Informational redundancy of tRNA(4Ser) and tRNA(7Ser) genes in Drosophila melanogaster and evidence for intergenic recombination.

Variant tRNA genes have been widely observed in multicellular eukaryotes. Recent biochemical studies have shown that some of them are expressed in a tissue- or a stage-specific manner. These findings would thus imply that certain modified tRNAs may be crucial for the development of the organism. Using Drosophila melanogaster as a model, we have taken a combined genetic and molecular approach to examine critically the possible biological functions of tRNA(4, 7Ser) genes. We showed that at least 50% of the total templates can be deleted from the genome without inducing abnormal phenotypes such as Minute, or a decrease in viability. In addition, two of the tRNASer variant genes that are unique in sequence are also completely dispensable. This strongly implies that even though they may be expressed in vivo, they play no essential role in the development of the fruitfly. By comparison with some of the corresponding tRNA genes in another sibling species, Drosophila erecta, our results suggest strongly that the variants are products non-reciprocal exchanges among the tRNA(4, 7Ser), genes. Such intergenic recombination events may have a major influence in the concerted evolution of the two gene families.

Overexpression of Cu-Zn superoxide dismutase in Drosophila does not affect life-span.

Aging and disease processes may be due to deleterious and irreversible changes produced by free radical reactions. The enzyme copper-zinc superoxide dismutase (Cu-Zn SOD; superoxide:superoxide oxidoreductase, EC 1.15.1.1) performs a protective function by scavenging superoxide radicals. The Cu-Zn SOD gene (Sod) cloned from Drosophila melanogaster was introduced via P element-mediated transformation into the germ line. Homozygous lines carrying additional copies of the Sod gene were recovered and characterized. Increases in Sod transcripts and enzyme activity were observed in the transformed lines, indicating that all of the sequence information required for gene expression is contained on the inserted gene fragment. The effects of additional SOD on oxygen free radical metabolism and longevity were investigated. Additional SOD did not markedly affect oxygen metabolism or longevity.

Cloning, sequence analysis and chromosomal localization of the Cu-Zn superoxide dismutase gene of Drosophila melanogaster.

The Cu-Zn superoxide dismutase (SOD) cDNA and genomic DNA from Drosophila melanogaster have been isolated. The sequence of the coding region for the Sod gene as well as 413 bp of the 5'-untranslated region, 247 bp of the 3'-flanking DNA, and the single 725-bp intron have been determined [Seto et al., Nucleic Acids Res. 15 (1987) 10601]. The sequence reveals an additional C-terminal triplet coding for valine not found in the mature SOD protein. The nucleotide sequence of the coding region has 56% and 57% homology compared with the corresponding human and rat Sod genes, respectively. The codon usage for the Sod gene is similar to that found for other Drosophila genes. The gene hybridizes to position 68A4-9 on Drosophila polytene chromosomes. In addition, in wild-type Drosophila the Sod cDNA hybridizes to a 0.7-0.8-kb transcript which is greatly diminished in a Sod 'null' mutant that produces only 3.5% of the SOD protein.

Nucleotide sequences of three tRNA(Ser) 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.

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.

N4-(6-aminohexyl)cytidine and -deoxycytidine nucleotides can be used to label DNA.

Bisulfite is known to catalyze transamination between cytidine derivatives and amines. Using 1,6-diaminohexane we describe the synthesis and recovery of the 5'-triphosphates of N4-(6-aminohexyl)cytidine and -deoxycytidine (dahCTP). Both may be incorporated into DNA by nick translation with DNA polymerase I of Escherichia coli to provide reactive sites for the attachment of immunological or other labels. Biotinyl dahCTP is actively incorporated into DNA by the same system and can be detected by the binding of streptavidin complexed to an indicator enzyme such as acid phosphatase. Such labeled DNA is a suitable nonradioactive probe for detection of related sequences by hydridization.

The nucleotide sequence of tRNAVal3a and tRNAVal3b from Drosophila melanogaster.

The nucleotide sequences of two valine tRNA's of Drosophila melanogaster are the following: tRNAVal3a, (sequence in text) tRNAVal3b, (sequence in text) tRNAVal3a shows greater similarity to prokaryotic and eukaryotic organelle tRNAVal's than does tRNAVal3b.

Drosophila melanogaster tRNAVal3b genes and their allogenes.

Drosophila tRNAVal3b genes have been analyzed with respect to their nucleotide sequence and in vitro transcription efficiency. Plasmid pDt78R contains a single tRNA gene derived from the major tRNAVal3b gene cluster at chromosome band 84D. Its sequence corresponds to that of the tRNAVal3b. Two other plasmids, pDt41R and pDt48, each contain a tRNAVal3b-like gene from the minor tRNAVal3b gene cluster at chromosome bands 90BC. They contain the expected CAC anticodon, but their sequence differs from the tRNA at four positions. In homologous cell-free extracts, the tRNAVal3b variant genes in pDt41R and pDt48 are transcribed an order of magnitude more efficiently than the tRNAVal3b gene in pDt78R. However, the variant genes do not appear to contribute significantly to the in vivo tRNA pool [Larsen et al.: Mol. Gen. Genet. 185 (1982) 390-396]. We propose the term allogenes to describe families of related DNA sequences that may code for variant forms of a standard tRNA isoaccepting species.

