Genetics of the fission yeast Schizosaccharomyces pombe.

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GENETICS OF THE FISSION YEAST Annu. Rev. Genet. 1992.26:373-402. Downloaded from www.annualreviews.org by Drexel University on 03/15/13. For personal use only.

SCHIZOSACCHAROMYCES POMBE Jacqueline Hayles and Paul Nurse Biochemistry Department, University of Oxford, South Parks Road, Oxford, OXl

3QU, U.K.

KEY WORDS; S. pomb e, signal transduction, genome, promoters, molecular genetics

CONTENTS

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . .

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375 375 375 376 376

RECOMB INAnON . ........... . . . . . .. . . . .... . . . . ..... . . . . . . Rela tionship between the Gene tical and the Physical Map . . . . . . . . . . . . . . . Genes Affecting Recombina tional Processes . . ... . . . . .. . .. . . .. . . ....

377 377 378

GENE STRUCTURE AND REGULATION . .. . ...... . ..... . . ...... . . 5' and 3' R egions of Genes . . . . . . . . . . . .. . . . . . . . . . . . . ... . . . . . . Nuclear Pre-mRNA Splicing . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . .

379 379 381

PROTEIN TRAFFICKING

381

GENOME ORGANIZATION DNA Conten t . . . . . . . . Chromosome Structure . Chromatin Organization . R epe ti tive S equences . . .

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MITOTIC CELL CYCLE . . ..... . . .... . . . .... . . . ... . . . . ... . ... Cell Cycle Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

382 382

SEXUAL DEVELOPMENT . . . . . . . . . . . . .. . . . . ... . . . . . . . . . . . . . .. . . . . . . . . . ... . . . . . . . . . . ..... . . . . . .. . .

383 383 387 392

MOLECULAR GENETICS . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . .. Introduction of DNA ...................................... Plasmid Episomes . . . ..... . . . .. . . .. . .. . . . . ... . . . . ... . . . . . . Manipulating Genomic Sequences . .. . . . .. . . . . . .. . .. . . .. . . . . . ...

392 392 393 394

CONCLUSIONS . . . . .. . . . .... . . . . . . . . . . .. . . . . .. . . . . . . . . . . . .

395

';!:��:�1,r:: ::::::::::::::::::::::::::::::::::::::::::: Meiosis and Sporulation

373 0066-4197/92/1215-0373$02.00

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Annu. Rev. Genet. 1992.26:373-402. Downloaded from www.annualreviews.org by Drexel University on 03/15/13. For personal use only.

INTRODUCTION The yeast Schizosaccharomyces pombe is a unicellular eukaryote that is becoming increasingly popular as a model organism for molecular genetical studies. It was originally isolated from the East African millet beer known as Pombe. The strains used in the laboratory today are derived from a Swiss isolate called Schizosaccharomyces liquifaciens Oswalder, now named S. pombe var. pombe. It is a member of the Ascomycota, although not closely related to other present day Ascomycetes. It probably diverged from the budding yeast Saccharomyces cerevisiae some 1000 million years ago, soon after the Ascomycota separated from the lineage leading to the Metazoa ( 1 38). S. pombe was developed as a genetically tractable organism in the early 90 1950s by Urs Leupold (104) . He isolated a homothallic strain h (so called because 90% of the cells could form spores) and two heterothallic strains of 90 opposite mating types, h+ and h -, which were derived from the h strain . It is from these original isolates that all present day S. pombe strains used for genetic analysis are derived. The haploid cells are rodlike and are 7-14j..Lm in length and 3 4j..Lm in diameter. In rich medium they grow by apical extension and divide by medial fission. When starved, cells enter stationary phase either from G l or G2, depending on which nutrient becomes limiting. Cells starved of nitrogen arrest predominantly in G l and if cells of both mating types are present they conjugate , forming a diploid zygote that undergoes meiosis and sporulates to generate four haploid spores contained within an ascus. S. pombe is normally haploid but can be induced to undergo a diploid mitotic cell cycle if cells are re-fed with nitrogen after conjugation but before meiosis has been initiated. If such heterozygous h+ Ih - diploids are nitrogen starved they are able to sporulate directly. It has also been observed that some cells in an h + Ih culture are able to conjugate with each other. Homozygous h -/h- and h + Ih + diploids may arise by endomitosis (104) . These cells are unable to sporulate directly but can mate with cells of the opposite mating type. These features of its life cycle make S. pombe amenable for genetical analysis. Mutants, for example, can be isolated from a haploid strain and tested for dominance or recessiveness using a diploid strain. Genes can be defined by both linkage analysis and complementation. Since the development of a transformation system for S. pombe, a range of molecular genetical techniques has been developed. Genes can be cloned by complementation of a mutant function and the genomic copy of a gene replaced by homologous recombination with alleles that have been mutated in vitro. We discuss recent advances in our knowledge of the biology of S. pombe that have been based primarily on genetical analysis. This review is intended to provide an introduction to current knowledge of S. pombe; for a more

GENETICS OF S. POMBE

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detailed discussion of various topics we have referred the reader to the appropriate reviews and experimental papers.

GENOME ORGANIZATION

DNA Content The DNA content of a 2e haploid cell of S. pombe is O.03pg, with


mitochondrial DNA comprising about 6% of total DNA in growing cells and

about 14% in stationary phase cells (10, lOa). The haploid genome of S. pombe is organized as three chromosomes that are visible microscopically when they become partially condensed during mitosis and meiosis ( 1 26). This organization is confirmed by the presence of three linkage groups (78). Pulse field gel (PFG) electrophoretic methods have visualized all three chromo somes, yielding size estimates of 5.7Mb, 4.7Mb, and 3.5Mb, respectively, for chromosomes I , II, and III, giving a total genome size of about 14Mb (29). The three chromosomes are linear, although occasionally a ring chromosome II can form by fusion of the ends, possibly by recombination at the telomeres (J-B . Fan, C. Gaillardin, C. Smith, manuscript submitted).

Chromosome Structure CENTROMERES

The centromeres of all three chromosomes of S. pombe have

been cloned and characterized. They span regions of about 40kb, 70kb, and 100kb for chromosomes I, II, and III, respectively (14, 16,46). They contain two major repeated motifs dg and dh (also called K and L) arranged as large

inverted repeats around a central core region. The actual number of repeated

dg, dh motifs and their organization within the palindromic arrangement varies at each centromere. tRNA genes are also clustered at all three centromeres

(79, 141). The size and repetitive nature of fission yeast centromeres suggest that they are more like those of higher eukaryotes than the centromeres of budding yeast and, in fact, the centromeres from the two yeasts do not function when transformed from one yeast to the other ( 1 1 8). This configuration of large inverted repeats around the central core region is likely to be important for complete centromere function. Such a structural arrangement is required to hold the sister chromatids together during meiosis

I. This has been shown with mini-chromosomes, partially deleted for one arm of the inverted repeat, which undergo sister chromatid segregation at meiosis

I. However, these deletions do not abolish mitotic stability, suggesting that spindle attachment can still take place (47, 110). The nucleosomal configu ration at the centromeric region may also be important for centromere function. The central core and the associated repeats in cenl and 2 show no regular

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nuc1eosomes. When these centromeres are introduced into S. eerevisiae (where they are nonfunctional) these regions are packaged into nuc1eosomes. This suggests that in S. pombe these regions are prevented from forming nuc1eosomes, perhaps by the binding of kinetochore proteins (1 18). ORIGINS OF REPLICATION The 14-Mb genome of S. pombe is replicated in about 10-20 min (10, 107b), indicating that it should contain at least one origin about every 80 kb of DNA, assuming average rates of DNA replication. It has been postulated that ARS elements that confer upon a plasmid the ability to transfQrm efficiently (see Plasmid Episomes), are origins of replication. ARS elements are present in the genome of S. pombe at a frequency of one per 20 kb of DNA (94), a frequency higher than that calculated for origin distribution.

The consensus telomeric sequence for S. pombe is (Cl-6GO--l TO--IGTAl-2)n. Linear minichromosomes generated in S. pombe have 300-bp telomeric sequences at their ends attached directly to the original breakage sites. This suggests that they did not result from recombination or transloca tion, but rather were synthesized de novo (92a). TELOMERES

Chromatin Organization In S. pombe the genes encoding the four core histones have been cloned (15, 92), but as yet there is no evidence for the presence of a histone HI gene or protein ( 157). There are t wo genes for H2A (htal and 2), one for H2B (htbl) three for H3 (hthl, 2, and 3) and three for H4 (hfo1 , 2, and 3). They are organized as a repeat of an H2A gene and H2B gene, three repeats of the H3 and H4 genes, and a single copy of an H2A gene (92). A histone variant encoded by the gene phtl is involved in chromosome stability (T. Carr, S. Dorrington, P. Nurse, in preparation). H igher order folding of chromatin occurs in S. pombe, but to a lesser degree than in higher eukaryotes (157). The changes in chromatin structure during interphase and mitosis have been described (147) and several genes involved in these changes have been identified. These include the crml and the top1 and top2 genes (1, 150, 151), the latter encoding DNA topoisomerases. The endl gene encodes a protein with similarity to 5' methylcytosine transferases (R. Bartlett, P. Nurse, in preparation). A end1 mutant has a disturbed nuclear structure and, although not yet assigned a function, this gene may possibly encode a protein influencing chromatin structure. Repetitive Sequences After digestion .of total cellular DNA and gel electrophoresis, repetItIve sequences such as r RNA and mitochondrial DNA can be visualized as distinct


377

bands against the background of digested DNA. Recombination can occur between repeated sequences leading to concerted evolution between gene families ( 1 03). Unequal crossing over can also lead to gene rearrangements such as amplification ( 1 3). The genes encoding 5 . 8S, 1 8S , and 25S ribosomal RNAs are organized as clusters of tandemly repeated units (5, 1 3 1 ) found at both ends of chromosome III (30, 1 52) . The repeat is contained on a lO.4-kb fragment with an estimated 100-1 50 copies in total, which suggests at least 1000 kb of the genome is in the form of rDNA. However, the amount of rDNA appears to vary from strain to strain (J-B. Fan, C. L. Smith, submitted; (30)). The rDNA repeat contains an ARS element and S. pombe has a naturally occurring plasmid of rDNA analogous to 3f..Lm DNA of S. cerevisiae (35). The rRNA genes are transcribed by POL l , the large subunit of which is + encoded by nuc1 (52). The synthesis of rRNA is reduced in a top] top2 double mutant, whereas mRNA and tRNA synthesis are relatively unaffected. This suggests that relaxation of supercoiled DNA is required for efficient + + + rRNA gene transcription. The nuc1 , top] and top2 gene functions are necessary for nucleolus formation (52). About 30 copies of the 5S rRNA genes are dispersed throughout the genome, and not within the rRNA repeats (90) . This arrangement of the rRNA genes of S. pombe is similar to that found in other fungi and higher eukaryotes.


rRNA GENES

Between 200-300 tRNA genes are dispersed throughout the genome, with clustering of tRNA genes at the centromeres (79, 141). The significance of this clustering is not known. tRNA genes are found individually or in a pairwise configuration (58).

tRNA GENES

There are two families of retrotransposons in S. pombe, called Tfl and Tf2. Both Tfl and 2 are found in three wild-type strains not extensively used for genetical analysis (85). Tfl is present in about 30-40 copies and is an active transposable element related to the gypsy family of retrotransposons (84). Strains derived from the Leupold isolates do not contain Tfl , except for the LTR, but do have Tf2. The restriction pattern of Tf2 in two of these strains is the same, suggesting that it is stably inherited (85). TRANSPOSONS

