Chromosoma

Meiotic behavior of a complex hexavalent in heterozygous mice for Robertsonian translocations: insights for synapsis dynamics Abstract Natural populations of the house mouse Mus musculus domesticus show great diversity in chromosomal number due to the presence of chromosomal rearrangements, mainly Robertsonian translocations. Breeding between two populations with different chromosomal configurations generates subfertile or sterile hybrid individuals due to impaired meiotic development. In this study, we have analyzed prophase-I spermatocytes of hybrids formed by crossing mice from Vulcano and Lipari island populations. Both populations have a 2n = 26 karyotype but different combinations of Robertsonian translocations. We studied the progress of synapsis, recombination, and meiotic silencing of unsynapsed chromosomes during prophase-I through the immunolocalization of the proteins SYCP3, SYCP1, γH2AX, RAD51, and MLH1. In these hybrids, a hexavalent is formed that, depending on the degree of synapsis between chromosomes, can adopt an open chain, a ring, or a closed configuration. The frequency of these configurations varies throughout meiosis, with the maximum degree of synapsis occurring at mid pachytene. In addition, we observed the appearance of heterologous synapsis between telocentric and metacentric chromosomes; however, this synapsis seems to be transient and unstable and unsynapsed regions are frequently observed in mid-late pachytene. Interestingly, we found that chiasmata are frequently located at the boundaries of unsynapsed chromosomal regions in the hexavalent during late pachytene. These results provide new clues about synapsis dynamics during meiosis. We propose that mechanical forces generated along chromosomes may induce premature desynapsis, which, in turn, might be counteracted by the location of chiasmata. Despite these and additional meiotic features, such as the accumulation of γH2AX on unsynapsed chromosome regions, we observed a large number of cells that progressed to late stages of prophase-I, indicating that synapsis defects may not trigger a meiotic crisis in these hybrids.

De novo genome assembly of the cichlid fish Astatotilapia latifasciata reveals a higher level of genomic polymorphism and genes related to B chromosomes Abstract Supernumerary B chromosomes (Bs) are accessory elements to the regular chromosome set (As) and have been observed in a huge diversity of eukaryotic species. Although extensively investigated, the biological significance of Bs remains enigmatic. Here, we present de novo genome assemblies for the cichlid fish Astatotilapia latifasciata, a well-known model to study Bs. High coverage data with Illumina sequencing was obtained for males and females with 0B (B−), 1B, and 2B (B+) chromosomes to provide information regarding the diversity among these genomes. The draft assemblies comprised 771 Mb for the B− genome and 781 Mb for the B+ genome. Comparative analysis of the B+ and B− assemblies reveals syntenic discontinuity, duplicated blocks and several insertions, deletions, and inversions indicative of rearrangements in the B+ genome. Hundreds of transposable elements and 1546 protein coding sequences were annotated in the duplicated B+ regions. Our work contributes a list of thousands of genes harbored on the B chromosome, with functions in several biological processes, including the cell cycle.

