Function In Meiosis Research Articles

Ecm11 protein of yeastSaccharomyces cerevisiaeis regulated by sumoylation during meiosis

Damaged regulation of the small ubiquitin-like modifier (SUMO) system contributes to some human diseases; therefore, it is very important to identify the SUMO targets and to determine the function of their sumoylation. In this study, it is shown that Ecm11 protein in Saccharomyces cerevisiae is modified by SUMO during meiosis. It is known that Ecm11 is required in the early stages of yeast meiosis where its function is related to DNA replication and crossing over. Here it is shown that the level of Ecm11 protein is low in mitosis, but high in meiosis. The highest level of Ecm11 is in the early-middle phase of sporulation. A specific site of sumoylation was identified in Ecm11 at Lys5 and evidence is provided that sumoylation at this site directly regulates Ecm11 function in meiosis. On the other hand, no relationship was observed between sumoylation of Ecm11 and its role during vegetative growth. It was shown that Ecm11 interacts with Siz2 SUMO ligase in a two-hybrid system; although Siz2 is not essential for the Ecm11 sumoylation.

Sequential Loading of Cohesin Subunits during the First Meiotic Prophase of Grasshoppers

The cohesin complexes play a key role in chromosome segregation during both mitosis and meiosis. They establish sister chromatid cohesion between duplicating DNA molecules during S-phase, but they also have an important role during postreplicative double-strand break repair in mitosis, as well as during recombination between homologous chromosomes in meiosis. An additional function in meiosis is related to the sister kinetochore cohesion, so they can be pulled by microtubules to the same pole at anaphase I. Data about the dynamics of cohesin subunits during meiosis are scarce; therefore, it is of great interest to characterize how the formation of the cohesin complexes is achieved in order to understand the roles of the different subunits within them. We have investigated the spatio-temporal distribution of three different cohesin subunits in prophase I grasshopper spermatocytes. We found that structural maintenance of chromosome protein 3 (SMC3) appears as early as preleptotene, and its localization resembles the location of the unsynapsed axial elements, whereas radiation-sensitive mutant 21 (RAD21) (sister chromatid cohesion protein 1, SCC1) and stromal antigen protein 1 (SA1) (sister chromatid cohesion protein 3, SCC3) are not visualized until zygotene, since they are located in the synapsed regions of the bivalents. During pachytene, the distribution of the three cohesin subunits is very similar and all appear along the trajectories of the lateral elements of the autosomal synaptonemal complexes. However, whereas SMC3 also appears over the single and unsynapsed X chromosome, RAD21 and SA1 do not. We conclude that the loading of SMC3 and the non-SMC subunits, RAD21 and SA1, occurs in different steps throughout prophase I grasshopper meiosis. These results strongly suggest the participation of SMC3 in the initial cohesin axis formation as early as preleptotene, thus contributing to sister chromatid cohesion, with a later association of both RAD21 and SA1 subunits at zygotene to reinforce and stabilize the bivalent structure. Therefore, we speculate that more than one cohesin complex participates in the sister chromatid cohesion at prophase I.

Cloning and expression analysis of TSK1, a wheat SKP1 homologue, and functional comparison with Arabidopsis ASK1 in male meiosis and auxin signalling

Plants possess multiple homologues of the SKP1 gene encoding an essential subunit of the SCF ubiquitin ligases, but only ASK1 (Arabidopsis SKP1-like 1) and ASK2 have been characterised genetically. In addition, little is known about the function of SKP1 homologues in monocots. Here we report on a winter wheat homologue of SKP1 named TSK1 (Triticum aestivum SKP1-like 1). Expression analyses revealed that it was expressed predominantly in young roots and floral buds. RNA in situ hybridisation showed that it was expressed in the shoot apical meristem (SAM) and anthers, especially the tapetum and microsporocytes at the time of meiosis. It was also expressed in almost the entire meristematic and elongation zones of the root. These observations indicated that TSK1 might function in dividing cells. The Arabidopsis ask1-1 mutant with overexpressed TSK1 driven by the CaMV 35S promoter exhibited partial fertility, suggesting that TSK1 could partially restore function in meiosis to the ask1-1 mutant. In addition, overexpression of TSK1 in wild type Arabidopsis resulted in changes in auxin responses and auxin-related phenotypes, consistent with a role of ASK1 in Arabidopsis auxin response. These results suggest possible functional conservation between TSK1 and ASK1.

