Meiotic recombination and chromosome segregation in Schizosaccharomyces pombe

In most organisms homologous recombination is vital for the proper segregation of chromosomes during meiosis, the formation of haploid sex cells from diploid precursors. This review compares meiotic recombination and chromosome segregation in the fission yeast Schizosaccharomyces pombe and the distantly related budding yeast Saccharomyces cerevisiae, two especially tractable microorganisms. Certain features, such as the occurrence of DNA breaks associated with recombination, appear similar, suggesting that these features may be common in eukaryotes. Other features, such as the role of these breaks and the ability of chromosomes to segregate faithfully in the absence of recombination, appear different, suggesting multiple solutions to the problems faced in meiosis.

Identification of an autoimmune serum containing antibodies against the Barr body

Transcriptional inactivation of one X chromosome in mammalian female somatic cells leads to condensation of the inactive X chromosome into the heterochromatic sex chromatin, or Barr body. Little is known about the molecular composition and structure of the Barr body or the mechanisms leading to its formation in female nuclei. Because human sera from patients with autoimmune diseases often contain antibodies against a variety of cellular components, we reasoned that some autoimmune sera may contain antibodies against proteins associated with the Barr body. Therefore, we screened autoimmune sera by immunofluorescence of human fibroblasts and identified one serum that immunostained a distinct nuclear structure with a size and nuclear localization consistent with the Barr body. The number of these structures was consistent with the number of Barr bodies expected in diploid female fibroblasts containing two to five X chromosomes. Immunostaining with the serum followed by fluorescence in situ hybridization with a probe against XIST RNA demonstrated that the major fluorescent signal from the autoantibody colocalized with XIST RNA. Further analysis of the serum showed that it stains human metaphase chromosomes and a nuclear structure consistent with the inactive X in female mouse fibroblasts. However, it does not exhibit localization to a Barr body-like structure in female mouse embryonic stem cells or in cells from female mouse E7.5 embryos. The lack of staining of the inactive X in cells from female E7.5 embryos suggests the antigen(s) may be involved in X inactivation at a stage subsequent to initiation of X inactivation. This demonstration of an autoantibody recognizing an antigen(s) associated with the Barr body presents a strategy for identifying molecular components of the Barr body and examining the molecular basis of X inactivation.

Molecular markers reveal that population structure of the human pathogen Candida albicans exhibits both clonality and recombination.

The life history of Candida albicans presents an enigma: this species is thought to be exclusively asexual, yet strains show extensive phenotypic variation. To address the population genetics of C. albicans, we developed a genetic typing method for codominant single-locus markers by screening randomly amplified DNA for single-strand conformation polymorphisms. DNA fragments amplified by arbitrary primers were initially screened for single-strand conformation polymorphisms and later sequenced using locus-specific primers. A total of 12 single base mutations and insertions were detected from six out of eight PCR fragments. Patterns of sequence-level polymorphism observed for individual strains detected considerable heterozygosity at the DNA sequence level, supporting the view that most C. albicans strains are diploid. Population genetic analyses of 52 natural isolates from Duke University Medical Center provide evidence for both clonality and recombination in C. albicans. Evidence for clonality is supported by the presence of several overrepresented genotypes, as well as by deviation of genotypic frequencies from random (Hardy-Weinberg) expectations. However, tests for nonrandom association of alleles across loci reveal less evidence for linkage disequilibrium than expected for strictly clonal populations. Although C. albicans populations are primarily clonal, evidence for recombination suggests that sexual reproduction or some other form of genetic exchange occurs in this species.

Half tetrad analysis in alfalfa using multiple restriction fragment length polymorphism markers.

