Drosophila melanogaster as a model for studying protein-encoding genes that are resident in constitutive heterochromatin.

The organization of chromosomes into euchromatin and heterochromatin is one of the most enigmatic aspects of genome evolution. For a long time, heterochromatin was considered to be a genomic wasteland, incompatible with gene expression. However, recent studies--primarily conducted in Drosophila melanogaster--have shown that this peculiar genomic component performs important cellular functions and carries essential genes. New research on the molecular organization, function and evolution of heterochromatin has been facilitated by the sequencing and annotation of heterochromatic DNA. About 450 predicted genes have been identified in the heterochromatin of D. melanogaster, indicating that the number of active genes is higher than had been suggested by genetic analysis. Most of the essential genes are still unknown at the molecular level, and a detailed functional analysis of the predicted genes is difficult owing to the lack of mutant alleles. Far from being a peculiarity of Drosophila, heterochromatic genes have also been found in Saccharomyces cerevisiae, Schizosaccharomyces pombe, Oryza sativa and Arabidopsis thaliana, as well as in humans. The presence of expressed genes in heterochromatin seems paradoxical because they appear to function in an environment that has been considered incompatible with gene expression. In the future, genetic, functional genomic and proteomic analyses will offer powerful approaches with which to explore the functions of heterochromatic genes and to elucidate the mechanisms driving their expression.

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.

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.

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.

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.

Segregation distortion in Drosophila melanogaster: genomic organization of Responder sequences.

The heterochromatic Responder (Rsp) locus of Drosophila melanogaster is the target of the two distorter loci Sd and E(SD). Rsp is located in a specific heterochromatic region of the second chromosome and is made up of AT-rich satellite sequences whose abundance is related to its sensitivity to the distorter chromosomes. Here we report that a cluster of Rsp sequences is also located in the third chromosome. The third-chromosome cluster has the same flanking sequences as the clone originally used to identify the Rsp elements, and one of the flanking sequences is a rearranged 412 retrotransposon. The presence of a second, unlinked Rsp-sequence cluster makes re-interpretation necessary for some earlier experiments in which segregation of the third chromosome had not been followed and raises interesting possibilities for the origin of the Rsp locus.

Cloning and chromosomal localization of a cDNA encoding a mitochondrial porin from Drosophila melanogaster.

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.

Transposable elements are stable structural components of Drosophila melanogaster heterochromatin.

We determined the distribution of 11 different transposable elements on Drosophila melanogaster mitotic chromosomes by using high-resolution fluorescent in situ hybridization (FISH) coupled with charge-coupled device camera analysis. Nine of these transposable elements (copia, gypsy, mdg-1, blood, Doc, I, F, G, and Bari-1) are preferentially clustered into one or more discrete heterochromatic regions in chromosomes of the Oregon-R laboratory stock. Moreover, FISH analysis of geographically distant strains revealed that the locations of these heterochromatic transposable element clusters are highly conserved. The P and hobo elements, which are likely to have invaded the D. melanogaster genome at the beginning of this century, are absent from Oregon-R heterochromatin but clearly exhibit heterochromatic clusters in certain natural populations. Together these data indicate that transposable elements are major structural components of Drosophila heterochromatin, and they change the current views on the role of transposable elements in host genome evolution.

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.

Interaction systems between heterochromatin and euchromatin in Drosophila melanogaster.

The constitutive heterochromatin is still one of the major unsolved problems in genetics. In Drosophila melanogaster three genetic systems involving specific interactions between heterochromatic and euchromatic genetic elements are known: the Segregation Distortion, the crystal-Stellate and the abo-ABO systems. The genetic and molecular analysis of each system will allow the identification of all the components and the elucidation of the mechanisms underlying their interactions. The results of this analysis should provide insights into the biological significance of heterochromatin and into the evolutionary forces that result in the maintainance and stability of this enigmatic genetic material.

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.

Bari-1, a new transposon-like family in Drosophila melanogaster with a unique heterochromatic organization.

We have identified a new middle repetitive DNA family in Drosophila melanogaster. This family is composed of a 1.7-kb element, called Bari-1, that shows common characteristics with many transposable elements. Bari-1 is present in a few euchromatic sites that vary in different stocks. However, it is peculiar in that most copies are homogeneously clustered with a unique location in a specific heterochromatic region close to the centromere of the second chromosome. The molecular analysis of different copies coming from the euchromatin and the heterochromatin has revealed that, independent of their location, all possess the same open reading frame. The putative protein encoded by Bari-1 shares similarity with the transposase of the Tc1 transposon of Caenorhabditis elegans. We compare the Bari-1 organization with other mobile DNA families and discuss the possibility of some functional role for the heterochromatic cluster.

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.

Homologous nuclear genes encode cytoplasmic and mitochondrial glutamine synthetase in Drosophila melanogaster.

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.

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.