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R E Esposito

Publications and source records attributed to R E Esposito.

At least 19 recordsLinked to original sources
PubMed2004

GermOnline, a cross-species community knowledgebase on germ cell differentiation.

GermOnline provides information and microarray expression data for genes involved in mitosis and meiosis, gamete formation and germ line development across species. The database has been developed, and is being curated and updated, by life scientists in cooperation with bioinformaticists. Information is contributed through an online form using free text, images and the controlled vocabulary developed by the GeneOntology Consortium. Authors provide up to three references in support of their contribution. The database is governed by an international board of scientists to ensure a standardized data format and the highest quality of GermOnline's information content. Release 2.0 provides exclusive access to microarray expression data from Saccharomyces cerevisiae and Rattus norvegicus, as well as curated information on approximately 700 genes from various organisms. The locus report pages include links to external databases that contain relevant annotation, microarray expression and proteome data. Conversely, the Saccharomyces Genome Database (SGD), S.cerevisiae GeneDB and Swiss-Prot link to the budding yeast section of GermOnline from their respective locus pages. GermOnline, a fully operational prototype subject-oriented knowledgebase designed for community annotation and array data visualization, is accessible at http://www.germonline.org. The target audience includes researchers who work on mitotic cell division, meiosis, gametogenesis, germ line development, human reproductive health and comparative genomics.

Animals↗
PubMed2001

A screen for genes required for meiosis and spore formation based on whole-genome expression.

BACKGROUND: Meiosis is the process by which gametes are generated with half the ploidy of somatic cells. This reduction is achieved by three major differences in chromosome behavior during meiosis as compared to mitosis: the production of chiasmata by recombination, the protection of centromere-proximal sister chromatid cohesion, and the monoorientation of sister kinetochores during meiosis I. Mistakes in any of these processes lead to chromosome missegregation. RESULTS: To identify genes involved in meiotic chromosome behavior in Saccharomyces cerevisiae, we deleted 301 open reading frames (ORFs) which are preferentially expressed in meiotic cells according to microarray gene expression data. To facilitate the detection of chromosome missegregation mutants, chromosome V of the parental strain was marked by GFP. Thirty-three ORFs were required for the formation of wild-type asci, eight of which were needed for proper chromosome segregation. One of these (MAM1) is essential for the monoorientation of sister kinetochores during meiosis I. Two genes (MND1 and MND2) are implicated in the recombination process and another two (SMA1 and SMA2) in prospore membrane formation. CONCLUSIONS: Reverse genetics using gene expression data is an effective method for identifying new genes involved in specific cellular processes.

Cell Nucleus↗
PubMed2001

Identification of the Sin3-binding site in Ume6 defines a two-step process for conversion of Ume6 from a transcriptional repressor to an activator in yeast.

The DNA-binding protein Ume6 is required for both repression and activation of meiosis-specific genes, through interaction with the Sin3 corepressor and Rpd3 histone deacetylase and the meiotic activator Ime1. Here we show that fusion of a heterologous activation domain to Ume6 is unable to convert it into a constitutive activator of early meiotic gene transcription, indicating that an additional function is needed to overcome repression at these promoters. Mutations in UME6 allowing the fusion to activate lie in a predicted amphipathic alpha helix and specifically disrupt interaction with Sin3 but not with Teal, an activator of Ty transcription also found to interact with Ume6 in a two-hybrid screen. The mutations cause a loss of repression by Ume6 and precisely identify the Ume6 Sin3-binding domain, which we show interacts with the paired amphipathic helix 2 region of Sin3. Analysis of these mutants indicates that conversion of Ume6 to an activator involves two genetically distinct steps that act to relieve Sin3-mediated repression and provide an activation domain to Ume6. The mutants further demonstrate that premature expression and lack of subsequent rerepression of Ume6-Sin3-regulated genes are not deleterious to meiotic progression and suggest that the essential role of Sin3 in meiosis is independent of Ume6. The model for Ume6 function arising from these studies indicates that Ume6 is similar in many respects to metazoan regulators that utilize Sin3, such as the Myc-Mad-Max system and nuclear hormone receptors, and provides new insights into the control of transcriptional repression and activation by the Ume6-URS1 regulatory complex in yeast.

Amino Acid Sequence↗
PubMed2000

Spo1, a phospholipase B homolog, is required for spindle pole body duplication during meiosis in Saccharomyces cerevisiae.

