TOP-2 is differentially required for the proper maintenance of cohesin on meiotic chromosomes in C. elegans spermatogenesis and oogenesis

During meiotic prophase I, accurate segregation of homologous chromosomes requires the establishment of a chromosomes with a meiosis-specific architecture. Sister chromatid cohesins and the enzyme Topoisomerase II are important components of meiotic chromosome axes, but the relationship of these proteins in the context of meiotic chromosome segregation is poorly defined. Here, we analyzed the role of Topoisomerase II (TOP-2) in the timely release of sister chromatid cohesins during spermatogenesis and oogenesis of Caenorhabditis elegans. We show that there is a different requirement for TOP-2 in meiosis of spermatogenesis and oogenesis. The loss-of-function mutation top-2(it7) results in premature REC-8 removal in spermatogenesis, but not oogenesis. This is due to a failure to maintain the HORMA-domain proteins HTP-1 and HTP-2 (HTP-1/2) on chromosome axes at diakinesis and mislocalization of the downstream components that control sister chromatid cohesion release including Aurora B kinase. In oogenesis, top-2(it7) causes a delay in the localization of Aurora B to oocyte chromosomes but can be rescued through premature activation of the maturation promoting factor via knock-down of the inhibitor kinase WEE-1.3. The delay in Aurora B localization is associated with an increase in the length of diakinesis chromosomes and wee-1.3 RNAi mediated rescue of Auorora B localization in top-2(it7) is associated with a decrease in chromosome length. Our results imply that the sex-specific effects of Topoisomerase II on sister chromatid cohesion release are due to differences in the temporal regulation of meiosis and chromosome structure in late prophase I in spermatogenesis and oogenesis.

HIM-17 regulates the position of recombination events and GSP-1/2 localization to establish short arm identity on bivalents in meiosis

The position of recombination events established along chromosomes in early prophase I and the chromosome remodeling that takes place in late prophase I are intrinsically linked steps of meiosis that need to be tightly regulated to ensure accurate chromosome segregation and haploid gamete formation. Here, we show that RAD-51 foci, which form at the sites of programmed meiotic DNA double-strand breaks (DSBs), exhibit a biased distribution toward off-centered positions along the chromosomes in wild-type Caenorhabditis elegans, and we identify two meiotic roles for chromatin-associated protein HIM-17 that ensure normal chromosome remodeling in late prophase I. During early prophase I, HIM-17 regulates the distribution of DSB-dependent RAD-51 foci and crossovers on chromosomes, which is critical for the formation of distinct chromosome subdomains (short and long arms of the bivalents) later during chromosome remodeling. During late prophase I, HIM-17 promotes the normal expression and localization of protein phosphatases GSP-1/2 to the surface of the bivalent chromosomes and may promote GSP-1 phosphorylation, thereby antagonizing Aurora B kinase AIR-2 loading on the long arms and preventing premature loss of sister chromatid cohesion. We propose that HIM-17 plays distinct roles at different stages during meiotic progression that converge to promote normal chromosome remodeling and accurate chromosome segregation.

Unusual Order of Chromosome Arrangement Location in Late Prophase - Early Prometaphase of Mitosis in Haploid Maize Plant Obtained With the Use of Mutation ig

Despite the huge amount of works devoted to the study of mitotic division, there is still a lot of unclear in its mechanisms. For example, insufficient attention has been paid to the processes of cell division in plant forms of different ploidy levels. The literature contains only fragmentary data on haploids in lower plants. This does not allow making any generalizations regarding mitosis in haploids of higher plants. This article presents the results of a cytological study of mitotically dividing cells of haploid maize plants. The article demonstrates the effectiveness of the well-known Chase method, based on the use of genetic markers and the ig mutation (indeterminate gametophyte) for obtaining and detecting haploids. An effective modification of a simple method of acetocarmine staining of cytological preparations is described. An essential result obtained is, in our opinion, the detection of a very brief moment in the state of chromosomes in a dividing cell of a haploid maize plant. This moment is characterized by the fact that the chromatids have already separated and turned into independent chromosomes, but have not yet begun their movement under the action of kinetochore microtubules. It is this feature that made it possible to designate this state as late prophase – early prometaphase of mitosis. An equally important feature of the detected moment is the unusual ordered arrangement of chromosomes, which lie parallel to each other close to each other along their entire length with the centromeres located on one line, which can be considered the equator of the fission spindle. The revealed fact allows us to assume that an essential role in the formation of such an arrangement of chromosomes is played by the bond of chromosomes with the equator of the nuclear membrane and their subsequent connection with the equator of the fission spindle.

