Phenotypic consequences of copper-zinc superoxide dismutase overexpression in Drosophila melanogaster

We report here the isolation of a tandem duplication of a small region of the third chromosome of Drosophila melanogaster containing the Cu-Zn superoxide dismutase (cSOD) gene. This duplication is associated with a dosage-dependent increase in cSOD activity. The biological consequences of hypermorphic levels of cSOD in genotypes carrying this duplication have been investigated under diverse conditions of oxygen stress imposed by acute exposure to ionizing radiation, chronic exposure to paraquat, and the normoxia of standard laboratory culture. We find that a 50% increase in cSOD activity above the normal diploid level confers increased resistance to ionizing radiation and, in contrast, confers decreased resistance to the superoxide-generating agent paraquat. The duplication is associated with a minor increase in adult life-span under conditions of normoxia. These results reveal important features of the biological function of cSOD within the context of the overall oxygen defense system of Drosophila.     

Cytogenetic analysis of the cSOD microregion in Drosophila melanogaster

This report describes the genetic organization of a euchromatic region on the third chromosome of Drosophila melanogaster extending cytologically from 68A2 to C1, an interval comprising 10 or 11 polytene chromosome bands. The gene for cytoplasmic superoxide dismutase (cSOD) maps within this interval, as does low xanthine dehydrogenase (lxd).--Recessive lethal mutations were generated within the region by ethyl methanesulfonate mutagenesis and by hybrid dysgenesis. These lethals fall into 11 functional groups, which were partially ordered by complementation with deletions having breakpoints within the region. The distribution of dysgenesis-induced mutations in the region is highly nonrandom, the majority being within a single group. The mutability of this gene is comparable to that of singed (sn), a documented "hot-spot" for P-element insertion.--One of the EMS-induced lethals, l-108, fulfills biochemical criteria expected of a hypomorphic allele of cSOD. To our knowledge this is the first such allele recovered of this gene, and it should prove very useful in an analysis of the in vivo function of cytoplasmic SOD. Indeed, it has been demonstrated that cSOD is almost certainly a vital gene.     

HeT-A_pi1, a piRNA target sequence in the Drosophila telomeric retrotransposon HeT-A, is extremely conserved across copies and species

The maintenance of the telomeres in Drosophila species depends on the transposition of the non-LTR retrotransposons HeT-A, TAHRE and TART. HeT-A and TART elements have been found in all studied species of Drosophila suggesting that their function has been maintained for more than 60 million years. Of the three elements, HeT-A is by far the main component of D. melanogaster telomeres and, unexpectedly for an element with an essential role in telomere elongation, the conservation of the nucleotide sequence of HeT-A is very low. In order to better understand the function of this telomeric retrotransposon, we studied the degree of conservation along HeT-A copies. We identified a small sequence within the 3' UTR of the element that is extremely conserved among copies of the element both, within D. melanogaster and related species from the melanogaster group. The sequence corresponds to a piRNA target in D. melanogaster that we named HeT-A_pi1. Comparison with piRNA target sequences from other Drosophila retrotransposons showed that HeT-A_pi1 is the piRNA target in the Drosophila genome with the highest degree of conservation among species from the melanogaster group. The high conservation of this piRNA target in contrast with the surrounding sequence, suggests an important function of the HeT-A_pi1 sequence in the co-evolution of the HeT-A retrotransposon and the Drosophila genome.     

Expression and translocation of Drosophila nuclear encoded cytochrome b(5) proteins to mitochondria

DNA sequence studies of cytochrome b(5) (Cyt-b) genes from Drosophila melanogaster and Drosophila virilis predict that the Drosophila Cyt-b proteins are extremely hydrophobic and have at least eight potential transmembrane spanning domains. Primary protein sequence analysis also predicts that the Cyt-b proteins have mitochondrial targeting sequences and they contain sites for potential post-translational modification similar to other cytochrome proteins. We report the characterization of the cytochrome b(5) proteins from Drosophila melanogaster and Drosophila virilis. We have used a Drosophila cytochrome b(5) specific antibody to demonstrate that cytochrome b(5) proteins are expressed in muscle-containing tissues in the fly. We also provide evidence that the nuclear encoded cytochrome b(5) protein that contains a mitochondrial targeting sequence is translocated to mitochondria.     

