A Double-Strand Break Repair Component Is Essential for S Phase Completion in Fission Yeast Cell Cycling

Fission yeast rad22+, a homologue of budding yeast RAD52, encodes a double-strand break repair component, which is dispensable for proliferation. We, however, have recently obtained a cell division cycle mutant with a temperature-sensitive allele of rad22+, designated rad22-H6, which resulted from a point mutation in the conserved coding sequence leading to one amino acid alteration. We have subsequently isolated rad22+ and its novel homologue rti1+ as multicopy suppressors of this mutant. rti1+ suppresses all the defects of cells lacking rad22+. Mating type switch-inactive heterothallic cells lacking either rad22+ or rti1+ are viable, but those lacking both genes are inviable and arrest proliferation with a cell division cycle phenotype. At the nonpermissive temperature, a synchronous culture of rad22-H6 cells performs DNA synthesis without delay and arrests with chromosomes seemingly intact and replication completed and with a high level of tyrosine-phosphorylated Cdc2. However, rad22-H6 cells show a typical S phase arrest phenotype if combined with the rad1-1 checkpoint mutation. rad22+ genetically interacts with rad11+, which encodes the large subunit of replication protein A. Deletion of rad22+/rti1+ or the presence of rad22-H6 mutation decreases the restriction temperature of rad11-A1 cells by 4–6°C and leads to cell cycle arrest with chromosomes incompletely replicated. Thus, in fission yeast a double-strand break repair component is required for a certain step of chromosome replication unlinked to repair, partly via interacting with replication protein A.

The Rho-GEF Rom2p Localizes to Sites of Polarized Cell Growth and Participates in Cytoskeletal Functions in Saccharomyces cerevisiae

Rom2p is a GDP/GTP exchange factor for Rho1p and Rho2p GTPases; Rho proteins have been implicated in control of actin cytoskeletal rearrangements. ROM2 and RHO2 were identified in a screen for high-copy number suppressors of cik1Δ, a mutant defective in microtubule-based processes in Saccharomyces cerevisiae. A Rom2p::3XHA fusion protein localizes to sites of polarized cell growth, including incipient bud sites, tips of small buds, and tips of mating projections. Disruption of ROM2 results in temperature-sensitive growth defects at 11°C and 37°C. rom2Δ cells exhibit morphological defects. At permissive temperatures, rom2Δ cells often form elongated buds and fail to form normal mating projections after exposure to pheromone; at the restrictive temperature, small budded cells accumulate. High-copy number plasmids containing either ROM2 or RHO2 suppress the temperature-sensitive growth defects of cik1Δ and kar3Δ strains. KAR3 encodes a kinesin-related protein that interacts with Cik1p. Furthermore, rom2Δ strains exhibit increased sensitivity to the microtubule depolymerizing drug benomyl. These results suggest a role for Rom2p in both polarized morphogenesis and functions of the microtubule cytoskeleton.

SET1, A Yeast Member of the Trithorax Family, Functions in Transcriptional Silencing and Diverse Cellular Processes

The trithorax gene family contains members implicated in the control of transcription, development, chromosome structure, and human leukemia. A feature shared by some family members, and by other proteins that function in chromatin-mediated transcriptional regulation, is the presence of a 130- to 140-amino acid motif dubbed the SET or Tromo domain. Here we present analysis of SET1, a yeast member of the trithorax gene family that was identified by sequence inspection to encode a 1080-amino acid protein with a C-terminal SET domain. In addition to its SET domain, which is 40–50% identical to those previously characterized, SET1 also shares dispersed but significant similarity to Drosophila and human trithorax homologues. To understand SET1 function(s), we created a null mutant. Mutant strains, although viable, are defective in transcriptional silencing of the silent mating-type loci and telomeres. The telomeric silencing defect is rescued not only by full-length episomal SET1 but also by the conserved SET domain of SET1. set1 mutant strains display other phenotypes including morphological abnormalities, stationary phase defects, and growth and sporulation defects. Candidate genes that may interact with SET1 include those with functions in transcription, growth, and cell cycle control. These data suggest that yeast SET1, like its SET domain counterparts in other organisms, functions in diverse biological processes including transcription and chromatin structure.

Canine sexual dimorphism in Egyptian Eocene anthropoid primates: Catopithecus and Proteopithecus

Two very small late Eocene anthropoid primates, Catopithecus browni and Proteopithecus sylviae, from Fayum, Egypt show evidence of substantial sexual dimorphism in canine teeth. The degree of dimorphism suggests that these early anthropoids lived in social groups with a polygynous mating system and intense male–male competition. Catopithecus and Proteopithecus are smaller in estimated body size than any living primates showing canine dimorphism. The origin of canine dimorphism and polygyny in anthropoids was not associated with the evolution of large body size.