The SPO1 gene was cloned and shown to encode an early meiotic transcript specifying a nuclear protein with extensive similarity to fungal and vertebrate phospholipase enzymes. Alteration of a conserved serine residue in the putative phospholipase active site, and presence of the spo1-1 temperature-sensitive mutation, which resides near this site, each result in loss of SPO1 function. The phenotype of a complete deletion indicates that SPO1 is dispensable for vegetative growth, premeiotic DNA synthesis and meiotic recombination. In contrast, it is required for Meiosis I (MI) and Meiosis II (MII) chromosome segregation and spore formation. In a null mutant approximately 75% of cells arrest early at MI spindle pole body (SPB) duplication, approximately 20% arrest at MII, and approximately 5% arrest at spore formation. Progression beyond the first arrest point suggests the existence of functions partially redundant to Spo1 and that Spo1 is required at multiple stages. At present SPO1 is the only known gene required for SPB duplication in meiosis but not in mitosis. Its product may thus play a regulatory (rather than a structural) role in SPB function. The transcriptional program in the spo1 null is similar to the wild type early in meiosis but is significantly delayed at later stages of sporulation. A single gene, CWP1, was recovered as a multicopy suppressor of the spo1 null. CWP1 encodes a cell wall protein with a glycolipid moiety. We propose that, when modified by other lipases, this moiety may substitute for the product(s) of Spo1p lipase activity in meiosis. Based on the similarity of Spo1p to phospholipase B enzymes, its unique role in SPB duplication, and pleiotropic effects on MII, late gene expression and spore formation, we propose that the Spo1 protein participates in a novel meiotic pathway that functions through the SPB to coordinate nuclear division with spore development.

Cell Nucleus↗
PubMed2000

The core meiotic transcriptome in budding yeasts.

We used high-density oligonucleotide microarrays to analyse the genomes and meiotic expression patterns of two yeast strains, SK1 and W303, that display distinct kinetics and efficiencies of sporulation. Hybridization of genomic DNA to arrays revealed numerous gene deletions and polymorphisms in both backgrounds. The expression analysis yielded approximately 1,600 meiotically regulated genes in each strain, with a core set of approximately 60% displaying similar patterns in both strains. Most of these (95%) are MATa/MATalpha-dependent and are not similarly expressed in near-isogenic meiosis-deficient controls. The transcript profiles correlate with the distribution of defined meiotic promoter elements and with the time of known gene function.

Binding Sites↗
PubMed2000

Recombination can partially substitute for SPO13 in regulating meiosis I in budding yeast.

Recombination and chromosome synapsis bring homologous chromosomes together, creating chiasmata that ensure accurate disjunction during reductional division. SPO13 is a key gene required for meiosis I (MI) reductional segregation, but dispensable for recombination, in Saccharomyces cerevisiae. Absence of SPO13 leads to single-division meiosis where reductional segregation is largely eliminated, but other meiotic events occur relatively normally. This phenotype allows haploids to produce viable meiotic products. Spo13p is thought to act by delaying nuclear division until sister centromeres/chromatids undergo proper cohesion for segregation to the same pole at MI. In the present study, a search for new spo13-like mutations that allow haploid meiosis recovered only new spo13 alleles. Unexpectedly, an unusual reduced-expression allele (spo13-23) was recovered that behaves similarly to a null mutant in haploids but to a wild-type allele in diploids, dependent on the presence of recombining homologs rather than on a diploid genome. This finding demonstrates that in addition to promoting accurate homolog disjunction, recombination can also function to partially substitute for SPO13 in promoting sister cohesion. Analysis of various recombination-defective mutants indicates that this contribution of recombination to reductional segregation requires full levels of crossing over. The implications of these results regarding SPO13 function are discussed.

Alleles↗
PubMed1995

UME6 is a central component of a developmental regulatory switch controlling meiosis-specific gene expression.

The UME6 gene of Saccharomyces cerevisiae was identified as a mitotic repressor of early meiosis-specific gene expression. It encodes a Zn2Cys6 DNA-binding protein which binds to URS1, a promoter element needed for both mitotic repression and meiotic induction of early meiotic genes. This paper demonstrates that a complete deletion of UME6 causes not only vegetative derepression of early meiotic genes during vegetative growth but also a significant reduction in induction of meiosis-specific genes, accompanied by a severe defect in meiotic progression. After initiating premeiotic DNA synthesis the vast majority of cells (approximately 85%) become arrested in prophase and fail to execute recombination; a minority of cells (approximately 15%) complete recombination and meiosis I, and half of these form asci. Quantitative analysis of the same early meiotic transcripts that are vegetatively derepressed in the ume6 mutant, SPO11, SPO13, IME2, and SPO1, indicates a low level of induction in meiosis above their vegetative derepressed levels. In addition, the expression of later meiotic transcripts, SPS2 and DIT1, is significantly delayed and reduced. The expression pattern of early meiotic genes in ume6-deleted cells is strikingly similar to that of early meiotic genes with promoter mutations in URS1. These results support the view that UME6 and URS1 are part of a developmental switch that controls both vegetative repression and meiotic induction of meiosis-specific genes.

