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Meiosis is a specialized form of cell division in which haploid gametes are produced from diploid progenitors. This reduction in ploidy results from proper meiotic chromosome segregation and is ensured by crossover recombination events. Given their importance, it is no surprise that crossover formation is regulated in most eukaryotes. Crossover assurance is a regulatory mechanism that helps to ensure that each pair of chromosomes gets at least one crossover during meiosis. We seek to better understand how crossover assurance works. To do so, we have developed a system in which crossover formation between a pair of chromosomes is restricted to a defined region. If crossover assurance functions in this context, then crossovers should frequently form in this defined region. Our experiments involve three yeast strains: Homolog: diploid Saccharomyces cerevisiae. Homeolog: Diploid S. cerevisiae, but with one copy of III derived from S. paradoxus and one from S. cerevisiae. Homo-meolog: The homeolog strain, but with the HIS4 region of the S. paradoxus III replaced with the corresponding S. cerevisiae sequence. S. cerevisiae and S. paradoxus are largely syntenic and have 80-90% sequence homology. This level of sequence divergence greatly reduces the incidence of meiotic crossing over. Thus, in the Homeolog strain chromosomes III will frequently fail to form crossovers. In the Homo-meolog strain, a defined region of homology surrounding HIS4 (a hotspot for meiotic recombination) exists in a chromosomal context of homeology. In the Homo-meolog strain, crossover assurance should result in a high incidence of crossover formation in the HIS4 region. By comparing the spectrum of meiotic recombination events in the HIS4 region in the three strains, we will gain insight into the means through which crossover assurance is enforced. These experiments are in the preliminary stage. Strain construction and data collection are ongoing, but our preliminary results demonstrate an elevated incidence of crossing over in the HIS4 region in the homo-meolog strain relative to both the homolog and homeolog strains. Spore viability patterns in the homo-meolog strain are not statistically distinguishable from that of the homolog strain, but are different from that of the homeolog strain. Taken together, these results suggest that the crossovers are targeted to the HIS4 region in the homo-meolog strain, possible through the action of a crossover assurance mechanism. Further analysis of the patterns of recombination in these strains may provide insight into the means through which this regulation is exerted.

Meiosis is a specialized cell division that leads to a reduction of ploidy in sexually reproducing organisms through segregation of homologous chromosomes at the first meiotic division. Improper segregation of chromosomes during meiosis results in anueploidy, which is usually fatal during embryonic development. The meiotic process is therefore tightly regulated. Typically, proper segregation of homologs at meiosis I requires pairing of homologous chromosomes, followed by crossover recombination between homologs. Crossovers enable proper chromosomal segregation during the first meiotic division in part by establishing tension in the meiotic spindle. However, in the absence of crossovers, some cells maintain the ability to direct homologous chromosomes to opposite spindle poles, through a poorly understood mechanism known as distributive segregation. We are using the common brewers yeast Saccharomyces cerevisiae to determine possible roles of two genes in distributive segregation. The genes of interest, Ndj1 and Tid1, have been previously demonstrated to play a role in crossover interference, but their roles in distributive segregation are not well understood. Ndj1 has been shown to function in the tethering of telomeres to the nuclear envelope and may aid in the homology search chromosomes undergo. Tid1 has been characterized as a recombination accessory factor and may stimulate crossovers by directing recombinases to double strand break sites early in meiosis. To assay distributive segregation, we use yeast in which crossing over between one chromosome pair is prevented (due to sequence divergence). Using this system, we can assay the ability of yeast to carry out distributive segregation. Our results indicate that mutations in Ndj1 impair the ability of yeast to carry out distributive segregation, while mutations in Tid1 do not affect distributive segregation. These results, in turn, suggest that Ndj1 may play a role in distributive segregation. This experiment is part of a larger question to determine whether crossover assurance and crossover interference are independent mechanisms.

Yes / A hallmark of the conserved ATM/ATR signalling is its ability to mediate a wide range of functions utilizing only a limited number of adaptors and effector kinases. During meiosis, Tel1 and Mec1, the budding yeast ATM and ATR, respectively, rely on a meiotic adaptor protein Hop1, a 53BP1/Rad9 functional analog, and its associated kinase Mek1, a CHK2/Rad53-paralog, to mediate multiple functions: control of the formation and repair of programmed meiotic DNA double strand breaks, enforcement of inter-homolog bias, regulation of meiotic progression, and implementation of checkpoint responses. Here, we present evidence that the multi-functionality of the Tel1/Mec1-to-Hop1/Mek1 signalling depends on stepwise activation of Mek1 that is mediated by Tel1/Mec1 phosphorylation of two specific residues within Hop1: phosphorylation at the threonine 318 (T318) ensures the transient basal level Mek1 activation required for viable spore formation during unperturbed meiosis. Phosphorylation at the serine 298 (S298) promotes stable Hop1-Mek1 interaction on chromosomes following the initial phospho-T318 mediated Mek1 recruitment. In the absence of Dmc1, the phospho-S298 also promotes Mek1 hyper-activation necessary for implementing meiotic checkpoint arrest. Taking these observations together, we propose that the Hop1 phospho-T318 and phospho-S298 constitute key components of the Tel1/Mec1- based meiotic recombination surveillance (MRS) network and facilitate effective coupling of meiotic recombination and progression during both unperturbed and challenged meiosis. / MRC program grant U1175.01.005.00005.01 from RSC and MRC centre grant G0801130 from JAC. / Erratum: 18 Apr 2016: Penedos A, Johnson AL, Strong E, Goldman AS et al (2016) Correction: Essential and Checkpoint Functions of Budding Yeast ATM and ATR during Meiotic Prophase Are Facilitated by Differential Phosphorylation of a Meiotic Adaptor Protein, Hop1. PLOS ONE. 11(4): e0154170. https://doi.org/10.1371/journal.pone.0154170.

