FB elements can promote exon shuffling: a promoter-less white allele can be reactivated by FB mediated transposition in Drosophila melanogaster.

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.

MAX, a novel retrotransposon of the BEL-Pao family, is nested within the Bari1 cluster at the heterochromatic h39 region of chromosome 2 in Drosophila melanogaster.

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.

A patchwork interspersed sequence is present in a high copy number in the sheep genome.

We have isolated a new interspersed sequence present in a high copy number in the ovine genome. This patchwork sequence, named 3.79 AS1, is part of a larger element encompassing similarities to constant region of reverse transcriptase and to art2 shared with the Bovine Dimer Driven Family (BDDF). The 3.79 AS1 sequence includes homologies to amplification promoting sequences (APS), to a potential origin of bidirectional DNA replication (OBR), to the Alu core sequence motif GGAGGC required for RNA polymerase III promoter function and to the ATGGCTGCCAT sequence that has been shown to be able to induce amplification-dependent transformation in murine cells. Fluorescent in situ hybridization experiments using probes derived from both ends of the 3.79 AS1 sequence showed a widespread signal over all sheep chromosomes, except the Y chromosome. We propose that the structural features of the 3.79 AS1 patchwork sequence, that is likely to be a subfamily of Bov B LINE that invaded the Artiodactyl genome prior to the separation of the Bovidae species, facilitated its massive amplification and dispersion in the ovine genome.

A genetic analysis of the porin gene encoding a voltage-dependent anion channel protein in Drosophila melanogaster.

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.

dtctex-1, the Drosophila melanogaster homolog of a putative murine t-complex distorter encoding a dynein light chain, is required for production of functional sperm.

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.

Total synthesis of (+)-zaragozic acid C.

A total synthesis of (+)-zaragozic acid C is described. Key features of the synthesis are the use of a double Sharpless asymmetric dihydroxylation reaction of diene 6 to control stereochemistry at four contiguous stereocenters from C3 to C6; the introduction of the C1-side chain by reaction between the anion derived from the dithiane monosulfoxide 27 and the core aldehyde 12; a high yielding, acid-mediated simultaneous acetonide deprotection-dithiane removal-ketalization procedure leading exclusively to the 2, 8-dioxabicyclo[3.2.1]octane core 34; and a novel triple oxidation procedure allowing installation of the tricarboxylic acid.

The complete Tirant transposable element in Drosophila melanogaster shows a structural relationship with retrovirus-like retrotransposons.

We have determined the structure and organization of Tirant, a retrotransposon of Drosophila melanogaster reported in literature to be responsible for four independent mutations. Tirant is a long terminal repeat (LTR) retrotransposon 8527bp long. It possesses three open reading frames (ORF) encoding Gag, Pol and Env proteins with a strong similarity with ZAM, a recently identified member of the gypsy class of retrovirus-like mobile elements. Molecular analysis of the Tirant genomic copies present in four D. melanogaster strains revealed that most of them are defective, non-autonomous elements that differ in the position and extension of the conserved internal portion. Defective elements lacking the Gag ORF but retaining the Env ORF are abundant in heterochromatin. Four discrete Tirant transcripts are observed during embryogenesis in the strain Oregon-R, the smaller of which, 1.8kb in size, originates from the splicing of a primary transcript and leads to a subgenomic RNA coding for the Env product.

The Drosophila melanogaster gene for the NADH:ubiquinone oxidoreductase acyl carrier protein: developmental expression analysis and evidence for alternatively spliced forms.

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.

Identification of nuclear genes encoding mitochondrial proteins: isolation of a collection of D. melanogaster cDNAs homologous to sequences in the Human Gene Index database.

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.

Intra- and interspecies variation among Bari-1 elements of the melanogaster species group.

We have investigated the distribution of sequences homologous to Bari-1, a Tc1-like transposable element first identified in Drosophila melanogaster, in 87 species of the Drosophila genus. We have also isolated and sequenced Bari-1 homologues from D. simulans, D. mauritiana, and D. sechellia, the species constituting with D. melanogaster the melanogaster complex, and from D. diplacantha and D. erecta, two phylogenetically more distant species of the melanogaster group. Within the melanogaster complex the Bari-1 elements are extremely similar to each other, showing nucleotide identity values of at least 99.3%. In contrast, Bari-1-like elements from D. diplacantha and D. erecta are on average only 70% similar to D. melanogaster Bari-1 and are usually defective due to nucleotide deletions and/or insertions in the ORFs encoding their transposases. In D. erecta the defective copies are all located in the chromocenter and on chromosome 4. Surprisingly, while D. melanogaster Bari-1 elements possess 26-bp inverted terminal repeats, their D. diplacantha and D. erecta homologues possess long inverted terminal repeats similar to the terminal structures observed in the S elements of D. melanogaster and in several other Tc1-like elements of different organisms. This finding, together with the nucleotide and amino acid identity level between D. diplacantha and D. erecta elements and Bari-1 of D. melanogaster, suggests a common evolutionary origin and a rapid diversification of the termini of these Drosophila Tc1-like elements.

