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.Communicated by G. P. Georgiev     

Vital genes in the heterochromatin of chromosomes 2 and 3 of Drosophila melanogaster

Heterochromatin has been traditionally regarded as a genomic wasteland, but in the last three decades extensive genetic and molecular studies have shown that this ubiquitous component of eukaryotic chromosomes may perform important biological functions. In D. melanogaster, about 30 genes that are essential for viability and/or fertility have been mapped to the heterochromatin of the major autosomes. Thus far, the known essential genes exhibit a peculiar molecular organization. They consist of single-copy exons, while their introns are comprised mainly of degenerate transposons. Moreover, about one hundred predicted genes that escaped previous genetic analyses have been associated with the proximal regions of chromosome arms but it remains to be determined how many of these genes are actually located within the heterochromatin. In this overview, we present available data on the mapping, molecular organization and function of known vital genes embedded in the heterochromatin of chromosomes 2 and 3. Repetitive loci, such as Responder and the ABO elements, which are also located in the heterochromatin of chromosome 2, are not discussed here because they have been reviewed in detail elsewhere.     

Intercalary heterochromatin in Drosophila melanogaster polytene chromosomes and the problem of genetic silencing

The morphological characteristics of intercalary heterochromatin (IH) are compared with those of other types of silenced chromatin in the Drosophila melanogaster genome: pericentric heterochromatin (PH) and regions subject to position effect variegation (PEV). We conclude that IH regions in polytene chromosomes are binding sites of silencing complexes such as PcG complexes and of SuUR protein. Binding of these proteins results in the appearance of condensed chromatin and late replication of DNA, which in turn may result in DNA underreplication. IH and PH as well as regions subject to PEV have in common the condensed chromatin appearance, the localization of specific proteins, late replication, underreplication in polytene chromosomes, and ectopic pairing.     

Carbazole/dioxin-degrading car gene cluster is located on the chromosome of Pseudomonas stutzeri strain OM1 in a form different from the simple transposition of Tn4676

The carbazole-degrading (car) operon on the chromosome of Pseudomonas stutzeri strain OM1 showed >99% identity to that in the 72.8 kb catabolic transposon, Tn4676, on plasmid pCAR1. Southern hybridization using probes prepared from the pCAR1 sequence and sequencing analyses showed that the OM1 chromosome contained the 55 kb DNA region, almost all of which was a part of Tn4676, flanked by two copies of novel insertion sequence, ISPst3, and included the car gene.     

Involvement of desat1 gene in the control of Drosophila melanogaster pheromone biosynthesis

Cuticular pheromones in Drosophila melanogaster are unsaturated hydrocarbons with at least one double bond in position 7: 7-tricosene and 7-pentacosene in males and 7,11-heptacosadiene and 7,11-nonacosadiene in females. We have previously shown that a desaturase gene, desat1, located in chromosome region 87 C could be involved in this process: the Desat1 enzyme preferentially leads to the synthesis of palmitoleic acid, a precursor of 7 fatty acids and 7-unsaturated hydrocarbons. Therefore, we have searched for P–elements in the 87 region and mapped them. One was found inserted into the first intron of the desat1 gene. Flies heterozygous for this insertion showed a large decrease in the level of 7-unsaturated hydrocarbons, comparable to that observed in flies heterozygous for a deficiency overlapping desat1. Less than 1% of flies homozygous for this insertion were viable. They were characterized by dramatic pheromone decreases. After excision of the transposon, the pheromone phenotype was reversed in 69% of the lines and the other excision lines had more or less decreased amounts of 7-unsaturated hydrocarbons. All these results implicate desat1 in the synthesis of Drosophila pheromones.     