Correlated effects of sperm competition and postmating female mortality

Adaptations in one sex may impair fitness in the opposite sex. Experiments with Drosophila melanogaster have shown that seminal fluid from the male accessory gland triggers a series of postmating responses in the female, including increased egg laying rate and lower remating propensity, but that accessory gland proteins also increase female death rate. Here, we tested the relationships among the longevity of females mated to males from 51 chromosome-extracted D. melanogaster lines, male-mating ability, and sperm-competitive ability. We found significant differences in longevity of females mated to males of different genotypes, and all mated females showed a higher death rate than control virgin females shortly after mating. Both the age-independent mortality parameter (the intercept of the female's survival function) and the slope of the mortality rate curve were significantly correlated with the proportion of progeny sired by the first male to mate relative to tester males (sperm-defense ability, P1). No significant correlation was found between the proportion of progeny sired by the second-mating male relative to tester males (sperm-offense ability, P2) and any mortality parameter. Our results support the hypothesis of a tradeoff between defensive sperm-competitive ability of males and life-history parameters of mated females.

A role for heterodimerization in nuclear localization of a homeodomain protein

The A mating type genes of the mushroom Coprinus cinereus encode two families of dissimilar homeodomain proteins (HD1 and HD2). The proteins heterodimerize when mating cells fuse to generate a transcriptional regulator that promotes expression of genes required for early steps in sexual development. In previous work we showed that heterodimerization brings together different functional domains of the HD1 and HD2 proteins; a potential activation domain at the C terminus of the HD1 protein and an essential HD2 DNA-binding motif. Two predicted nuclear localization signals (NLS) are present in the HD1 protein but none are in the HD2 protein. We deleted each NLS separately from an HD1 protein and showed that one (NLS1) is essential for normal heterodimer function. Fusion of the NLS sequences to the C terminus of an HD2 protein compensated for their deletion from the HD1 protein partner and permitted the two modified proteins to form a functional transcriptional regulator. The nuclear targeting properties of the A protein NLS sequences were demonstrated by fusing the region that encodes them to the bacterial uidA (β-glucuronidase) gene and showing that β-glucuronidase expression localized to the nuclei of onion epidermal cells. These observations lead to the proposal that heterodimerization regulates entry of the active transcription factor complex to the nucleus.

Quantitative assessment of enzyme specificity in vivo: P2 recognition by Kex2 protease defined in a genetic system

The specificity of the yeast proprotein-processing Kex2 protease was examined in vivo by using a sensitive, quantitative assay. A truncated prepro-α-factor gene encoding an α-factor precursor with a single α-factor repeat was constructed with restriction sites for cassette mutagenesis flanking the single Kex2 cleavage site (-SLDKR↓EAEA-). All of the 19 substitutions for the Lys (P2) residue in the cleavage site were made. The wild-type and mutant precursors were expressed in a yeast strain lacking the chromosomal genes encoding Kex2 and prepro-α-factor. Cleavage of the 20 sites by Kex2, expressed at the wild-type level, was assessed by using a quantitative-mating assay with an effective range greater than six orders of magnitude. All substitutions for Lys at P2 decreased mating, from 2-fold for Arg to >106-fold for Trp. Eviction of the Kex2-encoding plasmid indicated that cleavage of mutant sites by other cellular proteases was not a complicating factor. Mating efficiencies of strains expressing the mutant precursors correlated well with the specificity (kcat/KM) of purified Kex2 for comparable model peptide substrates, validating the in vivo approach as a quantitative method. The results support the conclusion that KM, which is heavily influenced by the nature of the P2 residue, is a major determinant of cleavage efficiency in vivo. P2 preference followed the rank order: Lys > Arg > Thr > Pro > Glu > Ile > Ser > Ala > Asn > Val > Cys > AsP > Gln > Gly > His > Met > Leu > Tyr > Phe > Trp.

