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Tctex-1 is a light chain of the cytoplasmic and flagellar dyneins and a candidate for one of the distorter products that cause transmission ratio distortion in mice. We report the identification, characterization, and a preliminary mutational analysis of the function of the Drosophila melanogaster dtctex-1 gene, the putative ortholog of the mammalian tctex-1 gene family. Four P-transposon insertions which disrupt the 5' untranslated region of dtctex-1 are viable in homozygous form but cause male sterility due to the production of non-motile sperm. In males homozygous for dtctex-1 mutant alleles the dtctex-1 transcript is undetectable, while in homozygous females transcripts of lower molecular weight are present. By secondary mobilization of P-element insertions several revertants and new mutant alleles carrying deletions in the 5' UTR region of the gene were produced and characterized by PCR and by Northern analysis.

We have isolated the Drosophila melanogaster gene encoding the mitochondrial acyl carrier protein (mtACP), a subunit of NADH:ubiquinone oxidoreductase involved in de novo fatty acid synthesis in the mitochondrion. This gene expresses two distinct mature transcripts by alternative splicing, which encode mature polypeptides of 86 (mtACP1A) and 88 (mtACP1B) amino acids, respectively. Drosophila mtACP1 is 72% identical to mammalian mtACP, 47% identical to Arabidopsis thaliana mtACP, and 46% identical to Neurospora crassa mtACP. The most highly conserved region encompasses the site that binds pantetheine-4'-phosphate in all known ACPs. Southern analysis of genomic DNA and in situ hybridization to salivary gland chromosomes indicate that a single gene (mtacp1), located at 61F6-8, encodes the two isoforms of D. melanogaster mtACP1. Sequence analysis revealed that the gene contains four exons and that exons IIIA and IIIB are alternatively spliced. A P-element-induced loss-of-function mutation in the mtacp1 gene causes lethality, indicating that the gene is essential for viability. Developmental Northern analysis shows that mtacp1 is expressed at higher levels during late embryogenesis, in the pupa and in the adult. RNA in situ hybridization on embryos indicates that the mtacp1 gene is highly expressed in the tracheal system. Zygotic mtacp1 function is required for both male and female gametogenesis.

The oxidative phosphorylation (OXPHOS) is the primary energy-producing process of all aerobic organisms and the only cellular function under the dual control of both the mitochondrial and the nuclear genomes. Functional characterization and evolutionary study of the OXPHOS system is of great importance for the understanding of many as yet unclear aspects of nucleus-mitochondrion genomic co-evolution and co-regulation gene networks. The MitoDrome database is a web-based database which provides genomic annotations about nuclear genes of Drosophila melanogaster encoding for mitochondrial proteins. Recently, MitoDrome has included a new section annotating genomic information about OXPHOS genes in Drosophila pseudoobscura and Anopheles gambiae and their comparative analysis with their Drosophila melanogaster and human counterparts. The introduction of this new comparative annotation section into MitoDrome is expected to be a useful resource for both functional and structural genomics related to the OXPHOS system.

A Tirant element, inserted at the 5′ end of the mitochondrial glutamine synthetase ( mt-gs ) gene in a mutant allele giving rise to a recessive female sterility phenotype, was cloned and utilized to characterize this novel retrotransposable element of the Drosophila melanogaster genome. The 5.3 kb element present in the fs(2)PM11-19 mt-gs allele possesses a 417 bp long terminal repeat (LTR) at both ends. There is a serine tRNA binding site downstream of the 5′ LTR sequence and a polypurine tract upstream of the 3′ LTR end. The insertion leads to the duplication of a host-site CGCG sequence. In situ hybridization to salivary glands chromosomes showed evidence of the mobile nature of the element. The DNA sequencing of the cloned 5.3 kb element revealed that Tirant possesses an open reading frame (ORF) that shows similarity with the envelope protein encoded by the gypsy and 297 retrotransposons. In addition, the cloned element appears to be a subgenomic fragment of a not yet identified complete element, because only the integrase domain of the reverse transcriptase gene is found.

S-adenosyl-L-homocysteine hydrolase (AdoHcyase, EC 3.3.1.1) catalyzes the hydrolysis of S-adeno-syl-L-homocysteine to adenosine and homocysteine and thus plays a crucial role in normal cellular metabolism. We have isolated the cDNA for Drosophila melanogaster AdoHcyase by screening a Drosophila ovarian expression library. The 1584-nucleotide cDNA encodes a protein of 431 amino acids, showing 80.5% identity with human AdoHcyase. Southern analysis of genomic DNA and in situ hybridization to salivary gland chromosomes indicate that a single gene encodes the D. melanogaster AdoHcyase. The gene resides in region 13C1-2 on the X chromosome. Transcript analysis shows a single AdoHcyase mRNA present in unfertilized eggs, and, at a more or less constant level of expression, in all developmental stages tested, ranging from early embryos to adults. The deduced amino acid sequence was compared to a putative AdoHcyase-like protein encoded by a cDNA mapping to the 89E region of the second chromosome and showing much lower similarity to known AdoHcyases. We discuss the hypothesis that a sequence that originated by duplication of an ancestral AdoHcyase gene has, in the course of evolution, been recruited to supply a different function.

