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armadillo, bazooka, and stardust are critical for early stages in formation of the zonula adherens and maintenance of the polarized blastoderm epithelium in Drosophila.

Cellularization of the Drosophila embryo results in the formation of a cell monolayer with many characteristics of a polarized epithelium. We have used antibodies specific to cellular junctions and nascent plasma membranes to study the formation of the zonula adherens (ZA) in relation to the establishment of basolateral membrane polarity. The same approach was then used as a test system to identify X-linked zygotically active genes required for ZA formation. We show that ZA formation begins during cellularization and that the basolateral membrane domain is established at mid-gastrulation. By creating deficiencies for defined regions of the X chromosome, we have identified genes that are required for the formation of the ZA and the generation of basolateral membrane polarity. We show that embryos mutant for both stardust (sdt) and bazooka (baz) fail to form a ZA. In addition to the failure to establish the ZA, the formation of the monolayered epithelium is disrupted after cellularization, resulting in formation of a multilayered cell sheet by mid-gastrulation. SEM analysis of mutant embryos revealed a conversion of cells exhibiting epithelial characteristics into cells exhibiting mesenchymal characteristics. To investigate how mutations that affect an integral component of the ZA itself influence ZA formation, we examined embryos with reduced maternal and zygotic supply of wild-type Arm protein. These embryos, like embryos mutant for both sdt and baz, exhibit an early disruption of ZA formation. These results suggest that early stages in the assembly of the ZA are critical for the stability of the polarized blastoderm epithelium.

Animals↗

[Model genetic system for analysis of attachment of HeT-A elements to terminal deletions in Drosophila melanogaster].

Telomeres of Drosophila consist of multiple copies of LINE-like transposable elements. These elements are assigned to two classes, HeT-A and TART. They are attached to terminal deletions at their 3' end, thus compensating for the absence of telomerase in Drosophila cells. The attachment of HeT-A elements to the X-chromosome terminal deletions of the regulatory region of the yellow gene was studied. It was shown that, in the case of degradation of the yellow promoter sequence (chromosome underreplication), the Het-A promoter located at the 3' end of this element can activate transcription of the gene. The minimal size of the 3'-end HeT-A element sequence sufficient for the yellow expression was shown to be 400 bp. Since the yellow mutation is expressed phenotypically and the gene impairment is not lethal, we created a convenient model genetic system based on this effect. Using this system, the frequency of attachments of the HeT-A elements to the chromosome end can be visually recorded. This frequency varied in a wide range (from 0.2 x 10(-4) to 2 x 10(-3)) and was strain-specific.

Animals↗

Baculovirus expression of functional P210 BCR-ABL oncogene product.

The chronic myelogenous leukemia-associated P210 BCR-ABL oncogene protein product has been produced using the baculovirus expression system. High-level expression of the P210 BCR-ABL protein required the removal of GC rich 5' non-coding sequences. P210 BCR-ABL synthesized in insect cells is an active tyrosine protein kinase indistinguishable from P210 BCR-ABL isolated from human cells. Both proteins utilize angiotensin II as a phosphate acceptor in vitro with a Km for ATP of approximately 1.5 microM. P210 BCR-ABL produced in insect cells undergoes autophosphorylation in vitro and in vivo. Gel filtration of P210 BCR-ABL reveals that the protein elutes as a high molecular weight complex of about 800 kD. Approximately 4 to 5 mg of P210 BCR-ABL is produced in one liter of infected insect cells. Following cell disruption and a three-step ion exchange and gel filtration purification procedure, 0.4 mg of soluble P210 BCR-ABL is obtained per liter of suspension culture. An alternative procedure employing detergent extraction and immunoaffinity chromatography gave higher yields and purity from smaller amounts of infected cell extracts. The availability of intact, soluble and enzymatically active P210 BCR-ABL represents a significant advance for studying the biochemical and biophysical properties of the ABL oncogene family of proteins.

Animals↗

Genomic sequence of a 320-kb segment of the Z chromosome of Bombyx mori containing a kettin ortholog.

