Rapid sequential walking from termini of cosmid, P1 and YAC inserts.
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Animals↗
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Results for “Chromosomes, Insect”
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Chromosomal translocation heterozygotes in the house fly. Egg hatch, sex ratios, and transmission to progeny of 193 translocations.
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Animals↗
The product of proliferation disrupter is concentrated at centromeres and required for mitotic chromosome condensation and cell proliferation in Drosophila.
Homozygosity for a null mutation in the proliferation disrupter (prod) gene of Drosophila causes decreased mitotic index, defects of anaphase chromatid separation, and imperfect chromosome condensation in larval neuroblasts and other proliferating cell populations. The defective condensation is especially obvious near the centromeres. Mutant larvae show slow growth and massive cell death in proliferating cell populations, followed by late larval lethality. Loss of prod function in mitotic clones leads to the arrest of oogenesis in the ovary and defective cuticle formation in imaginal disc derivatives. The prod gene encodes a novel 301-amino-acid protein that is ubiquitously expressed and highly concentrated at the centric heterochromatin of the second and third mitotic chromosomes, as well as at > 400 euchromatic loci on polytene chromosomes. We propose that Prod is a nonhistone protein essential for chromosome condensation and that the chromosomal and developmental defects are caused by incomplete centromere condensation in prod mutants.
Amino Acid Sequence↗
Maintenance of sister-chromatid cohesion at the centromere by the Drosophila MEI-S332 protein.
Sister-chromatid cohesion is essential for the faithful segregation of chromosomes during cell division. Recently biochemical analysis with Xenopus extracts suggests that cohesion is established during S phase by a cohesion complex but that other proteins must maintain it in mitosis. The Drosophila melanogaster MEI-S332 protein is present on centromeres in mitosis and meiosis and is essential for cohesion at the centromeres in meiosis II. Here, we analyze the timing of MEI-S332 assembly onto centromeres and the functional domains of the MEI-S332 protein. We find that MEI-S332 is first detectable on chromosomes during prometaphase, and this localization is independent of microtubules. MEI-S332 contains two separable functional domains, as mutations within these domains show intragenic complementation. The carboxy-terminal basic region is required for chromosomal localization. The amino-terminal coiled-coil domain may facilitate protein-protein interactions between MEI-S332 and male meiotic proteins. MEI-S332 interacts with itself in the yeast two-hybrid assay and in immunoprecipitates from Drosophila oocyte and embryo extracts. Thus it appears that MEI-S332 assembles into a multimeric protein complex that localizes to centromeric regions during prometaphase and is required for the maintenance of sister-chromatid cohesion until anaphase, rather than its establishment in S phase.
Amino Acid Substitution↗
Dosage compensation in high resolution: global up-regulation through local recruitment.
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Acetylation↗
c(3)G encodes a Drosophila synaptonemal complex protein.
The meiotic mutant c(3)G (crossover suppressor on 3 of Gowen) abolishes both synaptonemal complex (SC) formation and meiotic recombination, whereas mutations in the mei-W68 and mei-P22 genes prevent recombination but allow normal SC to form. These data, as well as a century of cytogenetic studies, support the argument that meiotic recombination between homologous chromosomes in Drosophila females requires synapsis and SC formation. We have cloned the c(3)G gene and shown that it encodes a protein that is structurally similar to SC proteins from yeast and mammals. Immunolocalization of the C(3)G protein, as well as the analysis of a C(3)G-eGFP expression construct, reveals that C(3)G is present in a thread-like pattern along the lengths of chromosomes in meiotic prophase, consistent with a role as an SC protein present on meiotic bivalents. The availability of a marker for SC in Drosophila allowed the investigation of the extent of synapsis in exchange-defective mutants. These studies indicate that SC formation is impaired in certain meiotic mutants and that the synaptic defect correlates with the exchange defects. Moreover, the observation of interference among the residual exchanges in these mutant oocytes implies that complete SC formation is not required for crossover interference in Drosophila.
Animals↗
Molecular polymorphism as a tool for differentiating ground beetles (Carabus species): application of ubiquitin PCR/SSCP analysis.