Maternal regulation of chromosomal imprinting in animals Abstract Chromosomal imprinting requires an epigenetic system that "imprints" one of the two parental chromosomes such that it results in a heritable (cell-to-cell) change in behavior of the "imprinted" chromosome. Imprinting takes place when the parental genomes are separate, which occurs during gamete formation in the respective germ-lines and post-fertilization during the period when the parental pro-nuclei lie separately within the ooplasm of the zygote. In the mouse, chromosomal imprinting is regulated by germ-line specific DNA methylation. But the methylation machinery in the respective germ-lines does not discriminate between imprinted and non-imprinted regions. As a consequence, the mouse oocyte nucleus contains over a thousand oocyte-specific germ-line differentially methylated regions (gDMRs). Upon fertilization, the sperm provides a few hundred sperm-specific gDMRs of its own. Combined, there are around 1600 imprinted and non-imprinted gDMRs in the pro-nuclei of the newly fertilized zygote. It is a remarkable fact that beginning in the maternal ooplasm, there are mechanisms that manage to preserve DNA methylation at ~ 26 known imprinted gDMRs in the face of the ongoing genome-wide DNA de-methylation that characterizes pre-implantation development. Specificity is achieved through the binding of KRAB-zinc finger proteins to their cognate recognition sequences within the gDMRs of imprinted genes. This in turn nucleates the assembly of localized heterochromatin-like complexes that preserve methylation at imprinted gDMRs through recruitment of the maintenance methyl transferase Dnmt1. These studies have shown that a germ-line imprint may cause parent-of-origin-specific behavior only if "licensed" by mechanisms that operate post-fertilization. Study of the germ-line and post-fertilization contributions to the imprinting of chromosomes in classical insect systems (Coccidae and Sciaridae) show that the ooplasm is the likely site where imprinting takes place. By comparing molecular and genetic studies across these three species, we suggest that mechanisms which operate post-fertilization play a key role in chromosomal imprinting phenomena in animals and conserved components of heterochromatin are shared by these mechanisms.

Molecular and genetic organization of bands and interbands in the dot chromosome of Drosophila melanogaster Abstract The fourth chromosome smallest in the genome of Drosophila melanogaster differs from other chromosomes in many ways. It has high repeat density in conditions of a large number of active genes. Gray bands represent a significant part of this polytene chromosome. Specific proteins including HP1a, POF, and dSETDB1 establish the epigenetic state of this unique chromatin domain. In order to compare maps of localization of genes, bands, and chromatin types of the fourth chromosome, we performed FISH analysis of 38 probes chosen according to the model of four chromatin types. It allowed clarifying the dot chromosome cytological map consisting of 16 loose gray bands, 11 dense black bands, and 26 interbands. We described the relation between chromatin states and bands. Open aquamarine chromatin mostly corresponds to interbands and it contains 5′UTRs of housekeeping genes. Their coding parts are embedded in gray bands substantially composed of lazurite chromatin of intermediate compaction. Polygenic black bands contain most of dense ruby chromatin, and also some malachite and lazurite. Having an accurate map of the fourth chromosome bands and its correspondence to physical map, we found that DNase I hypersensitivity sites, ORC2 protein, and P–elements are mainly located in open aquamarine chromatin, while element 1360, characteristic of the fourth chromosome, occupies band chromatin types. POF and HP1a proteins providing special organization of this chromosome are mostly located in aquamarine and lazurite chromatin. In general, band organization of the fourth chromosome shares the features of the whole Drosophila genome.

Spindle assembly without spindle pole body insertion into the nuclear envelope in fission yeast meiosis Abstract Centrosomes represent the major microtubule organizing center (MTOC) in eukaryotic cells and are responsible for nucleation of the spindle, the vehicle of chromosome segregation. In human female meiosis, however, spindle assembly occurs in the absence of centrosomes or other MTOCs and microtubules are nucleated around chromosomes. In yeast, spindle formation in mitosis and meiosis depends on the activity of spindle pole bodies (SPBs), the functional equivalents of centrosomes; thus, SPBs and centrosomes use similar machineries to assemble spindles. Here, we develop a system to explore the molecular mechanisms supporting acentrosomal spindle formation using fission yeast meiosis as a model scenario. We achieve this situation by removing access of the SPBs to the nucleus after their duplication. Under these conditions, we observe self-assembly-based spindle formation in the nuclear environment, conferring an ability to segregate chromosomes independently of the SPBs. Our results open the possibility to utilize the experimental advantages of fission yeast for insights into the molecular basis of acentrosomal spindle formation in meiosis.

