Paul Nurse

Molecular genetic analysis of fission yeast Schizosaccharomyces pombe.

Publisher Summary This chapter describes techniques concerned with classical and molecular genetics, cell biology, and biochemistry that can be used with Schizosaccharomyces pombe . Conjugation and sporulation cannot take place in S. pombe except under conditions of nutrient starvation. ME medium is generally used for genetic crosses. To cross two strains, a loopful of h – and a loopful of h + are mixed together on a ME plate. The cross is left to dry and is then incubated below 30°, as conjugation is severely reduced above this temperature. Fully formed four-spore asci can be seen after 2–3 days of incubation. A 2 day-old cross is usually used for tetrad analysis. Using a 3-day-old cross, one can check for the presence of asci under the light microscope. Random spore analysis allows many more spores to be examined than in tetrad analysis, and in this way recombination mapping and strain construction can be carried out. Diploid cells arise spontaneously in most S. pombe strains, this characteristic can be used to isolate homozygous diploids of any strain. For mutagenesis of yeast strains, ethylmethane sulfonate (EMS) and nitrosoguanidine is used.

Negative regulation of mitosis by wee1+, a gene encoding a protein kinase homolog

Fission yeast wee1- mutants initiate mitosis at half the cell size of wild type. The wee1+ activity is required to prevent lethal premature mitosis in cells that overproduce the mitotic inducer cdc25+. This lethal phenotype was used to clone wee1+ by complementation. When wee1+ expression is increased, mitosis is delayed until cells grow to a larger size. Thus wee1+ functions as a dose-dependent inhibitor of mitosis, the first such element to be specifically identified and cloned. The carboxy-terminal region of the predicted 112 kd wee1+ protein contains protein kinase consensus sequences, suggesting that negative regulation of mitosis involves protein phosphorylation. Genetic evidence indicates that wee1+ and cdc25+ compete in a control system regulating the cdc2+ protein kinase, which is required for mitotic initiation.

cdc25+ functions as an inducer in the mitotic control of fission yeast

In the fission yeast S. pombe the cdc25+ gene function is required to initiate mitosis. We have cloned the cdc25+ gene and have found that increased cdc25+ expression causes mitosis to initiate at a reduced cell size. This shows that cdc25+ functions as a dosage-dependent inducer in mitotic control, the first such mitotic control element to be specifically identified. DNA sequencing of the cdc25+ gene has shown that it can encode a protein of MW 67,000. Evidence is described showing that cdc25+ functions to counteract the activity of the mitotic inhibitor wee1+, and indicating that both mitotic control elements act independently to regulate the initiation of mitosis.

Purified maturation-promoting factor contains the product of a Xenopus homolog of the fission yeast cell cycle control gene cdc2+

In the fission yeast S. pombe, the Mr = 34 kd product of the cdc2+ gene (p34cdc2) is a protein kinase that controls entry into mitosis. In Xenopus oocytes and other cells, maturation-promoting factor (MPF) appears in late G2 phase and is able to cause entry into mitosis. Purified MPF consists of two major proteins of Mr approximately equal to 32 kd and 45 kd and expresses protein kinase activity. We report here that antibodies to S. pombe p34cdc2 are able to immunoblot and immunoprecipitate the approximately equal to 32 kd component of MPF from Xenopus eggs. The Mr approximately equal to 32 kd and 45 kd proteins exist as a complex that expresses protein kinase activity. These findings indicate that a Xenopus p34cdc2 homolog is present in purified MPF and suggest that p34cdc2 is a component of the control mechanism initiating mitosis generally in eukaryotic cells.

A Long Twentieth Century of the Cell Cycle and Beyond

Virchow who promoted the idea that all cells were produced by the fission of preexisting cells (for a pithy, and to predict the course of research for the next cen-1921), who recognized that eggs and sperm were single tury, and to do it all in 10 pages! It is unrealistic to try cells which became joined together at fertilization, so to be comprehensive in such a review, and so I will even the most complex multicellular organisms passed focus on what I consider to be the important principles through a single celled stage. Thus, cell division was underlying the cell cycle, with less emphasis on detailed established as the basis of growth and development of descriptions of molecular mechanisms which would de-both animals and plants. generate into lists of genes and proteins. Referencing Improvements in microscopes and microscopic tech-will be minimal, and will be restricted to reviews, a few niques led to a detailed description of the changes oc-key primary publications, and to books written in English curring to the chromosomes during mitosis, the most for summaries of the earlier literature. conspicuous event of cell division. A critical feature ob-Let us begin at the end of the century by summarizing served by Flemming and Strasburger during the 1880s what is now known about the cell cycle. We know that was the appearance of elongate chromosomal threads the cell cycle is the universal process by which cells formed from the nucleus, which then split lengthways reproduce, and that it underlies the growth and develop-before shortening and thickening later in mitosis (Wilson, ment of all living organisms. The most important events 1925; Flemming, 1965). Van Beneden later showed that of the cell cycle are those concerned with the copying the longitudinal halves of each split chromosome sepa-and partitioning of the hereditary material, that is repli-rated apart into the two daughter nuclei, and that the cating the chromosomal DNA during S phase and sepa-chromosomes of a fertilized nematode egg were derived rating the replicated chromosomes during mitosis. Con-in equal numbers from the egg and sperm. With this trols operate that regulate onset of these events and discovery, Weissman came to the important conclusion compensate for errors in their execution. The molecular that the chromosomes were the basis of heredity, and basis of these controls is highly conserved from simple that germ cells formed a continuous line of heredity unicellular eukaryotes such as yeast …