Nucleotide divergence of the rp49 gene region between Drosophila melanogaster and two species of the Obscura group of Drosophila

Summary A 2.1-kb SStI fragment including the rp49 gene and the 3 end of the -serendipity gene has been cloned and sequenced in Drosophila pseudoobscura. rp49 maps at region 62 on the tip of chromosome II of this species. Both the coding and flanking regions have been aligned and compared with those of D. subobscura. There is no evidence for heterogeneity in the rate of silent substitution between the rp49 coding region and the rate of substitutions in flanking regions, the overall silent divergence per site being 0.19. Noncoding regions also differ between both species by different insertions/deletions, some of which are related to repeated sequences. The rp49 region of D. pseudoobscura shows a strong codon bias similar to those of D. subobscura and D. melanogaster. Comparison of the rates of silent (K _S ) and nonsilent (K _a ) substitutions of the rp49 gene and other genes completely sequenced in D. pseudoobscura and D. melanogaster confirms previous results indicating that rp49 is evolving slowly both at silent and nonsilent sites. According to the data for the rp49 region, D. pseudoobscura and D. subobscura lineages would have diverged some 9 Myr ago, if one assumes a divergence time of 30 Myr for the melanogaster and obscura groups.Offprint requests to: C. Segarra     

Embryonic origin of the imaginal discs of the head of Drosophila melanogaster

The embryonic development of the primordia of the Drosophila head was studied by using an enhancer trap line expressed in these structures from embryonic stage 13 onward. Particular attention was given to the question of how the adult head primordia relate to the larval head segments. The clypeo-labral bud to the stage 13 embryo is located at a lateral position in the labrum adjacent to the labral sensory complex (epiphysis). Both clypeo-labral bud and sensory complex are located anterior to the engrailed-expression domain of the labrum. Throughout late embryogenesis and the larval period, the clypeo-labral bud forms integral part of the epithelium lining the roof of the atrium. The labial disc originates from the lateral labial segment adjacent to the labial sensory complex (hypophysis). It partially overlaps with the labial en-domain. After head involution, the labial disc forms a small pocket in the ventro-lateral wall of the atrium. The eye-antenna disc develops from a relatively large territory occupying the dorso-posterior part of the procephalic lobe, as well as parts of the dorsal gnathal segments. Cells in this territory are greatly reduced in number by cell death during stages 12–14. After head involution, the presumptive eye-antenna disc occupies a position in the lateral-posterior part of the dorsal pouch. Evagination of this tissue occurs during the first hours after hatching. In the embryo, no en-expression is present in the presumptive eye-antenna disc. en-expression starts in three separate regions in the third instar larva.     

A New Construct for Cloning DNA and Modeling the Structure of Drosophila melanogaster Polytene Chromosomes

Modification of P-element-based transformation vector pCaSpeR3 yielded a new construct, pICon, which contains the structural region of the Escherichia coli lacZ, the adjacent 5 and 3 regulatory regions of hsp70, pUC19, and two tandem FRTs. Owing to the hsp70 promoter, the pICon insertion site may be located on polytene chromosomes after heat shock by light or electron microscopy. The pUC19 sequence with a polylinker allows cloning of the genomic sequence adjacent to the 3 end of pICon by P-target rescue. Functional FRTs allow insertion or deletion of various DNA fragments. The construct is large (22,046 bp), forms easily detectable structures in polytene chromosomes, and may be used to study the structural and functional organization of the Drosophila melanogaster genome, in particular, to elucidate the causes of banding pattern formation. To map the molecular boundaries of interband 3C6/C7, the DNA sequence of this region was cloned between the two FRTs.     

