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Oligomerisation of Tube and Pelle leads to nuclear localisation of dorsal.

In the Drosophila embryo the nuclear localisation of Dorsal, a member of the Rel family, is regulated by an extracellular signal, which is transmitted to the interior of the egg cell by a cascade of proteins involving the novel protein Tube and the protein kinase Pelle. Here we analyse the activation mechanism of Tube and Pelle and the interaction between these two components. We show that both proteins, although having different biochemical activities, are activated by the same mechanism. Membrane association alone is not sufficient, but oligomerisation is required for full activation of Tube and Pelle. By deletion analysis we determined the domains of Tube and Pelle mediating the physical interaction and the signalling to downstream components. In order to investigate the link between Pelle and the target of the signalling cascade, the Dorsal/Cactus complex, we isolated and characterised the novel, but evolutionary conserved protein Pellino, which associates with the kinase domain of Pelle.

Animals↗

A role for Drosophila SMC4 in the resolution of sister chromatids in mitosis.

BACKGROUND: Faithful segregation of the genome during mitosis requires interphase chromatin to be condensed into well-defined chromosomes. Chromosome condensation involves a multiprotein complex known as condensin that associates with chromatin early in prophase. Until now, genetic analysis of SMC subunits of the condensin complex in higher eukaryotic cells has not been performed, and consequently the detailed contribution of different subunits to the formation of mitotic chromosome morphology is poorly understood. RESULTS: We show that the SMC4 subunit of condensin is encoded by the essential gluon locus in Drosophila. DmSMC4 contains all the conserved domains present in other members of the structural-maintenance-of-chromosomes protein family. DmSMC4 is both nuclear and cytoplasmic during interphase, concentrates on chromatin during prophase, and localizes to the axial chromosome core at metaphase and anaphase. During decondensation in telophase, most of the DmSMC4 leaves the chromosomes. An examination of gluon mutations indicates that SMC4 is required for chromosome condensation and segregation during different developmental stages. A detailed analysis of mitotic chromosome structure in mutant cells indicates that although the longitudinal axis can be shortened normally, sister chromatid resolution is strikingly disrupted. This phenotype then leads to severe chromosome segregation defects, chromosome breakage, and apoptosis. CONCLUSIONS: Our results demonstrate that SMC4 is critically important for the resolution of sister chromatids during mitosis prior to anaphase onset.

Alleles↗

Evidence for two distinct members of the amylase gene family in the yellow fever mosquito, Aedes aegypti.

Genomic DNA fragments encoding a salivary gland-specific alpha-amylase gene, Amylase I (Amy I), and an additional amylase, Amylase II (AmyII) of the yellow fever mosquito, Aedes aegypti, were isolated and characterized. Two independently isolated DNA fragments, G34-F and G34-14A, encode polymorphic alleles of Amy I. A 3.2 kilobase (kb) EcoR I fragment of G34-F, F2, has been sequenced in its entirety and contains 832 base pairs (bp) of the 5'-end, non-coding and putative promoter regions that are adjacent to 2.4 kb of the Amy I coding region. One intron, 59 bp in length, is found towards the 3'-end of the clone. A third genomic clone, 3A, corresponding to Amy II, was sequenced and shown not to contain the primary DNA sequence that encodes the 260 amino acid region that uniquely characterizes the amino terminal end of the Amy I product. Amy I was assigned by restriction fragment length polymorphism (RFLP) mapping to chromosome 2 (23.0 cM) and Amy II to chromosome 1 (44.0 cM). Amy I and Amy II are highly polymorphic and there may be multiple linked copies at each locus. Comparisons between Amy I and Amy II are presented for the putative promoter and conceptual translation products. The identification of two distinct amylase genes and their separate linkage assignments provides evidence for a multigene family of alpha-amylases in Ae. aegypti.

Aedes↗

A family of pupal-specific cuticular protein genes in the mosquito Anopheles gambiae.