RECOMBINATION Relationship Between the Genetical and Physical Maps A database of about 500 genes has been constructed as part of the fission yeast genome project (81), and about half of these have been mapped or partially mapped. The present genetic map length is 2100 cM ( 1 04) . On


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average, 1 cM is equivalent to about 6 kb of DNA, although this relationship is not constant throughout the genome because of gene conversion and the variation in the frequency of meiotic crossing over. The mating-type (mat) region affects the level of meiotic recombination between matI and his2. The double-strand break (DSB) associated with matl efficiently promotes meiotic recombination at this site (77). This region also contains a recombination cold spot between the mat2 and mat3 genes, which cannot be separated by meiotic recombination (23) although they are 1 5 kb apart (8) . Recombination in this region is suppressed by the rikl (recombi nation in K), swi6 (switch) and elrl (cryptic locus repression) gene functions (27, 75, 144). The centromeric regions also show reduced levels of meiotic recombination ( 107) . Certain mutations such as ade6-M26 give rise to hot spots of recombination. This mutation stimulates intragenic recombination at the ade6 locus during meiosis but not mitosis, possibly by creating a site of entry for meiotic-specific recombination enzymes (42). The chromosomal location of this mutation may also be important for this effect (119), and increased transcription of the ade6 mutant gene stimulates both mitotic and meiotic recombination, resulting in increased gene conversion (43). Mitotic recombination (non-sister chromatid exchange in a diploid) occurs naturally at 5-200-fold lower frequency than meiotic recombination. The level of recombination varies throughout the genome, being highest in regions adjacent to the centromere where it is only fivefold lower than during meiosis. It has been suggested that pairing occurs preferentially near the centromere during mitosis and that this pairing becomes extended in a small proportion of cells, resulting in recombination of genes distal to the centromere (98) . The matI locus is also a hot spot for mitotic recombination, leading to high frequency homozygosity of the distal arm of chromosome II (78).

Genes Affecting Recombinational Processes Compared to S. cerevisiae, S. pombe is very radiation resistant, possibly due to an efficient recombinational repair pathway (1 15). So far, 23 rad (radiation-sensitive) genes have been identified and many of these have been cloned and sequenced (See references in (32». These genes have been tentatively assigned to three groups according to their involvement in excision repair, recombination repair, and gamma ray repair (80). A study of the effects of these genes on meiotic, mitotic, and uv-induced mitotic recombination indicated that mutations in many of them did not affect recombination levels. However, mutations in rad2, 15, and 21 caused increased mitotic recombi nation levels. Furthermore , mutations in the radl , 2 and 8 genes resulted in reduced (radl ) or slightly increased (rad2 and 8) levels of uv-induced mitotic recombination (44).



379

The swi genes are involved in the formation and resolution of the double-strand break during mating-type switching (see Mating Type). Of mutants for eight swi genes tested, only a swi5 mutant showed reduced meiotic and mitotic recombination levels (132), and swi5, 9, and 10 mutants had increased radiation sensitivity ( 1 33). Mutations in nineteen rad genes were tested for their effects on mating-type switching; radI O, 1 6, and 20 were found to define the same gene (now called radI O), which is allelic to swi9. Rad22 was found to be a new swi gene ( 1 33). These results suggest that the swi and rad genes have overlapping functions, possibly in the processing of double-strand breaks. Of the mutations so far identified that affect recombination, many appear to be specific either for mitotic or meiotic recombination. The mitotic ree59, and edel 7 mutants show hyper-recombination whereas ree52, 53, 57, 58, and 60 mutants show reduced mitotic recombination frequencies (45, 139). Most of these mutants are affected in intergenic recombination and show no effect on intragenic recombination, suggesting that these two processes are not identical. The rec50, ree59, and edel l mutations also influence chromosome stability (9). The ree2, 3, and 5 mutants have either increased or decreased meiotic gene conversion between dispersed tRNA genes but are unaffected for intergenic recombination in meiosis. These genes may play a role in the illegitimate pairing and intragenic recombination between repeated sequences such as the tRNA gene families, which leads to concerted evolution between these unlinked genes ( 145) . Mutations in sixteen other ree genes, ree6 to 21, cause defects in meiotic recombination; the ree9, 1 7, and 1 9 mutants are also sensitive to DNA damage (20, 120) . The phenotype of these mutants suggests that although there are gene products involved generally in the process of recombination, other gene functions may also be required to promote or prevent specific types of recombination. GENE STRUCTURE AND REGULATION 5' and 3' Regions of Genes In S. pombe the organization of POLII transcribed protein-coding genes is typical of higher eukaryotes. The 5' regions of the more highly expressed genes have a TATA element (129), between 25 and 45 nuc1eotides upstream of the transcriptional start site. TFIID is a general transcription factor that binds to the TATA element and initiates assembly of the transcription complex (134) . The S. pombe gene encoding TFIlD has been cloned (33, 54). Like higher eukaryotes, transcripts of S. pombe are initiated within a limited region from the TATA box. The distance from the TATA element


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appears to be more important than the DNA sequence for determining the site of initiation of transcription. This site determination is different from s. cerevisiae, where transcription is initiated at a greater distance from the TATA element, perhaps explaining why promoters from each organism do not work particularly well in the heterologous system ( 1 30). The TATA box is less obvious in the 5' regions of low level transcribed genes, where other elements are presumably required for initiation ( 1 29) . Another sequence, CAGTCACA, has been found in some TATA-Iess genes at a similar site to the TATA box (I. Witt, N. F. Kaufer & T. Gross, manuscript submitted). This sequence may be involved in transcription initiation and is thought to bind a factor other than TFIID. The specific activation of gene transcription involves the binding of factors to upstream activating sequences. Several transcriptional activators found in other eukaryotes are also present in S. pombe, including the CCAAT binding complex ( 1 14), API (65 , 146), ATF (64), HSF (38, 66) , and GCN4 ( 1 28). The finding of these conserved activities and the fact that several different mammalian genes can be transcribed efficiently in S. pombe ( 149) suggests that fission yeast is a useful organism for genetical studies of higher eukaryotic promoters. The lack of suitable inducible genes has limited studies of transcriptional regulation in fission yeast. The recent cloning of }bpI and nmtI now make such studies possible. The }bpI gene encodes fructose 1, 6-bisphosphatase and cells deleted for this gene are unable to grow on glycerol (56). FbpI is transcriptionally regulated in response to glucose. The level of transcript varies lOO-fold and is repressed by high levels of cAMP. Mutations have been isolated that allow constitutive transcription of }bpI and ten git (glucose insensitive transcription) genes identified. One of these , git2, is allelic to cyrI , which encodes adenylate cyclase (55). The nmtl (no message in thiamine) gene is involved in thiamine biosynthesis and its transcription is tightly regulated by the level of intracellular thiamine (93). As with other strongly expressed genes in S. pombe, the TATA element occurs 25 bp upstream of the nmtI transcriptional start site. If this sequence is progressively truncated transcriptional activity is reduced, although it is still repressed by thiamine. Interestingly, when the TATA box is completely removed initiation still takes place at the same site, suggesting that, at least in this promoter, other elements may define the start of transcription (5a). The consensus sequence for polyadenylation in higher eukaryofes is AATAAA and is found at the 3' end of genes encoding for polyadenylated mRNAs. Although this sequence is often found in S. pombe, it is not utilized . The signals used b y fission yeast are more like those o f S. cerevisiae, as budding yeast transcripts can be polyadenylated accurately in S. pombe.


381

However, the polyadenylation sequence in S. pombe is not clearly defined other than as an AT-rich region (60). Nuclear pre-mRNA Splicing About 30-40% of POLII transcribed fission yeast genes contain introns, which are removed during the pre-mRNA splicing process. These introns are usually


less than 100 bp but range in size from 36-700 bp. About 50% of these genes

have multiple introns (124). The fission yeast consensus sequence for splicing is (5' splice site) GTA� GT, (branch site) CTAi , (3' splice site) Ai: AG. This sequence is similar to mammalian splice sequences and a mammalian intron has, in fact, been correctly spliced in S. pombe (67). The genes snul, 2, 4, 5, and 6, encoding the snRNAs found in the spliceosome, have been cloned , from S. pombe. Both snul and 4 are essential (17, 121). All five single-copy genes encode for snRNAs with similarities in both primary sequence and

secondary structure with mammalian snRNAs. VI, 2, 4, and 5 all have a tri-methylguanosine 5' cap and all five snRNAs can be i mmunoprecipi tated from snRNPs with antibodies to one of the core proteins, Sm (17). Mutants defective for pre-mRNA splicing have been isolated, defining four genes prpl-4 (123, 127). These mutants are defective at an early stage of processing and accumulate unspliced pre-mRNA; tRNA is not affected. Strains bearing mutations in the three genes, prpl-3, also have reduced U6 snRNA levels within spliceosomes and accumulate U6 pre-RNA. This suggests that V6 preRNA (125a) is processed like pre-mRNA even though it is a putative POLIII transcribed gene. A further gene, snml. has been identified and a snml mutant has reduced levels of VI, 2, 4, 5, and 6 snRNA and accumulates U2 and U4 with aberrant 3' ends; pre-mRNA splicing is also reduced (122).

PROTEIN TRAFFICKING The targeting of proteins to the lumen of the endoplasmic reticulum (ER) involves the binding of a signal recognition particle (SRP) to the signal

sequence peptide at the N-terminus of proteins destined for secretion. The SRP consists of a ribonucleoprotein containing a snRNA called 7SL encoded by the srp7 gene (11). In the ER lumen and the Golgi apparatus, which is morphologically very well developed in fission yeast, there are proteins responsible for protein modification and secretion. One of these proteins, BiP,

a member of the HSP70 family of proteins, is thought to be required for

correct protein folding. The predicted protein sequence for BiP from S. pombe

contains a putative signal peptide and N' glycosylation site. BiP is not secreted and the peptide sequence sufficient for its retention is ADEL. This differs


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from mammals (KDEL) and budding yeast (HDEL). However, S. pombe is able to recognize both these other sequences although less efficiently than ADEL (117). Two GTP-binding ypt proteins have been implicated in the protein secretory pathway. The yptl and ypt3 genes are both essential and encode functional homologues of the YPTI and SEC4 gene products from S. cerevisiae (9 9, 100). The ypt2 gene is also essential (48) and deletion of a fourth gene, rhyl , generates a temperature-sensitive growth defect that can be rescued by the rab6 gene from humans (49). As yet no function has been assigned to these gene products. MITOTIC CELL CYCLE

This topic, initially studied in S. pombe by Murdoch Mitchison in Edinburgh has been extensively reviewed recently (see 34, 111), and thus is only considered briefly here. Cell Cycle Controls

During rapid growth cells spend about 0.7 of a cycle in G2 while S phase, G1, and mitosis are about 0.1 of a cycle each. There are two major rate-limiting steps for progress through the cell cycle. One in late G1 is known as Start and is the point at which the cells become committed to the cell cycle as opposed to the alternative fate of conjugation. The second is known as the mitotic control and acts in late G2 to regulate the timing of entry into mitosis. The gene functions cdc2 and cdcl O act at Start. Cells blocked at Start are still able to conjugate, unlike cells blocked later in the cycle. The cdc2 gene encodes a 34-kd serine/threonine protein kinase required twice during each cell cycle, both at Start and at the mitotic control. The biochemical role of p34 cdc2 at Start still remains uncertain. The cdclO gene product, p87cdclO. may be required for transcriptional regulation of S-phase genes (86). START

The timing of mitotic onset is regulated by a protein kinase cascade that results in the activation of the p34cdC2/p56cdc13 protein 2 kinase by dephosphorylation of tyrosine (Y)15 of p34cdc (111). The weel gene encodes a serine/tyrosine kinase p107wee1 that inhibits p34cdc2 function, and a second weel-like gene (31), mikl , encoding a related kinase, may have a similar role (88). The cdc25 gene encodes a tyrosine phosphatase p80cdC25 c c2 that activates p34 d by dephosphorylating phosphorylated Y15 ( 1 1 1). Certain mutations in p34cdc2 or alterations in the expression of weel or cdc2 5 change the timing of entry into mitosis, which causes cells to delay mitotic onset or to enter mitosis prematurely (111).