The Sda1 Protein Is Required for Passage through Start

We have used affinity chromatography to identify proteins that interact with Nap1, a protein previously shown to play a role in mitosis. Our studies demonstrate that a highly conserved protein called Sda1 binds to Nap1 both in vitro and in vivo. Loss of Sda1 function causes cells to arrest uniformly as unbudded cells that do not increase significantly in size. Cells arrested by loss of Sda1 function have a 1N DNA content, fail to produce the G1 cyclin Cln2, and remain responsive to mating pheromone, indicating that they arrest in G1 before Start. Expression of CLN2 from a heterologous promoter in temperature-sensitive sda1 cells induces bud emergence and polarization of the actin cytoskeleton, but does not induce cell division, indicating that the sda1 cell cycle arrest phenotype is not due simply to a failure to produce the G1 cyclins. The Sda1 protein is absent from cells arrested in G0 and is expressed before Start when cells reenter the cell cycle, further suggesting that Sda1 functions before Start. Taken together, these findings reveal that Sda1 plays a critical role in G1 events. In addition, these findings suggest that Nap1 is likely to function during G1. Consistent with this, we have found that Nap1 is required for viability in cells lacking the redundant G1 cyclins Cln1 and Cln2. In contrast to a previous study, we have found no evidence that Sda1 is required for the assembly or function of the actin cytoskeleton. Further characterization of Sda1 is likely to provide important clues to the poorly understood mechanisms that control passage through G1.

Genomic evidence for a complete sexual cycle in Candida albicans

Candida albicans is a diploid fungus that has become a medically important opportunistic pathogen in immunocompromised individuals. We have sequenced the C. albicans genome to 10.4-fold coverage and performed a comparative genomic analysis between C. albicans and Saccharomyces cerevisiae with the objective of assessing whether Candida possesses a genetic repertoire that could support a complete sexual cycle. Analyzing over 500 genes important for sexual differentiation in S. cerevisiae, we find many homologues of genes that are implicated in the initiation of meiosis, chromosome recombination, and the formation of synaptonemal complexes. However, others are striking in their absence. C. albicans seems to have homologues of all of the elements of a functional pheromone response pathway involved in mating in S. cerevisiae but lacks many homologues of S. cerevisiae genes for meiosis. Other meiotic gene homologues in organisms ranging from filamentous fungi to Drosophila melanogaster and Caenorhabditis elegans were also found in the C. albicans genome, suggesting potential alternative mechanisms of genetic exchange.

The second STE12 homologue of Cryptococcus neoformans is MATa-specific and plays an important role in virulence

Cryptococcus neoformans STE12α, a homologue of Saccharomyces cerevisiae STE12, exists only in MATα strains. We identified another STE12 homologue, STE12a, which is MATa specific. As in the case with Δste12α, the mating efficiency for Δste12a was reduced significantly. The Δste12a strains surprisingly still mated with Δste12α strains. In MATα strains, STE12a functionally complemented STE12α for mating efficacy, haploid fruiting, and regulation of capsule size in the mouse brain. Furthermore, when STE12a was replaced with two copies of STE12α, the resulting MATa strain produced hyphae on filament agar. STE12a regulates mRNA levels of several genes that are important for virulence including CNLAC1 and CAP genes. STE12a also modulates enzyme activities of phospholipase and superoxide dismutase. Importantly, deletion of STE12a markedly reduced the virulence in mice, as is the case with STE12α. Brain smears of mice infected with the Δste12a strain showed yeast cells with a considerable reduction in capsule size compared with those infected with STE12a strains. When the disrupted locus of ste12a was replaced with a wild-type STE12a gene, both in vivo and in vitro mutant phenotypes were reversed. These results suggest that STE12a and STE12α have similar functions, and that the mating type of the cells influences the alleles to exert their biological effects. C. neoformans, thus, is the first fungal species that contains a mating-type-specific STE12 homologue in each mating type. Our results demonstrate that mating-type-specific genes are not only important for saprobic reproduction but also play an important role for survival of the organism in host tissue.

Characterization of Chlamydomonas reinhardtii Zygote-Specific cDNAs That Encode Novel Proteins Containing Ankyrin Repeats and WW Domains1

Genes that are expressed only in the young zygote are considered to be of great importance in the development of an isogamous green alga, Chlamydomonas reinhardtii. Clones representing the Zys3 gene were isolated from a cDNA library prepared using zygotes at 10 min after fertilization. Sequencing of Zys3 cDNA clones resulted in the isolation of two related molecular species. One of them encoded a protein that contained two kinds of protein-to-protein interaction motifs known as ankyrin repeats and WW domains. The other clone lacked the ankyrin repeats but was otherwise identical. These mRNA species began to accumulate simultaneously in cells beginning 10 min after fertilization, and reached maximum levels at about 4 h, after which time levels decreased markedly. Genomic DNA gel-blot analysis indicated that Zys3 was a single-copy gene. The Zys3 proteins exhibited parallel expression to the Zys3 mRNAs at first, appearing 2 h after mating, and reached maximum levels at more than 6 h, but persisted to at least 1 d. Immunocytochemical analysis revealed their localization in the endoplasmic reticulum, which suggests a role in the morphological changes of the endoplasmic reticulum or in the synthesis and transport of proteins to the Golgi apparatus or related vesicles.

