Cytomolecular investigations using repetitive DNA probes contribute to the identification and characterization of Characidium sp. aff. C. vidali (Teleostei: Characiformes)

Characidium sp. aff. C. vidali is a species found in coastal streams in southeastern Brazil, which has karyotypic explanatory elements as the occurrence of microstructural variations, keeping the chromosomal macrostructure of the genus. The objective of this study was to apply cytomolecular tools in the chromosomes of Characidium sp. aff. C. vidali to identify characteristics in their karyotype contributing to cytogenetic definition of this species, adding information about the evolution of the chromosomal structure of the group. The species showed 2n = 50 chromosomes and from 1 to 4 additional B microchromosomes. FISH technique showed histone H3 and H4 genes in the short arm of pair 10, and microsatellites (CA)15, (CG)15, (GA)15 and (TTA)10 clustered in the subtelomeric portions of all A chromosomes, with total accumulation by supernumerary. The telomeric probe marked terminal regions of all chromosomes, in addition to the interstitial portion of four pairs, called ITS sites, with these markings being duplicated in two pairs, hence the double-ITS classification. C-banding revealed that supernumerary chromosomes are completely heterochromatic, that ITS sites are C-banding positive, but double-ITS sites are C-banding negative. So, throughout the evolution to Characidium, genomic events are occurring and restructuring chromosomes in populations.

Comparative FISH mapping of ribosomal DNA clusters and TTAGG telomeric sequences to holokinetic chromosomes of eight species of the insect order Psocoptera

Repetitive DNAs are the main components of eukaryotic genome. We mapped the 18S rDNA and TTAGG telomeric probe sequences by FISH to meiotic chromosomes of eight species of the order Psocoptera considered a basal taxon of Paraneoptera: Valenzuela burmeisteri (Brauer, 1876), Stenopsocus lachlani Kolbe, 1960, Graphopsocus cruciatus (Linnaeus, 1768), Peripsocus phaeopterus (Stephens, 1836), Philotarsus picicornis (Fabricius, 1793), Amphigerontia bifasciata (Latreille, 1799), Psococerastis gibbosa (Sulzer, 1766), and Metylophorus nebulosus (Stephens, 1836). These species belong to five distantly related families of the largest psocid suborder Psocomorpha: Caeciliusidae, Stenopsocidae, Peripsocidae, Philotarsidae, and Psocidae. We show that all the examined species share a similar location of 18S rDNA on a medium-sized pair of autosomes. This is the first study of rDNA clusters in the order Psocoptera using FISH. We also demonstrate that these species have the classical insect (TTAGG)n telomere organization. Our results provide a foundation for further cytogenetic characterization and chromosome evolution studies in Psocoptera.

Differentiation of Sex Chromosomes and Karyotype Characterisation in the Dragonsnake Xenodermus javanicus (Squamata: Xenodermatidae)

Highly differentiated heteromorphic ZZ/ZW sex chromosomes with a heterochromatic W are a basic principle among advanced snakes of the lineage Colubroidea, while other snake lineages generally lack these characteristics. For the first time, we cytogenetically examined the dragonsnake, Xenodermus javanicus, a member of the family Xenodermatidae, which is phylogenetically nested between snake lineages with and without differentiated sex chromosomes. Although most snakes have a karyotype with a stable chromosomal number of 2n = 36, the dragonsnake has an unusual, derived karyotype with 2n = 32 chromosomes. We found that heteromorphic ZZ/ZW sex chromosomes with a heterochromatic W are present in the dragonsnake, which suggests that the emergence of a highly differentiated W sex chromosome within snakes predates the split of Xenodermatidae and the clade including families Pareatidae, Viperidae, Homalopsidae, Lamprophiidae, Elapidae, and Colubridae. Although accumulations of interstitial telomeric sequences have not been previously reported in snakes, by using FISH with a telomeric probe we discovered them in 6 pairs of autosomes as well as in the W sex chromosome of the dragonsnake. Similarly to advanced snakes, the sex chromosomes of the dragonsnake have a significant accumulation of repeats containing a (GATA)n sequence. The results facilitate the dating of the differentiation of sex chromosomes within snakes back to the split between Xenodermatidae and other advanced snakes, i.e. around 40-75 mya.