Yes / In the section “Generation of phospho-specific Hop1 antibodies” of the Materials and Methods, we made several mistakes when indicating the sequence of the peptides used for the generation of antibodies. / Erratum: Penedos A, Johnson AL, Strong E, Goldman AS, Carballo JA, Cha RS (2015) Essential and Checkpoint Functions of Budding Yeast ATM and ATR during Meiotic Prophase Are Facilitated by Differential Phosphorylation of a Meiotic Adaptor Protein, Hop1. PLoS ONE 10(7): e0134297. doi: 10.1371/ journal.pone.0134297

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Memoria para optar al Título Profesional de Médico Veterinario / El objetivo del presente estudio fue evaluar la distribución de los gránulos corticales (GC)en el citoplasma ovular, y su relación con el desarrollo meiótico en ovocitos caninos durante la maduración in vitro (MIV) a las 48, 72 y 96h, comparados con aquellos ovocitos no sometidos a cultivo (no madurados) y ovocitos madurados in vivo (ovulados). La distribución de los GC durante la MIV e in vivo fue evaluada a través de la tinción con la lectina lens culinaris conjugada con FITC mediante microscopia de epifluorescencia, en tanto, la configuración cromatínica se evaluó paralelamente mediante la tinción con DAPI y microscopia de epifluorescencia. A través del tiempo de incubación se pudieron establecer tres patrones de distribución de los GC que indicarían el movimiento de estas estructuras durante el proceso de maduración del ovocito. El patrón A, Homogéneo liso; en el cual la marca fluorescente se presentó fina y homogénea en todo el citoplasma ovular, Patrón B, Homogéneo granuloso; pequeñas agrupaciones o conglomerados de aspecto granular (“Clusters”) distribuidos uniformemente por todo el citoplasma, Patrón C, Granuloso cortical; de aspecto granular, en la cual los gránulos se ubicaron inmediatamente por debajo de la membrana plasmática del ovocito en todo su perímetro. El patrón A, se observó sólo en ovocitos en vesícula germinativa (VG) (19%), y fue encontrado sólo en ovocitos no madurados, no obstante la mayoría (p<0,05) de los ovocitos en VG presentó el patrón B, observándose mayoritariamente (p<0,05) en ovocitos no madurados (94%), también en ovocitos madurados en cultivo por 48h (4%), disminuyendo (P<0.05) al aumentar el tiempo de cultivo hasta las 96h. El patrón C se observó sólo en aquellos ovocitos sometidos a MIV aumentando significativamente con el tiempo de cultivo, además de observarse en todos (100%)los ovocitos ovulados. A medida que el tiempo transcurrió, la maduración meiótica progresó sólo con el tiempo de cultivo a una tasa menor que la alcanzada por los GC durante la MIV, a pesar de observar que ovocitos con una distribución cortical o periférica de los gránulos (patrón C), aumentaron en estados nucleares de metafase I (MI) y metafase II (MII) con el tiempo de cultivo. Además considerando que el desarrollo meiótico sólo alcanzó un 27 % de MII a las 96 h en comparación al 90% de ovocitos ovulados en MII, es posible establecer que sólo la distribución de los GC progresaría paralelamente al tiempo de cultivo indicando con esto que la maduración citoplasmática evaluada a través de la migración de estas vesículas no ocurre coordinadamente con la maduración nuclear

Thesis advisor: Laura Hake / The completion of meiosis in Xenopus oocytes requires the coordinated translation of stored mRNAs. CPEB, the cytoplasmic polyadenylation element binding protein, controls the translation of developmentally important early-class maternal mRNAs. Resumption of meiosis through stimulation with progesterone leads to the phosphorylation and activation of CPEB. This results in the lengthening of the poly(A) tails and translation of mRNAs containing the cytoplasmic polyadenylation element (CPE). XGef, a putative guanine nucleotide exchange factor, binds to and is required for CPEB activation. Translation of c-mos, a MAPK kinase kinase, is controlled by CPEB, and activation of the Mos/MAPK pathway is required for meiotic maturation. In addition, the synthesis of Ringo protein, an atypical cdk binding protein and activator, is required for progesterone-induced maturation, though Ringo is able to stimulate resumption of meiosis independent of progesterone. Although much work has been done to understand the key events leading to activation of maturation promoting factor (MPF) and meiotic maturation, the events immediately following progesterone stimulation remain unclear, particularly regarding the role of XGef. The work that follows describes experiments performed to further understand the role of XGef in meiotic maturation through both Ringo and MAPK activity. It was found that XGef and Ringo interact directly and form a complex throughout early meiosis. XGef is involved in Ringo’s influence during meiosis, specifically through MEK-activation of MAPK. Notably, XGef functions in a common pathway and complex with Ringo most likely to influence CPEB phosphorylation and activation. / Thesis (BS) — Boston College, 2009. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: College Honors Program. / Discipline: Biology.