Cloning of a polymorphic sequence from the nontranscribed spacer of horse rDNA

Abstract

Multiple rRNA genes are present in most genomes, and in eukaryotes they are arranged in clusters of tandemly repeating units. Each unit is composed of a transcribed region and of a nontranscribed spacer (NTS; Long and Dawid 1980). In different species the rDNA clusters are located on one or on several chromosome pairs and define the nucleolar organizing regions (NOR; Sollner-Webb and Mougey 1991). The regions transcribing the mature rRNA are conserved. On the contrary, the NTS regions are widely divergent in sequence length and composition (Long and Dawid 1980), and length variants have been observed in several species. In humans the NTS length polymorphism is due to a variable number of tandemly repeated modules in different rDNA units (La Volpe et al. 1985) and single variants seem to be spread on different chromosomes (Ranzani et al. 1984). Little is known about the horse rDNA. A restriction map of the transcribed region has been obtained with human rRNA to probe horse genomic DNA (Tanhauser et al. 1986), and three chromosome pairs were shown to be positive to NOR banding (Richer et al. 1990). A DNA library was constructed in the phage vector hGEM-I 1. Genomic DNA from two horses was partially digested with MboI and the 12to 23-kb fragments were ligated with BamHI cloning arms. The library was screened with a mixture of two plasmids (pLS6BB and pLS6BE) containing 3.3 kb of the human 28S region (Ranzani et al. 1984); XHNTS1 was isolated. The clone contains an insert of 15.9 kb, which is homologous to about 200 bp of the 3' end of the transcribed region and spans through the adjacent nontranscribed spacer (data not shown). Restriction maps of horse and human rDNA were obtained by cutting genomic DNA with three restriction enzymes (EcoRI, BamHI, BglII) in single and multiple digestions and subsequently hybridizing the DNA transfers with the two human probes, pLS6BB and pLS6BE, and with the horse hHNTS1 probe. The two restriction maps are shown in Fig. 1A and B. In the transcribed region the restriction maps of the two species are similar with minor differences in site position, while the nontranscribed region is divergent. The human map shown in Fig. IA is in agreement with that already published (Ranzani et al. 1984). The definition of the 18S, 5.8S, 28S, and spacer boxes was derived from La Volpe and associates (1985). When a restriction map of the hHNTS1 insert was generated (data not shown), no EcoRI sites were found. However, when the clone was used to probe genomic DNA, it detected many EcoRI fragments (Fig. 2). Most EcoRI fragments are comprised within the nontranscribed spacer and represent length variants, while the 7.6-kb fragment is contained in the transcribed region and the 9.2-kb fragment is distal to the variability regions. These results suggest that HNTS1 was derived from a rare variant lacking the