Pachytene karyotypes of 17 species of birds

Background: To date less than 10% of bird species have been karyotyped. They are rather conservative with diploid chromosome numbers about 78-80 in most species examined. Immunostaining of meiotic chromosomes at pachytene stage enables more precise estimates of the number, morphology and variability of macro- and microchromosomes than conventional analysis of mitotic metaphase chromosomes does. Analysis of pachytene chromosomes led to discovery of germline-restricted chromosome (GRC) that was present in germline cells and absent in somatic cells in all 16 species of passerine birds examined. GRC has not been found in any non-passerine bird. Results: In this study, using immunolocalization of SYCP3, the main protein of the lateral elements of the synaptonemal complex (SC) and centromere proteins we examined male pachytene karyotypes of sixteen passerine species and one outgroup species the Common cuckoo Cuculus canorus and provided their idiograms and precise estimates of their diploid chromosome numbers and the numbers of chromosome arms. We provided the first description of the karyotypes of three species, corrected the published data on the karyotypes of ten species and confirmed them for four species. The pachytene cells of the Gouldian finch, Brambling and Common linnet contained heteromorphic bivalents indicating heterozygosity for inversions or centromere shifts. The European pied flycatcher, Gouldian finch and Domestic canary have extended centromeres in several macro- and microchromosomes. GRCs of various sizes and shapes were detected in all passerine species examined. Their chromatin was heavily labeled by anticentromere antibodies. The lateral elements of the GRC SC varied in their size from the largest to the smallest element of the pachytene karyotype. They also varied in shape from continuous to fragmented. Conclusions: All songbirds examined, except the Eurasian skylark, have highly conservative karyotypes, 2n=80-82+GRC with seven pairs of macrochromosomes and 33-34 pairs of microchromosomes. The interspecies differences concern the sizes of the macrochromosomes, morphology of the microchromosomes and sizes of the centromeres. GRC is present in all songbird species examined, varying in size, morphology and SC structure even between closely related species. This indicates its fast evolution.

BRCA2 Haploinsufficiency in Telomere Maintenance

Our previous studies showed an association between monoallelic BRCA2 germline mutations and dysfunctional telomeres in epithelial mammary cell lines and increased risk of breast cancer diagnosis for women with BRCA2 999del5 germline mutation and short telomeres in blood cells. In the current study, we analyzed telomere dysfunction in lymphoid cell lines from five BRCA2 999del5 mutation carriers and three Fanconi Anemia D1 patients by fluorescence in situ hybridization (FISH). Metaphase chromosomes were harvested from ten lymphoid cell lines of different BRCA2 genotype origin and analyzed for telomere loss (TL), multitelomeric signals (MTS), interstitial telomere signals (ITS) and extra chromosomal telomere signals (ECTS). TL, ITS and ECTS were separately found to be significantly increased gradually between the BRCA2+/+, BRCA2+/- and BRCA2-/- lymphoid cell lines. MTS were found to be significantly increased between the BRCA2+/+ and the BRCA2+/- heterozygous (p < 0.0001) and the BRCA2-/- lymphoid cell lines (p < 0.0001) but not between the BRCA2 mutated genotypes. Dysfunctional telomeres were found to be significantly increased in a stepwise manner between the BRCA2 genotypes indicating an effect of BRCA2 haploinsufficiency on telomere maintenance.

Frequent numerical and structural chromosome changes in early generations of synthetic hexaploid wheat

Modern hexaploid wheat (Triticum aestivum L.; AABBDD) evolved from a hybrid of tetraploid wheat (closely related to Triticum turgidum L. ssp. durum (Desf.) Husn., AABB) and goatgrass (Aegilops tauschii Coss., DD). Variations in chromosome structure and ploidy played important roles in wheat evolution. How these variations occurred and their role in expanding the genetic diversity in modern wheat is mostly unknown. Synthetic hexaploid wheat (SHW) can be used to investigate chromosome variation that occurs during the early generations of existence. SHW lines derived by crossing durum wheat ‘Langdon’ with twelve Ae. tauschii accessions were analyzed using oligonucelotide probe multiplex fluorescence in situ hybridization (FISH) to metaphase chromosomes and SNP markers. Cluster analysis based on SNP markers categorized them into three groups. Among 702 plants from the S8 and S9 generations, 415 (59.12%) carried chromosome variations involving all 21 chromosomes but with different frequencies for each chromosome and sub-genome. Total chromosome variation frequencies varied between lines, but there was no significant difference among the three groups. The non-random chromosome variations in SHW lines detected in this research may be an indication that similar variations occurred in the early stages of wheat polyploidization and played important roles in wheat evolution.