DNA Replication↗
PubMed1995

UME6, a negative regulator of meiosis in Saccharomyces cerevisiae, contains a C-terminal Zn2Cys6 binuclear cluster that binds the URS1 DNA sequence in a zinc-dependent manner.

UME6 is a protein of 836 amino acids from Saccharomyces cerevisiae that acts as a repressor and activator of several early meiotic genes. UME6 contains, near the C-terminus, the amino acid sequence-771C-X2-C-X6-C-X6-C-X2-C-X6-C-, in which the spacings of the six Cys residues are identical to those found in 39 N-terminal Cys-rich DNA binding subdomains of fungal transcription factors. This sequence has been shown in GAL4 and other proteins to form a zinc binuclear cluster. In spite of the different location, the C-rich sequence, cloned and over-produced within the last 111 amino acid residues of UME6, UME6(111), forms a binuclear cluster and exhibits a Zn-dependent binding to the URS1 DNA sequence. The latter, TAGCCGCCGA, is required for the repression or activation of meiosis-specific genes by UME6. UME6(111) contains 1.8 +/- 0.4 mol Zn/mol protein and the Zn can be exchanged for Cd to yield a protein containing 1.9 +/- 0.1 mol Cd/mol protein. At 5 degrees C, 113Cd2UME6(111) shows two 113Cd NMR signals, with chemical shifts of 699 and 689 ppm, similar to those observed for 113Cd2GAL4(149). The magnitude of these chemical shifts suggests that each 113Cd nucleus is coordinated to four -S- ligands, compatible with a 113Cd2 cluster structure in which two thiolates from bridging ligands. The entire UME6 gene has been cloned and overexpressed and binds more tightly to the URS1 sequence than the zinc binuclear cluster domain alone. DNase I footprints of UME6 on URS1-containing DNA show that the protein protects the phosphodiesters of the 5'-CCGCCG-3' region within the URS1 sequence.

Amino Acid Sequence↗
PubMed1994

Reversal of cell determination in yeast meiosis: postcommitment arrest allows return to mitotic growth.

When yeast from the early stages of meiosis are transferred from sporulation to growth medium, they can reenter the mitotic cell cycle directly. In contrast, cells from later stages of meiosis (after the initiation of the first nuclear division) will complete meiosis and sporulation despite the shift to growth medium, a phenomenon known as "commitment to meiosis." This study reports the surprising finding that when the normal progression of meiosis is arrested, cells from later stages of meiosis can return to growth. Cells were arrested after the first or second meiotic division by three independent means: the spo14 mutation, the spo3-1 mutation, and a high-temperature arrest of wild-type cells. In every case, the arrested cells were able to form buds after transfer to growth medium. These cells, however, experienced a delay upon return to growth relative to uncommitted cells. We propose that the commitment phenomenon results from a transient delay of mitotic growth, which occurs specifically during meiosis, and that commitment does not involve an irreversible inhibition of mitosis as previously thought.

Cell Nucleus↗
PubMed1994

UME6 is a key regulator of nitrogen repression and meiotic development.

This report describes the identification, cloning, and molecular analysis of UME6 (CAR80/CARGRI), a key transcriptional regulator of early meiotic gene expression. Loss of UME6 function results in the accumulation of fully derepressed levels (70- to 100-fold increase above basal level) of early meiotic transcripts during vegetative growth. In contrast, mutations in five previously identified UME loci (UME1 to UME5), result in low to moderate derepression (2- to 10-fold increase) of early meiotic genes. The behavior of insertion and deletion alleles indicates that UME6 is dispensable for mitotic division but is required for meiosis and spore germination. Despite the high level of meiotic gene expression during vegetative growth, the generation times of ume6 mutant haploid and diploid cells are only slightly reduced. However, both ascus formation and spore viability are affected more severely. The UME6 gene encodes a 91-kD protein that contains a C6 zinc cluster motif similar to the DNA-binding domain of GAL4. The integrity of this domain is required for UME6 function. It has been reported recently that a mutation in CAR80 fails to complement an insertion allele of UME6. CAR80 is a gene required for nitrogen repression of the arginine catabolic enzymes. Here, through sequence analysis, we demonstrate that UME6 and CAR80 are identical. Analyses of UME6 mRNA during both nitrogen starvation and meiotic development indicate that its transcription is constitutive, suggesting that regulation of UME6 activity occurs at a post-transcriptional level.

Amino Acid Sequence↗
PubMed1994

SPO13 negatively regulates the progression of mitotic and meiotic nuclear division in Saccharomyces cerevisiae.