Telomeric Recombination in Lymphocytes Implicates ALT, an Alternative Mechanism for Telomere Length Maintenance, in Normal Human Hematopoietic Cells.

Telomeres are the very ends of chromosomes and protect the genome from recombination, end-to-end-fusion, and recognition as damaged DNA. Telomeres are eroded with each cell division, eventually reaching such critically short length as to cause cell cycle arrest, apoptosis, or genomic instability. In most highly proliferative cells, including hematopoietic stem cells and T lymphocytes, telomere attrition is countered by telomere extension by telomerase reverse transcriptase complex. The majority of cancer cells also express telomerase, which maintains telomere length and allows indefinite cell proliferation. However, about 10% of tumors maintain telomere length in the absence of telomerase by mechanisms collectively termed alternative lengthening of telomeres (ALT). ALT mainly acts through asymmetrical exchange of telomeric material between chromosomes or sister chromatids, producing one daughter-cell with short telomeres and a limited life-span and its sister with long telomeres and higher proliferative capacity. To date, ALT has only been reported in cancer cells or through genetic engineering of mammalian cells. Here we investigated whether ALT mechanisms were active in hematopoietic cells using chromosome orientation fluorescent in situ hybridization (CO-FISH). In standard FISH, a telomeric probe produces fours signals per chromosome, one at each end of the two chromatids. Using CO-FISH, the newly synthesized DNA strand is fragmented by BrdU incorporation and UV light exposure and then digested by exonucleases. In CO-FISH, a telomeric probe produces two signals only, one at each end of the chromosome; in the presence of telomeric recombination, the telomeric signal is split, generating more than two signals per chromosome. Peripheral blood lymphocytes from three healthy volunteers, normal human fibroblasts, K562 cells, telomerase-positive HeLa cells (known to be negative for ALT),and telomerase-negative VA13 cells (known to be positive for ALT) were investigated for telomeric sister chromatid exchange (t-SCE); at least 20 metaphases per cell type were examined. Cultured peripheral blood lymphocytes and VA13 cells both showed increased levels of telomeric sister chromatid exchange in comparison to the other cells (P=0.0001): telomeric probe generated 2.62±0.11 telomeric signals/chromosome in lymphocytes; 2.23±0.04 in VA13 cells; 2.09±0.01 in HeLa cells; 2.02±0.01 in K562 cells; and 2.02±0.01 in human skin fibroblasts. Staining incorporated-BrdU over 24 hours and evaluation of “harlequin” chromosomes point to a similar rate of genomic sister chromatid exchange in lymphocytes, VA13 cells, and HeLa cells, suggesting that high chromatid exchange is confined to the telomeric region. A physical association between promyelocytic leukemia protein (PML) and telomeres is characteristic of some ALT-positive cells, but confocal microscopy failed to co-localize the telomeric probe and anti-PML monoclonal antibody in peripheral blood lymphocytes, suggesting that t-SCE in lymphocytes is not mediated by PML. This is the first demonstration of ALT activation in normal mammalian cells. ALT may be activated in peripheral blood lymphocytes as a complementary mechanism to maintain telomere length, and may explain the differences in age-related telomere shortening observed between lymphocytes and granulocytes.

Localization of the repetitive telomeric sequence (TTAGGG)n in four salmonid species

We have analyzed the localization of the highly conserved telomeric sequence (TTAGGG)n in four salmonid species, two of the genus Salmo (Salmo trutta and Salmo salar) and two of the genus Oncorhynchus (Onchorhynchus mykiss and Onchorhynchus kisutch), by fluorescent in situ hybridization. As expected, the hybridization signal was mostly localized at the telomeres of all chromosomes in the four species. Two species evidenced special hybridization sites with the telomeric probe: (i) interstitial heterochromatic blocks in particular long chromosomes in S. salar, this observation supports tandem fusions as the karyotypic evolutionary mechanism leading to the formation of the long acrocentric and submetacentric chromosomes in the karyotype of S. salar; (ii) the whole NOR region in O. mykiss; this observation suggests that the (TTAGGG)n sequence is scattered all along this chromosome region. Key words : Salmo, Oncorhynchus, telomeres, in situ hybridization, evolution.