An Infertile Azoospermic Male with 45, X T(Yp;15) Karyotype

Objective: 45, X is a very rare condition that usually results from Y/autosomal translocations or insertions. Here we present an infertile azoospermic man who had 45, X t(Yp;15) karyotype and deletion of AZF (azoospermia factor) gene region. Case report: A 35-year-old infertile azoospermic man with a typical male appearance came for infertility genetic counseling. He was infertile for more than ten years and had short height. High-resolution of metaphase chromosomes of 50 peripheral white blood cells were analyzed for karyotyping. Fluorescence in situ hybridization (FISH) analysis and Polymerase chain reaction (PCR) were done for SRY and AZF gene localization. Karyotyping and FISH analysis revealed 45, X t(Yp;15) karyotype and no mosaicism. More investigation on the Y chromosome revealed no deletion in the SRY region, but AZF a/b/c were deleted. It was revealed that Yp's subtelomeric region but not Yq was translocated to chromosome 15. Conclusion: This study shows that despite the lack of a complete Y chromosome in this person, the occurrence of secondary male traits is a result of the short arm translocation of the Y chromosome, which contains the (ex-determining region Y) SRY gene. Infertility is also due to the Y chromosomes long arm's deletion containing the AZF gene region.

Statistical evaluation of chromosomes of some Lathyrus L. taxa growing in Turkey

In this study, statistical analysis was performed on mitotic metaphase chromosomes of 26 Lathyrus taxa, four of which are endemic. According to the findings obtained as a result of analysis, taxa close to each other were determined. Chromosome characteristics were revealed based on the arm lengths of taxa. Based on these, ANOVA, correlation analysis, PCA and cluster analysis were performed to determine the chromosomal relationships between taxa. Morphological similarities of plant taxa and chromosomal statistics results may not be always parallel to each other. According to Cluster Analysis, especially L. hirsutus - L. odoratus, L. brachypterus subsp. haussknechtii - L. phaselitanus, L. stenophyllus - L. chloranthus, L. gorgoni var. gorgoni, - L. nissolia - L. pratensis, L. tuberosus - L. annuus taxa are closely related.

Translesion polymerase eta both facilitates DNA replication and promotes increased human genetic variation at common fragile sites

Common fragile sites (CFSs) are difficult-to-replicate genomic regions that form gaps and breaks on metaphase chromosomes under replication stress. They are hotspots for chromosomal instability in cancer. Repetitive sequences located at CFS loci are inefficiently copied by replicative DNA polymerase (Pol) delta. However, translesion synthesis Pol eta has been shown to efficiently polymerize CFS-associated repetitive sequences in vitro and facilitate CFS stability by a mechanism that is not fully understood. Here, by locus-specific, single-molecule replication analysis, we identified a crucial role for Pol eta (encoded by the gene POLH) in the in vivo replication of CFSs, even without exogenous stress. We find that Pol eta deficiency induces replication pausing, increases initiation events, and alters the direction of replication-fork progression at CFS-FRA16D in both lymphoblasts and fibroblasts. Furthermore, certain replication pause sites at CFS-FRA16D were associated with the presence of non-B DNA-forming motifs, implying that non-B DNA structures could increase replication hindrance in the absence of Pol eta. Further, in Pol eta-deficient fibroblasts, there was an increase in fork pausing at fibroblast-specific CFSs. Importantly, while not all pause sites were associated with non-B DNA structures, they were embedded within regions of increased genetic variation in the healthy human population, with mutational spectra consistent with Pol eta activity. From these findings, we propose that Pol eta replicating through CFSs may result in genetic variations found in the human population at these sites.