The sex chromosome constitution of the silkworm, Bombyx mori, is ZW in the female and ZZ in the male. Very little molecular information is available about the Z chromosome in Lepidoptera, although the topic is interesting because of the absence of gene dosage compensation in this chromosome. We constructed a 320-kb BAC contig around the Bmkettin gene on the Z chromosome in Bombyx and determined its nucleotide sequence by the shotgun method. We found 13 novel protein-coding sequences in addition to Bmkettin. All the transposable elements detected in the region were truncated, and no LTR retrotransposons were found, in stark contrast to the situation on the W chromosome. In this 320-kb region, four genes for muscle proteins (Bmkettin, Bmtitin1, Bmtitin2, and Bmprojectin) are clustered, together with another gene (Bmmiple) on the Z chromosome in B. mori; their orthologs are also closely linked on chromosome 3 in Drosophila, suggesting a partial synteny. Real-time RT-PCR experiments demonstrated that transcripts of 13 genes of the 14 Z-linked genes found accumulated in larger amounts in males than in female moths, indicating the absence of gene dosage compensation. The implications of these findings for the evolution and function of the Z chromosome in Lepidoptera are discussed.

Amino Acid Motifs↗

Structure, chromosomal localization, and expression of the Drosophila melanogaster gene encoding sepiapterin reductase.

We have isolated and characterized a Drosophila melanogaster gene encoding the sepiapterin reductase (SR). The gene does not have introns. The 5'- and 3'-RACE analysis, which determined the transcription start point (tsp) and polyadenylation site, respectively, showed that the gene produces single mRNA species. The potential promoter region lacks distinct TATAAA or CCAAT box consensus sequences. RNA blot analysis revealed that the gene encodes a 1.4kb transcript that could be detected throughout development and in both heads and bodies of adults. The Drosophila SR gene maps to 15A on the X chromosome.

Alcohol Oxidoreductases↗

A selective sweep associated with a recent gene transposition in Drosophila miranda.

In Drosophila miranda, a chromosome fusion between the Y chromosome and the autosome corresponding to Muller's element C has created a new sex chromosome system. The chromosome attached to the ancestral Y chromosome is transmitted paternally and hence is not exposed to crossing over. This chromosome, conventionally called the neo-Y, and the homologous neo-X chromosome display many properties of evolving sex chromosomes. We report here the transposition of the exuperantia1 (exu1) locus from a neo-sex chromosome to the ancestral X chromosome of D. miranda. Exu1 is known to have several critical developmental functions, including a male-specific role in spermatogenesis. The ancestral location of exu1 is conserved in the sibling species of D. miranda, as well as in a more distantly related species. The transposition of exu1 can be interpreted as an adaptive fixation, driven by a selective advantage conferred by its effect on dosage compensation. This explanation is supported by the pattern of within-species sequence variation at exu1 and the nearby exu2 locus. The implications of this phenomenon for genome evolution are discussed.

Alleles↗

[Intratetrad mating and its genetic and evolutionary consequences].

Genetic characteristics of intratetrad mating, i.e., fusion of haploid products of one meiotic division, are considered. Upon intratetrad mating, the probability of homozygotization is lower than that upon self-fertilization, while heterozygosity at genes linked to the mating-type locus, which determines the possibility of cell fusion, is preserved. If the mating-type locus is linked to the centromere, the genome regions adjoining the centromeres of all chromosomes remain heterozygous. Intratetrad mating is characteristic of a number of fungi (Saccharomyces cerevisiae, Saccharomycodes ludwigii, Neurospora tetrasperma, Agaricus bisporus, Microbotrium violaceum, and others). Parthenogenetic reproduction in some insects also involves this type of fusion of nuclei. Intratetrad mating leads to the accumulation of haplolethals (i.e., lethals manifesting in haploid cells but not hindering their mating) in pericentric chromosome regions. Since heterozygosity increases viability of an organism, recombination has been suppressed during evolution in fungi characterized by intratetrad mating, which ensures heterozygosity of the most part of the genome.

Biological Evolution↗

A genome-wide survey of hybrid incompatibility factors by the introgression of marked segments of Drosophila mauritiana chromosomes into Drosophila simulans.