The Drosophila melanogaster LEM-domain protein MAN1

Here we describe the Drosophila melanogaster LEM-domain protein encoded by the annotated gene CG3167 which is the putative ortholog to vertebrate MAN1. MAN1 of Drosophila (dMAN1) and vertebrates have the following properties in common. Firstly, both molecules are integral membrane proteins of the inner nuclear membrane (INM) and share the same structural organization comprising an N-terminally located LEM motif, two transmembrane domains in the middle of the molecule, and a conserved RNA recognition motif in the C-terminal region. Secondly, dMAN1 has similar targeting domains as it has been reported for the human protein. Thirdly, immunoprecipitations with dMAN1-specific antibodies revealed that this Drosophila LEM-domain protein is contained in protein complexes together with lamins Dm0 and C. It has been previously shown that human MAN1 binds to A- and B-type lamins in vitro. During embryogenesis and early larval development LEM-domain proteins dMAN1 and otefin show the same expression pattern and are much more abundant in eggs and the first larval instar than in later larval stages and young pupae whereas the LEM-domain protein Bocksbeutel is uniformly expressed in all developmental stages. dMAN1 is detectable in the nuclear envelope of embryonic cells including the pole cells. In mitotic cells of embryos at metaphase and anaphase, LEM-domain proteins dMAN1, otefin and Bocksbeutel were predominantly localized in the region of the two spindle poles whereas the lamin B receptor and lamin Dm0 were more homogeneously distributed. Downregulation of dMAN1 by RNA interference (RNAi) in Drosophila cultured Kc167 cells has no obvious effect on nuclear architecture, viability of RNAi-treated cells and the intracellular distribution of the LEM-domain proteins Bocksbeutel and otefin. In contrast, the localization of dMAN1, Bocksbeutel and otefin at the INM is supported by lamin Dm0. We conclude that the dMAN1 protein is not a limiting component of the nuclear architecture in Drosophila cultured cells.     

Segmental organisation of the tail region in the embryo of Drosophila melanogaster

Summary The segmental organisation of the tail region in the embryo of Drosophila melanogaster, which is defined here as the epidermal region posterior to the boundary between abdominal segments A7 and A8, has been investigated by means of ultraviolet (UV) laser fate-mapping and phenotypic analysis of embryonic mutants that alter the segmental pattern of the larval cuticle. Wild-type embryos were irradiated in the presumptive tail region with a UV- laser microbeam of 20 m diameter at the blastoderm stage. The ensuing defects were scored in the cuticle pattern of the tail region of the first-instar larva, which is described in detail in this paper. The spatial distribution of defect frequencies was used to construct a blastoderm fate-map of the cuticle structures of the larval tail region. The segmental origin of the larval tail structures was inferred from the phenotypic analysis of segmentation and homoeotic mutants, which revealed pattern repetition throughout the embryonic tail region corresponding to four segment anlagen, A8 to A11, and a non-segmental telson. These data enabled the transformation of the blastoderm fate-map of cuticle structures into a map of tail segment anlagen. The tail anlage occupies about 10% of the egg length (EL), bounded by segment A7 anteriorly at 20% EL and by the proctodaeum posteriorly at 10% EL, as measured from the posterior pole. The anlagen of segments A8 and A9 appear to be narrow dorso-ventral strips of blastoderm cells similar to the anlagen of the trunk segments, whereas the anlagen of A10 and A11 are smaller and produce fewer pattern elements. The telson is represented in the cuticle by the tuft which derives from a very dorsal posterior position. The antero-posterior axis of the entire tail anlage appears curved upward posteriorly. Differences in the mode of development between tail and trunk segments are discussed, as are similarities of larval and imaginal tail development in Drosophila. Comparison with tail development in other insects suggests that, during evolution, the transition from semi-long-germ to long-germ development modified the organisation of the tail region without affecting its primary subdivision into metameric units.     

A strategy for mapping the heterochromatin of chromosome 2 of Drosophila melanogaster

The heterochromatin of chromosome 2 of Drosophila melanogaster has been among the best characterized models for functional studies of heterochromatin owing to its abundance of genetic markers. To determine whether it might also provide a favorable system for mapping extended regions of heterochromatin, we undertook a project to molecularly map the heterochromatin of the left arm of chromosome 2 (2Lh). In this paper, we describe a strategy that used clones and sequence information available from the Drosophila Genome Project and chromosome rearrangements to construct a map of the distal most portion of 2Lh. We also describe studies that used fluorescent in situ hybridization (FISH) to examine the resolution of this technique for cytologically resolving heterochromatic sequences on mitotic chromosomes. We discuss how these mapping studies can be extended to more proximal regions of the heterochromatin to determine the structural patterns and physical dimensions of 2Lh and the relationship of structure to function.     