Differentiation between Carabus species (ground beetle) and subspecies is difficult, although there have been extensive studies. To address this problem we have applied PCR in combination with SSCP analysis focussing on the evolutionally conservative ubiquitin gene to elaborate a new approach to molecular differentiation between species. We report that Carabidae possess an ubiquitin gene and that its gene has a multimeric structure. Differential SSCP analysis was performed with the monomeric form of the gene to generate a clear SSCP pattern. Such PCR/SSCP resulted in reproducible patterns throughout our experiments. Comparing different Carabus species (Carabus granulatus, C. irregularis, C. violaceus and C. auronitens) we could observe clear interspecies differences but no differences between genders. Some species showed some remarkable differences between the individuals. We suggest that the ubiquitin PCR-SSCP technique might be an additional tool for the differentiation of ground beetles.
Animals↗
Genetic, cytogenetic and developmental analysis of the Drosophila melanogaster tumor suppressor gene lethal(2)tumorous imaginal discs (1(2)tid).
Three of the twenty recessive-lethal tumor suppressor genes of Drosophila cause imaginal disc tumors in the homozygously mutated state. One of these is the lethal(2)tumorous imaginal discs (l(2)tid) gene. Histological preparations show the tumorous imaginal disc epithelium to consist of a mosaic of cells in monolayer and cells in clumped arrangement. In contrast, the wild-type imaginal disc epithelium is comprised exclusively of cells in monolayer arrangement. Mutant imaginal disc tissue pieces implanted into ready-to-pupariate wild-type larvae fail to differentiate. Implantation of l(2)tid imaginal disc tissue pieces in vivo into wild-type adult flies revealed a lethal, tumorous growth comparable to that in situ, thus characterizing the l(2)tid imaginal discs as truly malignant. The phenotypes of double mutants between two l(2)tid alleles and tumor suppressor genes, such as lethal(2)giant larvae and lethal(2)brain tumor, and the epithelial overgrowth mutant lethal(2)fat are described and discussed. Finally, we present the genetic, cytogenetic and molecular localization of the l(2)tid gene to the giant chromosome bands 59F4-6.
Alleles↗
Formation and morphology of dark puffs in Drosophila melanogaster polytene chromosomes.
The formation of unusual dark puffs in Drosophila melanogaster polytene chromosomes has been studied by electron microscopic (EM) analysis. Fly stocks transformed by the P[ry; Prat:bw] and P[hs-BRC-z1] constructs were used. In the former the bw gene is under the promoter of a housekeeping gene, Prat; in the latter the Br-C locus, mapping to the dark puff 2B, is under the promoter of a heat-shock gene, hsp70. Inserted into region 65A of the 3L chromosome, the Prat:bw copies give rise to structures which are morphologically reminiscent of the so-called "dark" puffs. In contrast, insertion of P[hs-BRC-z1] into region 99B of the 3R chromosome causes a regular "light" puff of form. Comparative analysis of the dark puffs--both transgenic and natural--suggests that there might be at least two mechanisms underlying their formation. One is a local incomplete decondensation of activated bands, characteristic of the so-called small puffs. The other is the formation of ectopic-looking contacts between the bands adjacent to the puffing zone. Transposition of the DNA, from which such a puff develops, causes a regular light puff to form at the new location. Heterochromatic regions do not appear to be directly involved in puffing.
Animals↗
Structure and function in the nucleus.
Current evidence suggests that the nucleus has a distinct substructure, albeit one that is dynamic rather than a rigid framework. Viral infection, oncogene expression, and inherited human disorders can each cause profound and specific changes in nuclear organization. This review summarizes recent progress in understanding nuclear organization, highlighting in particular the dynamic aspects of nuclear structure.
Animals↗
The Drosophila genome sequence: implications for biology and medicine.
The 120-megabase euchromatic portion of the Drosophila melanogaster genome has been sequenced. Because the genome is compact and many genetic tools are available, and because fly cell biology and development have much in common with mammals, this sequence may be the Rosetta stone for deciphering the human genome.