MITOTIC CONTROL

GENETICS

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The cdc2-3w mutant is de fective in the checkpoint control ensuring the dependency of mitosis upon completion of S phase. These cells enter mitosis in the presence of hydroxy urea (HU), which blocks DNA replication. A screen for further mutants that enter mitosis in the presence of HU and an analysis of rad mutants has shown that radl , 3, 9, and 1 7 and five hus (hydroxyurea sensitive) genes are involved in the control mechanism arresting cell cycle progress when S phase is incomplete (2, 28, 126a; T. Enoch, A. Carr, P. Nurse, submitted). There is also a dependency between entry into S phase and passage through the previous mitosis. Certain cdc2 mutants can re-replicate their DNA in the complete absence of mitosis, indicating that the cdc2 gene product also plays a central role in this dependency (12).


DEPENDENCIES BETWEEN S PHASE AND MITOSIS

SEXUAL DEVELOPMENT

Sexual development occurs when cells are deprived of nutrients and cease proliferation. A key role is played by the mat genes that determine the mating type of the cell. In response to mating factor (pheromone), a cell forms a conjugation tube and fuses with a cell of the opposite mating type, producing a diploid zygote that immediately undergoes meiosis and sporulation. Mating Type

Cells of S. pombe exist as one of two mating types, plus (P) or minus (M). The homothallic wild-type strain h90 switches its matin� type approximatel� once per generation. The two heterothallic strains h + (normal) and h (stable) are relatively stable with respect to switching. In an h +N strain only s about 10-5 cells switch per generation and the h - strain does not switch at all. Because homothallic cells can switch mating type, clonal cultures contain both h + and h - cells and in low nitrogen medium conjugate and sporulate. Heterothallic strains, on the other hand, only contain h + or h - cells and in low nitrogen simply enter stationary phase. These cells only conjugate if mixed with cells of opposite mating type (See references in (24)). The mating-type region consists of three loci, matI, 2, and 3. The essential 15-kb L region separates matI and mat2 and the 15-kb K region separates mat2 and mat3. Each locus is flanked by regions of homology known as HI and H2. Mat2 and 3 have a further flanking region of homology known as H3 (24, 70). Mat2 and 3 contain unexpressed plus and minus information cassettes, respectively. The expressed matl locus contains either plus or minus information that has been transferred from mat2 or 3 by copy transposition (6) . The h+N strain contains copies of mat2, K, and mat3 at the matI locus, a rearrangement that reduces the switching frequency. The h -s strain has a

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deletion covering mat2 and K and thus lacks plus information and so is completely stable (24). MATING-TYPE SWITCHING Switching is initiated by a DSB at matI. The DSB maps between matI and HI, but the exact site of the cut is only known for one strand. The other strand is tightly bound with proteins, hence the precise break site is not known ( l 08). A DNA-binding activity SAP! (switch activating protein) binds to SAS l . This site is found within 140 bp of the DSB and is essential for switching. A second site required for activity, SAS2, is also found in this region and overlaps with the smt (switching mating type) mutations. Both SAS! and SAS2 are required for efficient switching (4). The smt mutants, which are defective in switching, have small chromosomal deletions at matI (4), in the vicinity of the DSB. Other mutants defective in switching define eleven unlinked swi genes. The swi genes can be subdivided into three classes: Class Ia genes are involved in DSB formation; the class Ib genes in utilization of the cut for switching; and the class II genes in resolution of recombination intermediates (25). Individual swi mutants have only reduced switching, but switching is almost completely abolished in a triple mutant carrying a swi mutation from each class (132). Switching occurs in both haploids and diploids with each mat locus switching independently. A specific pattern of switching is followed such that only one cell among four granddaughters will be switched (the I in 4 pattern) and the sister of a newly switched cell will switch in consecutive generations (recurrent switching) (74a). The pattern of switching is believed to arise because of the inheritance of a specific DNA strand by daughter cells. Genetic evidence has shown that a strand-specific imprinting event is required for DSB formation (74). Although the molecular nature of the imprinting event is not known, DNA modifications such as methylation could be involved. In DNA prepared from a growing population, about 25% of the matI DNA contains a DSB. This level is constant throughout the cell cycle, suggesting that the cut is long lived (6, 108). DSB formation is independent of switching and if the cell fails to switch, the DSB is healed normally (77). A DSB is created one generation before it is utilized, probably in G2. Evidence from the switching pattern of smt mutants suggests that it is healed and remade each generation. DNA replication is thought to play a role in the switching process (76). The following model has been suggested to account for the observed pattern of switching (Figure 1; 76): (A) A newly imprinted P cell in G l has an imprinted chromosome without a DSB. After DNA replication one chromatid is imprinted and one is not imprinted. Only the imprinted chromatid can be cut to form a DSB; this occurs early in G2 or at the time of DNA replication. The unimprinted uncut chromatid becomes imprinted before DNA replication in the next cell cycle,


385

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'/

\

Mcell (U) *

Figure 1 A representation of the two DNA strands of the matl locus of a P cell during the G 1 and G2 phases of the cell cycle. A, B, and C show the segregation of the imprinted DNA strand and the DSB during the cell divisions that give rise to four granddaughter cells. The pattern of segregation gives rise to the I in 4 and the recurrent switching patterns. A, B and C are described in the text. (*) imprinting event on one strand of the DNA, (1/) DSB, (=) the switched matl locus, (S) a switchable cell, and (U) an unswitchable cell.

although the actual timing is not known. If imprinting occurs before cell division it must occur at a time such that the chromatid does not receive a DSB. (B) This uncut chromatid is segregated to one of the daughter cells. This daughter cell is P type and unswitchable and is the same as the cell described in (A). It thus follows the same fate, giving rise to two P-type daughters, of which one is switchable and one unswitchable. (C) The imprinted and cut chromatid is segregated to the other daughter cell. This cell is also P type, but is now switchable as it has inherited the DSB. During DNA replication the imprinted strand is sealed, replicated, then re-cleaved, while the other strand that is not imprinted is sealed and replicated by copy transposition from mat3 . Thus, after DNA replication, one chromatid has an imprinted strand with a DSB and one chromatid has switched and is not imprinted. Division


386

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of this P cell gives rise to one switched M daughter and one P daughter, which is competent to switch. From the original cell in (A) only one of the four granddaughters has switched mating type. The P-switchable granddaughters are the same as the cell in (C) and satisfy the recurrent switching pattern by giving rise to one M cell and one P cell. In contrast, the newly switched M cell and the unswitchable P cell follow the 1 in 4 pattern (ABC). In cells competent to switch, the level of switching to the opposite mating type is not 100%. Between 75% and 95% of unswitchable cells follow the I in 4 pattern and about 90% of switchable cells follow the recurrent pattern (76). There are several reasons for the less than 100% fidelity of these patterns: cells may occasionally switch to the same mating type, the DSB may be healed without switching, or the imprinting event or DSB formation may not occur with 100% efficiency. That the recurrent switching pattern leads to one switched daughter in most generations suggests that switching is not random; it shows a pronounced directionality. P cells nearly always switch to M and M cells nearly always switch to P. Random switching would lead to an apparently reduced level of switching. The position of mat2 and 3, rather than the information each contains, appears to be the important feature for switching, because strains with swapped cassettes (mat2-M, mat3-P) switch inefficiently (G. Thon, A. Klar, in preparation). The direction of switching may be influenced by the swi6 gene function, as in swi6 mutants switches from M to P are preferentially affected. A second gene, rikI, may also be important for switching. As discussed earlier, the K region is totally repressed for recombination; the swi6 and riki gene functions may be involved in this repression, as mutants of both genes show increased recombination in the K region (27 , 75). A third gene, elrl, which represses expression of mat2 and 3, also represses recombination in this region. It has been suggested that the chromatin organization of this region may regulate donor choice, donor expression, and recombination in the K region (144). MATING-TYPE GENES The mating-type genes are of fundamental importance in sexual differentiation. MatI-M encodes two genes, mati-Me and mati-Mi while MatI-P encodes mati-Pc and matI-Pi (c constitutive or conjugation, i inducible, M meiosis). Both matI-Me and matI-Pi have homology with DNA binding proteins (70, 140). MatI-Pc and matI-Me are sufficient for conjugation in the absence of matI-Pi or Mi function. Both mati-Pc and Me are expressed constituitively at low level in growing cells but their transcripts increase in level in nitrogen starvation conditions. The function of all four mat genes is required for sporulation. Transcription of matI-Pi and Mi is induced in the absence of nitrogen (70), but induction of matI-Pi is =

=

=


387

also dependent on the mating pheromone signal (73 , 82 , 109, 109a) . Thus mating pheromone response is required not only to bring about conjugation but also for later stages of sexual development. Either M or P factor is sufficient to generate this signal. This requirement may account for the sporulation defect seen in many conjugation-deficient mutants including matl -Pe and Me. The matl-Mi gene does not appear to be under the same regulation as the matI -Pi gene. Preliminary evidence suggests that its induction is independent of both matI -Me and pheromone induction (26) .


Conjugation The early stages of sexual differentiation leading to conjugation are brought about through a gene network that includes the matl-M and matl-P loci. In the absence of nitrogen and in the presence of pheromones, this network brings about inactivation of the patl (parthenogenesis, also known as rani) encoded protein kinase p52patl (95). This kinase represses sexual differentia tion by inhibition of the mei2 gene function. After completion of the mei2 function the cell becomes committed to meiosis and sporulation and is unable to re-enter vegetative growth (155) . In the following sections we cover only the principal gene functions involved in the gene network regulating sexual differentiation (Figure 2; for a more detailed review, see also (26». Conjugation occurs in low ni trogen in response to diffusible pheromones, the P and M factors. M factor has been partially purified; it induces the mating response in h + cells in low nitrogen but has no effect on exponentially growing cells (18). M factor is a Tyr Thr Pro Lys Val Pro Tyr Met Cys (S-famesyl )-OCH3 nonapeptide, with limited similarity to S. cerevisiae a factor. It is processed from a precursor encoded by two genes, mfml and mfm2 . These are transcribed only in h cells in response to nitrogen starvation. P factor has also been identified, but is not yet characterized (19). Two genes sxal (sexually activated) and sxa2 have been identified that may be involved in regulating the mating pheromone response (63). These genes encode products with similarity to an aspartyl protease and a serine car boxypeptidase, respectively. Mutants in sxal and 2 give mating-type specific defects; sxal mutants are P-cell specific and sxa2 mutants are M-cell specific. In sporulation conditions, both mutants form elongated conjugation tubes when cells of the opposite mating type are present , but they mate only inefficiently. The sxal transcript is present in all cell types and is increased in response to nitrogen starvation whereas the sxa2 transcript is only induced in response to mating pheromone. These gene products may be involved in degradation of the mating pheromone or in processes required for formation of the zygote. Unlike the situation in budding yeast, pheromones do not have any effect EARLY RESPONSES TO MATING PHEROMONES

388

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STE12 STE13 CYRl CAPl caSl CGS2 PACl