The recent origin of the Sdic gene cluster in the melanogaster subgroup

Gene families are composed of closely related genes and are an important part of eukaryotic genomes. In the proximal region of the X chromosome of Drosophila melanogaster there is a cluster of four tandem Sdic genes, located between the gene Cdic and the gene AnnX. Sdic is a chimeric gene that encodes a novel protein with sperm-specific expression. It had been hypothesized that the Sdic gene cluster was formed after the split of D. melanogaster and D. simulans. To study the evolution of this cluster, the sequence of this region was studied in several Drosophilidae species. In all species analyzed, Sdic genes are absent and AnnX and Cdic are adjacent to each other. The results allowed the inference of the ancestral situation and the reconstruction of the evolution of the cluster, and confirm that the Sdic cluster was indeed formed in the lineage that gave rise to D. melanogaster, being one of the youngest gene clusters known. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

A small genetic region that controls dihydroorotate dehydrogenase in Drosophila melanogaster

A locus is described that controls levels of mitochondrial dihydroorotate dehydrogenase (EC 1.3.3.1) in Drosophila melanogaster. The effects of alleles of the locus, Dhod, are manifest in preparations from whole organisms as well as in partially purified mitochondrial preparations; however, other mitochondrial functions do not appear to be appreciably affected by Dhod genotypes. The locus maps near p in the proximal portion of the right arm of chromosome 3. Flies trisomic for a chromosome segment including that region display elevated enzyme levels, implying that an enzyme structural gene is in that vicinity. Furthermore, Dhod alleles are semidominant in heterozygotes, suggesting that the dosage-sensitive element detected in the trisomics is actually the Dhod locus. These findings are discussed relative to the role of dihydroorotate dehydrogenase in the de novo pyrimidine biosynthetic pathway and relative to other pathway mutants that have been described in Drosophila.This work was supported by NSF Grants PCM 76-17214 to W. Cohen and PCM 78-14164 To J. Rawls, as well as NIH Research Career Development Award 1 KO4 AM00676 to J. Rawls.     

Embryonic cAMP and developmental potential in Drosophila melanogaster

Summary Measurements of cAMP in early embryos of Drosophila melanogaster demonstrate that the dunce gene plays a major role, and the rutabaga gene a secondary role, in maternal regulation of embryonic cAMP content. Studying the double mutant combination, we find that variability in elevated cAMP content between individual embryos is associated with a wide variability in developmental potential. Embryos with about five times the normal cAMP content define a threshold between apparently normal and abnormal development. Measurements of cAMP content in anterior and posterior halves of embryos indicate that the posterior embryonic region, which is developmentally more sensitive to the effects of elevated cAMP than the anterior region, does not contain more cAMP than the anterior region. The variety of developmental defects observed is discussed in relation to possible targets of cAMP action. Offprint requests to: J.A. Kiger, Jr     

Evolutionary pattern of the gtwin retrotransposon in the Drosophila melanogaster subgroup

The gtwin retrotransposon was recently discovered in the Drosophila melanogaster genome and it is evolutionarily closer to gypsy endogenous retrovirus. This study has identified gtwin homologous sequences in the genome of D. simulans, D. sechellia, D. erecta and D. yakuba by performing homology searches against the public genome database of Drosophila species. The phylogenetic analyses of the gtwin env gene sequences of these species have shown some incongruities with the host species phylogeny, suggesting some horizontal transfer events for this retroelement. Moreover, we reported the existence of DNA sequences putatively encoding full-length Env proteins in the genomes of Drosophila species other than D. melanogaster. The results suggest that the gtwin element may be an infectious retrovirus able to invade the genome of new species, supporting the gtwin evolutionary picture shown in this work.     

Analysis of morphogenetic movements in the development of the notum anlage of Drosophila melanogaster

Summary A comparison of the morphogenetic maps of the notum anlage of Drosophila melanogaster derived from the gynandromorph data and mosaics induced by somatic crossing-over during the first instar larval stage revealed that practically no major morphogenetic movements occur in the development of the anlage between the blastoderm and first instar larval stages and the adult stage. By comparing the morphogenetic map derived from gynandromorphs and the fate map derived from data on the transplantation of fragments of the mature wing imaginal disc, it was observed that no major morphogenetic movements occur in the notum anlage between the stages of the allocation of the disc and the mature disc. The results are consistent with the observations of other authors concerning the larval development of eye-antenna, wing and leg discs.