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The Localization of the Genes for tRNA4Glu and tRNA2Asp in Drosophila melanogaster by In Situ Hybridization

Eric Kubli, Thomas Schmidt, Albert H. Egg


The localization of the genes coding for tRNAs has been greatly simplified by the introduction of the method of in situ hybridization (Gall and Pardue 1969; John et al. 1969). Early attempts to localize the tRNA genes in Drosophila by hybridizing 3H-labeled total tRNA to polytene salivary gland chromosomes were hampered by the low specific activity of the RNA obtained after feeding 3H-labeled RNA precursors to Drosophila larvae (Steffensen and Wimber 1971). This difficulty was overcome by using the in vitro iodination procedure of RNA developed by Commerford (1971). The first successful attempt to localize the genes for a purified Drosophila tRNA was made by Grigliatti et al. (1974). These authors localized the genes for tRNA5Lys at the 48F–49A region. Some label, however, was also found at 56EF, the locus of the 5S RNA genes. The purification procedures used (RPC-5 chromatography) obviously did not completely eliminate traces of the 5S RNA from this tRNA sample. Therefore, we decided to use two more specific isolation procedures for the purification of Drosophila tRNA isoacceptors (Kubli and Schmidt 1978; Schmidt et al. 1978).

Grosjean et al. (1973) developed an anticodon-anticodon affinity chromatography for the isolation of tRNAGlu and tRNAPhe isoacceptors. This method has been applied to the isolation of a tRNAGlu isoacceptor from Sepharose 4B prefractionated Drosophila tRNA. Double-label experiments have shown that this isoacceptor corresponds to tRNA4Glu of Grigliatti et al. (1974).

After in vitro iodination, the purified tRNA4Glu was hybridized to salivary gland chromosomes...

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