LOW NITROGEN

•

LOW cAMP

STEll

i

MEIOSIS AND SPORULATION Figure 2

A model of the possible gene network in a P cell that acts in response to nitrogen

starvation and M factor, to bring about conjugation with an M cell. The diploid zygote thus formed immediately undergoes meiosis and sporulation. The major gene functions involved in the network of interactions are boxed. Other gene functions have been placed on the figure in the area where they probably function. The dotted line represents a less well understood

interaction.

in normal growth medium and cells must be starved for nitrogen to elicit a pheromone response ( 1 8). It is not known if pheromones induce cell cycle arrest in fission yeast. Nuclei must be in the G1 state before nuclear fusion can take place, but this occurs anyway in nitrogen-starved cells, so there is no need to invoke a separate mechanism to arrest cells in G 1. The mam2 (M-specific) and map3 (P-specific) genes encode the P- and putative M-factor receptors, respectively (73). These receptors are necessary for response to mating pheromone. The transcription of mam2 is dependent on reduced nitrogen levels and is transcribed in h - and h-Ih + cells. The gpa] gene encodes a G protein u subunit that probably interacts with both the M and P receptors as the transcript is found in cells of both mating types (112). A gpar mutant is defective in mating and sporulation and a nitrogen-starved activated gpa] mutant forms a conjugation tube in the absence of cells of the opposite mating type. The sporulation deficiency of gpa] mutants is probably due to lack of the pheromone signal required for matI-Pi transcription discussed earlier. Gpal transcripts are increased in low nitrogen conditions in homothallic and h + Ih diploid strains, suggesting that the presence of pheromone may also be required to increase the transcript level. The activating role of the Gu subunit is in contrast to its role in S. cerevisiae, where it is inhibitory for pheromone response (112). -


The ste1 2 and 13 gene func tions appear to act at a very early stage of sexual development. Homothallic cells of either mutant show no signs of conjugation and have poor viability in starvation conditions. This characteristic has led to the suggestion that these gene functions are required for entry into stationary phase. Both gene functions may affect transcriptional regulation of mei2, since homothallic ste12 and 13 mutants in sporulation conditions have reduced levels of mei2 transcript (72). cAMP levels also appear to influence this early stage of sexual development. Cells of S. pombe in low nitrogen medium have reduced levels of cAMP (10 I ). The cyrI (allelic to git2) gene encodes adenylate cyclase (68, 89) and capl encodes an adenyl cyclase interacting protein (cap) (69). Cyrl mutants have no measurable cAMP and are derepressed for mating even in rich medium. A complete deletion of the open reading frame of capl also allows cells to conjugate and sporulate in rich medium, although abnormal asci are formed. Mutations in either of these genes allow bypass of the nutritional starvation signal. However, the mating factor signal is still required as, in the absence of cells of opposite mating type, cyrl and capl mutants do not sporulate directly but continue to proliferate (68, 69, 89). The genes cgsl and cgs2/pdel have homology with the regulatory subunit of cAMP-dependent protein kinase (cAPK) and low affinity cAMP phosphodiesterase, respectively (21, 101). The lack of the regulatory subunit activates cAPK and lack of cgs2/pdel-encoded phosphodiesterase leads to increased levels of cAMP. Both cases should lead to elevated cAPK activity. Like ste12 and 13 these mutants are sterile and show variable but reduced viability when starved. The loss of cgsl and cgs2/pdel or the addition of cAMP inhibits sexual development and prevents an increase in mei2 transcripts during nitrogen starvation (21). The stell gene function plays a central role in sexual development. stell mutants are defective for conjugation and sporulation and do not secrete M or P factor. The low level of stell transcript found in growing cells is induced to a higher level in low nitrogen in homothallic or heterothallic strains (140). The induction of stell transcription is responsive to cAMP levels; it is not induced in the presence of high cAMP even at low nitrogen levels and is induced to high levels in a cyrl mutant even in rich medium (140). The stell gene encodes a putative DNA-binding protein containing a HMG (High Mobility Group) box that can bind in vitro to the DNA sequence TICTITGTIY. This T-rich (TR) box is found upstream of the matl-P, matl-M and mei2 genes and is essential for the induction of transcription by stell. The stell gene function is also thought to activate ste6 transcription in response to low nitrogen (see below). Interestingly, stell also has a TR-like sequence in its 5' upstream region, suggestive of autoregulation. The stell gene function is essential for the induction of the mati-P, mati-M and mei2 genes in low nitrogen. Transcription of the matl genes is also induced when

EARLY RESPONSES TO NUTRIENT DEPLETION


389

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HAYLES

&

NURSE

stel l + is moderately overexpressed in rich medium, resulting in a phenotype


similar to a eyrl mutant (140).

LATER SIGNAL TRANSDUCTION EVENTS Other members of the ste family of genes that act slightly later in sexual development encode proteins implicated in signal transduction. Rasl , which is allelic to ste5, encodes p25ras1 (87). A rasl- mutant is able to grow vegetatively but has a swollen cell morphology (36, 106). It produces both mating factors although the production of P factor is reduced (83). Both h + and h - rasr mutants are defective in their pheromone response and arrest in low nitrogen as spherical cells. The induction of mati-Me or Pc transcription is unaffected in a rasr mutant, but transcript levels of mati-Pi and, to a lesser extent, matl-Mi are reduced ( l 05). An h + Ih- homozygous rasr mutant is still partially sensitive to pheromone since it can still sporulate to some extent (36, 106). Cells with an activated rasl allele. rasl -vaI1,7 (previously referred to as rasl -vaI12(106» have increased levels of transcription from matI -Pi (109a) and appear to be hypersensitive to mating pheromone; they fonn long conjugation tubes but fail to mate efficiently (36, 106). This mutant may not be hypersensitive to pheromone, but may continue to grow in a polarized manner and thus prevent conjugation. An h + Ih- diploid homozygous for the rasl -val17 allele sporulates nonnally and is defective only for conjugation. P25ras1 probably does not mediate the pheromone response pathway as activated p25raSlvall7 cannot substitute for the pheromone signal. The r asl-val 17 mutant still needs to be stimulated by mating factor before cells can fonn a conjugation tube. It has been suggested that p25ras1 activity modulates pheromone response (109a, 112). The ste6 and gapl/sarl genes encode products with sequence similarities to a GDP-GTP exchange protein and to GAP (GTPase Activating Protein), respectively (59, 62, 153). The ste6 gene function is a putative activator and GAP is a putative inhibitor of sexual development. The ste6 function is only required for conjugation and may be transcriptionally regulated by the stel l HMG DNA-binding protein (140). The absence of GAP results in an activated ras phenotype indicating that GAP acts upstream of rasl. No other activating 2 r 1 gene functions appear to be required for the production of active p 5 as in the absence of GAP (62). A second gene ral2 (ras like) also encodes a protein that acts like an activator of p25ras1 . This gene is one of four rat genes that have a ras - phenotype when inactivated. Genetic evidence suggests that ral I , 2, and 3 are in a pathway leading to p25ras1 activation, with rail and 3 acting upstream of ral2 (37). It is possible that these proteins have different roles in the activation of p25ras1, perhaps responding to different signals or activating it to different levels.


39 1


Byrl (bypass of ras) is allelic to stel and byr2 is probably allelic to ste8. Both genes encode putative protein kinases whose function is required downstream of p25ras1 activity ( l 05 , 1 54). They may act on the pathway leading to matl-Pi activation in response to pheromone. The low sporulation levels of rasl - diploids can be completely abolished by mutations in byrl or 2. Other ste mutants have also been identified (ste 2, 3, 4, 7, 9, and 10) ( 137) . A ste10 mutant, like a ste6 mutant, has a ras -phenotype and is defective only for conjugation. The other ste mutants are defective for both conjugation and sporulation, but as yet their role in sexual development is unclear (83) . SO far we have considered gene functions required for conjugation of cells of opposite mating type in response to low nitrogen levels and the presence of mating pheromone. These gene functions relieve an inhibitory pathway that prevents conjugation and sporulation until it is inactivated under conditions that promote sexual development. The patl gene-encoded protein kinase, p52pat 1 , plays a central role in the inhibition of conjugation and sporulation ( 109 , 109a) . Patl null mutants undergo sporulation in rich medium even from the haploid state. However, these mutants cannot bypass the stell or mei2 gene functions since both stel l and mei2 mutants suppress patl mutants ( 1 36, 137) . Patl mutants can also be suppressed by addition of cAMP (7) , by increasing cAMP levels in cgsl and cgs2/pdel (21) mutants , or by overexpression of the gene pad (61) . The pad gene encodes a double-stranded ribonuclease III, suggesting that ribonuclease activity may also be involved in repressing sexual develop ment Partially inactive patJ mutants are derepressed for conjugation in rich medium and have increased transcription of matl -Pe and Pi and matl -Me even in heterothallic strains. If the patJ gene product is completely inactivated then haploid sporulation occurs ( l 09) . It has been proposed ( 1 09) that in low nitrogen and in response to pheromone, p52patl is partially inactivated. This allows increased transcription from matl-P and M genes in the appropriate cell type. Only after conjugation does patl become completely inactivated and allow activation of mei2 gene function and entry into meiosis. After conjugation, the presence of matl -Pi and Mi in the same cell allows transcription of the mei3 gene, although this is onlX seen in response to mel3 , nitrogen starvation (97) . The mei3 gene product, p2 1 leads to inactivation of the pS2pat1 kinase (96) . In the absence of pS2pat t activity the mei2 gene function is activated and leads to premeiotic DNA replication. The mei2 gene is therefore under positive transcriptional regulation by stel l and negative regulation involving the p52patl protein kinase. After activation of mei2 gene function the cell becomes committed to meiosis and sporulation.

RELIEF OF INHIBITION OF SEXUAL DEVELOPMENT

392

HAYLES & NURSE


Meiosis and Sporulation Several cdc gene functions may be required for the meiotic as well as the mitotic cell cycle and it is possible that control points similar to Start and the mitotic control point may act during meiosis. The two genes known to function at Start in the mitotic cell cycle are cdc2 and cdclO (34). Using a method for synchronizing the meiotic cell cycle in cdcis and cdclOts mutants, it has been suggested that the p87cdc lO function is required for premeiotic DNA replication 3 cdc2 but that p 4 function is not required until meiosis II (7). Other genetical data also indicate that p34cdc2 is required at meiosis II (40) . Premeiotic DNA synthesis is followed by meiosis I when homologous chromosomes pair and undergo exchange of genetic information. Electron microscopic analysis of the nucleus in meiosis I showed no evidence for the formation of a synaptonemal complex, but revealed structures, termed linear elements, that run parallel to the nuclear axis (53). The lack of a synaptonemal complex may account for the fact that no interference is observed during meiotic gene mapping (22) . The mei4 gene function is required during meiosis I and mei4 mutants arrest with an elongated nucleus ( 1 35). At completion of meiosis I, nuclear division takes place, giving two daughter nuclei. The nuclei proceed to meiosis II without an intervening S phase. Meiosis II is the more mitotic-like division, during which sister chromatids separate and segregate to the daughter nuclei. It requires the activities of the cdc2, 13, 25, mes1 , and mes2 genes (39). The mes1 and 2 functions are required earlier than the other gene functions (24), since in these mutants SPB duplication, meiosis II , and spore wall formation do not take place. Meiosis II is blocked in cdc2, 13, and 25 mutants but a spore wall forms, leading to the formation of two diploid spores. Modifications of the cytoplasmic face of the spindle pole body are necessary for forespore membrane and spore wall development ( 143). At least 20 spo (sporulation) genes have been identified (71 ) . Calmodulin has also been implicated i n the sporulation process because a specific cam1 (calmodulin) mutant is defective for sporulation (142) . Analysis of strains with mutations in the cdc and spo genes show that meiosis II and spore formation can proceed independently of each other. MOLECULAR GENETICS Introduction of DNA Three procedures have been devised for introducing DNA into cells of S. pombe. Using an ARS-containing plasmid (see below) , all three methods yield 4 in excess of 1 0 transformants/lLg DNA. The spheroplast method, the first 4 procedure devised, gives 1-2 x 10 transformants/lLg DNA (102); this fre quency varies little with different strains of S. pombe. The addition of lipofectin increases transformation frequency above 1 x 105/lLg and also allows


