes have also been examined with respect to their transcription during the mitotic cell cycle and so far, none have shown any dramatic changes in level. Only histone transcription and transcription from the histone-like gene next door to cdc2 have shown changes during the cell cycle. These peak during S-phase (Aves et al., 1985). Both the cdc2 and cdcl0 genes have now been sequenced and their predicted open reading frames compared to previously identified proteins using several data bases (Hindley & Phear, 1984; Aves et al., 1985). These comparisons have not identified any other proteins with similarities to the cdcl0 reading frame but there are a number of interesting comparisons with respect to cdc2. About 20% homology at the amino acid level is seen with cyclic AMP dependent protein kinase and several other putative protein kinases in the src family of oncogenes. A higher level of homology, around 60%, was seen with the budding yeast cell division cycle gene cdc28. This was a particularly interesting comparison because the cdc28 gene is also required for start and mitosis in budding yeast. The cdc2 and cdc28 genes are very closely related with respect to their molecular function. A cdc28 gene introduced into a fission yeast cdc2 mutant allows growth of these cells at their restrictive temperature, demonstrating the near functional equivalence of these two gene products (Beach et al., 1982~). The two yeasts are not closely related and indeed comparisons of 5s RNA sequences suggest that they may have diverged between 800 and 1200 million years ago. Therefore the finding that the cdc2 and cdc28 gene functions have been conserved over such long periods of time suggests that these functions may play important roles in all eukaryotic cells. To maximize the match between the cdc2 and cdc28 reading frames it was necessary to propose the presence of four introns in the cdc2 gene whereas the cdc28 gene has none. The presence of these introns was established by S1 mapping. Examination of the sequences around the intron-exon boundaries of cdc2 and other Schizosaccharomyces pombe genes indicates that the Schizosaccharomyces pornbe RN A processing signals are quite different from those of Saccharomyces cereuisiae and are more similar to those of the filamentous fungi and higher eukaryotes. Not all regions of the cdc2 and cdc28 proteins are equally conserved. Two regions which are amongst the most highly conserved parts prove to contain two interesting active sites (Nurse, 1985 b). One of these is an ATP binding site of the sort found in protein kinases and the other is a putative phosphorylation receptor site. The presence of these two sites strongly suggests that the cdc2/cdc28 gene product is a protein kinase which could be regulated by phosphorylation. Antibodies have now been raised against the cdc2 protein in an attempt to prove whether this hypothesis is correct. Synthetic peptides corresponding to regions of the cdc2 protein have been made and used for immunization of mice and rabbits. These antibodies detect a Schizosaccharomycespombe protein of 33 kdal. This has been shown to be the cdc2 gene product as its level is increased when the cdc2 gene is cloned behind a strong promoter. Preliminary evidence suggests that this protein does not change in level in the cell cycle and that it is probably phosphorylated. The antibodies can now be used as a probe to investigate the role of phosphorylation in controlling the function of the cdc2 gene product during the mitotic cycle and to establish whether it has protein kinase activity. Various mutants of the cdc2 gene have now been cloned and are being sequenced in an attempt to establish more precisely the relationships between the structure and function of the cdc2 protein. The good genetics of Schizosaccharomyces pornbe can be exploited in order to identify other gene functions with which the cdc2 gene protein interacts. Extragenic suppressor mutants have been isolated which allow temperature sensitive cdc2 mutants to grow at their restrictive temperature. These suppressor mutants define two genes called sucl and suc2. The sucl suppressor mutant has a direct effect on the mitotic cycle, delaying mitosis and cell division until cells reach an increased size. Mutants in both the sucl and suc2 genes have been found to have an effect on the meiotic cell cycle. They block the second meiotic nuclear division, thus forming a two-spored instead of the regular four-spored ascus. One of these genes, sucl, has been cloned.