SCO proteins are copper-donor chaperones involved in the assembly of mitochondrial cytochrome c oxidase (COX). Mutations in the two human SCO-encoding genes, SCO1 and SCO2, produce tissue-specific COX deficiencies associated with distinct clinical phenotypes. Here, we report the identification and characterization of scox, the single Drosophila melanogaster SCO-encoding gene. Null mutations of the scox gene are associated with larval lethality, while mutations in its 5'UTR are associated with motor dysfunction and female sterile phenotypes. All mutant phenotypes may be rescued by a transgene encompassing wild-type scox. The analysis of the phenotypes associated with the D. melanogaster scox mutations shows that unimpaired COX assembly and activity is required for biological processes that specifically depend on an adequate energy supply. Finally, we identified the SCO1 orthologs in 39 eukaryotic species informative for a tentative reconstruction of the evolutionary history of the SCO function. Comparison of the exon/intron structure and other key features suggest that eukaryotic SCO genes descend from an intron-rich ancestral gene already present in the last common ancestor of lineages that diverged as early as metazoans and flowering plants.

Molecular phylogenies based on the molecular clock require the comparison of orthologous genes. Orthologous and paralogous genes usually have very different evolutionary fates. In general, orthologs keep the same functions in species, whereas, particularly over a long time span, paralogs diverge functionally and may become pseudogenes or get lost. In eukaryotic genomes, because of the degree of redundancy of genetic information, homologous genes are grouped in gene families, the evolution of which may differ greatly between the various organisms. This implies that each gene in a species does not always have an ortholog in another species and thus, due to multiple duplication events following a speciation, many orthologous clades of paralogs are generated. We are often dealing with a one-to-many or many-to-many relationship between genes. In this paper, we analyze the evolution of two gene families, the p53 gene family and the porin gene family. The evolution of the p53 family shows a one-to-many gene relationship going from invertebrates to vertebrates. In invertebrates only a single gene has been found, while in vertebrates three members of the family, namely p53, p63, and p73, are present. The evolution of porin (VDAC) genes (VDAC1, VDAC2, and VDAC3) is an example of a many-to-many gene relationship going from yeast to mammals. However, the porin gene redundancy found in invertebrates and possibly in some fishes may indicate a tendency to duplicate the genetic material, rather than a real need for function innovation.

Foldback (FB) elements are transposable elements found in many eukaryotic genomes; they are thought to contribute significantly to genome plasticity. In Drosophila melanogaster, FBs have been shown to be involved in the transposition of large chromosomal regions and in the genetic instability of some alleles of the white gene. In this report we show that FB mediated transposition of w 67C23 , a mutation that deletes the promoter of the white gene and its first exon, containing the start codon, can restore expression of the white gene. We have characterized three independent events in which a 14-kb fragment from the w 67C23 locus was transposed into an intron region in three different genes. In each case a local promoter drives the expression of white, producing a chimeric mRNA. These findings suggest that, on an evolutionary timescale, FB elements may contribute to the creation of new genes via exon shuffling.

A homogeneous array of 80 tandem repeats of the Bari1 transposon is located in the pericentromeric h39 region of chromosome 2 of Drosophila melanogaster. Here, we report that the Bari1 cluster is interrupted by an 8556-bp insertion. DNA sequencing and database searches identified this insertion as a previously unannotated retrotransposon that we have named MAX. MAX possesses two ORFs; ORF1 putatively encodes a polyprotein comprising GAG and RT domains, while ORF2 could encode a 288-amino acid protein of unknown function. Alignment with the RT domains of known LTR retrotransposons shows that MAX belongs to the BEL-Pao family, which remarkable for its widespread presence in different taxa, including lower chordates. We have analyzed the distribution of MAX elements within representative species of the Sophophora subgroup and found that they are restricted to the species of the melanogaster complex, where they are heavily represented in the heterochromatin of all autosomes and on the Y chromosome.