393

the uptake of minichromosomes of up to 500 kb in size (3). A modified lithium acetate method (113) routinely gives between 1 X 104 and 1 x 106 trans formantsffl-g and electroporation, although not yet widely used, gives at least I X l OS transformants/j.tg and is very rapid (125). Plasmid Episomes


High levels of transformation are only possible using plasmids containing an

ARS element (156). Fragments of DNA with ARS activity from S. pombe are 0 . 8-1 . 8 kb long and contain sequences that are 69-7 5% A + T. All fragments have an AT-rich I l-bp core with the consensus sequence 5' (AlT)PuTTTATTTA(AlT) 3 ' , similar to the 5' (A/T)TTTATPuTTT(A/T) 3 ' sequence found i n budding yeast. Unlike budding yeast, this core sequence is not essential for ARS activity, indicating that other sequences found near to this core may be responsible for ARS activity (94). If so, they have only limited primary sequence similarity. The core sequence may perform some function in the chromosome that is irrelevant for ARS activity in a plasmid. Plasmids containing an ARS replicate autonomously and do not become rearranged. Generally copy number is about 5-30 per cell. Plasmids without an ARS can transform cells but do so at lower frequencies and colonies take longer to appear (41, 50, 156). Most of these transformants are unstable and often the plasmids become rearranged (156) . In some cases, the plasmid sequences become integrated at random locations in the genome, but nevertheless they are still unstable (156). In other cases, the plasmids appear to become multimeric and to have acquired an ARS element from the genome. Non-ARS plasmids may become integrated into the genome and then be fortuitously excised with an ARS and can therefore replicate autonomously. Unrearranged non-ARS plasmid can also be recovered from these trans formants (41, 50). This may be because mixed multimers, presumably formed by homologous recombination, allow non-ARS plasmids to replicate along with the ARS plasmid. However, the hypothesis that non-ARS containing plasmids can, on occasion, replicate autonomously cannot be completely ruled out. In S. pombe, autonomously replicating plasmids are rather unstable and without selection are rapidly lost from the population. Even under selection, only around 50% of the cells usually contain plasmid. Given the high copy number (5-30) of these plasmids, this instability indicates that the segregation pattern is probably nonrandom. It has been impossible to construct stable, low copy number CEN plasmids similar to those in S. cerevisiae because centromeres in S. pombe are so large. Centromeres in S. pombe do stabilize plasmids but can only be transformed into cells using YAC (Yeast Artificial Chromosome) vectors (46) or as minichromosomes (92). A noncentromeric sequence called STB also confers stability and results in about 80 copies of


394

HAYLES & NURSE

the plasmid per cell (50), but it is not clear if STB confers these properties on all plasmids. Selectable markers used in fission yeast rescue the leul, ura4 and ade6 2+ mutants ( 102). The LEU gene from S. cerevisiae rescues the leul -32 mutant + but may not always rescue as a single copy. The ura4 gene appears to work well even as a single copy, although expression may be dependent on the chromosomal location when integrated. The opal nonsense suppressor gene sER AUG tRNA sup3_5 has also been used with the nonsense mutation ade6-704 because it forms the basis of an easy red/white color screen ( 1 02). Sectoring due to plasmid loss is not seen in the same way with this system as in budding yeast (51), as the cells become mixed during formation of the fission yeast colony. Two strong constitutive promoters used for expression in S. pombe are those derived from the SV40 early region and the adhl (alcohol dehydroge nase) gene (102) . Two inducible expression systems have been developed using endogenous promoters from S. pombe. The pCHY I vector uses thejbpl promoter (56) and the pREP and pRIP series of autonomously replicating and integrating plasmids (92b) use the nmtl promoter (93). A third expression system uses the glucocorticoid response element (116). The jbp1 promoter is repressed in 8% glucose and induced maximally in 0. 1 % glucose + 3% glycerol. The thiamine repressible nmtl promoter ( 148) is strongly transcribed in minimal medium and repressed when cells are grown in the presence of thiamine. There are three different forms of this promoter, allowing transcrip tion to different levels. Manipulating Genome Sequences Generally, the one-step gene replacement technique is used to replace the Wild-type copy of a gene with a deleted or mutated copy. This technique involves transformation with a linear DNA fragment followed by a double crossover event to replace the chromosomal gene copy ( 1 02) . The procedure requires regions of homology on either side of the gene of interest to allow homologous recombination to take place. In S. pombe, at least 500 bp of homology is preferable on both sides of the gene of interest (H. Schmidt, J. Hayles , unpublished observations), although less on one side is sufficient in some cases (59, 1 12) . Integration does not always occur at the homologous site and for certain loci illegitimate integration events predominate. The modified lithium acetate transformation procedure may favor homologous gene replacement (B . Grallert, P. Nurse, personal communication) . Both homologous and nonhomologous integration events will give rise to stable colonies. However, not all colonies obtained after transformation with linear DNA are stable. Presumably in these unstable transformants the DNA has rearranged so that it can replicate autonomously, perhaps due to the same mechanism that occurs with the non-ARS plasmids (See above).

GENETICS OF S.

POMBE

395


CONCLUSIONS We conclude by considering the suitability of fission yeast as a model organism for studying problems of eukaryotic cell and molecular biology. Many similarities exist between fission yeast and higher eukaryotic cells , for example, in the areas of chromosome structure and function, gene regulation, cell cycle, and signal transduction. Of particular value has been the working out of the control that regulates mitosis in fission yeast and the demonstration that this control applies to higher eukaryotic cells. These studies establish fission yeast as an ideal model eukaryote for the genetical investigation of many such problems . I t is sometimes argued that fission yeast i s superior to budding yeast because it is more similar to higher eukaryotes . Although this may be a correct assumption for certain problems, e.g. the regulation of mitosis, there is no reason to believe it will generally be true. Both yeasts are about equally diverged from the Metazoa and, thus, are both excellent model eukaryotes.

Indeed, a comparison of how things work in both organisms by exploiting their excellent genetics and the ease of gene transfer from one to the other will be highly instructive. The sequencing of the genomes of both of these simple but rather diverged eukaryotes combined with the types of genetical analyses reviewed here, should advance our understanding of how the eukaryotic cell operates . ,

ACKNOWLEDGMENTS

We thank D . Hughes, I-B . Fan, N . Kaufer, A. Klar, H. Levine, K. Maundrell, C. Smith, and M. Yamamoto for providing unpublished data and commenting on various sections of the manuscript. We also thank our colleagues in the ICRF Cell Cycle Group for discussions and for reading of the manuscript, especially K . Labib, S . Martin, and C. Norbury , and S. MacNeill for critical reading of the manuscript. Literature Cited 1.

2.

3.

Adachi, Y . , Yanagida, M. 1989. Higher order chromosome structure is affected by cold-sensitive mutations in a Schizosaccharomyces pombe gene crml + which encodes a 1 15-kD protein preferentially localized in the nucleus and its periphery. J. Cell Bioi. 108: 1 195-207 Al-Khodairy, F . , Carr, A. M. 1992. Mutants defining the G2 checkpoint pathway in S. pombe. EMBO J. 1 1 : 1 343-50 Allshire, R. C. 1990. Introduction of large linear minichromosomes into

4.

5.

Schizosaccharomyces pumbe by an im proved transformation procedure . Pruc. Natl. Acad. Sci. USA 87:4043-47 Arcangioli, B . , Klar, A. J. S. 199 1 . A novel switch-activating site (SAS 1 ) and its cognate binding factor (SAP1) required for efficient matI switching in Schizosaccharomyces pombe. EMBO J. 199 1 :3025-32 Bamitz, J. T . , Cramer, J. H . , Rownd, R. H . , Cooley, L . , Soli, D. 1982. Arrangement of the ribosomal RNA genes in Schizosaccharomyces pombe. FEBS Lett. 1 43 : 1 29-32

396 5a.

6.

7.


8.

9.

10.

lOa.

11.

12.

13.

14.

15.

16.

HAYLES & NURSE Basi, G. ,Schmid, E . , Maundrell, K. 1992. TATA box mutations in the nmtI promoter of S. pombe affect transcription efficiency but not the site of transcription initiation nor the re pressibility by thiamine. G ene.In press Beach, D. 1983. Cell type switching by DNA transposition in fission yeast. Na tur e 305:682-88 Beach, D . , Rodgers, L . , Gould, J. 1985. r anI + controls the transition from mitotic division to meiosis in fission yeast. Curr o G enet. 10:297-3 1 1 Beach, D. H . , Klar, A . J . 1984. Rearrangements of the transposable mating-type cassettes of fission yeast. EMBO 1. 3:603-10 Bodi, Z., Gysler, J. A., Kohli, J. 1 99 1 . A quantitative assay to measure chromosome stability in Schizosac

17.

18.

19.

20.

charomyces pombe. Mol. Gen. Genet.

21 .

229:77- 80 Bostock, C. J. 1970. DNA synthesis in the fission yeast Sch i osac ch rom y c es pomb e. E xp . Cell Res . 60: 1 6-26 Bostock, C. J. 1969. Mitochondrial DNA in the fission yeast Sch izosac

22.

z

a

charomyces pombe. Biochim. Biophys. Acta 195: 579-8 1 Brennwald, P . , Liao, X . , Holm, K . , Porter, G . , Wise, J. A . 1988. Identi fication of an essential Sch izo saccharo myces pombe RNA homologous to the

7SL component of signal recognition particle. Mol. Cell. B io i . 8 : 1580-90 Broek, D . , Bartlett, R . , Crawford, c C Nurse, P. 1 99 1 . Involvement of p34 in establishing the dependency of S phase on mitosis. Na tur e 349:388-93 Carr, A. M . , MacNeill, S. A . , Hayles, J . , Nurse, P. 1989. Molecular cloning and sequence analysis of mutant alleles of the fission yeast cdc2 protlin kinase gene: implications for cdc2 protein structure and function. Mol . G en. G enet. 218:41--49 Chikashige , Y . , Kinoshita, N . , Naka seko, Y . , Matsumoto, T . , Murakami, S . , et al. 1989. Composite motifs and repeat symmetry in S. pomb e centro meres: direct analysis by integration of No tl restriction sites. Cell 57:739-5 1 Choe, J . , Schuster, T . , Grunstein, M . 1985. Organization, primary structure, and evolution of histone H2A and H2B genes of the fission yeast Sch izosacc haromyces pomb e. Mol . Cell . B ioi. 5:3261-69 Clarke, L . , Baum, M. P. 1990. Func tional analysis of a centromere from fission yeast: a role for centromere specific repeated DNA sequences. Mol. Cell. Bioi. 10: 1 863-72

I£.t

23.

24.

25.

26.

27.

28 .

29.

30.