Silencing of the Meiotic Genes SMC1β and STAG3 in Somatic Cells by E2F6

Silencing of the Meiotic Genes SMC1β and STAG3 in Somatic Cells by E2F6

The spindle assembly checkpoint is not essential for CSF arrest of mouse oocytes

In Xenopus oocytes, the spindle assembly checkpoint (SAC) kinase Bub1 is required for cytostatic factor (CSF)-induced metaphase arrest in meiosis II. To investigate whether matured mouse oocytes are kept in metaphase by a SAC-mediated inhibition of the anaphase-promoting complex/cyclosome (APC/C) complex, we injected a dominant-negative Bub1 mutant (Bub1dn) into mouse oocytes undergoing meiosis in vitro. Passage through meiosis I was accelerated, but even though the SAC was disrupted, injected oocytes still arrested at metaphase II. Bub1dn-injected oocytes released from CSF and treated with nocodazole to disrupt the second meiotic spindle proceeded into interphase, whereas noninjected control oocytes remained arrested at metaphase. Similar results were obtained using dominant-negative forms of Mad2 and BubR1, as well as checkpoint resistant dominant APC/C activating forms of Cdc20. Thus, SAC proteins are required for checkpoint functions in meiosis I and II, but, in contrast to frog eggs, the SAC is not required for establishing or maintaining the CSF arrest in mouse oocytes.

Telomere dynamics unique to meiotic prophase: formation and significance of the bouquet.

Telomeres carry out conserved and possibly ancient functions in meiosis. During the specialized prophase of meiosis I, meiotic prophase, telomeres cluster on the nuclear envelope and move the diploid genetic material around within the nucleus so that homologous chromosomes can align two by two and efficiently recombine with precision. This recombination is in turn required for proper segregation of the homologs into viable haploid daughter cells. The meiosis-specific telomere clustering on the nuclear envelope defines the bouquet stage, so named for its resemblance to the stems from a bouquet of cut flowers. Here, a comparative analysis of the literature on meiotic telomeres from a variety of different species illustrates that the bouquet is nearly universal among life cycles with sexual reproduction. The bouquet has been well documented for over 100 years, but our understanding of how it forms and how it functions has only recently begun to increase. Early and recent observations document the timing and provide clues about the functional significance of these striking telomere movements.

Whi3 binds the mRNA of the G1 cyclin CLN3 to modulate cell fate in budding yeast.

Eukaryotic cells commit in G1 to a new mitotic cycle or to diverse differentiation processes. Here we show that Whi3 is a negative regulator of Cln3, a G1 cyclin that promotes transcription of many genes to trigger the G1/S transition in budding yeast. Whi3 contains an RNA-recognition motif that specifically binds the CLN3 mRNA, with no obvious effects on Cln3 levels, and localizes the CLN3 mRNA into discrete cytoplasmic foci. This is the first indication that G1 events may be regulated by locally restricting the synthesis of a cyclin. Moreover, Whi3 is also required for restraining Cln3 function in meiosis, filamentation, and mating, thus playing a key role in cell fate determination in budding yeast.

Mammalian STAG3 is a cohesin specific to sister chromatid arms in meiosis I.

Cohesins, which have been characterized in budding yeast and Xenopus, are multisubunit protein complexes involved in sister chromatid cohesion. Regulation of the interactions among different cohesin subunits and the assembly/disassembly of the cohesin complex to chromatin are key steps in chromosome segregation. We previously characterized the mammalian STAG3 protein as a component of the synaptonemal complex that is specifically expressed in germinal cells, although its function in meiosis remains unknown. Here we show that STAG3 has a role in sister chromatid arm cohesion during mammalian meiosis I. Immunofluorescence results in prophase I cells suggest that STAG3 is a component of the axial/lateral element of the synaptonemal complex. In metaphase I, STAG3 is located at the interchromatid domain and is absent from the chiasma region. In late anaphase I and the later stages of meiosis, STAG3 is not detected. STAG3 interacts with the structural maintenance chromosome proteins SMC1 and SMC3, which have been reported to be subunits of the mitotic cohesin complex. We propose that STAG3 is a sister chromatid arm cohesin that is specific to mammalian meiosis I.