The voltage-dependent anion channel (VDAC, also known as porin) is an abundant protein in the outer mitochondrial membrane that forms transmembrane channels permeable to solutes. While in mammals at least three different porin genes have been found, only one VDAC-encoding gene, porin, has been described so far in Drosophila melanogaster. It produces transcripts with alternative untranslated sequences. Here we report the identification of two PlacW insertions in the porin gene among a set of P-element insertions that have been mapped to the 32B3-4 region on the second chromosome. Homozygotes, as well as trans-heterozygotes for these insertions, lack VDAC, and die during the late pupal stage. Function can be restored by precise excision of the P transposon, while most deletions in the porin locus, produced by imprecise excisions, display the recessive lethal effect of the original mutant alleles. However, one of the deletions was found to be a hypomorphic male-sterile allele producing low levels of the VDAC protein, indicating that the product of the porin gene is also essential for male fertility. Analysis of the new mutant alleles also showed that the untranslated exon 1B of the porin gene is not required for VDAC synthesis. In the course of our investigation, we found that immediately adjacent to the porin gene are three more genes encoding proteins that share homology with the VDAC protein. The possible evolutionary and functional relationships of the porin-like genes at 32B3-4 are discussed.

As a first step towards using cross-species comparison to complete the inventory of the nuclear genes that encode mitochondrial polypeptides, and ultimately to understand their function through systematic molecular and genetic analysis in a model organism of choice, we report here the characterization of 41 Drosophila melanogaster cDNAs. These cDNAs were isolated by screening an ovarian expression library with antibodies against mitochondrial proteins and identify 17 novel Drosophila genes. The deduced amino acid sequences encoded by the majority of these cDNAs turned out to show significant homology to mitochondrial proteins previously identified in other species. Among others, ORFs putatively encoding six different subunits of ATP synthase and three NADH:ubiquinone reductase subunits were detected. By in situ hybridization, all cDNAs were mapped to single bands on polytene chromosomes, thus identifying candidate Drosophila genes required for mitochondrial biogenesis and maintenance. A search of the Human Gene Index database made it possible in most cases to align the entire Drosophila coding sequence with a human consensus sequence, suggesting that the cDNAs originate from insect counterparts of expressed mammalian genes. Our experimental strategy represents an efficient approach to the identification and interspecies comparison of genes encoding products targeted to the mitochondrion.

We have recently cloned a cDNA encoding mitochondrial porin in Drosophila melanogaster and shown its chromosomal localization (Messina et al., FEBS Lett. (1996) 384, 9–13). Such cDNA was used as a probe for screening a genomic library. We thus cloned and sequenced a 4494-bp genomic region which contained the whole gene for the mitochondrial porin or VDAC. It was found that this D. melanogaster porin gene contains five exons, numbered IA (115 bp), IB (123 bp), II (320 bp), III (228 bp) and IV (752 bp). The exons II, III and IV contain the protein coding sequence and the 3′ untranslated sequence (3′-UTR). The first base in exon II precisely corresponds to the first base of the starting ATG codon. Exon IA corresponds to the 5′-UTR sequence reported in the published cDNA sequence. Exon IB corresponds to an alternative 5′-UTR sequence, demonstrated to be transcribed by 5′-RACE experiments. The exon-intron splicing borders and the length of the exon III perfectly match a homologous internal exon detected in the mouse genes. Such exon encodes a protein domain predicted by sequence transmembrane arrangement models to contain major hydrophilic loops and it is thus suspected to have a conserved distinct function. In situ hybridization experiments confirmed the localization of the genomic clone on the chromosome 2L at region 32B3-4. Together with genomic Southern blotting at various stringencies, the same experiment did not confirm the presence of a second genetic locus on D. melanogaster chromosomes. Northern blots demonstrated that the porin gene is a housekeeping one: three messages of approx. 1.2–1.6 kbp are transcribed in every fly developmental stage that was studied. They were shown to derive by an alternative usage of different promoters and polyadenylation sites.

A female-sterile mutation (fs(2) PM11-19) was recovered in a screen for P-M hybrid dysgenesis induced mutations uncovered by a deletion of region 21B and was identified as an allele of the gene encoding the Drosophila glutamine synthetase I (GSI) mitochondrial isozyme. Molecular analysis has shown that fs(2)PM11-19 contains a 5 kb insert within 500 bp upstream of the transcriptional start site of the gsI gene. Mutant flies have extremely low levels of gsl transcription and GSI activity. A pre-existing deficiency (Df(2L) netpm1) with a breakpoint near the transcription start site was also found to be a female-sterile allele of gsl. All eggs laid by PM11-19 homozygous females, as well as by females heterozygous for this mutation and a deletion or any of several recessive lethal alleles of the gsl gene, fail to hatch. We conclude that an adequate level of maternally supplied GSI activity is necessary in the early stages of Drosophila embryonic development. © 1992 Wiley-Liss, Inc.

We describe the cloning of the glutamine synthetase 1 (GS1) gene based on cross-homology with the glutamine synthetase 2 (GS2) gene in Drosophila melanogaster. We have determined the GS gene number in the Drosophila genome, and we describe the isolation of cDNA clones corresponding to the two isoforms, their entire sequence and their transcription pattern. We looked for subcellular localization of one enzymic isoform; in this way, we were able to locate the GS1 enzyme within the mitochondria of D. melanogaster. We have compared different GS sequences from plants and humans; emerging evolutionary implications are discussed. In addition, we have identified a certain highly stable secondary structure at the nucleotide level in the coding region of isoforms located in the organella.