Dandekar, T. , Tollervey, D. 1989. Cloning of Sch izosaccharomyces pombe genes encoding the U l , U2, U3 and U4 snRNAs. Gene 8 1 :227-35 Davey, J. 1 99 1 . Isolation and quanti tation of M-factor, a diffusible mating factor from the fission yeast Sch izosac charomyces pombe. Yeast 7:357-66 Davey, J. 1992. Mating pheromones of the fission yeast Sch izosaccharomy c es pombe: Purification and structural characterisation of M-factor and anal ysis of two genes encoding the pher omone. EMBO 1. 1 1 :951-60 DeVeaux, L. C . , Hoagland, N . A . , Smith, G. R . 1992. Seventeen com plementation groups of mutations de creasing meiotic recombination in Sch izosaccharomyces pombe. G enetics 130:25 1-62 DeVoti , J. , Seydoux , G . , Beach, D . Mc od, M . 1 99 1 . Interaction between ranI protein kinase and cAMP de pendent protein kinase as negative reg ulators of fission yeast meiosis. EMBO 1. 10:3759-68 Egel, R. 1978. Synaptonemal complex

4,

and crossing-over: Structural support

or interference . Her edity 4 1 :233-37 Egel, E. 1984. Two tightly linked silent cassettes in the mating type region of Sch izo saccharomyces pombe. Curro Genet. 8 : 1 99-203 Egel, R. 1989. Mating-type genes, meiosis and sporulation. See Ref. 107a, pp. 3 1-73 Egel, R. , Beach, D. H . , Klar, A. J. 1984. Genes required for initiation and resolution steps of mating-type switch ing in fission yeast. Pr oc . Na tl . Acad. Sc i . USA 8 1 :3481-85 Egel, R . , Nielsen, 0 . , Weilguny, D. 1990. Sexual differentiation in fission yeast. Tr ends G enet. 6:369-73 Egel, R . , Willer, M . , Nielsen, O . 1989. Unblocking o f meiotic crossing over between the silent mating-type cassettes of fission yeast, conditioned by the recessive, pleiotropic mutant r ikI . Curr o Genet. 15 :407-10 Enoch, T . , Gould, K . , Nurse, P. 1992. Mitotic checkpoint control in fission yeast. Col d Spr ing Harbor Symp . Quant. Bio i . 56:409-16 Fan, J. B., Chikashige, Y . , Smith, C. L., Niwa, 0., Yanagida, M . , et al. 1989. Construction of a No tndJl re striction map of the fission yeast Schizosaccharomyces pomb e genome. Nucl eic Ac ids Res. 1 7:2801 - 1 8 Fan, J-B . , Grothues, D . , Smith, C. L . 1992. Alignment of Sfil sites with the Notl restriction map of Sch izosacc-


31.

32.


33.

34.

35.

36.

37.

38.

39.

40.

41 .

42.

haromyces pombe genome. Nucleic Acids Res. 19:6289-94 Featherstone, C . , Rpssell, P. 1 99 1 . Fission yeast p1 07wee mitotic inhibitor is a tyrosine/serine kinase. Nature 349: 808-1 1 Fenech, M . , Carr, A. M . , Murray, J . M. , Lehmann A . R . 1 991 . Cloning and characterisation of the rad4 gene of Schizosaccharomyces pombe; a gene showing similarity to the human XRCCI gene. Nucleic Acids Res. 19: 6737-41 Fikes, J. D . , Becker, D. M. , Winston, F. , Guarente, L. 1 990. Striking con servation of TFIID in Schizosaccharo myces pombe and Saccharomyces cerevisiae. Nature 346:291-94 Forsburg, S. L . , Nurse, P. 1 991 . Cell cycle regulation in the yeasts Saccharo myces cerevisiae and Schizosaccharo myces pombe. Annu. Rev. Cell Bioi. 7:227-56 Fournier, P . , Gaillardin, c . , de Lou vencourt, L . , Heslot H. ,Lang, B . F. , Kaudewitz, F. 1982. r-DNA plasmid from Schizosaccharomyces pombe: Cloning and use in yeast transforma tion. Curro Genet. 6:31-38 Fukui, Y . , Kozasa, T . , Kaziro, Y . , Takeda, T . , Yamamoto, M . 1986. Role of a ras homolog in the life cycle of Schizosaccharomyces pombe. Cell 44: 329-36 Fukui, Y. , Miyake, S . , Satoh, M . , Yamamoto, M. 1989. Characterization of the Schizosaccharomyces pombe ral2 gene implicated in activation of the rasi gene product. Mol. Cell. Bioi. 9:5617-22 Gallo, G. J . , Schuetz, T. J . , Kingston, R. E. 1 991 . Regulation of heat shock factor in Schizosaccharomyces pombe more closely resembles regulation in mammals than in Saccharomyces cerevisiae. Mol. Cell. Bioi. 1 1 : 281-88 Grallert, B . , Sipiczki, M. 1991 . Com mon genes and pathways in the reg ulation of the mitotic and meiotic cell cycles of Schizosaccharomyces pombe. Curro Genet. 20:1 99-204 GraUert, B . , Sipiczki, M. 1 990. Dis sociation of meiotic and mitotic roles of the fission yeast cdc2 gene. Mol. Gen. Genet. 222:473-75 Grimm, C . , Kohli, J. 1988. Observa tions on integrative transformation in Schizosaccharomyces pombe. Mol. Gen. Genet. 215:87-93 Grimm, C. , Munz, P. , Kohli, J. 1990. The recombinational hot spot mutation ade6-M26 of Schizosaccharomyces pombe stimulates recombination at sites

43.

44.

45.

46.

47.

48.

49.

50.

51 .

52.

53 .

397

in a nearby interval. CU". Genet. 1 8 : 193-97 Grimm, C . , Schaer, P. , Munz, P . , Kohli, J. 1 99 1 . The strong adhl pro moter stimulates mitotic and meiotic recombination at the ade6 gene of Schizosaccharomyces pombe. Mol. Cell. Bioi . 1 1 :289-98 Grossenbacher-Grunder, A-M. 1985. Spontaneous mitotic recombination in Schizosaccharomyces pombe. Curr Genet 10:95-101 Gysler, J. A . , Bodi, Z . , Kohli, J . 199 1 . Isolation and characterization of Schizosaccharomyces pombe mutants affected in mitotic recombination. Ge netics 128: 495-504 Hahnenberger, K. M . , Baum, M. P. , Polizzi, C. M . , Carbon, J . , Clarke, L. 1989. Construction of functional artificial minichromosomes in the fis sion yeast Schizosaccharomyces pombe . Proc. Natl. Acad. Sci. USA 86:577-81 Hahnenberger, K. M . , Carbon, J . , Clarke, L. 1 991 . Identification of DNA regions required for mitotic and meiotic functions within the centromere of Schizosaccharomyces pombe chromo some 1. Mol. Cell. BioI. 1 l :2206- 1 5 Haubruck, H . , Engelke, U . , Mertins, P. , Gallwitz, D. 1990. Structural and functional analysis of ypt2 , an essential ras-related gene in the fission yeast Schizosaccharomyces pombe encoding a SEC4 protein homologue. EMBO J

9 : 1 957-62

Hengst, L . , Lehmeier, T . , Gallwitz, D. 1990. The ryhi gene in the fission yeast Schizosaccharomyces pombe en coding a GTP-binding protein related to ras, rho and ypt: structure , expression and identification of its human homo logue. EMBO J. 9:1949-55 Heyer, W. D . , Sipiczki, M . , Kohli, J. 1986. Replicating plasmids in Schiz osaccharomyces pombe: improvement of symmetric segregation by a new genetic element. Mol. Cell. Bioi. 6:8089 Hieter, P . , Mann, C . , Synder, M . , Davis, R. W. 1985. Mitotic stability of yeast chromosomes: A colony color assay that measures nondisjunction and chromosome loss. Cell 40:381-92 Hirano, T . , Konoha, G., Toda, T . , Yanagida, M. 1989. Essential roles of the RNA polymerase I largest subunit and DNA topoisomerases in the for mation of fission yeast nucleolus. J. Cell Bioi. 108:243-53 Hirata, A . , Tanaka, K. 1982. Nuclear behaviour during conjugation and mei-

398

54.


55.

56.

57 .

58.

59.

60.

61.

62.

63.

64.

HAYLES

&

NURSE

osis in the fission yeast Schizosac h aromyce s pombe. J. G en . App l. M icrobio l. 28:263-74 Hoffmann, A . , Horikoshi, M . , Wang, C. K . , Schroeder, S . , Weil, P. A . , e t al. 1 990. Cloning o f the Schizosa c charomyces pombe TFIID gene reveals a strong conservation of functional do mains present in Saccharomyces cerevisiae TFIID. Genes Dev. 4 : 1 1 4 148 Hoffman, C. S . , Winston, F. 1 99 1 . Glucose repression o f transcription of the Schizosaccharomyces pombe ]bp i gene occurs by a cAMP signalling pathway. Genes Dev. 5:561-71 Hoffman, C. S . , Winston, F. 1990. Isolation and characterization of mu tants constitutive for expression of the tbpl gene of Schizo saccharomyces p om be. Gen etics 1 24:807-1 6 Hoffman, C . S . , Winston, F. 1989. A transcriptionally regulated expression vector for the fission yeast Schizo sacc haromyces pombe . Gene 84:473-79 Hottinger, W. A., Schaack, J . , Lapointe, J . , Mao, J . , Nichols, M o , et a1. 1985. Dimeric tRNA gene ar rangement in Schizosaccharomyces pombe allows increased expression of the downstream gene. N uc leic Acids R es. 1 3 :8739-47 Hughes, D. A . , Fukui, Y . , Yamamoto, M. 1990. Homologous activators of ras in fission and budding yeast. Nature 344:355-57 Humphrey, T . , Sadhale, P . , Platt, T . , Proudfoot, N . 1 99 1 . Homologous mRNA 3 ' end formation in fission yeast and budding yeast. EMBO 1. 10:3503-1 1 lino, Y . , Sugimoto, A . , Yamamoto, + M. 1 99.1 0 S . pombe pac1 , whose overexpression inhibits sexual devel opment, encodes a ribonuclease III-like RNase . EMBO 1. 10:221-26 lmai, Y o , Miyake, S . , Hughes, D. A . , Yamamoto, M . 1 99 1 . Identification of a GTPase-activating protein homolog in Schizosaccharomyc es pombe. M ol. C ell. Bio i. 1 1 :3088-94 Imai, Y . , Yamamoto M. 1 2. S chizo.; saccharomyc es pombe sxa l and sxa 2 encode putative proteases involved in the mating response. Mol. Cell. Bio i. 1 2 : 1 827-34 Jones, R. H . , Jones, N. C. 1989. Mammalian cAMP-responsive element can activate transcription in yeast and binds a yeast factor(s) that resembles the mammalian transcription factor ATF. Proc . Natl. Acad . Sci . USA 86:21 76-80

65.

66.

67.

68.

69.

70.

71.

72.

73.

74.