Outer kinetochore protein Dam1 promotes centromere clustering in parallel with Slk19 in budding yeast Abstract A higher order organization of the centromeres in the form of clustering of these DNA loci has been observed in many organisms. While centromere clustering is biologically significant to achieve faithful chromosome segregation, the underlying molecular mechanism is yet to be fully understood. In budding yeast, a kinetochore-associated protein Slk19 is shown to have a role in clustering in association with the microtubules whereas removal of either Slk19 or microtubules alone does not have any effect on the centromere clustering. Furthermore, Slk19 is non-essential for growth and becomes cleaved during anaphase whereas clustering being an essential event occurs throughout the cell cycle. Hence, we searched for an additional factor involved in the clustering and since the integrity of the kinetochore complex is shown to be crucial for centromere clustering, we restricted our search within the complex. We observed that the outermost kinetochore protein Dam1 promotes centromere clustering through stabilization of the kinetochore integrity. While in the absence of Dam1 we failed to detect Slk19 at the centromere, on the other hand, we found almost no Dam1 at the centromere in the absence of Slk19 and microtubules suggesting interdependency between these two pathways. Strikingly, we observed that overexpression of Dam1 or Slk19 could restore the centromere clustering largely in the cells devoid of Slk19 and microtubules or Dam1, respectively. Thus, we propose that in budding yeast, centromere clustering is achieved at least by two parallel pathways, through Dam1 and another via Slk19, in concert with the microtubules suggesting that having a dual mechanism may be crucial for ensuring microtubule capture by the point centromeres where each attaches to only one microtubule.

ChIP-cloning analysis uncovers centromere-specific retrotransposons in Brassica nigra and reveals their rapid diversification in Brassica allotetraploids Abstract Centromeres are indispensable functional units of chromosomes. The evolutionary mechanisms underlying the rapid evolution of centromeric repeats, especially those following polyploidy, remain unknown. In this study, we isolated centromeric sequences of Brassica nigra, a model diploid progenitor (B genome) of the allopolyploid species B. juncea (AB genome) and B. carinata (BC genome) by chromatin immunoprecipitation of nucleosomes containing the centromere-specific histone CENH3. Sequence analysis detected no centromeric satellite DNAs, and most B. nigra centromeric repeats were found to originate from Tyl/copia-class retrotransposons. In cytological analyses, six of the seven analyzed repeat clusters had no FISH signals in A or C genomes of the related diploid species B. rapa and B. oleracea. Notably, five repeat clusters had FISH signals in both A and B subgenomes in the tetraploid B. juncea. In the tetraploid B. carinata, only CL23 displayed three pairs of signals in terminal or interstitial regions of the C-derived chromosome, and no evidence of colonization of CLs onto C-subgenome centromeres was found in B. carinata. This observation suggests that centromeric repeats spread and proliferated between genomes after polyploidization. CL3 and CRB are likely ancient centromeric sequences arising prior to the divergence of diploid Brassica which have detected signals across the genus. And in allotetraploids B. juncea and B. carinata, the FISH signal intensity of CL3 and CRB differed among subgenomes. We discussed possible mechanisms for centromeric repeat divergence during Brassica speciation and polyploid evolution, thus providing insights into centromeric repeat establishment and targeting.

How dynamic could be the 45S rDNA cistron? An intriguing variability in a grasshopper species revealed by integration of chromosomal and genomic data Abstract To better understand the structure and variability of the 45S rDNA cistron and its evolutionary dynamics in grasshoppers, we performed a detailed analysis combining classical and molecular cytogenetic data with whole-genome sequencing in Abracris flavolienata, which shows extraordinary variability in the chromosomal distribution for this element. We found astonishing variability in the number and size of rDNA clusters at intra- and inter-population levels. Interestingly, FISH using distinct parts of 45S rDNA cistron (18S rDNA, 28S rDNA, and ITS1) as probes revealed a distinct number of clusters, suggesting independent mobility and amplification of the 45S rDNA components. This hypothesis is consistent with the higher genomic coverage of almost the entire cistron of 45S rDNA observed in A. flavolineata compared to other grasshoppers, besides coverage variability along the 45S rDNA cistron in the species. In addition, these differences in coverage for distinct components of the 45S rDNA cistron indicate emergence of pseudogenes evidenced by existence of truncated sequences, demonstrating the rDNA dynamics in the species. Although the chromosomal distribution of 18S rDNA was highly variable, the chromosomes 1, 3, 6, and 9 harbored rDNA clusters in all individuals with the occurrence of NOR activity in pair 9, suggesting ancestry or selective pressures to prevent pseudogenization of rDNA sequences in this chromosome pair. Additionally, small NORs and cryptic rDNA loci were observed. Finally, there was no evidence of enrichment and association of transposable elements, at least, inside or nearby rDNA cistron. These findings broaden our knowledge of rDNA dynamics, revealing an independent movement and amplification of segments of 45S rDNA cistron, which in A. flavolineata could be attributed to ectopic recombination.