Sister chromatid repair maintains genomic integrity during meiosis in Caenorhabditis elegans

SummaryDuring meiosis, the maintenance of genome integrity is critical for generating viable haploid gametes [1]. In meiotic prophase I, double-strand DNA breaks (DSBs) are induced and a subset of these DSBs are repaired as interhomolog crossovers to ensure proper chromosome segregation. DSBs in excess of the permitted number of crossovers must be repaired by other pathways to ensure genome integrity [2]. To determine if the sister chromatid is engaged for meiotic DSB repair during oogenesis, we developed an assay to detect sister chromatid repair events at a defined DSB site during Caenorhabditis elegans meiosis. Using this assay, we directly demonstrate that the sister chromatid is available as a meiotic repair template for both crossover and noncrossover recombination, with noncrossovers being the predominant recombination outcome. We additionally find that the sister chromatid is the exclusive recombination partner for DSBs during late meiotic prophase I. Analysis of noncrossover conversion tract sequences reveals that DSBs are processed similarly throughout prophase I and recombination intermediates remain central around the DSB site. Further, we demonstrate that the SMC-5/6 complex is required for long conversion tracts in early prophase I and intersister crossovers during late meiotic prophase I; whereas, the XPF-1 nuclease is required only in late prophase to promote sister chromatid repair. In response to exogenous DNA damage at different stages of meiosis, we find that mutants for SMC-5/6 and XPF-1 have differential effects on progeny viability. Overall, we propose that SMC-5/6 both processes recombination intermediates and promotes sister chromatid repair within meiotic prophase I, while XPF-1 is required as an intersister resolvase only in late prophase I.

The Synaptonemal Complex Central Region Modulates Crossover Pathways and Feedback Control of Meiotic Double-strand Break Formation

SummaryThe synaptonemal complex (SC) is a proteinaceous structure that mediates homolog engagement and genetic recombination during meiosis. Zip-Mer-Msh (ZMM) proteins promote crossover (CO) formation and initiate SC formation. In SC elongation, the SUMOylated SC component Ecm11 and its interacting protein Gmc2 facilitate the polymerization of Zip1, a SC-central region component in budding yeast. Through physical recombination, cytological, and genetic analyses, we here demonstrate that ecm11 and gmc2 mutants exhibit chromosome-specific defects in meiotic recombination. CO frequencies were reduced on a short chromosome (chromosome III), whereas CO and non-crossover (NCO) frequencies were increased on a long chromosome (chromosome VII). Further, persistent double-strand breaks (DSBs) occurred in unsynapsed chromosome regions during the late prophase, suggesting the presence of a negative regulation of DSB formation. The Ecm11-Gmc2 (EG) complex could participate in joint molecule (JM) processing and/or double-Holliday junction resolution for CO-designated recombination of the ZMM-dependent pathway. However, absence of the EG complex ameliorated the JM-processing defect in zmm mutants, suggesting a role of these proteins in suppression of ZMM-independent recombination. Therefore, the EG complex fosters ZMM-dependent processing and resolution of JMs while suppressing ZMM-independent JM processing and late DSB formation. Hence, EG-mediated SC central regions, which display properties similar to those of liquid crystals, may function as a compartment for sequestering recombination proteins in and out of the process to ensure meiosis specificity during recombination.

State changes of the HORMA protein ASY1 are mediated by an interplay between its closure motif and PCH2

HORMA domain-containing proteins (HORMADs) play an essential role in meiosis in many organisms. The meiotic HORMADs, including yeast Hop1, mouse HORMAD1 and HORMAD2, and Arabidopsis ASY1, assemble along chromosomes at early prophase and the closure motif at their C-termini has been hypothesized to be instrumental for this step by promoting HORMAD oligomerization. In late prophase, ASY1 and its homologs are progressively removed from synapsed chromosomes promoting chromosome synapsis and recombination. The conserved AAA+ ATPase PCH2/TRIP13 has been intensively studied for its role in removing HORMADs from synapsed chromosomes. In contrast, not much is known about how HORMADs are loaded onto chromosomes. Here, we reveal that the PCH2-mediated dissociation of the HORMA domain of ASY1 from its closure motif is important for the nuclear targeting and subsequent chromosomal loading of ASY1. This indicates that the promotion of ASY1 to an ‘unlocked’ state is a prerequisite for its nuclear localization and chromosomal assembly. Likewise, we find that the closure motif is also necessary for the removal of ASY1 by PCH2 later in prophase. Our work results in a unified new model for PCH2 and HORMADs function in meiosis and suggests a mechanism to contribute to unidirectionality in meiosis.

Closure motif-mediated state changes of the HORMA protein ASY1

Meiotic HORMA domain-containing proteins (HORMADs) are key components of the chromosome axis and play an essential role in meiosis in many organisms. The meiotic HORMADs, including yeast Hop1, mouse HORMAD1 and HORMAD2, and Arabidopsis ASY1, assemble along chromosomes at early prophase and the closure motif at their C-termini has been hypothesized to be instrumental for this step by promoting HORMAD oligomerization. In late prophase, ASY1 and its homologs are progressively removed from synapsed chromosomes promoting chromosome synapsis and recombination. The conserved AAA+ ATPase PCH2/TRIP13 has been intensively studied for its role in removing HORMADs from synapsed chromosomes. In contrast, not much is known about how HORMADs are loaded onto chromosomes. Here, we reveal that the PCH2-mediated dissociation of the HORMA domain of ASY1 from its closure motif is important for the nuclear targeting and subsequent chromosomal loading of ASY1. This indicates that the promotion of ASY1 to an ‘unlocked’ state is a prerequisite for its nuclear localization and chromosomal assembly. Likewise, we find that the closure motif is also necessary for the removal of ASY1 by PCH2 later in prophase. Our work results in a grand unified model for PCH2 and HORMADs function in meiosis.