�

Jones, R. H . , Moreno, S . , Nurse, P . , Jones, N. C . 1988 . Expression of the SV40 promoter in fission yeast: iden tification and characterization of an AP- l -1ike factor. Cell 53:659-67 Kasai, H . , Isono, Ko 1 99 1 . Dual modes of transcriptional and translational ini tiation of SSP I , the gene for mito chrondrial HSP70 responding to heat shock in Schizosaccharomyces pombe. Nucleic Acids Res. 19:533 1-37 Kaufer, N. F . , Simanis, V . , Nurse, P. 1 985. Fission yeast Schizo sac charomyces pombe correctly excises a mammalian RNA transcript intervening sequence. Nature 3 1 8:78-80 Kawamukai , M., Ferguson , K., Wigler, M . , Young, D . 1 991 . Genetic and biochemical analysis of the ad enylyl cyclase of Schizosaccharomyc es p ombe. Cell R egul. 2: 155-64 Kawamukai, M . , Gerst, J . , Field, J . , Riggs, M . , Rodgers, L . , e t al. 1992. Genetic and biochemical analysis of the adenyl cyclase-associated protein, cap, in Schizo saccharomyces pom be. Mo l. Bio I. C ell 3 : 1 67-80 Kelly, M . , Burke, J . , Smith, M o , Klar, A . , Beach, D. 1988 . Four mating-type genes control sexual differentiation in the fission yeast. EMBO J. 7 : 1 537-47 Kishida, M . , Shimoda, Co 1 986. Ge netic mapping of eleven spo genes essential for ascospore formation in the fission yeast Schizosaccharomyces pombe. Curr o G en et. 10:443-47 Kitamura, K . , Nakagawa, T . , Shi moda, C. 1990. Novel sterile mutants of the fission yeast Schizo saccha romy ces p ombe which are defective in their response to starvation. Curr o G en et. 1 8 : 3 1 5-21 Kitamura, K., Shimoda, C. 1 99 1 . The Schizosaccharomyc es pombe mam2 gene encodes a putative pheromone receptor which has signific ant homol ogy with the Saccharomyces c er evi sia e STE2 protein . EMBO J . 10:3743-5 1 Klar, A. Jo 1 990. The developmental fate of fission yeast cells is determined by the pattern of inheritance of parental and grandparental DNA strands. EMBO

J. 9 : 1 407- 1 5 74a.

75.

76.

Klar, A . 1 992. Mechanisms of devel opmental choices in mating-type inter conversion of fission yeast. Trends Genet. 8:208- 1 3 Klar, A . J . S o , Bonaduce, M . J . 1 99 1 . swi6, a gene required for mating-type switching, prohibits meiotic recombi nation in the mat2-mat3 "cold spot" of fission yeast. G en etics 1 29: 1 033-42 Klar, A. J . , Bonaduce , M. J . ,


77.


78.

79.

80.

81.

82.

83.

84.

85.

86.

Cafferkey, R. 1 99 1 . The mechanism of fission yeast mating type intercon version: seal/replicate/cleave model of replication across the double-stranded break site at matI . Genetics 127:489-96 Klar, A . J . , Miglio, L. M . 1986. Initiation of meiotic recombination by double-strand DNA breaks in S. pombe. Cell 46:725-3 1 Kohli, J. Hottinger, H. H . , Munz, P . Strauss, A . , Thuriaux, P . 1 9 7 7 . Genetic mapping in Schizosaccharomyces pombe by mitotic and meiotic analysis and induced haploidisation. Genetics 87:471-89 Kuhn, R. M . , Clarke, L . , Carbon, J . 1 99 1 . Clustered tRNA genes i n Schizo saccharomyces pombe centromeric DNA sequence repeats. Proc. Natl. Acad. Sci. USA 88: 1 306-10 Lehman, A . R., Carr, A . M . , Watts, F. Z . , Murray, J. M. 1 99 1 . DNA repair in the fission yeast, Schizosaccharomyces pombe. Murat. Res. 250:205-1 0 Lennon, G . G . , Lehrach, H . 1992. Gene database for the fission yeast Schizosaccharomyces pombe. Curro Genet. 2 1 : 1- 1 1 Leupold, U . , Neilsen, 0 . , Egel, R . 1989. Pheromone-induced meiosis in P-specific mutants of fission yeast. Curro Genet. 1 5:403-5 Leupold, U . , Sipiczki, M . , Egel, R . 1 99 1 . Pheromone production and re sponse in sterile mutants of fission yeast. Curro Genet. 20:79-85 Levin, H. L . , Boeke, J. D. 1 992. Demonstration of retrotransposition of the Tfl element in fission yeast. EMBO J. 1 1 : 1 145-53 Levin, H. L . , Weaver, D. c . , Boeke, J. D. 1 990. Two related families of retrotransposons from Schizosaccharo myces pombe. Mol. Cell. Bioi. 1 0 : 6791-98 Lowndes, N. F . , Mcinerny, C. J . , Johnson, A . L . , Fantes, P. A . , Johnston L. H. 1992. Control of DNA synthesis genes in fis on yeast by the cell-cycle gene cdclO . Nature 355:449-53 Lund, P. M . , Hasegawa, Y . , Kitamura, K . , Shimoda, c . , Fukui, Y . , et al. 1987. Mapping of the rasl gene of Schizosaccharomyces pombe. Mol. Gen. Genet. 209:627-29 Lundgren, K . , Walworth, N . , Booher, R . , Dembski, M . , Kirschner, M . , et al. 1 99 1 . mikI and weeI cooperate in the inhibitory tyrosine phosphorylation of cdc2. Cell 64: 1 1 1 1-22 Maeda, T. , Mochizuki, N . , Yamamoto, M. 1 990. Adenylyl cyclase is dispens-

90.

92.

92a.

92b.

93 .

94.

95.

96.

97.

98 .

�

87.

88.

89.

99.

100.

101.

399

able for vegetative cell growth in the fission yeast Schizosaccharomyces pombe. Proc. Natl. Acad. Sci. USA 87:7814-- 1 8 Mao, J . , Appel, B . , Schaack, J . , Sharp, S . , Yamada, H . , et al. 1982. The 5S RNA genes of Schizosaccharomyces pombe. Nucleic Acids Res. 1 0:487-500 Matsumoto, S . , Yanagida, M. 1985. Histone gene organization of fission yeast: a common upstream sequence. EMBO J. 4:353 1-38 Matsumoto, T. , Fukui, K . , Niwa, 0 . , Sugawara, N . , Szostak, J . W. , et al. 1987 . Identification of healed terminal DNA fragments in linear mlll! chromosomes of Schizosaccharomyces pombe. Mol. Cell. Bioi. 7:4424-30 Maundrell, K. 1992. Construction of the pREP and pRIP thiamine repressible expression vectors for fission yeast. Gene. In press Maundrell, K. 1 990. nmtl of fission yeast. A highly transcribed gene com pletely repressed by thiamine . J. Bioi. Chern. 265: 10857-64 Maundrell , K . , Hutchison, A . , Shall, S. 1988. Sequence analysis of ARS elements in fission yeast. EMBO J 7 :2203-9 McLeod, M . , Beach, D. 986. Ho mology between the ranI gene of fission yeast and protein kinases. EMBO J. 5:3665-7 1 McLeod, M . , Beach, D. 988. A specific inhibitor of the ranI protein kinase regulates entry into meiosis in Schizosaccharomyces pombe. Nature 332:509-14 McLeod, M . , Stein, M. , Be!fh, D. 1987. The product of the mei3 gene, expressed under control of the mating type locus, induces meiosis and spor ulation in fission yeast. EMBO J. 6: 729-36 Minet, M . , Grossenbacher-Grunder, A M . , Thuriaux , P. 1980. The origin of a centromere effect on mitotic recom bination. A study in the fission yeast Schizosaccharomyces pornbe. Curro Genet. 2:53-60 Miyake, S . , Tanaka, A . , Yamamoto, M. 199 1 . Mapping of four ras super family genes by physical and genetic means in Schizosaccharomyces pombe. Curro Genet 20:277-8 1 Miyake, S . , Yamamoto, M. 1990. Identification of ras-related, YPT fam ily genes in Schizosaccharomyces pombe. EMBO J. 9 : 1 4 1 7-22 Mochizuki, N . , Yamamoto, M. 1 992. Reduction in the intracellular cAMP level triggers initiation of sexual de-

J

1

400

HAYLES & NURSE velopment in fission yeast. Mol. Gen.

Genet. 233: 1 7-24

102.

103.

Moreno, S . , Klar, A . , Nurse, P. 1991 . Molecular genetic analysis of fission yeast Schizosaccharomyces pombe. Method. Enzymol. 194:795-23 Munz, P. , Amstutz, H . , Kohli, I . , Leupold, U. 1982. Recombination be tween dispersed serine tRNA genes in


Schizosaccharomyces pombe.

1 13.

Nature

300:225-3 1 104. Munz, P. W . , H . , Kohli, I . , Leupold, U. 1989. Genetics overview. See Ref. 107a, pp. 1-30 105. Nadin-Davis, S . , Nasim, A. 1 990. Schizosaccharomyces pombe ras} and byr} are functionally related genes of the ste family that affect starvation-in duced transcription of mating-type genes. Mol. Cell. Bioi. 10:549--60 106. Nadin-Davis, S. A . , Nasim, A . , Beach, D. 1986. Involvement of ras in sexual differentiation but not in growth control in fission yeast. EMBO J. 5:2963-71 107. Nakaseko, Y . , Adachi, Y . , Funahashi, S-1 . , Niwa, 0., Yanagida, M. 1 986. Chromosome walking shows a highly homologous repetitive sequence present in all the centromere regions of fission yeast. EMBO J. 5: 101 1-21 107a. Nasim, A., Young, P. , Johnson, B. F. , eds. 1989. Molecular Biology of Fission Yeast. New York: Academic. 469 pp. 107b. Nasmyth, K . , Nurse, P., Fraser, R. S. S. 1979. The effect of cell mass on the cell cycle timing and duration of S-phase in fission yeast. J. Cell Sci. 39:215 108. Nielsen, 0., Egel, R . 1989. Mapping the double-strand breaks at the mat ing-type locus in fission yeast by ge nomic sequencing. EMBO J. 8: 26976 109. Nielsen, 0 . , Egel, R. 1990. The patl protein kinase controls transcription of the mating-type genes in fission yeast. EMBO J. 9: 1 401-6 109a. Nielsen, 0., Davey, I., Egel, R. 1 992. The ras} function of Schizosaccharo myces cerevisiae mediates pheromone induced transcription. EMBO J. 1 1 : 1 39 1-95 1 10 . Niwa, 0 . , Matsumoto, T . , Chikashige, Y . , Yanagida, M. 1989. Characteriza tion of Schizosaccharomyces pombe minichromosome deletion derivatives and a functional allocation of their centromere. EMBO J 8:3045-52 1 1 1 . Nurse, P. 1990. Universal control mechanism regulating onset of M phase. Nature 344:503-8 1 12 . Obara, T. , Nakafuku, M . , Yamamoto,

1 14 .

1 15 .

1 16.

1 1 7.

1 18.

1 1 9.

120.

121.

122.

123.