Animals↗
Functional mapping of a trans-activating gene required for expression of a baculovirus delayed-early gene.
The temporal regulation of an early gene of the baculovirus Autographa californica nuclear polyhedrosis virus was examined. We constructed a plasmid (plasmid 39CAT) in which the bacterial gene for chloramphenicol acetyltransferase was placed under the control of the promoter for the gene for a A. californica nuclear polyhedrosis virus 39,000-dalton protein (39K). A transient expression assay of plasmid 39CAT revealed that the 39K gene was expressed in infected cells but not in uninfected cells, indicating that the 39K gene should be classified as a delayed-early gene. The 39K promoter also efficiently directed the synthesis of chloramphenicol acetyltransferase when the plasmid was cotransfected with viral DNA which had been restricted with several restriction enzymes. To map the location of the gene(s) required for the synthesis of 39K, plasmid 39CAT was cotransfected with purified restriction fragments of A. californica nuclear polyhedrosis virus DNA. Fragments which mapped between 90.7 and 100.8 map units induced plasmid 39CAT. Plasmid pEcoRI-B, containing EcoRI fragment B (90 to 100 map units), activated plasmid 39CAT. Functional mapping of plasmid pEcoRI-B indicated that the essential region was located between 95.0 and 97.5 map units. The 5' end of this gene was mapped, and the chloramphenicol acetyltransferase gene was inserted under the control of its promoter. Transient assay experiments indicated that the trans-acting regulatory gene was expressed in uninfected cells and is therefore an immediate-early gene. This gene was named IE-1.
Animals↗
Linkage and evolutionary diversification of developmentally regulated multigene families: tandem arrays of the 401/18 chorion gene pair in silkmoths.
The coordinately expressed silkmoth chorion genes, 401 and 18, are closely linked as a pair, in divergent orientation. Analysis of overlapping clones (chromosomal "walk") demonstrated that each of the multiple copies of this gene pair is embedded within a larger deoxyribonucleic acid unit, which is tandemly repeated in a few arrays or possibly a single array. Southern analysis and examination of clones from a single individual moth demonstrated that the repeat units are extensively polymorphic in restriction sites, length, and possibly number, no differential amplification was evident during choriogenesis. Intron and 5'-flanking sequences were shown to be specific for the 401/18 gene pair and not to be present elsewhere in the genome. The spatial distribution of variations in the genes and their flanking sequences were examined.
Animals↗
RNA binding by Sxl proteins in vitro and in vivo.
Sxl has been proposed to regulate splicing of specific target genes by directly interacting with their pre-mRNAs. We have therefore examined the RNA-binding properties of Sxl protein in vitro and in vivo. Gel shift and UV cross-linking assays with a purified recombinant MBP-Sxl fusion protein demonstrated preferential binding to RNAs containing poly(U) tracts, and the protein footprinted over the poly(U) region. The protein did not appear to recognize either branch point or AG dinucleotide sequences, but an adenosine residue at the 5' end of the poly(U) tract enhanced binding severalfold. MBP-Sxl formed two shifted complexes on a tra regulated acceptor site RNA; the doubly shifted form may have been stabilized by protein-protein interactions. Consistent with its proposed role in pre-mRNA processing, in nuclear extracts Sxl was found in large ribonucleoprotein (RNP) complexes which sedimented significantly faster than bulk heterogeneous nuclear RNP and small nuclear RNPs. Anti-Sxl staining of polytene chromosomes showed Sxl protein at a number of chromosomal locations, among which was the Sxl locus itself. Sxl protein could also be targeted to a new chromosomal site carrying a transgene containing splicing regulatory sequences from the Sxl gene, following transcriptional induction. After prolonged heat shock, all Sxl protein was restricted to the heat-induced puff at the hs93D locus. In contrast, a presumptive small nuclear RNP protein was observed at several heat puffs following shock.
Animals↗
Studies of the role of the Drosophila scs and scs' insulators in defining boundaries of a chromosome puff.