A maximum likelihood approach of half tetrad analysis (HTA) based on multiple restriction fragment length polymorphism (RFLP) markers was developed. This procedure estimates the relative frequencies of 2n gametes produced by mechanisms genetically equivalent to first division restitution (FDR) or second division restitution and simultaneously locates the centromere within a linkage group of RFLP marker loci. The method was applied to the diploid alfalfa clone PG-F9 (2n = 2x = 16) previously selected because of its high frequency of 2n egg production. HTA was based on four RFLP loci for which PG-F9 was heterozygous with codominant alleles that were absent in the tetraploid tester. Models including three linked and one unlinked RFLP loci were developed and tested. Results of the HTA showed that PG-F9 produced 6% FDR and 94% second division restitution 2n eggs. Information from a marker locus belonging to one linkage group was used to more precisely locate the centromere on a different linkage group. HTA, together with previous cytological analysis, indicated that in PG-F9, FDR 2n eggs are likely produced by diplospory, a mechanism common among apomictic species. The occurrence of FDR 2n eggs in plant species and their importance for crop evolution and breeding is discussed together with the potential applicability of multilocus HTA in the study of reproductive mutants.

Muc1, a mucin-like protein that is regulated by Mss10, is critical for pseudohyphal differentiation in yeast.

Pseudohyphal differentiation in Saccharomyces cerevisiae was first described as a response of diploid cells to nitrogen limitation. Here we report that haploid and diploid starch-degrading S. cerevisiae strains were able to switch from a yeast form to a filamentous pseudohyphal form in response to carbon limitation in the presence of an ample supply of nitrogen. Two genes, MSS10 and MUC1, were cloned and shown to be involved in pseudohyphal differentiation and invasive growth. The deletion of MSS10 resulted in extremely reduced amounts of pseudohyphal differentiation and invasive growth, whereas the deletion of MUC1 abolished pseudohyphal differentiation and invasive growth completely. Mss10 appears to be a transcriptional activator that responds to nutrient limitation and coregulates the expression of MUC1 and the STA1-3 glucoamylase genes, which are involved in starch degradation. MUC1 encodes a 1367-amino acid protein, containing several serine/threonine-rich repeats. Muc1 is a putative integral membrane-bound protein, similar to mammalian mucin-like membrane proteins that have been implicated to play a role in the ability of cancer cells to invade other tissues.

A change of ploidy can modify epigenetic silencing.

A silent transgene in Arabidopsis thaliana was reactivated in an outcross but not upon selfing of hemizygous plants. This result could only be explained by assuming a genetic difference between the transgene-free gametes of the wild-type and hemizygous transgenic plants, respectively, and led to the discovery of ploidy differences between the parental plants. To investigate whether a change of ploidy by itself can indeed influence gene expression, we performed crosses of diploid or tetraploid plants with a strain containing a single copy of a transgenic resistance gene in an active state. We observed reduced gene expression of the transgene in triploid compared with diploid hybrids. This led to loss of the resistant phenotype at various stages of seedling development in part of the population. The gene inactivation was reversible. Thus, an increased number of chromosomes can result in a new type of epigenetic gene inactivation, creating differences in gene expression patterns. We discuss the possible impact of this finding for genetic diploidization in the light of widespread, naturally occurring polyploidy and polysomaty in plants.

Double-strand break repair in the absence of RAD51 in yeast: a possible role for break-induced DNA replication.

In wild-type diploid cells of Saccharomyces cerevisiae, an HO endonuclease-induced double-strand break (DSB) at the MAT locus can be efficiently repaired by gene conversion using the homologous chromosome sequences. Repair of the broken chromosome was nearly eliminated in rad52delta diploids; 99% lost the broken chromosome. However, in rad51delta diploids, the broken chromosomes were repaired approximately 35% of the time. None of these repair events were simple gene conversions or gene conversions with an associated crossover, instead, they created diploids homozygous for the MAT locus and all markers in the 100-kb region distal to the site of the DSB. In rad51delta diploids, the broken chromosome can apparently be inherited for several generations, as many of these repair events are found as sectored colonies, with one part being repaired and the other part being lost the broken chromosome. Similar events occur in about 2% of wild-type cells. We propose that a broken chromosome end can invade a homologous template in the absence of RAD51 and initiate DNA replication that may extend to the telomere, 100 or more kb away. Such break-induced replication appears to be similar to recombination-initiated replication in bacteria.