M . , Kaziro, Y. 1992. Isolation and characterisation of a gene encoding a G-protein a subunit from Schizosac charomyces pombe: Involvement in mating and sporulation pathways. Proc. Natl. Acad. Sci. USA 88:5877-8 1 Okazaki, K . , Okazaki, N . , Kume, K . , Jinno, S . , Tanaka, K . , e t al. 1990. High-frequency transformation method and library transducing vectors for clon ing mammalian cDNAs by trans-com plementation of Schizosaccharomyces pombe. Nucleic Acids Res. 1 8:6485-89 Olesen, J. T . , Fikes, J. D . , Guarente, L. 1 99 1 . The Schizosaccharomyces pombe homolog of Saccharomyces cerevisiae HAP2 reveals selective and stringent conservation of the small es sential core protein domain. Mol. Cell. Bioi. 1 1 :61 1-19 Phipps, I., Nasim, A., Miller, D. R. 1985. Recovery, repair, and mutagen esis in Schizosaccharomyces pombe. Adv. Genet. 23: 1-72 Picard, D . , Schena, M . , Yamamoto, K. R. 1 990. An inducible expression vector for both fission and budding yeast. Gene 86:257-6 1 Pidoux, A. L . , Armstrong, I 1992. Analysis of the BiP gene and identi fication of an ER retention signal in Schizosaccharomyces pombe. EMBO J. 1 1 : 1 583-91 Polizzi, C . , Clarke, L. 1 99 1 . The chromatin structure of centromeres from fission yeast: differentiation of the central core that correlates with function. J. Cell Bioi. 1 12: 1 9 1-201 Ponticelli, A. S . , Smith, G. R. 1992. Chromosomal context depen dence of a eukaryotic recombinational hotspot. Proc. Natl. Acad. Sci. USA 89:227-31 Ponticelli, A. S . , Smith, G. R. 1989. Meiotic recombination-deficient mu tants of Schizosaccharomyces pombe. Genetics 123:45-54 Porter, G. , Brennwald, P. , Wise, J. A. 1990. U l small nuclear RNA from Schizosaccharomyces pombe has unique and conserved features and is encoded by an essential single copy gene. Mol. Cell. Bioi. 1 0:287481 Potashkin, J . , Frendewey, D. 1990. A mutation in a single gene of Schizosaccharomyces pombe affects the expression of several snRNAs and causes defects in RNA processing. EMBO J. 9:525-34 Potashkin, I . , Li, R . , Frendewey, D. 1989. Pre-mRNA splicing mutants of


124.

125.


125a.

126.

1 26a.

127.

128.

129.

130.

131.

132.

133.

134. 1 35 .

Schizosaccharomyces pombe. EMBO J. 8:55 1-59 Prabhala, G . , Rosenberg, G. H . , Kau fer, N. F. 1992. Architectural features of pre-mRNA introns in the fission yeast Schizosaccharomyces pombe. Yeast 8: 17 1-82 Prentice, H. 1992. High efficiency transformation of Schizosaccharomyces pombe. Nucleic Acids Res. 20:621 Reich, C . , Wise, J. A. 1990. Evolu tionary origin of the U6 small nuclear RNA intron. Mol. Cell. BioI. 10:554852 Robinow, C. F. 1977. The number of chromosomes in S. pombe: Light mi croscopy of stained preparations. Ge netics 87:49 1-97 Rowley, R . , Subramani, S . , Young, P. G. 1992. Checkpoint controls in Schizosaccharomyces pombe: radI o EMBO 1. 1 1 : 1 335-42 Rosenberg, G. H., Alahari, S. K. , Kau fer, N. F. 199 1 . prp4 from Schizosac charomyces pombe, a mutant deficient in pre-mRNA splicing isolated using genes containing artificial introns. Mol. Gen. Genet. 226:305-9 Ruden, D. M. 1990. Identification of Schizosaccharomyces pombe transcrip tion factor PGA4, which binds coop eratively to Saccharomyces cerevisiae GAL4-binding sites. Mol. Cell. Bioi. 10:1432-38 Russell, P. 1989. Gene cloning and expression. See Ref. 107a, pp. 24371 Russell, P. 1983 . Evolutionary diver gence of the mRNA transcription ini tiation in yeast. Nature 30 1 : 1 67-69 Schaak, J . , Mao, J . , Soli, D. 1982. The 5 . 8S RNA gene sequence and the ribosomal repeat of Schizosaccharomy ces pombe. Nucleic Acids Res. 10: 285 1-64 Schmidt, H. K . , P. , Gutz, H. 1987. Switching genes in Schizosaccharomy ces pombe: their influence on cell viability and recombination. Curro Genet. 1 1 :303-8 Schmidt, H . , Kapitza, F. P . , Stephen, E. R . , Gutz, H. 1989. Some of the swi genes of Schizosaccharomyces pombe also have a function in the repair of radiation damage. Curro Genet. 16:89-94 ' Sharp, P. A. 1992. TATA-binding protein is a classless factor. Cell 68: 8 1 9-21 Shimoda, C . , Hirata, A . , Kishida, M . , Hashida, T. , Tanaka, K. 1985. Char acterization of meiosis-deficient mu tants by electron microscopy and map-

136.

137.

138. 139.

140.

401

ping of four essential genes in the fission yeast Schizosaccharomyces pombe. Mol. Gen . Genet. 200:25257 Shimoda, C., Uehira, M . , Kishida, M . , Fujioka, H . , lino, Y . , et al. 1987. Cloning and analysis of transcription of the mei2 �ene responsible for ini tiation of meIosis in the fission yeast Schizosaccharomyces pombe. J Bacte riol 1 69:93-6 Sipiczki, M. 1988. The role of sterility genes (ste and aft) in the initiation of sexual development in Schizosaccharo myces pombe. Mol. Gen. Genet. 2 1 3 : 529-34 Sipiczki, M. 1989. Taxonomy and phy logenesis. See Ref. 107a, pp. 431-52 Sipiczki, M . , Grossenbacher-Grunder, A-M . , Bodi, Z. 1 990. Recombination and mating type switching in a ligase defective mutant of Schizosaccharomy ces pombe. Mol. Gen. Genet. 220: 307- 1 3 Sugimoto, A . , lino, Y . , Wantanabe, c ' + r s transcription factor with an HMG motif that is a critical regulator of sexual development. Genes Dev. 5 : 1 990-99 Takahashi, K . , Murakami, S . , Chika shige, Y . , Niwa, 0 . , Yanagida, M. 199 1 . A large number of tRNA genes are symmetrically located in fission yeast centromeres. 1. Mol. Bioi. 2 1 8 : 1 3-17 Takeda, T . , Imai, Y., Yamamoto, M. 1989. Substitution at position 1 16 of Schizosaccharomyces pombe calmodulin decreases its stability under nitrogen starvation and results in a sporulation-deficient phenotype. Proc. Natl. Acad. Sci. USA 86:973741 Tanaka, K . , Hirata, A. 1982. Asco spore development in the fission yeasts Schizosaccharomyces pombe and S. japonicus. J. Cell Sci. 56:263-79 Thon, G . , Klar, A. J. 1992. The clrI locus regulates the expression of cryptic mating type loci of fission yeast. Ge netics. In press Thuriaux, P. 1985. Direct selection of mutants influencing gene conversion in the yeast Schizosaccharomyces pombe. Mol. Gen. Genet. 1 99 : 36571 Toda, T., Shimanuki, M . , Yanagida, M. 199 1 . Fission yeast genes that confer resistance to staurosporine en code an AP- l-Iike transcription factor and a protein kinase related to the mammalian ERKIIMAP2 and budding

�� ;=��:nb'; ::l/ ��%:: �

141 .

142.

143.

144.

145.

146.

402

147.


148.

149.

150.

HAYLES & NURSE yeast FUS3 and KSSI kinases. Genes Dev. 5:60-73 Toda, T. , Yamamoto, M. , Yanagida, M. 1 98 1 . Sequential alterations in the nuclear chromatin region during mitosis of the fission yeast Schizosaccharomy ces pombe: video fluorescence micros copy of synchronously growing wild type and cold-sensitive cdc mutants by using a DNA-binding fluorescent probe. J. Cell Sci. 52:271-87 Tommasino, M . , Maundrell, K. 199 1 . Uptake of thiamine b y Schizosaccharo myces pombe and its effect as a tran scriptional regulator of thiamine sen sitive genes. Curr o Genet. 20:63-66 Toyama, R. , Okayama, H. 1 990. Human chorionic gonadotropin alpha and human cytomegalovirus promoters are extremely active in the fission yeast Schizosaccharomyces pombe. FEBS Lett. 268:217-21 Uemura, T., Ohkura , H. , Adachi, Y. , Morino, K . , Shiozaki, K . , et al. 1987. DNA topoisomerase II is required for condensation and separation of mitotic chromosomes in S. pombe. Cell 50:

chromatin organization. EMBO J. 3:

1737-44 152.

Uzawa, S. , Yanagida, M. 1992. Vi sualization of centromeric and nucleolar DNA in fission yeast by fluoresence in situ hybridization. J. Cell Sci. 101 :

153.

Wang, Y. , Boguski, M. , Riggs, M . , Rodgers , L . , Wigler, M. 199 1 . sarI . a gene from Schizosaccharomyces pombe encoding a protein that regulates rasl . Cell Regul. 2:453-65 Wang, Y. , Xu, H. P. , Riggs, M. , Rodgers, L . , Wigler, M. 1 99 1 . byr2 , a Schizosaccharomyces pombe gene en coding a protein kinase capable of partial suppression of the ras] mutant phenotype. Mol. Cell. Bioi. 1 1 :3554-

267-75

154.

63

155.

156.

9 1 7-25

151.

Uemura, T . , Yanagida, M. 1984. Iso lation of type I and II DNA topoiso merase mutants from fission yeast: single and double mutants show dif ferent phenotypes in cell growth and

157.

Watanabe, Y. , Lino, Y. , Furuhata, K. , Shimoda, C . , Yamamoto, M. 1 988. The S. pombe mei2 gene encoding a crucial molecule for commitment to meiosis is under the regulation of cAMP. EMBO J. 7:761-67 Wright, A. P . , Maundrell, K . , Shall, s. 1986. Transformation of Schizosac charomyces pombe by non-homolo gous, unstable integration of plasmids in the genome. Curro Genet. 10:503-8 Yanagida, M. 1990. Higher-order chro mosome structure in yeast. J. Cell Sci.

96:1-3

The fission yeast, Schizosaccharomyces pombe.

DNA repair in the fission yeast, Schizosaccharomyces pombe.

Synchronization of the fission yeast Schizosaccharomyces pombe using heat shocks.

A revised chromosome map of the fission yeast Schizosaccharomyces pombe.

Molecular genetic analysis of fission yeast Schizosaccharomyces pombe.

The mitochondrial genome of the fission yeast Schizosaccharomyces pombe : 5. Characterization of mitochondrial deletion mutants.

Cloning of the gene for ribosomal protein S13 from the fission yeast Schizosaccharomyces pombe.

Enzyme activities of D-glucose metabolism in the fission yeast Schizosaccharomyces pombe.

Detection and characterization of a ring chromosome in the fission yeast Schizosaccharomyces pombe.

Organisation of the complex locus trp1 in the fission yeast Schizosaccharomyces pombe.

Microtubules in the fission yeast Schizosaccharomyces pombe contain only the tyrosinated form of alpha-tubulin.

Visualization of chromosomes in mitotically arrested cells of the fission yeast Schizosaccharomyces pombe.

The critical glucose concentration for respiration-independent proliferation of fission yeast, Schizosaccharomyces pombe.

Genetic studies of purine breakdown in the fission yeast Schizosaccharomyces pombe.

Transcriptional activity of the human immunodeficiency virus-1 LTR promoter in fission yeast Schizosaccharomyces pombe.

Adenylyl cyclase activity of the fission yeast Schizosaccharomyces pombe is not regulated by guanyl nucleotides.

The nucleotide sequence of tRNA tyrosine from the fission yeast Schizosaccharomyces pombe.

Uncontrolled septation in a cell division cycle mutant of the fission yeast Schizosaccharomyces pombe.

Genetic analysis of antisuppressor mutants in the fission yeast Schizosaccharomyces pombe.

Regulation of oxidative stress-induced cytotoxic processes of citrinin in the fission yeast Schizosaccharomyces pombe.

Electron microscopic examination of sporulation-deficient mutants of the fission yeast Schizosaccharomyces pombe.

Absolute proteome and phosphoproteome dynamics during the cell cycle of Schizosaccharomyces pombe (Fission Yeast).

Transport of L-glutamic acid in the fission yeast Schizosaccharomyces pombe.

The Reporter System for GPCR Assay with the Fission Yeast Schizosaccharomyces pombe.