Physical relationship between satellite I and II DNA in centromeric regions of sheep chromosomes.

Fluorescence in situ hybridization (FISH) with probes representing sheep satellite I and satellite II DNAs shows a different distribution of the two repetitive DNA families in the centromeric region of most chromosomes. The single signal per chromosome produced by the satellite I probe suggests close proximity of this DNA family to the primary constriction. Satellite II produces two separate signals on the sister chromatids, and large blocks of satellite II DNA constitute most of the short arm of all acrocentric chromosomes. We have isolated and sequenced a phage clone containing a junction between discrete blocks of satellite I and satellite II sequences. The junction is characterized by an abrupt juxtaposition of arrays of the two satellites. The possibility that the peculiar structural features of this junction could have a functional significance is discussed.

Cloning and characterization of a copy of Tirant transposable element in Drosophila melanogaster.

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.

The S-adenosyl-L-homocysteine hydrolase of Drosophila melanogaster: identification, deduced amino acid sequence and cytological localization of the structural gene.

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.

The distribution of the transposable element Bari-1 in the Drosophila melanogaster and Drosophila simulans genomes.

The distribution of the transposable element Bari-1 in D. melanogaster and D. simulans was examined by Southern blot analysis and by in situ hybridization in a large number of strains of different geographical origins and established at different times. Bari-1 copies mostly homogeneous in size and physical map are detected in all strains tested. Both in D. melanogaster and in D. simulans a relatively high level of intraspecific insertion site polymorphism is detectable, suggesting that in both species Bari-1 is or has been actively transposing. The main difference between the two sibling species is the presence of a large tandem array of the element in a well-defined heterochromatic location of the D. melanogaster genome, whereas such a cluster is absent in D. simulans. The presence of Bari-1 elements with apparently identical physical maps in all D. melanogaster and D. simulans strains examined suggests that Bari-1 is not a recent introduction in the genome of the melanogaster complex. Structural analysis reveals unusual features that distinguish it from other inverted repeat transposons, whereas many aspects are similar to the widely distributed Tc1 element of C. elegans.

Genetic, molecular and developmental analysis of the glutamine synthetase isozymes of Drosophila melanogaster.

The glutamine synthetase isozymes of Drosophila melanogaster offer an attractive model for the study of the molecular genetics and evolution of a small gene family encoding enzymatic isoforms that evolved to assume a variety of specific and sometimes essential biological functions. In Drosophila melanogaster two GS isozymes have been described which exhibit different cellular localisation and are coded by a two-member gene family. The mitochondrial GS structural gene resides at the 21B region of the second chromosome, the structural gene for the cytosolic isoform at the 10B region of the X chromosome. cDNA clones corresponding to the two genes have been isolated and sequenced. Evolutionary analysis data are in accord with the hypothesis that the two Drosophila glutamine synthetase genes are derived from a duplication event that occurred near the time of divergence between Insecta and Vertebrata. Both isoforms catalyse all reactions catalysed by other glutamine synthetases, but the different kinetic parameters and the different cellular compartmentalisation suggest strong functional specialisation. In fact, mutations of the mitochondrial GS gene produce embryo-lethal female sterility, defining a function of the gene product essential for the early stages of embryonic development. Preliminary results show strikingly distinct spatial and temporal patterns of expression of the two isoforms at later stages of development.

Mutations in the glutamine synthetase I (gsI) gene produce embryo-lethal female sterility in Drosophila melanogaster.

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 gsI 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 gsI. 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 gsI 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.

Genetic determinants of glutamine synthetase in Drosophila melanogaster: a gene for glutamine synthetase I resides in the 21B3-6 region.

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.

Individual quantitative rDNA variation in three species of the Cucurbitaceae family.

Individual quantitative variation in rDNA content within three species of the Cucurbitaceae family has been studied by rRNA/DNA filter hybridization experiments. The results showed a 2.3-fold range of variation in the number of ribosomal cistrons per diploid cell in an Ecballium elaterium natural population. This range of variation is compared with the smaller range observed in three Cucumis sativus and in two Cucurbita pepo varieties obtained as F1 hybrids between pure lines.

Location and underreplication of satellite DNA in Drosophila melanogaster.

The two light nuclear satellites (PCsC1 = 1.672 and PCsC1 = 1.687) have been quantified in DNA isolated from the larvel imaginal discs and brains of Drosophila melanogaster with the genotypes X/O, X/X and X/Y. By comparing the results from these different genotypes, the amounts of the two satellites in the X and Y chromosomes and in the autosomes have been determined. The lightest satellite is not located to any appreciable extent in the X chromosome. The heterochromatic regions are not completely filled by these satellites. --Satellite DNA has also been quantified in DNA isolated from adults containing different genotypes. The two satellites are underreplicated to different extents. The apparent amount of underreplication for one of the satellites is different in different parts of the genome.