S. cerevisiae Srs2 helicase ensures normal recombination intermediate metabolism during meiosis and prevents accumulation of Rad51 aggregates Abstract We investigated the meiotic role of Srs2, a multi-functional DNA helicase/translocase that destabilises Rad51-DNA filaments and is thought to regulate strand invasion and prevent hyper-recombination during the mitotic cell cycle. We find that Srs2 activity is required for normal meiotic progression and spore viability. A significant fraction of srs2 mutant cells progress through both meiotic divisions without separating the bulk of their chromatin, although in such cells sister centromeres often separate. Undivided nuclei contain aggregates of Rad51 colocalised with the ssDNA-binding protein RPA, suggesting the presence of persistent single-strand DNA. Rad51 aggregate formation requires Spo11-induced DSBs, Rad51 strand-invasion activity and progression past the pachytene stage of meiosis, but not the DSB end-resection or the bias towards interhomologue strand invasion characteristic of normal meiosis. srs2 mutants also display altered meiotic recombination intermediate metabolism, revealed by defects in the formation of stable joint molecules. We suggest that Srs2, by limiting Rad51 accumulation on DNA, prevents the formation of aberrant recombination intermediates that otherwise would persist and interfere with normal chromosome segregation and nuclear division.

The molecular basis of monopolin recruitment to the kinetochore Abstract The monopolin complex is a multifunctional molecular crosslinker, which in S. pombe binds and organises mitotic kinetochores to prevent aberrant kinetochore-microtubule interactions. In the budding yeast S. cerevisiae, whose kinetochores bind a single microtubule, the monopolin complex crosslinks and mono-orients sister kinetochores in meiosis I, enabling the biorientation and segregation of homologs. Here, we show that both the monopolin complex subunit Csm1 and its binding site on the kinetochore protein Dsn1 are broadly distributed throughout eukaryotes, suggesting a conserved role in kinetochore organisation and function. We find that budding yeast Csm1 binds two conserved motifs in Dsn1, one (termed Box 1) representing the ancestral, widely conserved monopolin binding motif and a second (termed Box 2-3) with a likely role in enforcing specificity of sister kinetochore crosslinking. We find that Box 1 and Box 2-3 bind the same conserved hydrophobic cavity on Csm1, suggesting competition or handoff between these motifs. Using structure-based mutants, we also find that both Box 1 and Box 2-3 are critical for monopolin function in meiosis. We identify two conserved serine residues in Box 2-3 that are phosphorylated in meiosis and whose mutation to aspartate stabilises Csm1-Dsn1 binding, suggesting that regulated phosphorylation of these residues may play a role in sister kinetochore crosslinking specificity. Overall, our results reveal the monopolin complex as a broadly conserved kinetochore organiser in eukaryotes, which budding yeast have co-opted to mediate sister kinetochore crosslinking through the addition of a second, regulatable monopolin binding interface.

Alexandros Sfakianakis
Anapafseos 5 . Agios Nikolaos
Crete.Greece.72100
2841026182

6948891480

alsfakia@gmail.com