The meiosis-specific yeast gene SPO13 has been previously shown to be required to obtain two successive divisions in meiosis. We report here that vegetative expression of this gene causes a CDC28-dependent cell-cycle arrest at mitosis. Overexpression of SPO13 during meiosis causes a transient block to completion of the meiosis I division and suppresses the inability of cdc28ts strains to execute meiosis II. The spo13 defect can be partially suppressed by conditions that slow progression of the first meiotic division. Based on the results presented below, we propose that SPO13 acts as a meiotic timing function by transiently blocking progression through the meiosis I division, thereby allowing (1) coordination of the first division with assembly of the reductional segregation apparatus, and (2) subsequent entry into a second round of segregation to separate replicated sister chromatids without an intervening S-phase.

Cell Cycle↗
PubMed1994

The yeast UME5 gene regulates the stability of meiotic mRNAs in response to glucose.

We reported previously that early meiotic transcripts are highly unstable. These mRNAs exhibit half-lives of approximately 3 min when expressed during vegetative growth in glucose medium and are stabilized twofold during sporulation in acetate medium. Two genes, UME2 and UME5, that regulate the stability of meiosis-specific transcripts have been identified. The wild-type UME5 gene, which has been analyzed in detail, decreases the stability of all meiotic mRNAs tested approximately twofold when expressed during vegetative growth but has no effect on the half-lives of a number of vegetative mRNAs examined. The UME5 gene is dispensable for mitotic and meiotic development. Cells in which the entire UME5 gene has been deleted are viable, although the generation time is slightly longer and sporulation is less efficient. The UME5 transcript is constitutively expressed, and its stability is not autoregulated. The UME5 gene encodes a predicted 63-kDa protein with homology to the family of CDC28 serine/threonine-specific protein kinases. The kinase activity appears to be central to the function of the UME5 protein, since alteration of a highly conserved amino acid in the kinase domain results in a phenotype identical to that of a ume5 deletion. Genetic epistasis studies suggest that the UME2 and UME5 gene products act in the same pathway to regulate meiotic transcript stability. This pathway is independent of deadenylation and translation, two factors known to be important in regulating mRNA turnover. Significantly, the UME5-mediated destabilization of meiotic mRNAs occurs in glucose- but not in acetate-containing medium. Thus, the UME5 gene appears to participate in a glucose signal transduction pathway governing message stability.

Amino Acid Sequence↗
PubMed1993

Regulatory mechanisms in meiosis.

Meiosis can be viewed both as a process of cell differentiation and as a modification of the mitotic cell cycle. Here we describe recent progress in defining a variety of regulatory mechanisms that govern the meiotic divisions. Studies in the yeast Saccharomyces cerevisiae and in higher organisms have led to complementary insights into these controls.

Animals↗
PubMed1993

Meiotic recombination in yeast.

Over the past several years, the yeast Saccharomyces cerevisiae has proven to be an extremely useful model system for understanding how cells acquire high recombinational ability during meiosis. Due to recent advances in the physical monitoring of DNA intermediates during meiosis, new cytological methods for visualization of chromosomes during pairing and exchange, and progress in the identification and analysis of recombination-defective mutants, a general picture of the order and dependencies of specific recombination events is now emerging.

Meiosis↗
PubMed1992

Early meiotic transcripts are highly unstable in Saccharomyces cerevisiae.

Meiosis in Saccharomyces cerevisiae requires the induction of a large number of genes whose mRNAs accumulate at specific times during meiotic development. This study addresses the role of mRNA stability in the regulation of meiosis-specific gene expression. Evidence is provided below demonstrating that the levels of meiotic mRNAs are exquisitely regulated by both transcriptional control and RNA turnover. The data show that (i) early meiotic transcripts are extremely unstable when expressed during either vegetative growth or sporulation, and (ii) transcriptional induction, rather than RNA turnover, is the predominant mechanism responsible for meiosis-specific transcript accumulation. When genes encoding the early meiotic mRNAs are fused to other promoters and expressed during vegetative growth, their mRNA half-lives, of under 3 min, are among the shortest known in S. cerevisiae. Since these mRNAs are only twofold more stable when expressed during sporulation, we conclude that developmental regulation of mRNA turnover can be eliminated as a major contributor to meiosis-specific mRNA accumulation. The rapid degradation of the early mRNAs at all stages of the yeast life cycle, however, suggests that a specific RNA degradation system operates to maintain very low basal levels of these transcripts during vegetative growth and after their transient transcriptional induction in meiosis. Studies to identify specific cis-acting elements required for the rapid degradation of early meiotic transcripts support this idea. A series of deletion derivatives of one early meiosis-specific gene, SPO13, indicate that its mRNA contains determinants, located within the coding region, which contribute to the high instability of this transcript. Translation is another component of the degradation mechanism since frameshift and nonsense mutations within the SPO13 mRNA stabilize the transcript.

Cloning, Molecular↗