Sequence, organization, and evolution of the A+T region of Drosophila melanogaster mitochondrial DNA

The long (4.6-kb) A+T region of Drosophila melanogaster mitochondrial DNA has been cloned and sequenced. The A+T region is organized in two large arrays of tandemly repeated DNA sequence elements, with nonrepetitive intervening and flanking sequences comprising only 22% of its length. The first repeat array consists of five repeats of 338-373 bp. The second consists of four intact 464-bp repeats and a fifth partial repeat of 137 bp. Three DNA sequence elements are found to be highly conserved in D. melanogaster and in several Drosophila species with short A+T regions. These include a 300-bp DNA sequence element that overlaps the DNA replication origin and two thymidylate stretches identified on opposite DNA strands. We conclude that the length heterogeneity observed in the A+T regulatory region in mitochondrial DNAs from the genus Drosophila results from the expansion (and contraction) of the number of repeated DNA sequence elements. We also propose that the 300-bp conserved DNA sequence element, in conjunction with another primary sequence determinant, perhaps the adjacent thymidylate stretch, functions in the regulation of mitochondrial DNA replication.      

Drosophila melanogaster has only one myosin alkali light-chain gene which encodes a protein with considerable amino acid sequence homology to chicken myosin alkali light chains.

A chimeric lambda DNA molecule containing the myosin alkali light-chain gene of Drosophila melanogaster was isolated. The encoded amino acid sequence was determined from the nucleic acid sequence of a cDNA homologous to the genomic clone. The identity of the encoded protein was established by two criteria: (i) sequence homology with the chicken alkali light-chain proteins and (ii) comparison of the two-dimensional gel electrophoretic pattern of the peptides synthesized by in vitro translation of hybrid-selected RNA to that of myosin alkali light-chain peptides extracted from Drosophila myofibrils. There is only one myosin alkali light-chain in D. melanogaster; its chromosomal location is region 98B . This gene is abundantly expressed during the development of larval as well as adult muscles. The Drosophila protein appears to contain one putative divalent cation-binding domain (an EF hand) as compared with the three EF hands present in chicken alkali light chains.     

A 9.6 kb intervening sequence in D. virilis rDNA, and sequence homology in rDNA interruptions of diverse species of Drosophila and other diptera.

A large proportion of the 28S ribosomal RNA genes in Drosophila virilis are interrupted by a DNA sequence 9.6 kilobase pairs long. As regards both its presence and its position in the 28S gene (about two thirds of the way in), the D. virilis rDNA intervening sequence is similar to that found in D. melanogaster rDNA, but lengths differ markedly between the two species. Degrees of nucleotide sequence homology have been detected bewteen rDNA interruptions of the two species. This homology extends to putative rDNA intervening sequences in diverse higher diptera (other Drosophila species, the house fly and the flesh fly), but hybridization of cloned D. melanogaster and D. virilis rDNA interruption segments to DNA of several lower diptera has been negative. As is the case with melanogaster rDNA interruptions, segments of the virilis rDNA intervening sequence hybridize with non-rDNA components of the virilis genome, and interspecific homology may involve these non-rDNA sequences as well as rDNA interruptions. There is, however, evidence from buoyant density fractionation of DNA that the distributions of interruption-related sequences are distinct in D. melanogaster and D. virilis genomes. Moreover, thermal denaturation studies have indicated differing extents of homology between hybridizable sequences in D. virilis DNA and different segments of the D. melanogaster rDNA intervening sequence. We infer from our studies that rDNA intervening sequences are prevalent among higher diptera; that in the course of the evolution of these organisms, elements of the intervening sequences have been moderately to highly conserved; and that this conservation extends in at least two distantly related species of Drosophila to similar sequences found elsewhere in the genomes.     

A Drosophila melanogaster H3.3 cDNA encodes a histone variant identical with the vertebrate H3.3.

