Chromosoma

The chromosome complex of Dall's porpoise Phocoenoides dallii (True), a species of the Delphinidae (Cetacea), was investigated in male germ cells during the course of spermatogenesis. The diploid number of chromosomes in this species was 44 in the spermatogonia and the haploid number was 22 in both primary and secondary spermatocytes. Sex chromosomes of the typical XY-type were found to occur in this species. The X element is represented by one of the medium-sized chromosomes of rod-type characterized by a globular body located at its inner extremity, while the Y is very minute, attaining a size approximately one third that of the smallest autosome. Morphological analysis of the chromosomes shows the chromosome complement of this species to be strikingly characterized by the prevalence of medium-sized elements having subterminal fibre attachments. Comparison of the chromosomes with those of related forms of mammals shows that the chromosome constitution of this species approximates closely that of the pig. The question of the phylogenetical affinity of the Cetacea was discussed on the basis of the karyological evidence here reported.

During early development in female mammals, most genes on one of the two X-chromosomes undergo transcriptional silencing. In the extraembryonic lineages of some eutherian species, imprinted X-inactivation of the paternal X-chromosome occurs. In the cells of the embryo proper, the choice of the future inactive X-chromosome is random. We mapped several genes on the X-chromosomes of five common vole species and compared their expression and methylation patterns in somatic and extraembryonic tissues, where random and imprinted X-inactivation occurs, respectively. In extraembryonic tissues, more genes were expressed on the inactive X-chromosome than in somatic tissues. We also found that the methylation status of the X-linked genes was always in accordance with their expression pattern in somatic, but not in extraembryonic tissues. The data provide new evidence that imprinted X-inactivation is less complete and/or stable than the random form and DNA methylation contributes less to its maintenance.

Chromosome scaffolds are chromosome-shaped bodies, composed of non-histone proteins, which remain when the histones are extracted from chromosomes. Because of the well-known tendency of chromosomal proteins to aggregate, we have tested the possibility that chromosome scaffolds might be produced by aggregation of proteins during the preparation of scaffolds. Extraction of histones in the presence of sucrose, which inhibits aggregation, results in a much looser structure lacking the characteristic appearance of a scaffold, although sucrose does not extract any extra proteins. Extraction of histones from chromosomes in situ on EM grids produced only a network of fine fibres without a scaffold-like structure, while digestion of DNA from typical chromosome scaffolds in situ left only discrete particles of protein and not a continuous structure. We conclude, therefore, that the typical appearance of chromosome scaffolds produced by histone extraction may well represent an artefact resulting from protein aggregation. Our experiments suggest further that DNA, as well as protein, is a structural component of whatever type of core structure is responsible for maintaining the form of chromosome.

The condensed heterochromatic domains are known to be associated with transcriptional repression and cell differentiation. Here, we investigate the function of heterochromatin protein HP1b, a member of the HP1 family in Drosophila melanogaster, in transcription and development. Both knockdown and overexpression of HP1b resulted in partial lethality, indicating that HP1b is essential for the normal development. In contrast to the positive role of HP1a in heterochromatin formation, overexpression of HP1b decondensed the pericentromeric heterochromatin and reduced the association of HP1a and H3K9me2 with it, both known markers of pericentric heterochromatin. Interestingly, the structure of the heterochromatic fourth chromosome appeared not to be affected. Further experiments showed that the presence of HP1a partially rescued the lethality caused by HP1b overexpression in males, and it fully rescued the lethality in females. Consistent with this observation, the defective transcription of heterochromatic genes was also partially restored in the presence of HP1a. Overall, this study argues that HP1b counteracts HP1a function both in heterochromatin formation and in the transcriptional regulation of euchromatic genes.

Late prophase stages of Pales ferruginea (Tipulidae) spermatocytes were examined by means of conventional electron microscopic section technique, combined with cytochemical methods. The cytoplasm of cells in diakinesis contains nucleolus-like bodies (NLB) 1 μm in diameter which are formed in diplotene at the pores of the nuclear membrane. They are compound structures consisting of fibro-granular RNP material which is associated with one or two electron-dense globules. The RNP material has a hollow core which contains polyribosomes. The NLBs possibly indicate rRNA gene amplification. At diakinesis the nucleus contains numerous electron-dense RNP particles scattered throughout the chromatin-free karyoplasm, and associated with the condensed chromosomes. The diameter of the chromatinassociated particles is markedly higher (mean 630 Å) than that of the free particles (mean 540 Å). The RNP particles seem to be aggregates of 200 Å subunits. They are regarded as transcription products of chromosomal genes.