Insulators are DNA elements that establish independent transcriptional domains within eukaryotic genomes. The Drosophila scs and scs' insulators localize near the borders of a structural domain in the polytene chromosomes, known as a puff, produced by transcription of the 87A heat shock protein (hsp) genes. It has been suggested that scs and scs' are boundary elements that delimit this decondensed chromatin domain, reflecting the mechanism by which these sequences act to constrain regulatory interactions. This model was tested using transposons that carried a yellow gene to assess enhancer blocking and an hsp70-lacZ gene to examine the structure of a heat shock puff in the presence and absence of insulators. We found that although scs and scs' blocked enhancer function, these sequences did not prevent the spread of decondensation resulting from hsp70-lacZ transcription. Further analysis of the endogenous 87A locus demonstrated that scs and scs' reside within, not at, the borders of the puff. Taken together, our studies suggest that scs and scs' are not boundary elements that block the propagation of an altered chromatin state associated with puff formation. We propose that these insulators may have a direct role in limiting regulatory interactions in the gene-dense 87A region.
Animals↗
Structure and organization of the P element related sequences in Drosophila madeirensis.
The P element homologous sequences of the two closely related species Drosophila guanche and Drosophila subobscura represent a very special case of transposable-element derivatives. Although they have lost the regions known to be essential for P transposition by random mutations, all of them have selectively conserved the coding capacity for "P-repressor-like" proteins during the past few millions years. In both species, they are tandemly amplified in a single euchromatic gene cluster at equivalent chromosomal positions. In contrast, Drosophila madeirensis, an endemic species that is very closely related to both D. subobscura and D. guanche, harbours an additional P homologous site. Several mechanisms can be invoked to explain the generation of the new site in this species. In this work we present several molecular and cytological data in order to elucidate the possible evolutionary origin of the P derivatives of D. madeirensis.
Amino Acid Sequence↗
Systematics, evolution, and biology of scelionid and platygastrid wasps.
The Platygastroidea comprises two families of parasitoids, Scelionidae and Platygastridae, and nearly 4500 described species. They parasitize a diverse array of insects as well as spiders. Idiobiont endoparasitism of eggs is the putative ground plan biology, as reflected by all scelionids, but most Platygastridae are koinobiont endoparasitoids of immature Auchenorrhyncha, Sternorrhyncha, and Cecidomyiidae. The superfamily is demonstrably monophyletic but its phylogenetic position remains uncertain. Relationships within the Platygastroidea are also poorly known and the group is in need of comprehensive phylogenetic study. Significant information is available on host relationships and biology, although much of this is biased to a few genera of Telenominae that are employed as biocontrol agents. Hosts for many genera are unknown, in particular those that inhabit leaf litter or parasitize solitary host eggs. The Trissolcus basalis-Nezara viridula parasitoid-host association has become a favored model system in ecological, behavioral, and physiological research on insects.
Animals↗
Isolation and chronobiological analysis of a neuropeptide pigment-dispersing factor gene in Drosophila melanogaster.
In this article, the authors isolate a gene encoding a neuropeptide in Drosophila melanogaster. The substance is called pigment-dispersing factor (PDF), based on one of the roles it plays in crustaceans (the arthropods in which this factor was initially discovered). The PDF-encoding Drosophila gene (pdf) is intronless and present in a single copy per haploid genome. The cytological location of pdf is 97B on the third chromosome. The putative 102-amino-acid precursor (prepro-PDF) consists of a signal peptide and a PDF-associated peptide, followed by the mature PDF. The PDF-associated peptide region of the precursor is highly diverged from those of the crustacean precursors, whereas the primary structure of the mature PDF is conserved in other members of the pigment-dispersing hormone family. A single pdf transcript (ca. 0.8 kb) is expressed predominantly in the head; the expression levels of pdf mRNA are consistently higher in males than in females. Putative pdf homologous transcripts are present in other Drosophila species, which exhibit similar sexual dimorphic expression patterns. Cyclic expression of pdf over the course of the day and night was assessed, but the mRNA exhibited at best very gentle cycling. The pdf expression in two behaviorally arrhythmic mutants were examined; the expression was intact in a period0 mutant but absent in the disconnected mutant.
Amino Acid Sequence↗