UBC Theses and Dissertations
A Drosophila tRNA gene family Newton, Craig Hunter
This thesis describes a tRNA[sup Arg] gene family in the fruit fly D. melanogaster. The study was initiated in order to better understand the gene organization of a subset of this family. Of a total of 10 tRNA[sup Arg] gene copies that comprise this family, four genes are arranged tandemly on repeated sequences 200 bp and 600 bp in length. This organization suggests these four genes have undergone recent gene duplication. To investigate the significance of such events, these tRNA[sup Arg] genes were compared to other members of this gene family in regards their structure, in vitro function, and organization between different D. melanogaster strains and sibling species. The results show that the four repeated genes differ in sequence at a single nucleotide (CI3) relative to six additional gene copies. Five of these additional genes are identical in sequence and one differs at two nucleotides (A16, A37). The gene family is organized at four different chromosomal sites. Six of the 10 gene copies occur at polytene region 12E1-2 on the X chromosome. These include the four repeated genes (R12.1-R12.4) and two additional gene copies (R12.5-R12.6). A single gene occurs at another X-linked locus located at 19F (R19.1). The three remaining gene copies occur on chromosome 3R as a gene doublet at 85C (R85.1-R85.2) and as a single gene at 83AB (R83.1). All 10 genes in this family are active as templates for in vitro transcription in. homologous Drosophila extracts. The predicted 5' initiation sites are all very similar and occur at conserved nucleotides 4-5 bp upstream from the mature 5' ends of the genes. Six of the ten gene copies are transcribed efficiently in vitro. The four repeated gene copies however, have novel transcription properties; they are much less efficient templates in Drosophila extracts (2-5 fold) and are inhibited at KCl concentrations where other gene copies transcribe optimally. These properties do not result from the single nucleotide difference in coding sequence or an inability to form stable pre-initiation complexes. Instead they appear to result from the upstream 5' flanking sequence. These novel transcription properties are not observed in heterologous transcription systems containing human cell extracts. Instead they are transcribed efficiently relative to other members of the gene family and no longer exhibit sensitivity to KCl. Comparison of the repeated tRNA[sup Arg] gene locus between several D. melanogaster strains indicates that this is the predominant form in the majority of wild and laboratory stocks. However, a fraction of populations (5/45) contain a variant locus that consists of only three gene copies. These have apparently lost, or not gained, one of the repeats found in the majority of populations. Similar comparisons between D. melanogaster sibling species (D. simualns, D. teissieri, D. erecta, and D. yakuba) show that only the D. melanogaster lines contain the repeated genes. Thus in the time since the divergence from its closest related sibling species (D. simulans ), D . melanogaster lines have acquired three new gene copies by duplication of a putative ancestral single gene. Analysis of the four, three, and single gene loci isolated from D. melanogaster (pDt27R, p27ry2) and D. simulans (p27simC), respectively, at the nucleotide level suggests a model to account for the evolution of this gene cluster. One additional sequence associated with this gene family consists of a half tRNA[sup Arg] gene composed of the 3' 37 bp. This half gene contains a 3' CCA sequence and is flanked on one side by a region that is > 90% homologous to the LTR of the Drosophila retrotransposon mdg 1. It is not clear if this half gene is itself part of a related retrotransposon or has originated by recombination or aberrant reverse transcription with mdg 1. The 3' ends tRNA[sup Arg] have been proposed to function as primers for at least two classes of retrotransposon, mdg 1 and 412 (Yuki et al., 1986).
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