In hybrids between Drosophila simulans and D. mauritiana, males are sterile and females are fertile, in compliance with HALDANE's rule. The genetic basis of this phenomenon was investigated by introgression of segments of the mauritiana genome into a simulans background. A total of 87 positions throughout the mauritiana genome were marked with P-element insertions and replicate introgressions were made by repeated backcrossing to simulans for 15 generations. The fraction of hemizgyous X chromosomal introgressions that are male sterile is approximately 50% greater than the fraction of homozygous autosomal segments. This result suggests that male sterility factors have evolved at a higher rate on the X, but chromosomal differences in segment length cannot be ruled out. The fraction of homozygous autosomal introgression that are male sterile is several times greater than the fraction that are either female sterile or inviable. This observation strongly indicates that male sterility factors have evolved more rapidly than either female sterility or inviability factors. These results, combined with previous work on these and other species, suggest that HALDANE's rule has at least two causes: recessivity of incompatibility factors and differential accumulation of sterility factors affecting males and females.

Animals↗

Partial characterization of a fatty acid desaturase gene in Drosophila melanogaster.

The open reading frame of a gene encoding a protein homologous to vertebrate fatty acid desaturases has been characterized in Drosophila melanogaster. This is the first description of a desaturase sequence in insects. Two cDNAs were cloned: the combined 1.5 kb nucleotide sequences indicate that the gene encodes a polypeptide of 383 amino acid residues with a high sequence similarity to the well defined delta 9 desaturases from rat and yeast and desaturases from carp and tick. The Drosophila protein contains two histidine cluster motifs (HXXHH) and two hydrophobic regions which are conserved among all desaturases, whereas the amino and carboxy ends look more variable. The desaturase gene seems to be expressed in adults at a low level, with a greater prevalence in females than in males. Two genomic sequences have been amplified by polymerase chain reaction (PCR), and have a very similar open reading frame but a different organization; one with three introns and the other without introns. Both seem to correspond to two distinct genes and have been named "desat locus". They are localized to a single locus, 87C, on the right arm of the third chromosome. The possible involvement of the desat product in the biosynthesis of D. melanogaster contact pheromones is discussed.

Amino Acid Sequence↗

Genetic method for the preferential elimination of females of anopheles albimanus.

Recent field experiments demonstrated the possibility of using the sterile male method for the control of Anopheles albimanus Wiedemann, the most important vector of human malaria in Central America. Until now there was no practical method for excluding females from the releases of sterile males. A genetic method was developed for the preferential elimination of females during any of the four life stages. This genetic sexing system utilizes propoxur (o-isopropoxyphenyl methyl-carbamate) susceptibility as a recessive conditional lethal a T(Y:2R) translocation, and an In(2R)inversion. The propoxur resistance allele (dominant) was linked to the Y chromosome via a radiation-induced translocation, and genetic recombination was suppressed by inversions. In one of the strains produced, 99.7 percent of the females are eliminated when treated with propoxur, without male loss.

Animals↗

Use of meiotic pachytene stage of spermatocytes for karyotypic studies in insects.

Coleopterans represent by far the largest animal group, with more than 300,000 identified species. Only little progress in their chromosome analysis has been accomplished during recent decades, compared with that made in vertebrate cytogenetics. Both the small size of their genome and the difficulty of obtaining mitotic cells with nice chromosomes have limited the application of conventional techniques, such as chromosome banding. A method for obtaining chromosome banding on well-spread bivalents from the pachytene stage of the meiotic prophase, the most frequent stage in young imagines, is described. It makes possible the identification of all bivalents and the establishment of the karyotype with greater ease and accuracy than with mitotic cells. In addition, it gives some insight into chromosome organization at a stage when autosomes are assumed to undergo an intense transcriptional activity. The results of the technique, which was successfully applied to many species, are described here in two of them, Cetonia aurata and Adesmia montana as examples.

Animals↗

Identification and characterization of a putative sevenless homologue in the malaria vector Anopheles gambiae.