A cDNA encoding an H3.3 histone variant in Drosophila melanogaster predicts a protein with an amino acid (aa) sequence identical with that in vertebrates. The D. melanogaster H3.3 nucleotide (nt) sequence has diverged significantly from that of both the H3.3 gene of vertebrates and the H3.1 gene of D. melanogaster, largely through third nt changes in its codons. The perfect H3.3 aa sequence conservation between organisms as phylogenetically divergent as vertebrates and flies suggests that the H3.3 histone variant itself is an important structural component of chromatin, apart from the value of its replication-independent expression pattern.     

Functional conservation of the<Emphasis Type="Italic"> Sex-lethal</Emphasis> sex determining promoter,<Emphasis Type="Italic"> Sxl-Pe</Emphasis>, in<Emphasis Type="Italic"> Drosophila virilis</Emphasis>

The primary sex determination signal in Drosophila melanogaster, the ratio of X chromosomes to autosomes, sets the activity state of the switch gene, Sex-lethal ( Sxl), by regulating the establishment promoter, m-Sxl-Pe. We have identified and characterized the establishment promoter, v-Sxl-Pe, of the distantly related species Drosophila virilis. Like melanogaster, the virilis Sxl-Pe is organized into four sub-domains: the Sxl-Pe mRNA leader and exon E1 of Sxl protein, the core promoter, the sex-specific element and the augmentation element. The core promoter and sex-specific element of v-Sxl-Pe show considerable sequence similarity to m-Sxl-Pe and contain target sites for components of the X/A signaling system. While the augmentation element of v-Sxl-Pe also has sequence motifs that could function as target sites for the X/A signaling system, it shows little similarity to the melanogaster augmentation element. Functional studies reveal that v-Sxl-Pe drives sex-specific expression in D. melanogaster embryos and that the activity of the virilis promoter is controlled by known components of the melanogaster X/A counting system. Although v-Sxl-Pe responds appropriately to the melanogaster sex determination signal, it is less active than Sxl-Pe from melanogaster. Unexpectedly, the reduced activity is due to differences in the activity of the conserved core promoter, while the non-conserved augmentation element functions effectively. These findings suggest that low-affinity target sites for the X/A counting system are critical for the functioning of Sxl-Pe.     

Analysis of a fushi tarazu autoregulatory element: multiple sequence elements contribute to enhancer activity.

Regulatory sequences or factors involved in the regulation of target genes of Drosophila homeodomain proteins are largely unknown. Here, we identify sequence elements that are involved in the function of the fushi tarazu (ftz) autoregulatory element AE, a direct in vivo target of the homeodomain protein ftz. A systematic deletion analysis of AE in transgenic embryos defines multiple elements that are redundantly involved in enhancer activity. Sequences juxtaposed to ftz binding sites are not strictly required for enhancer function. Several sequence motifs are conserved in other developmentally regulated genes of Drosophila melanogaster and in the AE homologue of Drosophila virilis. The D. virilis AE is functional in D. melanogaster. The sequence motifs identified here are candidate elements contributing to the target specificity of the homeodomain protein ftz.     

Promoter analysis of the Drosophila melanogaster gene encoding the pterin 4alpha-carbinolamine dehydratase.

Pterin-4alpha-carbinolamine dehydratase (PCD) is a key enzyme in the regeneration pathway of tetrahydrobiopterin. Previously, we isolated and reported the Drosophila melanogaster gene encoding PCD. In the present study, we isolated and characterized the Drosophila virilis gene encoding PCD. The Drosophila virilis PCD gene has two introns and an open reading frame to encode a protein of 101 amino acids. The amino acid sequence of Drosophila virilis PCD shows a 83% homology to that of the Drosophila melanogaster PCD protein. From the alignment of the nucleotide sequence in the 5'-flanking region of the Drosophila melanogaster and Drosophila virilis PCD genes, we found four conserved sequences. Using a transient transfection assay, we showed that one of the conserved sequences (-127 to approximately -115) is critical for expression, also the minimal promoter region between -127 and +51 is necessary for the efficient expression of Drosophila melanogaster PCD.     