Periodic gene expression program of the fission yeast cell cycle

Cell-cycle control of transcription seems to be universal, but little is known about its global conservation and biological significance. We report on the genome-wide transcriptional program of the Schizosaccharomyces pombe cell cycle, identifying 407 periodically expressed genes of which 136 show high-amplitude changes. These genes cluster in four major waves of expression. The forkhead protein Sep1p regulates mitotic genes in the first cluster, including Ace2p, which activates transcription in the second cluster during the M-G1 transition and cytokinesis. Other genes in the second cluster, which are required for G1-S progression, are regulated by the MBF complex independently of Sep1p and Ace2p. The third cluster coincides with S phase and a fourth cluster contains genes weakly regulated during G2 phase. Despite conserved cell-cycle transcription factors, differences in regulatory circuits between fission and budding yeasts are evident, revealing evolutionary plasticity of transcriptional control. Periodic transcription of most genes is not conserved between the two yeasts, except for a core set of ∼40 genes that seem to be universally regulated during the eukaryotic cell cycle and may have key roles in cell-cycle progression.

Cohesin Rec8 is required for reductional chromosome segregation at meiosis

When cells exit from mitotic cell division, their sister chromatids lose cohesion and separate to opposite poles of the dividing cell, resulting in equational chromosome segregation. In contrast, the reductional segregation of the first stage of meiotic cell division (meiosis I) requires that sister chromatids remain associated through their centromeres and move together to the same pole. Centromeric cohesion is lost as cells exit from meiosis II and sister chromatids can then separate,,,. The fission yeast cohesin protein Rec8 is specific to and required for meiosis,,,. Here we show that Rec8 appears in the centromeres and adjacent chromosome arms during the pre-meiotic S phase. Centromeric Rec8 persists throughout meiosis I and disappears at anaphase of meiosis II. When the rec8 gene is deleted, sister chromatids separate at meiosis I, resulting in equational rather than reductional chromosome segregation. We propose that the persistence of Rec8 at centromeres during meiosis I maintains sister-chromatid cohesion, and that its presence in the centromere-adjacent regions orients the kinetochores so that sister chromatids move to the same pole. This results in the reductional pattern of chromosome segregation necessary to reduce a diploid zygote to haploid gametes.

Analysis of a genome-wide set of gene deletions in the fission yeast Schizosaccharomyces pombe.

We report the construction and analysis of 4,836 heterozygous diploid deletion mutants covering 98.4% of the fission yeast genome providing a tool for studying eukaryotic biology. Comprehensive gene dispensability comparisons with budding yeast—the only other eukaryote for which a comprehensive knockout library exists—revealed that 83% of single-copy orthologs in the two yeasts had conserved dispensability. Gene dispensability differed for certain pathways between the two yeasts, including mitochondrial translation and cell cycle checkpoint control. We show that fission yeast has more essential genes than budding yeast and that essential genes are more likely than nonessential genes to be present in a single copy, to be broadly conserved and to contain introns. Growth fitness analyses determined sets of haploinsufficient and haploproficient genes for fission yeast, and comparisons with budding yeast identified specific ribosomal proteins and RNA polymerase subunits, which may act more generally to regulate eukaryotic cell growth.

The fission yeast cdc18+ gene product couples S phase to START and mitosis

Commitment to the cell cycle in fission yeast requires the function of the cdc10+ transcriptional activator at START. The product of the cdc18+ gene is a major downstream target of cdc10+, and transcription of cdc18+ is activated by cdc10+ during passage through START. The cdc18+ function is required for entry into S phase. In addition, the product of the cdc18+ gene is part of the checkpoint control that prevents mitosis from occurring until S phase is completed. Thus, cdc18+ plays a key role in coupling S phase to START and mitosis.

Regulation of p34cdc2 protein kinase during mitosis

The cell-cycle timing of mitosis in fission yeast is determined by the cdc25+ gene product activating the p34cdc2 protein kinase leading to mitotic initiation. Protein kinase activity remains high in metaphase and then declines during anaphase. Activation of the protein kinase also requires the cyclin homolog p56cdc13, which also functions post activation at a later stage of mitosis. The continuing function of p56cdc13 during mitosis is consistent with its high level until the metaphase/anaphase transition. At anaphase the p56cdc13 level falls dramatically just before the decline in p34cdc2 protein kinase activity. The behavior of p56cdc13 is similar to that observed for cyclins in oocytes. p13suc1 interacts closely with p34cdc2; it is required during the process of mitosis and may play a role in the inactivation of the p34cdc2 protein kinase. Therefore, the cdc25+, cdc13+, and suc1+ gene products are important for regulating p34cdc2 protein kinase activity during entry into, progress through, and exit from mitosis.