In this work we have used a method that allows a rapid and precise quantification of rRNA genes. With the purpose of examining small numbers of rRNA genes, we used Drosophila melanogaster embryos which are inviable because their sex chromosomes carry extensive rDNA deletions. Two of the mutants, B s Ybb 1 and Ybb l , appear to be completely devoid of rDNA. The third, Ybb -, contains no more than five genes.

In Drosophila rnelanogaster there are two glutamine synthetase (GS) (EC 6.3.1.2) isozymes. They are called GSI and GSII. The two enzymes have different subunits and different genetic determination. A DNA fragment that comprises 80% of the coding region of the glutamine synthetase gene of Chinese hamster ovary (CHO) cells allowed the identification and cloning of an homologous DNA fragment of Drosophila. This sequence is located at the 10B8-11 region on the X chromosome. Dose variation of a chromosomal segment from 9F3 to 10C1-2, which encompasses the 10B region, leads to proportional variations of GSII without apparently influencing the amount of GSI.

We have raised polyclonal antibodies against purified the Drosphila melanogaster mitochondrial porin. They showed high titre and specificity and were thus used as a tool for screening an expression library. The isolated clone 1T1 showed 74% sequence identity in the last 19 residues at the C-terminus of human porin. A subclone of 1T1, containing the porin-like sequence, was thus used as a probe for re-screening a cDNA library and several positive clones were plaque-purified. We present here the sequence of a 1363 bp cDNA encoding a protein of 279 amino acids. Its identity with porin was also confirmed by N-terminal Edman degradation of the purified protein. The D. melanogaster porin shows an overall 51.8% identity with human porin isoform 1 (porin 31HL or HVDAC1) and an overall 55.7% identity with human porin isoform 2 (HVDAC2). Hydrophobicity plots and secondary structure predictions showed a very high similarity with data obtained from known porin sequences. The D. melanogaster porin cDNA was used as a probe for in situ hybridization to polytenic salivar gland chromosomes. It hybridizes with different intensities in two sites, in chromosome 2L, at region 31E and in chromosome 3L at region 79D. Thus, also in Drosophila melanogaster porin polypeptide(s) belong(s) to a multigene family.

Glutamate dehydrogenase has been purified to near-homogeneity from mature larvae of Drosophila melanogaster. The enzyme has a molecular weight of 347,000 measured by sucrose gradient sedimentation and 343,000 measured by variable-porosity acrylamide gel electrophoresis. Electrophoresis under denaturing conditions showed that the enzyme consists of six subunits of molecular weight 57,000. The structural gene for GDH has been mapped at 81.7 +/- 0.8 on the third chromosome by means of an electrophoretic variant.

Glutamine synthetase II was purified from Drosophila melanogaster adults. It was completely separable from the isozyme glutamine synthetase I by means of DEAE chromatography. The complete enzyme has an apparent molecular weight of 360,000. After two-dimensional electrophoresis it gave a single molecular species with an apparent molecular weight of 42,000. Structural analysis of the two isozymes showed that they are different both in subunit molecular weight and in isoelectric point. Peptide maps of the purified subunits showed considerable dissimilarity. Glutamine synthetase II is more active than glutamine synthetase I in the transferase assay, while the opposite is true in the biosynthetic assay. The kinetic parameters were determined, showing again noteworthy differences between the two isozymes. We therefore conclude that two forms of glutamine synthetase are present in Drosophila, with different primary structures, different kinetic behavior, and the possibility of different functional properties.

Recombinational and deletion mapping of electrophoretic variants of the glutamine synthetase I isozyme (GSI) in Drosophila melanogaster locates the gene in the 21B region on the second chromosome. We have conducted a genetic analysis of the region extending cytologically from 21A to 21B4-6. Recessive lethal mutations were generated by ethyl methanesulfonate (EMS) and ethyl nitrosourea (ENU) mutagenesis and by hybrid dysgenesis (HD). These lethals fall into seven functional groups, which were partially ordered by complementation with cytologically defined deficiencies of this region generated by hybrid dysgenesis. Two of the EMS- and two of the ENU-induced lethals fulfill biochemical criteria expected for null alleles of the GSI gene.

The vertebrate steroid hormone hydrocortisone-21-sodium succinate induces in isolated salivary glands of Drosophila melanogaster 3rd instar larvae a protein identified as the 70K heat-shock polypeptide by 1- and 2-dimensional gel electrophoresis analysis. This response is accompanied by significant induction of the puffs 87A and 87C.