CLONING AND SOME PROPERTIES OF DNA-SEQUENCES OF HOP (HUMULUS-LUPULUS L.) AMPLIFIED BY PCR USING CONSERVATIVE MOTIFS OF 7SL RNA GENES

PCR products amplified from hop genomic DNAs using conserved motifs of 7SL RNA genes were partially characterized. Direct primers, overlappi ng RNA polymerase III promoter-like box (box A) on the 5' end and the conserved sequence designated B element on the 3' end of 7SL DNA, yiel ded 7SL-specific sequences, which showed conservative micro-RFLP patte rns. Reverse primers designated RevA and RevB, which were complementar y to A and B elements, respectively, led to amplification of variable PCR products. These products did not hybridize with a 7SL RNA specific probe. A PCR priming either by single RevA or by RevB or by the combi nation of these two primers was analyzed. While RevA primer led to amp lification of relatively broad spectrum of DNA products ranging from 1 80 bp to more than 2 kb, the spectrum characteristic for the RevB prim er was more simple. Besides several (0.5 to 1 kb) products, there was a distinct product about 260 bp. A series of new bands, which appeared only when RevA and RevB were used in combination, were analyzed in mo re detail on acrylamide gels. These distinct bands, which represent pu tative intergenic spacers, ranged from 50 to 270 bp. Comparisons of th ese spacers characteristic for different hop genotypes showed minor di fferences in their electrophoretic mobility, suggesting a very narrow range of fragment length polymorphism of some of these sequences, from single to several nucleotides. The HindIII pattern of genomic 7SL DNA sequences from Osvald clone 72 exhibited bands corresponding to restr iction fragment lengths of approximately 7.09, 1.72, 0.78, 0.55, 0.29 and 0.2 kb. A plasmid library enriched for these fragments was establi shed and two 7SL DNA clones were analyzed by TGGE. These sequences had melting points similar to that, described previously (Matousek, Trnen a, 1996) and identified in the GenBank as 7SL RNA from cv. Spalter (AC X65985). partial sequencing of these two clones upstream promoter Box A, i.e. in the position of the intergenic spacers, revealed only 56% homology between them and the presence of several stop codons, confirm ing non-coding DNA regions.