4 2126 P. NURSE The wild-type version of this gene rescues certain mutants of cdc2 when present on a high copy number plasmid. Presumably overexpression of the wild-type version of sucl allows certain temperature sensitive mutants of cdc2 to function at their restrictive temperature. These data suggest that the sucl gene product suppresses mutant cdc2 gene function by protein-protein interaction and has roles in its own right in both the meiotic and mitotic cell cycles. Another gene function, cdc25, also appears to interact with cdc2. Mutants in cdc25 are temperature sensitive lethals which are unable to complete mitosis at their restrictive temperature (Thuriaux et al., 1980; Nurse & Thuriaux, 1984). Their mutant phenotype can be suppressed by wee mutants in the cdc2 gene (Fantes, 1979). This suggests that cdc25 gene function is not required for mitosis in wee mutants of cdc2. The cdc25 gene has been cloned by complementation of mutant function, and when overexpressed on a high copy number plasmid it advances cells into mitosis and cell division at a reduced size. This indicates that the cdc2.5 gene function is involved in regulating that of cdc2. It is hoped that this type of approach will identify the genes coding for the substrates and regulators of the cdc2 gehe function. They can then be used to elucidate the molecular function of cdc2 more fully, and thus determine how it brings about the transition of the cell through the commitment control in G 1, and controls the initiation of mitosis in late G2. It is a great pleasure in a lecture of this sort to be able to acknowledge and thank all the various collaborators with whom I have had the privilege to work. Some of these I have mentioned during the lecture as I was describing earlier work carried out in Edinburgh and Sussex. More recently in 1984 I moved to the Imperial Cancer Research Fund in London and the later work I described has been carried out by various people in my group there. The work on cdcl0 has been done by Steve Aves, on the cdc2 gene and its transcripts by Tony Carr, on the cdc2 protein and antibodies by Viesturs Simanis, on the sucl gene by Jackie Hayles and on the cdc25 gene by Paul Russell. REFERENCES AVES, S. J., DURKACZ, B. W., CARR, A. & NURSE, P. HINDLEY, J. & PHEAR, G. (1984). Sequence of the cell (1985). Cloning, sequencing and transcriptional division gene cdc2 from Schizosaccharomyces pombe; control of the Schizosaccharomyces pombe cdclo patterns of splicing and homology to protein kinases. start gene. EMBO Journal 4, Gene 31, BEACH, D. & NURSE, P. (1981). High frequency IINO, Y. & YAMAMOTO, M. (1985). Mutants of transformation of the fission yeast S. pombe. Nature, Schizosaccharomyces pombe which sporulate in the London 290, haploid state. Molecular and General Genetics 198, BEACH, D., PIPER, P. & NURSE, P. (1982~). Construc tion of a Schizosaccharomyces pombe gene bank in a MITCHISON, J. M. (1970). Physiological and cytological yeast bacterial shuttle vector and its use to isolate methods in Schizosaccharomyces pombe. Methods in genes by complementation. Molecular and General Cell Physiology 4, Genetics 187, MITCHISON, J. M. (1971). Biology of the CeN Cycle. BEACH, D., NURSE, P. & EGEL, R. (19823). Molecular Cambridge : Cambridge University Press. rearrangement of mating-type genes in fission yeast. Nature, London 2%, BEACH, D., DURKACZ, B. & NURSE, P. (1982~). Functionally homologous cell cycle control genes in budding and fission yeast. Nature, London 300, FANTES, P. (1979). Epistatic gene interactions in the control of division in fission yeast. Nature, London 279, FANTES, P. & NURSE, P. (1977). Control of cell size at division in fission yeast by growth-modulated size control over nuclear division. Experimental Cell Research 107, FANTES, P. & NURSE, P. (1978). Control of the timing of the cell division in fission yeast. Cell size mutants reveal a second control pathway. Experimental Cell Research 115, HARTWELL, L. 4. (1974). Saccharomyces cerevisiae cell cycle. Bacteriological Reviews 38, NASMYTH, K. & NURSE, P. (1981). Cell division cycle mutants altered in DNA replication and mitosis in the fission yeast Schizosaccharomyces pombe. Molecular and General Genetics 182, NURSE, P. (1975). Genetic control of cell size at cell division in yeast. Nature, London 256, NURSE, P. (1981). Genetic analysis of the cell cycle in micro-organisms. Symposia of the Society for General Microbiology 31, NURSE, P. (1985~). Mutants of the fission yeast Schizosaccharomyces pombe which alter the shift between cell proliferation and sporulation. Molecular and General Genetics 198, NURSE, P. (1985b). Cell cycle control genes in yeast. Trends in Genetics 1, NURSE, P. & BISSETT, Y. (1981). Gene required in G1 for commitment to cell cycle and in G2 for control of mitosis in fission yeast. Nature, London 292,

5 The Tenth Fleming Lecture 2127 NURSE, P. & THURIAUX, P. (1977). Controls over the timing of DNA replication during the cell cycle of fission yeast. E.xperimenta1 Cell Research 107, NURSE, P. & THURIAUX, P. (1980). Regulatory genes controlling mitosis in the fission yeast Schizosaccharomyces pombe. Genetics 96, NURSE, P. & THURIAUX, P. (1984). Temperature sensitive allosuppressor mutants of the fission yeast S. pombe influence cell cycle control over mitosis. Molecular and General Genetics 196, NURSE, P., THURIAUX, P. & NASMYTH, K. (1976). Genetic control of the cell division cycle in the fission yeast Schizosaccharomyces pombe. Molecular and General Genetics 146, THURIAUX, P., NURSE, P. & CARTER, B. (1978). Mutants altered in the control co-ordinating cell division with cell growth in the fission yeast Schizosaccharomyces pombe. Molecular and General Genetics 161, THURIAUX, P., SIPIZKI, M. & FANTES, P. (1980). Genetical analysis of a sterile mutant by protoplast fusion in the fission yeast Schizosaccharomyces pombe. Journal ojgeneral Microbiology 116,