INDEKS MITOSIS PUCUK DAUN Hibiscus rosa-sinensis L. VARIASI SINGLE PINK PADA BEBERAPA VARIASI WAKTU

AbstrakHibiscus rosa-sinensis L. atau kembang sepatu merupakan tanaman hias yang memiliki banyak manfaat dan merepresentasikan sifat poliploidi. Tujuan penelitian ini yaitu untuk mengetahui Indeks Mitosis (IM) dan jumlah kromosom pucuk daun H. rosa-sinensis pada beberapa variasi waktu. Indeks Mitosis dan waktu pengambilan pucuk sangat diperlukan untuk studi kromosom karena pada tahap tersebut karakter-karakter kromosom dapat diamati dengan jelas dan mudah dihitung. Waktu pengambilan pucuk yang dilakukan yaitu pada 08:00, 10:00, 12:00, 14:00, 16:00 WIB. Pembuatan sediaan kromosom dilakukan menggunakan metode squash menggunakan pewarna Aceto-orcein. Tahapan perlakuan meliputi perendaman pucuk daun di dalam air dingin selama 3 jam, fiksasi dalam larutan Carnoy selama ±24 jam, dan hidrolisis dalam larutan HCl 5N selama 30 menit. Hasil penelitian menunjukkan nilai IM tertinggi meristem pucuk daun H. rosa-sinensis variasi single pink besar muncul pada pukul 10:00 sebesar 94%. Berdasarkan hasil tersebut dapat disimpulkan bahwa waktu sampling yang optimal untuk analisis kromosom H. rosa-sinensis L. variasi single pink besar adalah pukul 10:00 dengan jumlah kromosom 2n= ca. 69-111. Hasil penelitian ini dapat dijadikan dasar untuk studi kromosom selanjutnya serta acuan untuk sampling variasi lainnya.Abstract Hibiscus rosa-sinensis L. is an ornamental plant that has many benefits and represents the character of polyploidy. The purpose of this study is to find out the Mitotic Index of leaf shoots Hibiscus rosa-sinensis on several shoots sampling times. The Mitotic Index and the timing of shoots sampling time are very necessary for chromosome studies because at this stage chromosomes characters can be clearly observed and easily calculated. Period time of collection the leaf shoots is from 08:00 AM to 16:00 PM, with two hours interval each at 08:00, 10:00, 12:00, 14:00, 16:00 . The chromosome preparation was carried out by using the squash method and aceto-orcein staining. The treatment steps included soaking the leaf shoots in cold water for 3 hours, fixation in Carnoy solution for ± 24 hours, and hydrolysis in 5N HCl solution for 30 minutes. The results showed that chromosomes were clearly visualized during the phase with the highest Mitotic Index. In addition, the percentage of Mitotic Index was found to be in line with the percentage of cells in late prophase. Among several sampling time variations, the highest Mitotic Index of Hibiscus rosa-sinensis leaf shoots appeared at 10:00 at 94% with the chromosome numbers of 2n= ca. 69-111. According to the data obtained, it is concluded that 10 AM is the most optimum sampling time that can be used as the basic information for further chromosome studies.

Sex Chromosome Pairing Mediated by Euchromatic Homology in Drosophila Male Meiosis

Diploid germline cells must undergo two consecutive meiotic divisions before differentiating as haploid sex cells. During meiosis I, homologs pair and remain conjoined until segregation at anaphase. Drosophila melanogaster spermatocytes are unique in that the canonical events of meiosis I including synaptonemal complex formation, double-strand DNA breaks, and chiasmata are absent. Sex chromosomes pair at intergenic spacer sequences within the ribosomal DNA (rDNA). Autosomes pair at numerous euchromatic homologies, but not at heterochromatin, suggesting that pairing may be limited to specific sequences. However, previous work generated from genetic segregation assays or observations of late prophase I/prometaphase I chromosome associations fail to differentiate pairing from maintenance of pairing (conjunction). Here, we separately examined the capability of X euchromatin to pair and conjoin using an rDNA-deficient X and a series of Dp(1;Y) chromosomes. Genetic assays showed that duplicated X euchromatin can substitute for endogenous rDNA pairing sites. Segregation was not proportional to homology length, and pairing could be mapped to nonoverlapping sequences within a single Dp(1;Y). Using fluorescence in situ hybridization to early prophase I spermatocytes, we showed that pairing occurred with high fidelity at all homologies tested. Pairing was unaffected by the presence of X rDNA, nor could it be explained by rDNA magnification. By comparing genetic and cytological data, we determined that centromere proximal pairings were best at segregation. Segregation was dependent on the conjunction protein Stromalin in Meiosis, while the autosomal-specific Teflon was dispensable. Overall, our results suggest that pairing may occur at all homologies, but there may be sequence or positional requirements for conjunction.