G. M. Tener

Analysis of tRNAs during the development of Drosophila

Abstract Conditions for the chromatography of radioactively labeled Drosophila aminoacyl-tRNAs on reversed-phase 5 columns have been established. All 20 aminoacyl-tRNAs from first instar, third instar, and adult flies were compared. While some of the aminoacyl-tRNAs remain essentially unchanged during the development of Drosophila, others show marked quantitative changes. These changes are discussed in relation to possible control mechanisms during development.

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.

Properties of tRNAPhe from Drosophila

Abstract 1. Drosophila tRNAPhe is not retarded on BD-cellulose columns as are other cytoplasmic tRNAPhes from eukaryotes. 2. Neither purified tRNAPhe nor crude tRNA has the characteristic fluorescence of tRNAs containing Y-type bases. A nucleotide analysis of purified tRNAPhe detected no Y-type base and in this respect it is similar to prokaryotic and organelle tRNAPhes. 3. Aminoacylations of Drosophila tRNA with [14C]phenylalanine using heterologous enzymes suggest that Drosophila tRNAPhe is functionally similar to yeast tRNAPhe but different from Escherichia coli tRNAPhe.

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.

Extensive microheterogeneity of serine tRNA genes from Drosophila melanogaster

The nucleotide sequences of nine genes corresponding to tRNA(Ser)4 or tRNA(Ser)7 of Drosophila melanogaster were determined. Eight of the genes compose the major tRNA(Ser)4,7 cluster at 12DE on the X chromosome, while the other is from 23E on the left arm of chromosome 2. Among the eight X-linked genes, five different, interrelated, classes of sequence were found. Four of the eight genes correspond to tRNA(Ser)4 and tRNA(Ser)7 (which are 96% homologous), two appear to result from single crossovers between tRNA(Ser)4 and tRNA(Ser)7 genes, one is an apparent double crossover product, and the last differs from a tRNA(Ser)4 gene by a single C to T transition at position 50. The single autosomal gene corresponds to tRNA(Ser)7. Comparison of a pair of genes corresponding to tRNA(Ser)4 from D. melanogaster and Drosophila simulans showed that, while gene flanking sequences may diverge considerably by accumulation of point changes, gene sequences are maintained intact. Our data indicate that recombination occurs between non-allelic tRNA(Ser) genes, and suggest that at least some recombinational events may be intergenic conversions.

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.

RNA-DNA hybridization analyses of tRNA 3b Val in Drosophila melanogaster

SummaryTransfer RNA was extracted from 50–300 mg of adult flies and specifically labeled in vitro. The level of individual isoacceptors was quantitated by efficient annealing to Drosphila tRNA genes carried on recombinant DNA plasmids immobilized on nitrocellulose filters. The level of tRNA3bValin the tRNA isolated from flies deficient in the major tRNA3bValloci has been examined. The results show that deletion of the major tRNA3bValloci resulted in a reduction of approximately 50% in the level of tRNA3bValbut did not produce the Minute phenotype; furthermore the effects of deficiencies at two loci were approximately additive.