Human sperm telomere-binding complex involves histone H2B and secures telomere membrane attachment.

Telomeres are unique chromatin domains located at the ends of eukaryotic chromosomes. Telomere functions in somatic cells involve complexes between telomere proteins and TTAGGG DNA repeats. During the differentiation of germ-line cells, telomeres undergo significant reorganization most likely required for additional specific functions in meiosis and fertilization. A telomere-binding protein complex from human sperm (hSTBP) has been isolated by detergent treatment and was partially purified. hSTBP specifically binds double-stranded telomeric DNA and does not contain known somatic telomere proteins TRF1, TRF2, and Ku. Surprisingly, the essential component of this complex has been identified as a specific variant of histone H2B. Indirect immunofluorescence shows punctate localization of H2B in sperm nuclei, which in part coincides with telomeric DNA localization established by fluorescent in situ hybridization. Anti-H2B antibodies block interactions of hSTBP with telomere DNA, and spH2B forms specific complex with this DNA in vitro, indicating that this protein plays a role in telomere DNA recognition. We propose that hSTBP participates in the membrane attachment of telomeres that may be important for ordered chromosome withdrawal after fertilization.

Dmc1 of Schizosaccharomyces pombe plays a role in meiotic recombination.

We report here a Schizosaccharomyces pombe gene (dmc1(+)) that resembles budding yeast DMC1 in the region immediately upstream of the rad24(+) gene. We showed by northern and Southern blot analysis that dmc1(+) and rad24(+) are co-transcribed as a bicistronic mRNA of 2.8 kb with meiotic specificity, whereas rad24(+) itself is constitutively transcribed as a 1.0-kb mRNA species during meiosis. Induction of the bicistronic transcript is under the control of a meiosis-specific transcription factor, Ste11. Disruption of both dmc1(+) and rad24(+) had no effect on mitosis or spore formation, and dmc1Delta cells displayed no change in sensitivity to UV or gamma irradiation relative to the wild type. Tetrad analysis indicated that Dmc1 is involved in meiotic recombination. Analysis of gene conversion frequencies using single and double mutants of dmc1 and rhp51 indicated that both Dmc1 and Rhp51 function in meiotic gene conversion. These observations, together with a high level of sequence identity, indicate that the dmc1(+) gene of S. POMBE: is a structural homolog of budding yeast DMC1, sharing both similar and distinct functions in meiosis.

Increased telomere size in sperm cells of mammals with long terminal (TTAGGG)n arrays.

An increase in the length of telomeres in human sperm compared to somatic cells has long been noted and considered within a popular hypothesis involving telomere shortening and cell aging. In the present study we determined telomere length in two species with long terminal TTAGGG arrays--bovine and porcine. Using several independent methods we demonstrate that the telomeres in the sperm of human, porcine and bovine are elongated by 69%, 24%, and 14%, respectively, in comparison with somatic tissues. Therefore, increased sperm telomere length is a feature preserved throughout mammalian evolution. The biological role of this phenomenon is discussed in the context of telomere functions in meiosis and fertilization.

Increased telomere size in sperm cells of mammals with long terminal (TTAGGG)n arrays

An increase in the length of telomeres in human sperm compared to somatic cells has long been noted and considered within a popular hypothesis involving telomere shortening and cell aging. In the present study we determined telomere length in two species with long terminal TTAGGG arrays—bovine and porcine. Using several independent methods we demonstrate that the telomeres in the sperm of human, porcine and bovine are elongated by 69%, 24%, and 14%, respectively, in comparison with somatic tissues. Therefore, increased sperm telomere length is a feature preserved throughout mammalian evolution. The biological role of this phenomenon is discussed in the context of telomere functions in meiosis and fertilization. Mol. Reprod. Dev. 51:98–104, 1998.© 1998 Wiley-Liss, Inc.