Dynamic methylation adjustment and counting as part of imprinting mechanisms.

Monoallelic expression in diploid mammalian cells appears to be a widespread phenomenon, with the most studied examples being X-chromosome inactivation in eutherian female cells and genomic imprinting in the mouse and human. Silencing and methylation of certain sites on one of the two alleles in somatic cells is specific with respect to parental source for imprinted genes and random for X-linked genes. We report here evidence indicating that: (i) differential methylation patterns of imprinted genes are not simply copied from the gametes, but rather established gradually after fertilization; (ii) very similar methylation patterns are observed for diploid, tetraploid, parthenogenic, and androgenic preimplantation mouse embryos, as well as parthenogenic and androgenic mouse embryonic stem cells; (iii) haploid parthenogenic embryos do not show methylation adjustment as seen in diploid or tetraploid embryos, but rather retain the maternal pattern. These observations suggest that differential methylation in imprinted genes is achieved by a dynamic process that senses gene dosage and adjusts methylation similar to X-chromosome inactivation.

Significant competitive advantage conferred by meiosis and syngamy in the yeast Saccharomyces cerevisiae.

The presumed advantages of genetic recombinations are difficult to demonstrate directly. To investigate the effects of recombination and background heterozygosity on competitive ability, we have performed serial-transfer competition experiments between isogenic sexual and asexual strains of the yeast Saccharomyces cerevisiae. The members of these diploid pairs of strains differed only in being heterozygous (sexual) or homozygous (asexual) at the mating type or MAT locus. Competing pairs had either a completely homozygous or a heterozygous genetic background, the latter being heterozygous at many different loci throughout the genome. A round of meiotic recombination (automixis) conferred a large and statistically significant enhancement of competitive ability on sexual strains with a heterozygous genetic background. By contrast, in homozygous background competitions, meiosis decreased the sexual strains' initial relative competitive ability. In all cases, however, the sexual strains outcompeted their isogenic asexual counterparts, whether meiotic recombination had occurred or not. In some genetic backgrounds, this was due in part to an overdominance effect on competitive advantage of heterozygosity at the MAT locus. The advantage of the sexual strains also increased significantly during the course of the homozygous background competitions, particularly when meiosis had occurred. This latter effect either did not occur or was very weak in heterozygous background competitions. Overall, sexual strains with heterozygous genetic backgrounds had a significantly higher initial relative competitive ability than those with homozygous backgrounds. The advantage of mating type heterozygosity in this organism extends far beyond the ability to recombine meiotically.

Molecular cloning of a protein kinase whose phosphorylation is regulated by genetic adhesion during Chlamydomonas fertilization.

Fertilization in Chlamydomonas is initiated by adhesive interactions between gametes of opposite mating types through flagellar glycoproteins called agglutinins. Interactions between these cell adhesion molecules signal for the activation of adenylyl cyclase through an interplay of protein kinases and ultimately result in formation of a diploid zygote. One of the early events during adhesion-induced signal transduction is the rapid inactivation of a flagellar protein kinase that phosphorylates a 48-kDa protein in the flagella. We report the biochemical and molecular characterization of the 48-kDa protein. Experiments using a bacterially expressed fusion protein show that the 48-kDa protein is capable of autophosphorylation on serine and tyrosine and phosphorylation of bovine beta-casein on serine, confirming that the 48-kDa protein itself has protein kinase activity. This protein kinase exhibits limited homology to members of the eukaryotic protein kinase superfamily and may be an important element in a signaling pathway in fertilization.

Genetic heterogeneity in isogenic homozygous clonal zebrafish.

