I. C. Gillam

Hybridization of tRNAs of Drosophila melanogaster to polytene chromosomes.

Highly purified tRNAs from Drosophila melanogaster were iodinated with 125I and hybridized to squashes of polytene chromosomes of Drosophila salivary glands followed by autoradiography to localize binding sites. Most tRNAs hybridize strongly to more than one site and weakly to one or more additional sites. The major sites for various tRNAs are the following: tRNA2Arg, 42A, 84F1,2; tRNA2Asp, 29DE; tRNA3Gly, 22BC, 35BC, 57BC; tRNA2Lys, 42A, 42E; tRNA5Lys, 84AB, 87B; tRNA2Met, 48B5–7, 72F1–2, 83F-84A; tRNA3Met, 46A1–2, 61D1–2, 70F1–2; tRNA4Ser, 12DE, 23E; tRNA7Ser, 12DE, 23E; tRNA3aVal, 64D; tRNA3bVal, 84D3–4, 92B1–9; tRNA4Val, 56D3–7, 70BC.

Isolation and characterization of recombinant DNA plasmids carrying Drosophila tRNA genes

Recombinant plasmids carrying Drosophila melanogaster tRNA genes were constructed by ligation of HindIII-cleaved Drosophila DNA to HindIII cut pBR322 DNA. 90 clones were isolated that contained genes for one or more of eleven tRNAs. 43 of the plasmids were characterized by a number of methods: restriction nuclease digestion; agarose gel electrophoresis; hybridization with individual, purified, 125I-labelled Drosophila tRNA molecules and in situ hybridization to Drosophila chromosomes. The results show that several different tRNA genes have been isolated which code for single, specific isoacceptors. The DNAs from 8 plasmids each hybridize to single sites on Drosophila polytene chromosomes. In addition, the data show examples of two different plasmids hybridizing to different loci coding for the same tRNA; this means that we have isolated representatives of tRNA genes which map at widely separated points on the Drosophila genome.

Cytokinins: Isolation and identification of 6-(3-methyl-2-butenylamino)-9-β-D-ribofuranosylpurine (2iPA) from yeast cysteine tRNA

Abstract A cytokinin in yeast cysteine tRNA has been isolated as the riboside and has been shown to have uv and mass spectra identical with those of synthetic 6-(3- methyl-2-butenylamino)-9-β - D -ribofuranosylpurine.

Genes coding for valine transfer ribonucleic acid-3b in Drosophila melanogaster.

Abstract The genes for tRNA3bval were localized to 84D and 92B on the polytene chromosomes of Drosophila melanogaster with a possible minor site at 90B-C by hybridization in situ and autoradiography with 125I-labeled tRNA3bval. Flies carrying a duplication of the 84D region had increased amounts (30%) of tRNA3bval in proportion to the increased number of genes. While a proportional decrease in the amount of tRNAval3b in flies bearing a deletion of the same region was found, the total acceptance of valine remained at the level found in the wild type.

Hybridization of tRNAs of Drosophila melanogaster to the region of the 5S RNA genes of the polytene chromosomes

We have previously reported that four tRNAs of Drosophila melanogaster randomly labeled with iodine-125 hybridize in part to the 56EF region of polytene chromosomes where 5S RNA genes occur. In the presence of a 100-fold excess of unlabeled 5S RNA no hybridization of randomly labeled 125I-tRNAAsp2γ occurred at 56EF although hybridization elsewhere was not affected. In addition, tRNAAsp2γ labeled by introducing 125I-5-iodocytidylyl residues into the 3′-CCA end with tRNA nucleotidyl transferase did not hybridize to 56EF but did hybridize to its other sites. The hybridization of tRNALys2, tRNAGly3 and tRNAMet3 at 56EF was not eliminated by a 25 to 100-fold excess of unlabeled 5S RNA. When these tRNAs were labeled at the -CCA terminus they hybridized to 56EF as well as to their other sites with the exception that terminally labeled tRNALys2 no longer hybridized to 62A. The hybridization of the latter three species of tRNA to the region of the 5S genes, amongst other sites, is confirmed. The previously observed hybridization of tRNAAsp2γ in this region appears to have been due to contamination of the tRNA sample with traces of material derived from 5S RNA.

Nucleotide sequences of three tRNASer from Drosophila melanogaster reading the six serine codons

The nucleotide sequences of three serine tRNAs from Drosophila melanogaster, together capable of decoding the six serine codons, were determined. tRNA(Ser)2b has the anticodon GCU, tRNA(Ser)4 has CGA and tRNA(Ser)7 has IGA. tRNA(Ser)2b differs from the last two by about 25%. However, tRNA(Ser)4 and tRNA(Ser)7 are 96% homologous, differing only at the first position of the anticodon and two other sites. This unusual sequence relationship suggests, together with similar pairs in the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae, that eukaryotic tRNA(Ser)UCN may be undergoing concerted evolution.

tRNA Genes of Drosophila melanogaster

Although the organization of tRNA genes in Drosophila has not been studied as extensively as those of the genes in Xenopus (Birnstiel et al. 1972; Clarkson et al. 1973a,b, 1978; Clarkson and Kurer 1976) and Saccharomyces (Olson et al. 1977; Goodman et al. 1977; Valenzuela et al. 1978; O’Farrell et al. 1978), we are nevertheless beginning to get a much clearer picture of this organization. The reason for this is that in addition to the techniques of genetic analysis, in vitro hybridization, and incorporation of DNA fragments into plasmids, it is possible with Drosophila to localize genes by in situ hybridization of radioactive tRNA to its polytene chromosomes. After autoradiography, tRNA genes may be assigned visually to specific sites on the chromosomes. THE NUMBER OF tRNA GENES DETERMINED BY HYBRIDIZATION One of the first questions asked about tRNA genes is how many of them exist in the haploid genome for each tRNA. Ritossa et al. (1966) estimated that there are 750 genes for all species of tRNA by observing the saturation level on hybridizing labeled tRNA to Drosophila DNA. More recently, the hybridization results of Weber and Berger (1976) suggested that Drosophila 4S RNA is composed of approximately 59 families of sequences encoded for by 590 genes. As they pointed out, the proposed number of different sets of tRNA genes is based on several assumptions and, at best, is an average value. The 4S RNA fraction contains not only tRNAs but also fragments of rRNA and other RNAs. Because of...

Localization of tRNA genes of Drosophila melanogaster by in situ hybridization

Six purified tRNAs labeled with 125I by chemical or enzymatic methods were hybridized to polytene chromosomes of Drosophila melanogaster. The main chromosomal regions of hybridization were: tRNAGGAGly, 58A, 84C, and 90E; tRNA2Leu, 44E, 66B5-8, and 79F; tRNA2bSer, 86A, 88A9-12, and 94A6-8; tRNA3Thr, 47F and 87B; tRNA4Thr, 93A1-2; and tRNA1γTyr, 19F, 22F-23A, 41, 50C1-4 and 85A. At 50C the hybridization of tRNA1γTyrwas polymorphic in the giant strains. When the hybridization of three valine isoacceptors studied previously was re-investigated, it was found that only one hybridization site, 90BC, was shared between tRNA3bValand tRNA4Val. tRNA3aValdid not have any sites in common with the other two.