Protein Phosphatase Type 1 Interacts with Proteins Required for Meiosis and Other Cellular Processes in Saccharomyces cerevisiae

Protein phosphatase type I (PP1) is involved in diverse cellular processes, and its activity toward specific substrates is thought to be controlled by different regulatory or targeting subunits. To identify regulatory subunits and substrates of the Saccharomyces cerevisiae PP1, encoded by GLC7, we used the two-hybrid system to detect interacting proteins. Among the many proteins identified were Gac1, a known glycogen regulatory subunit, and a protein with homology to Gac1. We also characterized a new gene designated GIP1, for Glc7-interacting protein. We show that a Gip1 fusion protein coimmunoprecipitates with PP1 from cell extracts. Molecular and genetic analyses indicate that GIP1 is expressed specifically during meiosis, affects transcription of late meiotic genes, and is essential for sporulation. Thus, the Gip1 protein is a candidate for a meiosis-specific substrate or regulator of PP1. Finally, we recovered two genes, RED1 and SCD5, with roles in meiosis and the vesicular secretory pathway, respectively. These results provide strong evidence implicating PP1 function in meiosis. In addition, this study indicates that the two-hybrid system offers a promising approach to understanding the multiple roles and interactions of PP1 in cellular regulation.

IME1 gene encodes a transcription factor which is required to induce meiosis in Saccharomyces cerevisiae

Previous studies have shown that the IME1 gene is required for sporulation and the expression of meiosis specific genes in Saccharomyces cerevisiae. However, sequence analysis has not revealed the precise functional role of the Ime1 protein. By engineering constructs which express various portions of the Ime1p fused to either the DNA binding or transcriptional activation domains of GAL4, we have conclusively demonstrated that IME1 is a transcription factor, apparently required for sporulation to activate the transcription of meiosis specific genes. The full Ime1p, when fused to the GAL4 DNA binding domain, can both activate GAL1-lacZ expression, and complement ime1-0 (a null allele) for the ability to sporulate, and transcriptionally activate IME2, a meiosis specific gene. As successively larger portions of the encoded Ime1p N-terminus are deleted from the GAL4(bd)-IME1 construct, the encoded fusion proteins retain the ability to complement an ime1 null allele, despite a decreasing ability to activate GAL1-lacZ transcription. However, a fusion construct which retains only the last 45 C-terminal amino acids of IME1 provides neither transcriptional activation of GAL1-lacZ nor complementation of ime1-0. Fusion of a GAL4 activation domain to this portion of IME1, results in a construct with a restored ability to complement an ime1-0 allele. This restored ability is dependent upon galactose induction. We conclude, therefore, that IME1 functions in meiosis as a transcriptional activator.

The role of the ameiotic1 gene in the initiation of meiosis and in subsequent meiotic events in maize.

Understanding the initiation of meiosis and the relationship of this event with other key cytogenetic processes are major goals in studying the genetic control of meiosis in higher plants. Our genetic and structural analysis of two mutant alleles of the ameiotic1 gene (am1 and am1-praI) suggest that this locus plays an essential role in the initiation of meiosis in maize. The product of the ameiotic1 gene affects an earlier stage in the meiotic sequence than any other known gene in maize and is important for the irreversible commitment of cells to meiosis and for crucial events marking the passage from premeiotic interphase into prophase I including chromosome synapsis. It appears that the period of ameiotic1 gene function in meiosis at a minimum covers the interval from some point during premeiotic interphase until the early zygotene stage of meiosis. To study the interaction of genes in the progression of meiosis, several double meiotic mutants were constructed. In these double mutants (i) the ameiotic1 mutant allele was brought together with the meiotic mutation (afd1) responsible for the fixation of centromeres in meiosis; and with the mutant alleles of the three meiotic genes that control homologous chromosome segregation (dv1, ms43 and ms28), which impair microtubule organizing center organization, the orientation of the spindle fiber apparatus, and the depolymerization of spindle filaments after the first meiotic division, respectively; (ii) the afd1 mutation was combined with two mutations (dsy1 and as1) affecting homologous pairing; (iii) the ms43 mutation was combined with the as1, the ms28 and the dv1 mutations; and (iv) the ms28 mutation was combined with the dv1 mutation and the ms4 (polymitotic1) mutations. An analysis of gene interaction in the double mutants led us to conclude that the ameiotic1 gene is epistatic over the afd1, the dv1, the ms43 and the ms28 genes but the significance of this relationship requires further analysis. The afd gene appears to function from premeiotic interphase throughout the first meiotic division, but it is likely that its function begins after the start of the ameiotic1 gene expression. The afd1 gene is epistatic over the two synaptic mutations dsy1 and as1 and also over the dv1 mutation. The new ameiotic*-485 and leptotene arrest*-487 mutations isolated from an active Robertson's Mutator stocks take part in the control of the initiation of meiosis.