We have cloned and sequenced members of a cuticular protein multi-gene family from the mosquito Anopheles gambiae. Three genes (agcp2a-c), each approximately 1 kb in length, were found in a 17.4 kb genomic phage clone. Analysis of ten cDNAs revealed that at least four related genes are present. The open reading frame of the genes and cDNAs showed 95% sequence identity. Divergence was observed in the sequence of the 3' ends and the number of copies of two repeated coding sequences. In situ hybridizations with a probe prepared from one of these circular protein genes physically mapped to two loci, 26B on chromosome 2L and 37A on 3R. Transcription of these An. gambiae cuticular protein genes appears to be limited to pharate pupae and the expressed protein(s) is found in early pupae. The deduced amino acid sequence of these proteins contains a hydrophilic region with significant similarity to other cuticular proteins including the pupal-specific cuticular protein, EDG84, of Drosophila melanogaster (Apple and Fristrom).

Amino Acid Sequence↗

Mapping quantitative trait loci affecting sternopleural bristle number in Drosophila melanogaster using changes of marker allele frequencies in divergently selected lines.

Quantitative trait loci (QTLs) responsible for variation in sternopleural bristle number in crosses between the laboratory lines of Drosophila melanogaster OregonR and CantonS were mapped using information from allele frequency changes of two families of retrotransposon markers in divergently selected populations. QTL effects and positions were inferred by likelihood, using transition matrix iteration and Monte Carlo interval mapping. Individuals from the selected population were genotyped for markers spaced at an average distance 4.4 cM. Four QTLs of moderate effect ranging from 0.6 to 1.32 bristles accounted for most of the selection response. A permutation test of the correspondence between the mapped QTLs and the positions of bristle number candidate genes suggested that alleles at these candidate genes were no more strongly associated with selected changes in marker allele frequency than were randomly chosen positions in the genome.

Alleles↗

Terminal pattern elements in Drosophila embryo induced by the torso-like protein.

The genes torso (tor) and torso-like (tsl) are two of the Drosophila maternal group genes implicated in a receptor tyrosine kinase signalling pathway that specifies terminal cell fate (reviewed in ref. 3). Loss-of-function mutations in these loci cause an identical phenotype in which pattern elements from the anterior (acron) and posterior (telson) ends have been deleted. We have cloned the tsl gene and demonstrate here that, in agreement with previous genetic data, it encodes a protein that is secreted and whose transcription is restricted to specialized categories of follicle cells localized at the poles of the egg chamber. At early blastoderm stage, tsl protein forms a symmetrical concentration gradient at the poles on the surface of the devitellinized embryo. Unrestricted expression of the tsl protein in tsl female mutants induces terminal pattern elements and suppresses the formation of abdomen in embryos. These results suggest that the tsl protein is the ligand that binds to the torso receptor.

Amino Acid Sequence↗

Measuring selective effects of modifier gene polymorphisms on the Bare locus of Drosophila subobscura.

An attempt to quantify the effects of modifier gene polymorphisms on the operation of natural selection on a major locus has been carried out. The modifier system we have investigated is constituted by a set of polygenic modifier loci affecting the morphological expression of the Bare (Ba) bristle mutant of Drosophila subobscura. Ba is a dominant mutant that is lethal in homozygous condition and both the polygenic modifiers and Ba are located on the O chromosome of this species. Experimental populations were found with Ba/+ individuals and two different types of populations were started according to their modifier genetic background: populations with wild O chromosomes of either high or low modifier effect (cages H and L, respectively). Fitness estimates (total fitness, viability and fertility) for genotypes of the Ba locus were obtained under the two different modifier backgrounds. In the populations with high modifier background the total fitness of the Ba/+ heterozygote was very similar to that of the +/+ homozygote (fitness equal to 1). However, in cages with low modifier background a strong selection against the Ba/+ heterozygote was detected (average of total fitnesses over generations was 0.66 +/- 0.10), and fertility appears to be the fitness component responsible for this effect (mean fertility was 0.55 +/- 0.08). These findings demonstrate that modifier gene polymorphisms affecting the expression of the Ba mutant may be associated with large selective effects on the major locus.

