DAPI staining of the mutant g48 embryos revealed that both the sperm chromosomes and oocyte chromosomes remain condensed and positioned near the cortex. This persistent pattern, a cluster of tightly arrayed oocyte chromosomes and a single sperm chromatin mass Fig. Likewise, the eggshells of emb embryos remain osmotically sensitive, another characteristic of wild-type meiotic one-cell embryos. These features distinguish emb embryos from unfertilized oocytes, which remain spherical in shape, fail to form eggshells, and contain a single, centrally located mass of endoreduplicating oocyte chromosomes.
Genetic and physical map data positioned emb within a three cosmid region on chromosome II Materials and Methods. Because our attempts to identify emb by germline transformation rescue proved inconclusive, information from the C. Of these genes, only F10B5. This result prompted us to sequence F10B5. This change results in a HisTyr alteration in the amino acid sequence Fig. The C. The mutated His is conserved in all Cdc16 proteins sequenced to date, but it is not one of the conserved hydrophobic residues that defines the TPR motif itself.
Taken together, these data indicate that emb encodes the C. Using the emb g48 phenotype as a guide, we screened preexisting collections of ts Mel mutants for additional members of this class. From several different mutant collections Miwa et al. Although emb-1 has not yet been cloned, emb encodes an ortholog of APC4 Furuta et al. We also initiated a large scale screen for new one-cell arrested ts mutants. Our goal was to isolate additional alleles of emb-1 , emb , and emb , as well as novel members of this phenotypic class. To facilitate the isolation of Mel mutants, screens were carried out in a genetic background of an egg laying—defective mutant lin-2 Kemphues et al.
Mapping crosses and complementation tests revealed that seven of these mutants are new alleles of emb 6 alleles and emb 1 allele , whereas the remaining 25 alleles define three new complementation groups: mat-1 6 alleles , mat-2 7 alleles , and mat-3 12 alleles. The genetic map positions of these genes are shown in Fig.
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Between fertilization and the first mitotic division, the one-cell stage of C. Before fertilization, the primary oocytes undergo a prolonged pause in diakinesis of meiosis I. At this stage, they contain six highly condensed bivalents within a nuclear envelope and a uniform meshwork of cytoplasmic tubulin Fig. B Albertson Immediately before fertilization, they undergo meiotic maturation, which includes nuclear envelope breakdown McCarter et al.
After fertilization, the oocyte-derived bivalents align and congress to form a metaphase plate with the five autosomal bivalents surrounding the sex chromosome bivalents in an axial orientation.
The associated meiotic spindle is a barrel-shaped structure that lies adjacent and parallel to the cortex Fig. During this time, the quiescent sperm chromatin remains as a single mass of condensed DNA near its site of entry Sadler and Shakes During anaphase, the spindle rotates to its telophase position, perpendicular to the cortex Fig. F , before extruding one set of oocyte chromosomes into the polar body Fig. Meiosis II proceeds in a similar manner Fig. H Albertson After these meiotic divisions, the maternal and paternal chromosomes decondense, are encased within nuclear envelopes, and undergo DNA synthesis Sadler and Shakes During this same period, the sperm centrioles duplicate and begin nucleating microtubules Fig.
Subsequently, the zygote enters a period of transitional prophase during which the maternal pronucleus migrates posteriorly to meet the paternal pronucleus Fig. The joined pronuclei then move centrally before initiating mitosis Fig. Tubulin and DAPI localization during meiotic progression of wild-type oocytes before and after fertilization. An oocyte in diakinesis of meiotic prophase I diak I is shown in A and B. During rotation, chromosomes enter anaphase and often up to 12 distinguishable homologues can be visualized at this time.
By the time rotation is complete E and F , the homologues have moved apart towards the opposite spindle poles. Univalents are no longer distinguishable. The oocyte meiotic chromosomes are indicated by double arrows C and G. Polar bodies, the discarded meiotic products, are indicated by vertical arrows G and I.
The duplicated sperm asters are indicated by arrows J. To further investigate the mutant defects, oocytes and embryos of adult-upshift mutant mothers were examined. No prefertilization defects were detected; DAPI staining revealed the expected six bivalents per oocyte and maturation defects were undetectable by DIC optics. To analyze defects after fertilization, wild-type and mutant embryos were costained with DAPI and anti-tubulin antibodies. Embryos within the uteri of wild-type mothers ranged in age from the 1—cell stage Fig.
In contrast, each of the mutant mothers were filled with 1-cell embryos, all of which were arrested at the same stage of development Fig. Within the mutant embryos, the oocyte chromosomes had properly congressed and aligned on an acentriolar, barrel-shaped meiotic spindle. However, no evidence of a metaphase to anaphase transition was observed: the homologues remained attached, and the spindle failed to rotate from its initial position parallel to the cortex. On the other side of the embryo, the sperm chromatin mass remained hypercondensed, and the sperm centrosomes remained quiescent.
D , with Fig. Tubulin and DAPI localization in one-cell arrested mat embryos. Wild-type and mat mutant adults were shifted to Shown in A and B are a spread of embryos from a wild-type mother. Wild-type animals display a range of developmental stages from meiotic and mitotic one-cells to multicellular.
