Nucleolus organizer regions, types of association and identification of carrier chromosomes in domestic sheep

The frequency of chromosomes carrying nucleolus organizer regions (NOR-positive) and the percentage of association between these chromosomes were determined in sheep metaphase spreads. The sequential R-NOR banding technique permitted identification of the chromosome regions involved as the telomeric ends of chromosomes 1 (lp), 2 (2q), 3 (2q), 4 and 25. nucleolus organizer regions / sequential R-NOR banding / sheep Résumé-Régions de l'organisateur nucléolaire, types d'associations et d'identification des chromosomes porteurs chez le mouton. La fréquence de chromosomes porteurs de la région d'organisation nucléolaire (NOR) et le pourcentage d'association entre chromosomes NOR-positifs ont été étudiés chez le mouton. La méthode de marquage séquentiel R-NOR a permis d'identifier comme régions chromosomiques impliquées, les télomères des chromosomes 1 (1p), 2 (2q), 3 (3q), 4 et !5.


INTRODUCTION
The silver-staining technique developed by Goodpasture and Bloom (1975) permitted the differential staining of chromosome proteins from particular regions of the chromosome, the nucleolus organizer regions. Miller et al (1976) showed that only the active NORs were stained with silver in human-mouse somatic cell hybrids.
In domestic sheep, the NORs are located in the telomeric regions of 5 pairs of chromosomes. In this paper, the frequency distribution of these chromosomes is determined, and the NOR-carrier chromosomes are identified using the R-NOR sequential banding technique.

MATERIALS AND METHODS
Blood samples were collected from the jugular vein of 12 Spanish Merino sheep and cultured with autologous plasma as a modification of the original whole blood culture method described by De Grouchy et al (1964). The metaphase spreads were banding using the R-NOR banding technique developed by Di Berardino et al (1985). The number of metaphase spreads examined per animal varied from 10-30, and for each spread, the number of NOR-positive chromosomes was recorded. The preparations were examined under UV light and micrographs of the best R-banded metaphases were taken using Kodalith film and printed on Valca No 2 paper.

RESULTS AND DISCUSSION
The R-NOR sequential banding technique permitted identification of the positive Ag-NOR chromosomes of sheep (fig 1). The nucleolus organizer regions are located on the telomeric ends of 2 acrocentric (4 and 25) and 3 metacentric chromosomes (1p, 2q, and 3q).
The frequency of NOR positive chromosomes varied between individuals (table I). The number of NORs per metaphase varied from 5-7.71, with an average of 6.31 NORs/metaphase. These results are similar to those reported in cattle (Mayr et al, 1987), and in goats (Moreno-Millin and Rodero, 1988a), but appear lower than those reported in sheep (8 NORs/metaphase) by Henderson and Bruere (1977).
Variations in the number of chromosomes showing positive NOR-staining occurred also in the metaphase spreads from the same animal (table I). In some metaphase spreads, both homologous chromosomes were stained, whereas in others only one chromosome of a given pair was NOR-positive. This variability has already been observed in cattle, goats, horses and humans (Mikelsaar et al, 1977;Mayr et al, 1987;Kopp et al, 1988;Moreno-Millin and Rodero, 1988a).
The frequency of total NORs per metaphase showed a trimodal distribution of 4, 6 and 8 positive NORs per cell (table II). This suggests the presence of different peripheral blood lymphocyte (PBL) populations, with a selective advantage given to the 3 types noted in culture. Similar results were obtained in cattle (Mayr et al, 1987), but as noted in this study, this trimodal distribution did not occur in specific animals.
The average rate of association between Ag-NOR-positive chromosomes was 0.30 per metaphase, and could be divided into associations between 2 or 3 chromosomes (average rates of 0.28 and 0.20, respectively). This association rate is lower than that observed in goats (Moreno-Mill g n and Rodero, 1988b). Preferential association between certain chromosomes was noted (table III) whereas random association had been noted by Henderson and Bruere (1977). In cattle, Mayr and Schleger (1982) observed random association of chromosomes in only 1 of the 3 breeds investigated.
Associations between chromosomes appears to reflect the dynamic formation of the nucleolus (Henderson and Bruere, 1977). However, an increase in the amount of rDNA in the chromosomes seems to be correlated with an increased frequency of association, resulting also in a greater activity and increased silver staining (Henderson et al, 1973;Warburton et al, 1976;Millet et al, 1977). These aspects are currently being investigated, in association with studies on the transmission patterns of NOR frequency distribution in sheep, to give an overall picture of NORs in sheep karyotype.