Animals↗

QTL analysis of variation in male courtship song characters in Drosophila virilis.

We have used a quantitative trait locus (QTL) mapping approach to study the genetic basis of differences between two Drosophila virilis strains representing extreme phenotypes in two song characters, the number of pulses in a pulse train (PN) and the length of a pulse train (PTL). Variation in these characters among 520 F2 males was studied by single-marker analysis and composite interval mapping (CIM) using a recombination linkage map constructed for 26 microsatellite markers. In single-marker analysis, two adjacent microsatellite markers on the third chromosome, msat19 and vir84 explained 13.8 and 12.4% of the variation in PN and 9.9 and 6.5% of the variation in PTL, respectively. CIM analysis revealed significant QTLs affecting PN, located on the X and the second, third and fourth chromosome of D. virilis, while variation in PTL was attributable to QTLs located only on the third chromosome.

Animals↗

An easter-like serine protease from Anopheles gambiae exhibits changes in transcript abundance following immune challenge.

The nucleotide and deduced amino acid sequence of a serine protease (AgSp14D1) from the human malaria vector, Anopheles gambiae, is presented. The gene product is a 360 amino acid protein that contains two domains and has the highest sequence similarity to the Drosophila melanogaster serine protease easter and to prophenol oxidase activating enzyme (pPAE) from Manduca sexta. The catalytic domain is at the carboxy terminus and has the conserved serine, histidine and aspartic acid residues found in serine proteases as well as six cysteines common to invertebrate enzymes. The amino terminus contains critical cysteines that define a clip (=disulphide knot) domain which places this gene product in a subfamily of regulatory serine proteases that includes not only easter and pPAE but also the Drosophila proteins masquerade, stubble and snake as well as proclotting enzyme and factor B from the horseshoe crab. In situ hybridization to the polytene chromosomes detects a single band at 14D and Southern analysis with a probe from the 5' end of the gene confirms the single copy status of this gene. Northern analysis reveals changes in transcript abundance during development and following blood feeding. Interestingly, this analysis also shows an increase in transcript levels following wounding or injection of bacteria.

Amino Acid Sequence↗

The nucleotide changes governing cuticular hydrocarbon variation and their evolution in Drosophila melanogaster.

The cuticular hydrocarbon (CH) pheromones in Drosophila melanogaster exhibit strong geographic variation. African and Caribbean populations have a high ratio of 5,9 heptacosadiene/7,11 heptacosadiene (the "High" CH type), whereas populations from all other areas have a low ratio ("Low" CH type). Based on previous genetic mapping, DNA markers were developed that localized the genetic basis of this CH polymorphism to within a 13-kb region. We then carried out a hierarchical search for diagnostic nucleotide sites starting with four lines, and increasing to 24 and 43 lines from a worldwide collection. Within the 13-kb region, only one variable site shows a complete concordance with the CH phenotype. This is a 16-bp deletion in the 5' region of a desaturase gene (desat2) that was recently suggested to be responsible for the CH polymorphism on the basis of its expression [Dallerac, R., Labeur, C., Jallon, J.-M., Knipple, D. C., Roelofs, W. L. & Wicker-Thomas, C. (2000) Proc. Natl. Acad. Sci. 97, 9449--9454]. The cosmopolitan Low type is derived from the ancestral High type, and DNA sequence variations suggest that the former spread worldwide with the aid of positive selection. Whether this CH variation could be a component of the sexual isolation between Zimbabwe and other cosmopolitan populations remains an interesting and unresolved question.

Animals↗

Genotoxic evaluation of the biocomponents of the cricket, Gryllus bimaculatus, using three mutagenicity tests.

The mutagenic potential of the extracted components of Gryllus bimaculatus, a species of cricket, was evaluated using short-term genotoxicity tests including the Ames, chromosome aberration, and micronuclei tests. In a Salmonella typhimurium assay, G. bimaculatus extract did not produce any mutagenic response in the absence or presence of S9 mix with TA98, TA100, TA1535, and TA1537. Chromosome aberration testing showed that G. bimaculatus had no significant effect on Chinese hamster ovary (CHO) cells. In the mouse micronucleus test, no significant alteration in occurrence of micronucleated polychromatic erythrocytes was observed in ICR male mice intraperitoneally administered with G. bimaculatus extract at doses of 15, 150, or 1500 mg/kg. These results indicate that G. bimaculatus extract exerts no mutagenic effect in these in vitro and in vivo systems.

