In situ hybridization of three transfer RNAs to the polytene chromosomes of Drosophila melanogaster

R. T. Elder, P. Szabo, O. C. Uhlenbeck

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10 Scopus citations

Abstract

Three Drosophila transfer RNAs, tRNA2Met, tRNA3Met and tRNA2Arg, were purified from larvae using a new fractionation procedure. After enzymatic aminoacylation, the amino group of aminoacyl-tRNA was coupled covalently to activated CH-Sepharose 4B (the N-hydroxysuccinimide ester of 6-aminohexanoic acid coupled to Sepharose 4B). The resin was washed extensively to remove the unaminoacylated tRNAs and the tRNA specific for the amino acid was eluted at high pH. Addition purification and separation of isoacceptors was done by RPC-5 chromatography. In situ hybridization of 125I-labeled tRNA2Met identifies 48AB on chromosome 2 and 72F on chromosome 3 as gene loci. The rate of in situ hybridization at these two sites is the same and corresponds to the rate expected for a pure tRNA. Hybridization to several other sites occurs at slower rates and is interpreted as due to impurities in the tRNA preparation. Based on in situ, filter, and gel transfer hybridizations, there are two genes for tRNA2Met at 48AB and two at 72F. Similar experiments with tRNA2Arg indicate about eight genes at 42A on chromosome 2 and about five genes at 84EF on chromosome 3. In situ hybridization of tRNA3Met labels 61D, 70DE, 42A and 42E. The rapid rate of labeling at 61D and 70DE establishes these as sites with tRNA3Met genes. The precision of the in situ hybridization measurements is too low to determine whether 42A and 42E are tRNA3Met genes or sites labeled by impurities.

Original languageEnglish (US)
Pages (from-to)1-17
Number of pages17
JournalJournal of Molecular Biology
Volume142
Issue number1
DOIs
StatePublished - 1980

ASJC Scopus subject areas

  • Structural Biology
  • Molecular Biology

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