Roles of a Fimbrin and an α-Actinin-like Protein in Fission Yeast Cell Polarization and Cytokinesis

Eukaryotic cells contain many actin-interacting proteins, including the α-actinins and the fimbrins, both of which have actin cross-linking activity in vitro. We report here the identification and characterization of both an α-actinin-like protein (Ain1p) and a fimbrin (Fim1p) in the fission yeast Schizosaccharomyces pombe. Ain1p localizes to the actomyosin-containing medial ring in an F-actin–dependent manner, and the Ain1p ring contracts during cytokinesis. ain1 deletion cells have no obvious defects under normal growth conditions but display severe cytokinesis defects, associated with defects in medial-ring and septum formation, under certain stress conditions. Overexpression of Ain1p also causes cytokinesis defects, and the ain1 deletion shows synthetic effects with other mutations known to affect medial-ring positioning and/or organization. Fim1p localizes both to the cortical actin patches and to the medial ring in an F-actin–dependent manner, and several lines of evidence suggest that Fim1p is involved in polarization of the actin cytoskeleton. Although a fim1 deletion strain has no detectable defect in cytokinesis, overexpression of Fim1p causes a lethal cytokinesis defect associated with a failure to form the medial ring and concentrate actin patches at the cell middle. Moreover, an ain1 fim1 double mutant has a synthetical-lethal defect in medial-ring assembly and cell division. Thus, Ain1p and Fim1p appear to have an overlapping and essential function in fission yeast cytokinesis. In addition, protein-localization and mutant-phenotype data suggest that Fim1p, but not Ain1p, plays important roles in mating and in spore formation.

Y chromosome polymorphism is a strong determinant of male fitness in Drosophila melanogaster

In many species, the Y (or W) chromosome carries relatively few functional genes. This observation motivates the null hypothesis that the Y will be a minor contributor to genetic variation for fitness. Previous data and theory supported the null hypothesis, but evidence presented here shows that the Y of Drosophila melanogaster is a major determinant of a male's total fitness, with standing genetic variation estimated to be 68% of that of an entire X/autosome genomic haplotype. Most Y-linked genes are expressed during spermatogenesis, and correspondingly, we found that the Y influences fitness primarily through its effect on a male's reproductive success (sperm competition and/or mating success) rather than his egg-to-adult viability. But the fitness of a Y highly depended on the genetic makeup of its bearer, reverting from high to low in different genetic backgrounds. This pattern leads to large epistatic (inconsistent among backgrounds) but no additive (consistent among backgrounds) Y-linked genetic variance for fitness. On a microevolutionary scale, the observed large epistatic variation on the Y substantially reduces heritable variation for fitness among males, and on a macroevolutionary scale, the Y produces strong selection for genomic rearrangements that move interacting genes onto the nonrecombining region of the Y.

Traumatic insemination and sexual conflict in the bed bug Cimex lectularius

The bed bug, Cimex lectularius, has a unique mode of copulation termed “traumatic” insemination [Carayon, J. (1966) in Monograph of the Cimicidae, ed. Usinger, R. (Entomol. Soc. Am., Philadelphia), pp. 81–167] during which the male pierces the female's abdominal wall with his external genitalia and inseminates into her body cavity [Carayon, J. (1966) in Monograph of the Cimicidae, ed. Usinger, R. (Entomol. Soc. Am., Philadelphia), pp. 81–167]. Under controlled natural conditions, traumatic insemination was frequent and temporally restricted. We show for the first time, to our knowledge, that traumatic insemination results in (i) last-male sperm precedence, (ii) suboptimal remating frequencies for the maintenance of female fertility, and (iii) reduced longevity and reproductive success in females. Experimental females did not receive indirect benefits from multiple mating. We conclude that traumatic insemination is probably a coercive male copulatory strategy that results in a sexual conflict of interests.

Assortative fertilization in Drosophila

The concept of gametic isolation has its origins in the 1937 edition of T. Dobzhansky’s Genetics and the Origin of Species. Involving either positive assortative fertilization (as opposed to self-incompatibility) or negative assortative fertilization, it occurs after mating but prior to fertilization. Gametic isolation is generally subsumed under either prezygotic or postmating isolation and thus has not been the subject of extensive investigation. Examples of assortative fertilization in Drosophila are reviewed and compared with those of other organisms. Potential mechanisms leading to assortative fertilization are discussed, as are their evolutionary implications.