Stochastic chromatin packing of 3D mitotic chromosomes revealed by coherent X-rays

DNA molecules are atomic-scale information storage molecules that promote reliable information transfer via fault-free repetitions of replications and transcriptions. Remarkable accuracy of compacting a few-meters-long DNA into a micrometer-scale object, and the reverse, makes the chromosome one of the most intriguing structures from both physical and biological viewpoints. However, its three-dimensional (3D) structure remains elusive with challenges in observing native structures of specimens at tens-of-nanometers resolution. Here, using cryogenic coherent X-ray diffraction imaging, we succeeded in obtaining nanoscale 3D structures of metaphase chromosomes that exhibited a random distribution of electron density without characteristics of high-order folding structures. Scaling analysis of the chromosomes, compared with a model structure having the same density profile as the experimental results, has discovered the fractal nature of density distributions. Quantitative 3D density maps, corroborated by molecular dynamics simulations, reveal that internal structures of chromosomes conform to diffusion-limited aggregation behavior, which indicates that 3D chromatin packing occurs via stochastic processes.

Differentially accessible, single copy sequences form contiguous domains along metaphase chromosomes that are conserved among multiple tissues

Background During mitosis, chromatin engages in a dynamic cycle of condensation and decondensation. Condensation into distinct units to ensure high fidelity segregation is followed by rapid and reproducible decondensation to produce functional daughter cells. Factors contributing to the reproducibility of chromatin structure between cell generations are not well understood. We investigated local metaphase chromosome condensation along mitotic chromosomes within genomic intervals showing differential accessibility (DA) between homologs. DA was originally identified using short sequence-defined single copy (sc) DNA probes of < 5 kb in length by fluorescence in situ hybridization (scFISH) in peripheral lymphocytes. These structural differences between metaphase homologs are non-random, stable, and heritable epigenetic marks which have led to the proposed function of DA as a marker of chromatin memory. Here, we characterize the organization of DA intervals into chromosomal domains by identifying multiple DA loci in close proximity to each other and examine the conservation of DA between tissues. Results We evaluated multiple adjacent scFISH probes at 6 different DA loci from chromosomal regions 2p23, 3p24, 12p12, 15q22, 15q24 and 20q13 within peripheral blood T-lymphocytes. DA was organized within domains that extend beyond the defined boundaries of individual scFISH probes. Based on hybridizations of 2 to 4 scFISH probes per domain, domains ranged in length from 16.0 kb to 129.6 kb. Transcriptionally inert chromosomal DA regions in T-lymphocytes also demonstrated conservation of DA in bone marrow and fibroblast cells. Conclusions We identified novel chromosomal regions with allelic differences in metaphase chromosome accessibility and demonstrated that these accessibility differences appear to be aggregated into contiguous domains extending beyond individual scFISH probes. These domains are encompassed by previously established topologically associated domain (TAD) boundaries. DA appears to be a conserved feature of human metaphase chromosomes across different stages of lymphocyte differentiation and germ cell origin, consistent with its proposed role in maintenance of intergenerational cellular chromosome memory.

Chromosome characterization and physical mapping of 18S rDNA in Lilium longiflorum originated interspecific hybrids using combined genomic and fluorescent in situ hybridization

This study was aimed at differentiating parental genomes, examining intergenomic composition, and mapping mitotic metaphase chromosomes by localizing parental and 18S rDNA probes in seven interspecific hybrid progenies that originated from Lilium longiflorum. Since in situ hybridization has not been previously used in lily breeding, flow cytometry was used in conjunction with genomic and fluorescent in situ hybridization to determine the genomic contribution of each parent to the interspecific progenies. A significant variation was observed in the DNA content, chromosome length, and 18S loci in F1 as compared to the female and male parents. L. longiflorum showed nearly two times higher DNA content than the male parents and L. longiflorum × Asiatic progenies, but eight times higher than L. longiflorum × L. hansonii. Genomic in situ hybridization results revealed that both female and male parents contributed an equal number of chromosomes to their interspecific F1 offspring. Fluorescent in situ hybridization mapping revealed that 18S rDNA had 8, 6 and 7 loci in L. longiflorum parents, i.e., White heaven, Bright tower, and White tower, respectively, whereas each Asiatic cultivar and L. hansonii used as male showed 8 and 12 loci respectively. Interspecific progenies showed 8 and 7 loci in LA, and 10–11 in LM hybrids. These cytogenetic results implied equal genetic and chromosomal contribution from both parents to their intergenomic progenies. Therefore, this combined (Schwarzacher et al., 1992)cytogenetic method has the potential to be an affordable and time-saving approach in lily breeding that could determine the status of hybrids and their genomic origin while achieving physical mapping and detecting genes in different genomes.