Animals↗

Mouse notch: expression in hair follicles correlates with cell fate determination.

Many vertebrate tissues, including skin, are known to develop as a consequence of epithelial-mesenchymal interactions. Much less is known about the role of cell-cell interaction within the epithelial or the mesenchymal compartments in morphogenesis. To investigate cell-cell interactions during skin development, and the potential role of the Notch homolog in this process, we cloned the mouse homolog of Notch (mNotch) and studied its expression pattern, starting as early as mesoderm formation. The novel application of double-labeled in situ hybridization in vertebrates allowed high resolution analysis to follow the fate of mNotch expressing cells directly. In comparison with the distribution of Id mRNA, analysis confirmed that in the hair follicle high levels of mNotch are expressed exclusively in the epithelial compartment. Hair follicle matrix cells start expressing mNotch as different cell types become distinguishable in the developing follicle. mNotch mRNA expression persists throughout the growth phase of the follicle and maintains the same expression profile in the second hair cycle. The cells in the follicle that undergo a phase of high level mNotch expression are in transition from mitotic precursors to several discreet, differentiating cell types. Our observations point out that both in time (during development) and in space (by being removed one cell layer from the dermal papilla) mNotch expression is clearly separated from the inductive interactions. This is a novel finding and suggests that mNotch is important for follicular differentiation and possibly cell fate selection within the follicle.

Amino Acid Sequence↗

Chromosome association of minichromosome maintenance proteins in Drosophila endoreplication cycles.

Minichromosome maintenance (MCM) proteins are essential eukaryotic DNA replication factors. The binding of MCMs to chromatin oscillates in conjunction with progress through the mitotic cell cycle. This oscillation is thought to play an important role in coupling DNA replication to mitosis and limiting chromosome duplication to once per cell cycle. The coupling of DNA replication to mitosis is absent in Drosophila endoreplication cycles (endocycles), during which discrete rounds of chromosome duplication occur without intervening mitoses. We examined the behavior of MCM proteins in endoreplicating larval salivary glands, to determine whether oscillation of MCM-chromosome localization occurs in conjunction with passage through an endocycle S phase. We found that MCMs in polytene nuclei exist in two states: associated with or dissociated from chromosomes. We demonstrate that cyclin E can drive chromosome association of DmMCM2 and that DNA synthesis erases this association. We conclude that mitosis is not required for oscillations in chromosome binding of MCMs and propose that cycles of MCM-chromosome association normally occur in endocycles. These results are discussed in a model in which the cycle of MCM-chromosome associations is uncoupled from mitosis because of the distinctive program of cyclin expression in endocycles.

Animals↗

Influence of chromosomal position and copy number of a white-directed ribozyme gene on the suppression of eye pigmentation in Drosophila melanogaster.

Different strains of transgenic Drosophila melanogaster carrying one, two, or three copies of a heat-shock promoter 70 (hsp70)-driven catalytic antisense RNA gene, directed against the white gene, were investigated for the expression level of ribozyme RNA. It was found that the steady-state concentrations of the hammerhead ribozyme were proportional to the copy number of the genes and that the suppressive effect on eye pigment accumulation was dosage dependent. In a further experiment, a D. melanogaster strain, deficient in eye pigmentation caused by a deletion of the white gene, was used for P element-mediated germline transformation: the transposon used contained the hsp70-driven, white-directed ribozyme gene and, on the same DNA, the mini-white gene under its own promoter. The spatial coupling of the transcription of ribozyme and target RNA resulted in more effective ribozyme-mediated inhibition of eye pigmentation under heat-shock conditions. These effects were dependent on the chromosomal integration site of the transposon.