Stage-specific effects of X-irradiation on yeast meiosis.

Previous work has shown that cdc13 causes meiotic arrest of Saccharomyces cerevisiae following DNA replication by a RAD9-dependent mechanism. In the present work, we have further investigated the implicit effects of chromosomal lesions on progression through meiosis by exposing yeast cells to X-irradiation at various times during sporulation. We find that exposure of RAD9 cells to X-irradiation early in meiosis prevents sporulation, arresting the cells at a stage prior to premeiotic DNA replication. rad9 meiotic cells are much less responsive to X-irradiation damage, completing sporulation after treatment with doses sufficient to cause arrest of RAD9 strains. These findings thereby reveal a RAD9-dependent checkpoint function in meiosis that is distinct from the G2 arrest previously shown to result from cdc13 dysfunction. Analysis of the spores that continued to be produced by either RAD9 or rad9 cultures that were X-irradiated in later stages of sporulation revealed most spores to be viable, even after exposure to radiation doses sufficient to kill most vegetative cells. This finding demonstrates that the lesions induced by X-irradiation at later times fail to trigger the checkpoint function revealed by cdc13 arrest and suggests that the lesions may be subject to repair by serving as intermediates in the recombination process. Strains mutant for chromosomal synapsis and recombination, and therefore defective in meiotic disjunction, were tested for evidence that X-ray-induced lesions might alleviate inviability by promoting recombination. Enhancement of spore viability when spo11 (but not hop 1) diploids were X-irradiated during meiosis indicates that induced lesions may partially substitute for SPO11-dependent functions that are required for the initiation of recombination.

Complementary transcripts from two genes necessary for normal meiosis in the yeast Saccharomyces cerevisiae.

The SPO12 gene, which is required for meiosis I chromosome division during sporulation of the yeast Saccharomyces cerevisiae, has been isolated. DNA sequencing has identified an open reading frame of 173 codons that encodes the putative SPO12 protein and has no significant sequence similarities to known genes. The last 15 amino acids of this putative protein have a high negative charge, which appears to be required for function. A second sporulation-specific gene, designated SPO16, was found adjacent to SPO12 and shown to be necessary for efficient spore formation. The two genes are encoded on opposite DNA strands with only 103 nucleotides between the termination codons. Up to 700 nucleotides of the SPO12 and SPO16 transcripts are complementary, and the 3' untranslated region of the longest SPO16 transcript is complementary to all or nearly all of the SPO12 mRNA. A strain homozygous for an insertion which removes the complementarity between the SPO12 and SPO16 mRNAs has an efficiency of sporulation, number of spores per ascus, and spore viability identical to those of a wild-type strain. The complementarity therefore has either no function or only a subtle function in meiosis and sporulation.

Complementary Transcripts from Two Genes Necessary for Normal Meiosis in the Yeast Saccharomyces cerevisiae

The SPO12 gene, which is required for meiosis I chromosome division during sporulation of the yeast Saccharomyces cerevisiae, has been isolated. DNA sequencing has identified an open reading frame of 173 codons that encodes the putative SPO12 protein and has no significant sequence similarities to known genes. The last 15 amino acids of this putative protein have a high negative charge, which appears to be required for function. A second sporulation-specific gene, designated SPO16, was found adjacent to SPO12 and shown to be necessary for efficient spore formation. The two genes are encoded on opposite DNA strands with only 103 nucleotides between the termination codons. Up to 700 nucleotides of the SPO12 and SPO16 transcripts are complementary, and the 3' untranslated region of the longest SPO16 transcript is complementary to all or nearly all of the SPO12 mRNA. A strain homozygous for an insertion which removes the complementarity between the SPO12 and SPO16 mRNAs has an efficiency of sporulation, number of spores per ascus, and spore viability identical to those of a wild-type strain. The complementarity therefore has either no function or only a subtle function in meiosis and sporulation.