The location of highly reiterated nucleotide sequences on the chromosomes has been studied by the technique of in situ hybridisation between the DNA of either Drosophila melanogaster salivary gland chromosomes or mouse chromosomes and tritium labelled complementary RNA (c-RNA) transcribed in vitro from appropriate templates with the aid of DNA dependent RNA polymerase extracted from Micrococcus lysodeikticus. The location of the hybrid material was identified by autoradiography after RNase treatment. — When Drosophila c-RNA, transcribed from whole DNA, was annealed with homologous salivary chromosomes in the presence of formamide the well defined labelling was confined to the chromocentre. With heat instead of formamide denaturation there was evidence of discontinuous labelling in various chromosome regions as well, apparently associated with banding. Xenopus ribosomal RNA showed no evidence of annealing to Drosophila chromosomes with the comparatively short exposure times used here. — When mouse satellite DNA was used as template the resulting c-RNA showed no hybridisation to Drosophila chromosomes but, when annealed with mouse chromosomes, the centromeric regions were intensely labelled. The interphase nuclei showed several distinct regions of high activity which suggested aggregation of centromeric regions of both homologous and non-homologous chromosomes. The results of annealing either c-RNA or labelled satellite DNA to homologous chromosomes were virtually indistinguishable. Incubation of Drosophila c-RNA with mouse chromosomes provided no evidence of localisation of grains. — It is inferred that both in mouse and Drosophila the centromeric regions of all chromosomes are enriched in highly reiterated sequences. This may be a general phenomenon and it might be tentatively suggested that the highly reiterated sequences play some role in promoting the close physical approximation of homologous and non-homologous chromosomes or chromosome regions to facilitate regulation of function.

The induction and distribution of chromosomal transcripts in the polytene chromosomes of D. melanogaster and D. hydei has been investigated by indirect immunofluorescence using an antiserum directed against DNA/RNA hybrids. The fluorescence was intense and occurred in most of the chromosomal subdivisions when the chromosomes were exposed to denaturing conditions and then allowed to reanneal. The extent of hybrid formation depended both on the extent of DNA denaturation and on the maintenance of RNA integrity. Fluorescence was absent from chromosomes treated with pancreatic RNase before denaturation. The velocity of the chromosomal DNA/RNA hybridization reaction and the effects of the initiation inhibitor of RNA synthesis, DRB, suggest that in order to hybridize the RNA has to be located in its transcriptional compartment. Even though overall patterns of fluorescence seem to be similar during a developmental stage, variations were observed, particularly some correlated with puff induction after ecdysone stimulation.

Ribosomal DNA (rDNA) loci encoding 5S and 45S (18S-5.8S-28S) rRNAs are important components of eukaryotic chromosomes. Here, we set up the animal rDNA database containing cytogenetic information about these loci in 1343 animal species (264 families) collected from 542 publications. The data are based on in situ hybridisation studies (both radioactive and fluorescent) carried out in major groups of vertebrates (fish, reptiles, amphibians, birds, and mammals) and invertebrates (mostly insects and mollusks). The database is accessible online at www.animalrdnadatabase.com . The median number of 45S and 5S sites was close to two per diploid chromosome set for both rDNAs despite large variation (1–74 for 5S and 1–54 for 45S sites). No significant correlation between the number of 5S and 45S rDNA loci was observed, suggesting that their distribution and amplification across the chromosomes follow independent evolutionary trajectories. Each group, irrespective of taxonomic classification, contained rDNA sites at any chromosome location. However, the distal and pericentromeric positions were the most prevalent (> 75% karyotypes) for 45S loci, while the position of 5S loci was more variable. We also examined potential relationships between molecular attributes of rDNA (homogenisation and expression) and cytogenetic parameters such as rDNA positions, chromosome number, and morphology.