HE ROLE OF REPETITIVE SEQUENCES IN THE EVOLUTION OF SEX CHROMOSOMES IN BIRDS.

In this paper, we analyzed the distribution and the transcriptional activity of different repetitive elements in the sex chromosomes of chicken at the lampbrush stage. Based on these results, we suggest participation of interspersed repeats in the maintenance evolutionarily significant level of variability of heteromorphic sex chromosomes in birds. Analysis of the organization peculiarities of chicken sex chromosome W specific repeats allowed us to hypothesize that the accumulation of tandem repeats enriched with homopurine tracks is significant for the evolution of sex chromosomes.

A highly repeated FCP centromeric sequence from chaffinch (Fringilla coelebs: Aves) genome is revealed within interchromosomal connectives during mitosis.

A highly repeated FCP (Fringilla coelebs PstI element) sequence was localized by FISH in centromeric regions of all chromosomes of the chaffinch. Besides, FISH signal was found also in interchromosomal connectives linking centromeres of non-homologous chromosomes in mitotic cells. The presence of DNA in the connectives was confirmed by immunostaining with anti-dsDNA antibodies as well as in experiments on nick-translation and random primed labeling in situ. Non-denaturing FISH with FCP probe and random primed labeling of non-denatured chromosomes resulted in fluorescence signal on both centromeres and intercentromeric connectives, thus providing evidence for the availability of single-strand DNA tracts in FCP sequence. It is suggested that the highly repeated FCP centromeric sequence may be respondible for interconnection of mitotic chromosomes and may by involved in nuclear architecture maintenance in the chaffinch.

The arrangement and transcription of telomere DNA sequences at the ends of lampbrush chromosomes of birds.

The arrangement of loops and chromomeres at the ends of lampbrush chromosomes in four species of bird is described with reference to chromomeres, loops and transcription units. Unlike the situation described in lampbrush chromosomes of amphibians, the lampbrush chromosomes of birds end in a terminal chromosome with conspicuous loops emerging from it. The fine-scale morphology of the ribonuclear protein matrix of these terminal loops is different from that of the majority of loops elsewhere on the chromosomes. In many cases the loops associated with the terminal chromomere are open ended, emerging from the chromomere but not returning to it at the other end. The distal ends of terminal open-ended loops therefore represent the true ends of the chromatids that make up a lampbrush half-bivalent. The pattern of binding of three telomeric DNA sequence probes to the terminal regions of bird lampbrush chromosomes, under conditions of DNA/DNA and DNA/RNA transcript in situ hybridization has been investigated by fluorescence in situ hybridization. All three probes gave the same results. With DNA/DNA and DNA/RNA transcript hybridization, three classes of structure were labelled: the terminal chromomere, a small number of interstitial chromomeres and the terminal transcription unit on telomere loops. Labelling of telomere loops, but not of terminal or interstitial chromomeres, was eliminated by ribonuclease treatment before in situ hybridization. The labelled regions of telomere loops were spaced away from the labelled terminal chromomere by an unlabelled sub telomeric transcription unit. After DNA/DNA in situ hybridization, no labelled loops were seen. DNA/RNA transcript in situ hybridization with single-stranded hexamers of each strand of telomeric DNA showed that the terminal transcription unit on telomere loops represents transcription exclusively from the C-rich strand of the repeat outwards towards the end of the chromosome. It is concluded that transcription specifically of the C-rich strand of strictly terminal clusters of telomere repeats is an obligatory event on the lampbrush chromosomes of birds and is unlikely to represent indiscriminate readthrough from proximally located gene elements.

Amplification of ribosomal genes and formation of extrachromosomal nucleoli in oocytes of starfish Henricia hayashi (Asteroidea: Echinasteridae).

DNA and RNA specific dyes, Ag-NOR staining and in situ hybridization were used for studying the nucleolar apparatus in the growing oocytes of Henricia hayashi (Asteroidea: Echinasteridae). A plasmid containing ribosomal genes of Drosophila melanogaster (Kolchinsky et al., 1980) labelled with 3H by nick-translation served as an rDNA probe. Multiple extrachromosomal nucleoli are formed by the cascade type as a result of growth and subsequent fragmentation of the chromosomal (primary) rDNA body and its derivative extrachromosomal (secondary) rDNA bodies. Ribosomal genes were shown in all nucleolar structures. Argentophilia of the primary and secondary DNA bodies appears to be due to the dense packing of the rDNA-containing material. Ag(+) NORs were detected in the extrachromosomal multiple nucleoli and NOR complexes. Amplification of rDNA is a highly probable conclusion from the existing data.

Transcription of lampbrush chromosomes of a centromerically localized highly repeated DNA in pigeon (Columba) relates to sequence arrangement.

A highly repetitive, centromerically localized DNA sequence (PR1) has been isolated from the genomic DNA of two species of pigeon (Columba livia and C. palumbus). PR1 is approximately 900 bp long. It includes a sequence that is similar to the CENP-B box of mammals. It represents about 5% of the genome in C. livia and 2% in C. palumbus. In both species, tandem arrays of PR1 form part of larger repeating units. The organization of PR1 repeats and the larger repeating units is strikingly different in the two species. The large repeating units in C. livia include long (at least 14 units) tandem arrays of PR1 interspersed with relatively short intervening sequences. The large repeats of C. palumbus have much shorter (4 units or fewer) PR1 arrays interspersed with longer sections of non-PR1 DNA. PR1 is transcribed on short lampbrush loops in the centromeric regions of all lampbrush bivalents of C. palumbus. In C. livia, it is not transcribed at any of the major pericentromeric sites at which it is known to be present, although it is transcribed at one minor centromeric site on chromosome 2. It is proposed that transcription of the noncoding PR1 sequence on lampbrush chromosomes of pigeons relates to its genomic organization. The proposal is discussed with regard to the 'read-through' hypothesis for transcription on lampbrush loops.

Centromeric tandem repeat from the chaffinch genome: isolation and molecular characterization.

A new family of avian centromeric satellites is described. The highly repeated sequence, designated FCP (Fringilla coelebs PstI element), was cloned from the 500-bp PstI digest fraction of the chaffinch (Fringilla coelebs L.) genomic DNA, sequenced, and characterized. The FCP repeat was found to have 505-506 bp length of monomer, 57% content of GC, to compose about 0.9% of the chaffinch genome, and to be highly methylated. Results of Southern-blot hybridization of cloned FCP element onto genomic DNA digested with different restriction enzymes, and sequencing directly from total genomic DNA using FCP-specific primers and ThermoFidelase enzyme (Fidelity Systems Inc.) were in agreement with a tandem arrangement of this repeat in the chaffinch genome. Five positions of single-nucleotide polymorphism (SNP) were found in the FCP monomers using direct genomic sequencing. Fluorescence in situ hybridization (FISH) with FCP probe and primed in situ labelling (PRINS) with FCP specific primers showed that the FCP elements occupy pericentric regions of all chaffinch chromosomes. On chromosome spreads, the fluorescent signals were also observed in the intercentromeric connectives between nonhomologous chromosomes. The results suggest that the centromeric FCP repeat is responsible for chromosome ordering during mitosis in chaffinch.