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An embryo in metaphase of meiosis I is visible on the left in A; an arrow marks its chromosomes in B. Shown in C and D are a spread of embryos from a mat-1 ax mother in which all the embryos are arrested in metaphase of meiosis I. The corresponding DAPI image is shown below each tubulin image. Anterior is to the left. Of these mutants, only emb-1 hc62 was not identified in our genetic screens.
In most images, the hypercondensed sperm chromatin mass is visible at the right end of each embryo. Because cell cycle dynamics are ultimately regulated by the activation and inactivation of cyclin-dependent kinases CDKs Gitig and Koff , the phosphorylation state of direct and indirect CDK targets can also be used to determine the stage of cell cycle arrest. For instance, histone H3 is phosphorylated during M phase entry and subsequently dephosphorylated before M phase exit Su et al.
Therefore, to test whether the mutant embryos remain in a prolonged M phase state, embryos were analyzed using an antibody against phosphorylated histone H3 phospho-H3. In both wild-type and mutant embryos, anti—phospho-H3 antibodies intensely stained oocyte chromosomes in their metaphase configuration Fig.
In the mutants, the oocyte chromosomes continued to stain even in the oldest in utero embryos, suggesting that the mutant embryos enter, but never leave, M phase. Note that this antibody does not recognize the highly condensed sperm chromatin mass in either wild-type or mutant embryos Fig.
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The three wild-type, one-cell stage embryos are in meiosis I metaphase left , meiosis II metaphase middle , and S phase right , based on their DNA staining patterns B. In the wild-type panels, phosphohistone H3 stains the pentagonal array of the oocyte metaphase meiosis I chromosomes arrow , but it does not stain the sperm chromatin mass on the other side of the embryo. In the middle embryo, the sperm chromatin mass is out of focus. Phosphohistone H3 brightly stains both the oocyte meiosis II chromosomes arrowhead and the chromatin within the adjacent polar body.
In the S phase embryo, phosphohistone H3 stains the two polar bodies and the oocyte pronucleus, but not the male pronucleus, which, in this particular embryo, lies next to the oocyte pronucleus. All three mat-2 ax embryos are in metaphase of meiosis I D , and their congressed chromosomes D, arrows stain brightly C.
Congressed chromosomes also stain in mat-1 ax E and mat-3 ax68 F. F The bottom mat-3 embryo is older and displays the terminal phenotype; the chromosomes continue to stain with phospho-H3 antibody, but have moved centrally and lost their condensed metaphase chromosome morphology. Mutant embryos were also analyzed using the MPM-2 monoclonal antibody that recognizes various mitotic phosphoproteins Davis et al.
The MPM-2 epitope is widely conserved, as this antibody stains kinetochores, centrosomes, and microtubules during early, but not late, M phase Vandre et al. As shown in Fig.
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MPM-2 also stains the sperm chromatin mass Fig. In the mutants, MPM-2 continued to stain the chromosomes in older embryos, a further indication that these aging embryos remain blocked in M phase. Note, anterior is to the left.
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A magnified view of the oocyte chromosomes are shown in each panel. Under stringent conditions, every allele from this ts mutant collection exhibits a block in both the meiosis I metaphase to anaphase transition and in M phase exit. Nevertheless, after 3—4 h at When mutant mothers were grown under suboptimal conditions e. Other embryos even underwent a few rounds of mitotic cell divisions. The particular conditions required to produce these hypomorphic phenotypes are allele specific, and the hypomorphic defects themselves are consistent with the previously reported phenotypes of both emb g48 and emb g53 Cassada et al.
In contrast, the first meiotic division of a primary spermatocyte takes place on a centriole-based, mitotic-like spindle, and, though cytokinesis is often incomplete, the division culminates in the symmetric division of the primary spermatocyte into two secondary spermatocytes Ward et al. Unlike oocytes, in which the first meiotic division only occurs after fertilization, spermatocytes initiate their first meiotic division autonomously as soon as they mature and separate from the syncytial gonad.
This first meiotic division is followed by a round of centriole duplication and the assembly of a bipolar meiosis II spindle, orthogonal to the first division axis. The separation of sister chromatids during meiosis II is accompanied by an unusual asymmetric division spermatid budding during which the two haploid sperm bud from a large residual cytoplast Fig.
Tubulin and DAPI localization in wild-type and mutant spermatocytes. Shown in A is a depiction of wild-type spermatogenesis. Secondary spermatocytes subsequently undergo a polarized budding division during which two haploid sperm separate from a central residual body. All meiotic stages can be seen.
A budding figure BF is visible in the lower right corner. Haploid sperm S and residual bodies RB are also indicated. Abnormal budding figures are also present in which all the chromosomes remain in the center of the developing residual body.
In the mat mutants, normal meiosis I—like spindles form, but anaphase figures are never observed F, G, J, and K. Given these striking differences between oocyte and spermatocyte meiosis, we investigated whether the mat genes functioned in both processes.