Isolation and characterization of Drosophila melanogaster U2 small nuclear RNA genes

We describe here the organization of DNA sequences complementary to Drosophila melanogaster U2 small nuclear (sn) RNA. From a genomic library we isolated two recombinants containing two genes each. Genomic reconstruction experiments and Southern analysis revealed that D. melanogaster possesses only four to five U2 snRNA genes or very closely related sequences. The nucleotide sequence of one of the clones analysed shows 77% homology with rat U2 snRNA. A stretch of 12 nucleotides that has been implicated in heterogeneous nuclear RNA splicing is conserved between rat and Drosophila. The genomic organization of these genes is very similar in different melanogaster strains but diverges highly in different Drosophila species.     

Drosophila ARSs contain the yeast ARS consensus sequence and a replication enhancer.

A number of restriction fragments that function as autonomously replicating sequences (ARSs) in yeast have been isolated from Drosophila melanogaster DNA. The behaviour in yeast of plasmids containing Drosophila ARS elements was studied and compared to that exhibited by the archetypal yeast ARS-1 plasmid. ARS functions were localised by subcloning and BAL-31 deletion analysis. These studies demonstrated the structural and functional complexity of Drosophila ARSs. Each Drosophila ARS element has at least two domains, one essential for replication (the replication sequence, RS) and a second (the replication enhancer, RE) which is essential for maximum function of the RS. The RS of three Drosophila ARSs was shown to contain a sequence identical to an 11 bp yeast ARS consensus sequence (5' A/T TTTATPuTTT A/T 3'). These observations lend support to the hypothesis that heterologous ARS elements may be of biological significance.     

Microevolutionary divergence pattern of the segmentation gene hunchback in Drosophila

To study the microevolutionary processes shaping the evolution of the segmentation gene hunchback (hb) from Drosophila melanogaster, we cloned and sequenced the gene from 12 isofemale lines representing wild-type populations of D. melanogaster, as well as from the closely related species Drosophila sechellia, Drosophila orena, and Drosophila yakuba. We find a relatively low degree of sequence variation in D. melanogaster (theta = 0.0017), which is, however, consistent with its chromosomal location in a region of low recombination. Tests of neutrality do not reject a neutral-evolution model for the whole region. However, pairwise tests with different subregions indicate that there is a relative excess of polymorphic sites in the leader and the intron. Codon usage pattern analysis shows a particularly biased codon usage in the highly conserved regions, which is in line with the hypothesis that selection on translational accuracy is the driving force behind such a bias. A comparison of the expression pattern of hb in different sibling species of D. melanogaster reveals some regulatory changes in D. yakuba, which could be interpreted as changes in the timing of secondary expression domains.     

The nucleotide sequence of a glutamine tRNA from rat liver.

A glutamate tRNA from rat liver was purified. By means of post-labeling techniques, its nucleotide sequence was shown to be: pU-C-C-C-A-C-A-U-m1G-G-U-C-psi-A-G-C- G-G-D-D-A-G-G-A-U-U-C-C-U-G-G-psi-U-mcm5S2U-U-C-A-C-C-C-A-G-G-C-G- G-C-m5C-m5C-G-G-G-Tm-psi-C-G-A-C-U-C-C-C-G-G-U-G-U-G-G-G-A-A-C-C-AOH. The sequence is remarkably similar to that of tRNAGlu from Drosophila melanogaster. Only 10 out of 75 nucleotides in the two tRNAs are different.     

The Drosophila virilis dopa decarboxylase gene is developmentally regulated when integrated into Drosophila melanogaster.

The dopa decarboxylase gene (Ddc) has been isolated from Drosophila virilis and introduced into the germ-line of Drosophila melanogaster by P-element mediated transformation. The integrated gene is induced at the correct stages during development with apparently normal tissue specificity, indicating that cis-acting elements required for regulation are functionally conserved between the two species. A comparison of the DNA sequences from the 5' flanking regions reveals a cluster of small (8-16 bp) conserved sequence elements within 150 bp upstream of the RNA startpoint, a region required for normal expression of the D. melanogaster Ddc gene.