4S RNA Gene Organization in Drosophila melanogaster

Abstract

Drosophila melanogaster has 300–750 genes for tRNAs per haploid genome (Ritossa et al. 1966; Tartof and Perry 1970). Since the kinetic complexity of the mixture suggests 60 different species (Weber and Berger 1976), each tRNA sequence is present an average of 5–12 times per haploid genome. We have studied the organization of the genes for several purified tRNAs. When combined with experiments using the unfractionated 4S RNA mixture, we can obtain an overall view of the organization of tRNA genes in Drosophila.

A major advantage of studying Drosophila is that with the high specific activities obtained by iodination of tRNA with ¹²⁵I (Prensky 1975), the tRNA genes can be located directly on the salivary polytene chromosomes by in situ hybridization. This technique is carried out by treating chromosomes fixed to a microscope slide under conditions that will denature some of their DNA without greatly disrupting their cytological structure. Radioactive RNA is then hybridized to the chromosomes and unhybridized RNA is removed by ribonuclease treatment and extensive washing. After autoradiography, the silver grains resulting from radioactive decay localize the hybridized tRNA molecules on the cytogenetic map (Gall and Pardue 1969). By counting the number of grains at a chromosomal site on many chromosomes, one can determine the amount of RNA-DNA hybrid that is formed at that site. In this way, the rate of the in situ hybridization reaction and the amount of hybrid at saturation can be determined. Such a quantitative approach has been used successfully for the analysis...