Animals↗

Evidence that the Drosophila olfactory mutant smellblind defines a novel class of sodium channel mutation.

The smellblind (sbl) gene of Drosophila is associated with olfactory defects, and the paralytic (para) gene encodes a voltage-gated sodium channel. sbl and para have similar genetic map positions, many combinations of sbl and para mutations fail to complement, and two sbl mutations contain molecular lesions within the para transcription unit. sbl mutations also behave like para mutations in that they are enhanced by the mutation no action potential temperature-sensitive (mlenapts1). The simplest interpretation of these results is that sbl and para are the same gene. Two sbl mutations produce olfactory defects not characteristic of classic sodium channel mutations and do not show typical heat-sensitive paralysis, suggesting that these sbl mutants define a novel class of sodium channel mutation.

Alleles↗

Linkage map of the honey bee, Apis mellifera, based on RAPD markers.

A linkage map was constructed for the honey bee based on the segregation of 365 random amplified polymorphic DNA (RAPD) markers in haploid male progeny of a single female bee. The X locus for sex determination and genes for black body color and malate dehydrogenase were mapped to separate linkage groups. RAPD markers were very efficient for mapping, with an average of about 2.8 loci mapped for each 10-nucleotide primer that was used in polymerase chain reactions. The mean interval size between markers on the map was 9.1 cM. The map covered 3110 cM of linked markers on 26 linkage groups. We estimate the total genome size to be approximately 3450 cM. The size of the map indicated a very high recombination rate for the honey bee. The relationship of physical to genetic distance was estimated at 52 kb/cM, suggesting that map-based cloning of genes will be feasible for this species.

Animals↗

Genetic characterization of ms (3) K81, a paternal effect gene of Drosophila melanogaster.

The vast majority of known male sterile mutants of Drosophila melanogaster fail to produce mature sperm or mate properly. The ms(3) K81(1) mutation is one of a rare class of male sterile mutations in which sterility is caused by developmental arrest after sperm entry into the egg. Previous studies showed that males homozygous for the K81(1) mutation produce progeny that arrest at either of two developmental stages. Most embryos arrest during early nuclear cycles, whereas the remainder are haploid embryos that arrest at a later stage. This description of the mutant phenotype was based on the analysis of a single allele isolated from a natural population. It was therefore unclear whether this unique paternal effect phenotype reflected the normal function of the gene. The genetic analysis and initial molecular characterization of five new K81 mutations are described here. Hemizygous conditions and heteroallelic combinations of the alleles were associated with male sterility caused by defects in embryogenesis. No other mutant phenotypes were observed. Thus, the K81 gene acted as a strict paternal effect gene. Moreover, the biphasic pattern of developmental arrest was common to all the alleles. These findings strongly suggested that the unusual embryonic phenotype caused by all five new alleles was due to loss of function of the K81+ gene. The K81 gene is therefore the first clear example of a strict paternal effect gene in Drosophila. Based on the embryonic lethal phenotypes, we suggest that the K81+ gene encodes a sperm-specific product that is essential for the male pronucleus to participate in the first few embryonic nuclear divisions.

Alleles↗

Inversion monophyly in African anopheline malaria vectors.

The African Anopheles gambiae complex of six sibling species has many polymorphic and fixed paracentric inversions detectable in polytene chromosomes. These have been used to infer phylogenetic relationships as classically done with Drosophila. Two species, A. gambiae and A. merus, were thought to be sister taxa based on a shared X inversion designated Xag. Recent DNA data have conflicted with this phylogenetic inference as they have supported a sister taxa relationship of A. gambiae and A. arabiensis. A possible explanation is that the Xag is not monophyletic. Here we present data from a gene (soluble guanylate cyclase) within the Xag that strongly supports the monophyly of the Xag. We conjecture that introgression may be occurring between the widely sympatric species A. gambiae and A. arabiensis and that the previous DNA phylogenies have been detecting the introgression. Evidently, introgression is not uniform across the genome, and species-specific regions, like the X-chromosome inversions, do not introgress probably due to selective elimination in hybrids and backcrosses.

Animals↗