Genes for tRNALys5 from Drosophila melanogaster.

The sequences of two cloned genes from Drosophila which hybridize with tRNALys5 are reported. One gene, in plasmid pDt39, has a sequence which corresponds to the sequence of tRNA. The other gene, in pDt59R, differs in three nucleotides pairs. Both plasmids are transcribed in vitro with extracts of Drosophila Kc cells to give full-sized tRNA precursors with four additional nucleotides at the 5'-end as well as truncated molecules containing 35 nucleotides. This premature termination occurs in a block of four T residues within the mature coding region. Sequences flanking the tRNA genes show little in common except for the blocks of five or more T-residues beyond the 3'-end of the gene. pDt39 hybridizes to 84AB on the polytene chromosomes of Drosophila and pDt59R hybridizes to 29A.

The structure of tRNA 5 Lys from Drosophila melanogaster.

The nucleotide sequence of Drosophila melanogaster tRNA 5 Lys is pGCCCGGAUAm2GCUCAGDCGGDAGAGCA psi psi GGACUsU*UUt6A*A psi CCAAGGm7GDm5CCAGGGTm psi CAm1AGUCCCUGUUCGGGCGCCA. The sU* is probably 5-methylcarboxymethyl-2-thiouridine and t6A* is a mixture of modified derivatives of t6A including t6A itself and a component sensitive to treatment with cyanogen bromide. This tRNA 5 Lys is 95% homologous to the rabbit liver tRNA 5 Lys.

The structures of genes hybridizing with tRNA4Val from Drosophila melanogaster.

Segments of cloned Drosophila DNA from four recombinant plasmids that hybridize with tRNA4Val have been sequenced. The segments from pDt92R and pDt120R that hybridize to 90C on the third polytene chromosome appear to be either repeats or alleles. They contain one structural gene each of identical sequence but differ at eight sites in 506 base pairs. The structural genes differ at four sites from the sequence expected from that of tRNA4Val. A third plasmid, pDt14, which hybridizes to 89BC on the third chromosome, also contains a structural gene with the same sequence as those in pDt92R and pDt120R. In addition, pDt14 has a gene for tRNA2Phe 214 base pairs upstream and with the same polarity as the tRNA4Val gene. The tRNA2Phe gene contains a 23-base pair segment identical with the corresponding segment in the tRNA4Val genes except for one base pair. The fourth plasmid investigated, pDt55, hybridizes to 70BC. It contains two tRNA4Val genes 525 base pairs apart with opposite polarity. These genes have identical sequences, which corresponds to that expected from the sequence of tRNA4Val. There is no evidence that the first three tRNA4Val genes are expressed at any stage during the development of Drosophila.

The nucleotide sequence of tRNA4Val of Drosophila melanogaster. Chloroacetaldehyde modification as an aid to RNA sequencing.

The nucleotide sequence of tRNA4Val from Drosophila melanogaster was determined to be pGUUUmCCGUm1GGUG psi AGCGGDU(acp3U)AUCACA psi CUGCCmUIACAm5CGCAGAAGm7GCCCCCGGT psi CGm1AUCCCGGGCGGAAACACCA. It is probable that residue C 49 is modified to m5C. The use of tRNA modified with chloroacetaldehyde to overcome secondary structure problems in sequencing is described.

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

Transfer 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 Drosophila tRNA genes carried on recombinant DNA plasmids immobilized on nitrocellulose filters. The level of tRNAVal3b in the tRNA isolated from flies deficient in the major tRNAVal3b loci has been examined. The results show that deletion of the major tRNAVal3b loci resulted in a reduction of approximately 50% in the level of tRNAVal3b but did not produce the Minute phenotype; furthermore the effects of deficiencies at two loci were approximately additive.

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 wer: tRNAGly(GGA), 58A, 84C, and 90E; tRNALeu(2), 44E, 66B5-8, and 79F; tRNASer(2b), 86A, 88A9-12, and 94A6-8; tRNAThr(3), 47F and 87B; tRNAThr(4), 93A1-2; and tRNATyr(1 gamma), 19F, 22F-23A, 41, 50C1-4 and 85A. At 50C the hybridization of tRNATyr(1 gamma) was 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 tRNAVal(3b) and tRNAVal(4). tRNAVal(3a) did not have any sites in common with the other two.

Heteroduplex analysis of tRNAVal3b genes from the 90BC and 84D sites of Drosophila melanogaster.

Two recombinant DNA plasmids coding for Drosophila tRNAVal3b hybridize to two different loci on the drosophila genome. Plasmid pDt41R hydridizines at the 90BC site, and pDt78R hybridizes at the 84D site. Heteroduplexes of the two plasmids were examined by electron microscopy. The study shows that the Drosophila segments have significant homology only over the length of the tRNAVal3b genes.

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-tRNA Asp2 gamma occurred at 56EF although hybridization elsewhere was not affected. In addition, tRNAAsp2 gamma 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 other sites. The hybridization of tRNALys2, tRNA3Gly 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 tRNAAsp2 gamma in this region appears to have been due to contamination of the tRNA sample with traces of material derived from 5S RNA.