The C32 isogenic homozygous diploid (IHD) strain of the zebrafish (Danio rerio) was found to be polyallelic at a malate dehydrogenase locus (sMdh-A). A variant allele is thought to have arisen via mutation within the past 10 bisexual generations that have maintained the strain since its last gynogenetic cloning event; this unique allele now predominates at the sMdh-A locus. The estimated mutation rate in this species is sufficiently high that long-term genetic homogeneity of its IHD clones cannot be assumed. Researchers using such bisexually maintained clones should be aware that they are not necessarily using genetically uniform subjects. Genetic uniformity of cloned IHD zebrafish will be maximized if experimental subjects are obtained soon after a cloning event.

Haploidy and androgenesis in Drosophila.

Adrogenesis, development from paternal but not maternal chromosomes, can be induced to occur in some organisms, including vertebrates, but has only been reported to occur naturally in interspecific hybrids of the Sicilian stick insect. Androgenesis has not been described previously in Drosophila. We now report the recovery of androgenetic offspring from Drosophila melanogaster females mutant for a gene that affects an oocyte- and embryo-specific alpha-tubulin. The androgenetic exceptions are X,X diploid females that develop from haploid embryos and express paternal markers on all 4 chromosomes. The exceptional females arise by fusion of haploid cleavage nuclei or failure of newly replicated haploid chromosomes to segregate, rather than fusion of two inseminating sperm. The frequency of androgenetic offspring is greatly enhanced by a partial loss-of-function mutant of the NCD (nonclaret disjunctional) microtubule motor protein, suggesting that wild-type NCD functions is pronuclear fusion. Diploidization of haploid paternal chromosome complements results in complete genetic homozygosity, which could facilitate studies of gene variation and mutational load in populations.

The genome of Arabidopsis thaliana.

Arabidopsis thaliana is a small flowering plant that is a member of the family cruciferae. It has many characteristics--diploid genetics, rapid growth cycle, relatively low repetitive DNA content, and small genome size--that recommend it as the model for a plant genome project. The current status of the genetic and physical maps, as well as efforts to sequence the genome, are presented. Examples are given of genes isolated by using map-based cloning. The importance of the Arabidopsis project for plant biology in general is discussed.

Functional haplodiploidy: a mechanism for the spread of insecticide resistance in an important international insect pest.

The coffee berry borer, Hypothenemus hampei, is the most important insect pest of coffee worldwide and has an unusual life history that ensures a high degree of inbreeding. Individual females lay a predominantly female brood within individual coffee berries and because males are flightless there is almost entirely full sib mating. We investigated the genetics associated with this interesting life history after the important discovery of resistance to the cyclodiene type insecticide endosulfan. Both the inheritance of the resistance phenotype and the resistance-associated point mutation in the gamma-aminobutyric acid receptor gene Rdl were examined. Consistent with haplodiploidy, males failed to express and transmit paternally derived resistance alleles. Furthermore, while cytological examination revealed that males are diploid, one set of chromosomes was condensed, and probably nonfunctional, in the somatic cells of all males examined. Moreover, although two sets of chromosomes were present in primary spermatocytes, the chromosomes failed to pair before the single meiotic division, and only one set was packaged in sperm. Thus, the coffee berry borer is "functionally" haplodiploid. Its genetics and life history may therefore represent an interesting intermediate step in the evolution of true haplodiploidy. The influence of this breeding system on the spread of insecticide resistance is discussed.

Increased activity of p53 in senescing fibroblasts.

The p53 tumor-suppressor protein binds DNA and activates the expression of a 21-kDa protein that inhibits both the activity of cyclin-dependent kinases and the function of proliferating cell nuclear antigen. Since p21 expression has been reported to increase 10- to 20-fold as human diploid fibroblasts lose the ability to replicate, we examined the expression and activity of p53 during replicative aging. Similar levels of total p53 mRNA and protein were expressed in low-passage (young) and high-passage (old) cells but both DNA binding activity in vitro and transcriptional activity of p53 in vivo were increased severalfold in high-passage cells. While the basis of increased p53 activity is presently unclear, it is not correlated with differential phosphorylation or changes in p53-mouse double minute 2 gene product interactions. These results provide evidence for the activation of a protein involved in the control of cell cycle checkpoints during cellular aging, in the absence of increased expression.