The interstitial telomeric sequences distribution and their relationship to recombination hot spots in the chicken meiosis

N. A. Lukina, S. A. Galkina, K. V. Zakharova, A. V. Rodionov
Laboratory of Chromosome Structure and Function
Biological Institute of Saint-Petersburg University, Saint-Petersburg, RUSSIA

The chromosomes of all Vertebratae are terminated by the clusters of the highly conserved telomeric sequence (TTAGGG)n, but some of these sequences are also found at distinct intrachromosomal sites. Based on the observations from different animal models it has been speculated that the presence of TTAGGG stretches other than the telomeric sites is related to Robertsonian and tandem fusions of ancestral chromosomes (Meyne et al., 1989, 1990; Lee et al., 1993; Schmid et al., 1994). Several studies have found that these interstitial telomeric repeat sequences can promote chromosome instability and may be hot spots for chromosomal rearrangement and fragility (Dosmari et al., 1999). More over, there is increasing evidence that interstitial telomere sites function as recombinational hot-spots. In particular, study of Armenian hamster spermatocyte meiotic chromosomes showed the correlation between chiasma and interstitial chromosome TTAGGG-position (Ashley, Ward, 1993).

To study the role of TTAGGG-sequences in promoting of crossingover in chicken female meiosis we have been studied the localization of (TTAGGG)n telomere repeat sequences and chiasma distribution in the chicken lampbrush chromosomes (LBC).

The LBC were isolated manually from chicken oocytes of 0,5 mm–2,5 mm diameter, employing the standard lampbrush technique (Solovei et.al, 1993) with some modifications. Preparations were stained with Coomassi Blue R 250 (Serva) and then well-spreaded LBC 1, 2, 3 were selected and photographed to study chiasma distribution within bivalents.

The sites of the (TTAGGG)n in chicken chromosomes were detected by using the plasmid pHuR 93 that contains 40 copies of the TTAGGG unit (Moyzis et al.,1988). It was labelled with biotin-16-dUTP by the nick-translation kit (both from Boehringer Mannheim). The hybridization mixture contained 20 ng/µl probe DNA in 50% formamide, 2xSSC, 10% dextran sulfate, 100 g/ml Escherichia coli tRNA, 20 g/ml GC-enriched Micrococcus carrier DNA (Sigma) at pH 7.0. The FISH protocol followed that of Solovei et al. (1993). The percent of bivalents exhibiting the signals was estimated.

Lampbrush chromosomes are giant diplotene bivalents that appear in the growing oocytes of hatching fowls. At the lampbrush chromosome stage each chicken macrobivalent exhibit a distinct longitudinal differentiation, a specific loop-chromomere pattern. On this basis it has been possible to draw the cytological maps of 1, 2, 3 macrobivalents (Fig. 1c, 2c, 3c).

Each chicken lampbrush bivalent possesses one or few chiasmata. (Fig. 1a, 2a, 3a). Chiasmata in diplotene bivalents are a reliable indicator of reciprocal recombination events (crossing-overs) (Carpenter, 1994; Tease and Jones, 1995). We showed that the chiasmata frequency varies in different chromosome regions. The frequency of chiasmata in the chicken macro-LBCs was counted and their distribution was studied to map the recombinational hot spots (Fig. 1c, 2c, 3c).

Using fluorescence in situ hybridization we have shown that telomere repeat sequences are situated at the ends of all chromosomes as well as in few internal sites on the macro-chromosomes 1–3 (Fig. 1b, 2b, 3b).

A cluster of the interstitial (TTAGGG)n-sequence, ITS11, was found in the region 1B. It was observed in 4 (19%) of all 21 studied bivalents. Another one called ITS12 was localized on the border of subregions 1C1–1C2 (19 (90.5%) of 21 examined bivalents). The third site, ITS13, was found in 1C5 region (9.5% of labelled bivalents). Two ITS-sites, ITS14 and ITS15, were found in the region 1E (86% and 9.5% of labelled bivalents, respectively). Last chromomere of the region 1J1 carries the signal in 19 (90.5%) of LBC1 studied. In a few preparations (2 of 21) there was also a weak TTAGGG-positive signal within region 1L1, ITS17.

Unlike bivalent 1, the bivalent 2 has only one interstitial TTAGGG-site (ITS21) in D region (Fig. 2b).

Bivalent 3 has a single interstitial TTAGGG-site (ITS31) in A region (Fig. 3b).

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Fig. 1. Chicken lampbrush chromosome 1 (LBC 1).
  1. LBC 1 stained by Coomassie Blue R250. Arrows show chiasmata.
  2. DAPI stained LBC 1 hybridized to the (TTAGGG)n sequence. Red signals are due to fluorochrome Cy3. Major chromosome TTAGGG-sites are indicated by arrows.
  3. Cytological map of the chicken LBC 1. Chromosome regions and markers are indicated.Chiasma distribution along 100 bivalents of the chicken bivalent 1. TTAGGG — major telomeric and internal sites of TTAGGG-repeat; ttaggg — minor internal sites of TTAGGG-repeat.

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Fig. 2. Chicken lampbrush chromosome 2. All indications as for Fig. 1.

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Fig. 3. Chicken lampbrush chromosome 3. All indications as for Fig. 1.

According to our data, the positions of chiasmata and the TTAGGG-positive interstitial chromomeres in chicken have no coincide, that disagree with the data on Armenian hamster spermatocytes (Ashley, Ward, 1993).

Our findings show that at least in chicken meiosis a role for interstitial telomeric sequences in promoting of crossing overs is not confirmed.

Acknowledgements

Different part of this work were supported by The Russian Basic Research Foundation (grant # 00-04-49327). Authors thank Prof. E.R.Gaginskaya of the St.Petersburg State University, Microscopic Research Center “Chromas” (RBRF # 00-04-55013) and the Ministry of Science and Research Programme “Comus-1” (# 06-03) for technical supporting.

© Laboratory of Chromosome Structure and Function, 2001