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Characterization of recombinant plasmids carrying Drosphila melanogaster tRNA Serine7 genes and their preparation for DNA sequencing Spurr, Mark Gregory


Specific Drosphila tRNA Ser4,7 plasmids were identified and isolated by hybridization with purified [¹²⁵I] tRNA Ser4,7 molecules. Seven clones were isolated carrying the Drosphila Ser tRNA Ser4,7 gene and were further characterized by restriction endonuclease digestion; agarose gel electrophoresis and hybridization with individual purified [¹²⁵I] tRNA Ser4,7 molecules. The results show that five different DNA fragments have been isolated, four which code for a single, specific isoacceptor, and one which appears to code for two different isoacceptors. Two plasmids which initially contained multiple Hind III inserts upon primary isolation were recloned to contain single Hind III inserts containing the tRNA Ser4,7 gene. One of these recloned plasmids contained a smaller tRNA Ser4,7 gene carrying insert than did its original multiple insert isolate. Small tRNA Ser4,7 gene carrying restriction fragments were labelled with T4 polynucleotide kinase and [³²P] ATP, strand separated, and electroeluted, in preparation for nucleotide sequencing.

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