Tyrosine kinase sequences were identified and characterized in Anopheles gambiae, the major vector of malaria in subsaharan Africa. One of these sequences has the characteristics expected for a homologue of the Drosophila sevenless gene, which is necessary for R7 photoreceptor cell fate determination in the developing compound eye. The putative Anopheles sevenless gene homologue is located in a telomeric region of the X chromosome and is expressed in the head of late larval and pupal stage mosquitoes. Identification of the Anopheles homologue of the sevenless gene is a first step towards the development of a dominant phenotypic marker that could be used for detecting transformed Anopheles mosquitoes in a wide variety of genetic backgrounds and, as such, could be used in the development of transgenic mosquitoes for the control of parasite transmission. Preliminary evidence for sevenless sequences were also found in DNA from blackfly, Mediterranean fruit fly and the honeybee.

Amino Acid Sequence↗

Nanos (nos) genes of the vector mosquitoes, Anopheles gambiae, Anopheles stephensi and Aedes aegypti.

A number of genetics-based strategies for the control of vector-borne diseases require the development of genetic drive systems for introgressing antipathogen effector genes into wild populations of insects. Modified transposons whose mobilization is controlled by the DNA elements of developmentally regulated genes offer a potential solution for introducing effector genes into mosquitoes. Such elements could exhibit sex-, stage- and species-specific transposition, thus mitigating some of the concerns associated with autonomous transposition. Hybridizations in situ show that the transcription products of the nanos orthologous genes of Anopheles gambiae (Anga nos), An. stephensi (Anst nos) and Aedes aegypti (Aeae nos) accumulate in developing oocytes in adult females and localize to the posterior pole in early embryos. These features make nos genes promising candidates for donating control sequences to modified transposons.

Aedes↗

Detailed characterization of polydnavirus immunoevasive proteins in an endoparasitoid wasp.

Polydnaviruses are a unique group of insect viruses in terms of their obligate and symbiotic associations with some parasitic wasps. The Cotesia kariyai polydnavirus (CkPDV) replicates only in ovarian calyx cells of C. kariyai female wasps and is injected into the wasp's host, the armyworm Pseudaletia separata, along with the eggs. A previous study indicated the possibility that one of the CkPDV surface proteins mediates immunoevasion by the wasp from the encapsulation reaction of the host insect's hemocytes. This protein was named immunoevasive protein (IEP). The present studies substantially confirmed the previous observation by showing that an anti-IEP IgG neutralizes immunoevasive activity on the wasp eggs. Further, we isolated the IEP homologue (IEP-2) cDNA and IEP (IEP-1) cDNA, sequenced them and found that both are cysteine-rich proteins, each containing epidermal growth factor (EGF)-like repeats. IEP genes were not found to reside in the CkPDV genome, but in the wasp chromosomal DNA. IEPs are synthesized in the female reproductive tract and their expression was detected from 4 days after pupation, 1 day later than expression of the virus capsid proteins. In situ hybridization and immunocytochemistry indicated that the lateral oviduct cells of the reproductive tracts produce IEP-1/IEP-2 mRNAs and secrete the proteins into the oviduct. These data suggest that the expression pattern and localization of IEPs are different from other components of CkPDV virions.

Amino Acid Sequence↗

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.

Adenosylhomocysteinase↗

DNA topoisomerase I is essential in Drosophila melanogaster.

Both biochemical and genetic experiments suggest that the type I DNA topoisomerase may participate in DNA replication, recombination, transcription, and other aspects of DNA metabolism. Despite its apparent importance, genetic studies in unicellular organisms including eubacteria and yeasts indicate that topoisomerase I is not essential for viability. We have previously isolated the cDNA clone encoding DNA topoisomerase I from Drosophila melanogaster. We report here the cytogenetic mapping of top1 to the X chromosome at 13C1 and isolation of top1 genomic DNA. Using P-element mutagenesis, we have isolated a mutant deficient in Drosophila topoisomerase I functions. Genetic studies of this mutant show that topoisomerase I is essential for the growth and development of the fruit fly, a multicellular organism. The biological functions of topoisomerase I are inferred from our analysis of the regulation of topoisomerase I expression during Drosophila development.

Animals↗

Degenerative evolution and functional diversification of type-III secretion systems in the insect endosymbiont Sodalis glossinidius.

Sodalis glossinidius, a maternally transmitted endosymbiont of tsetse flies, maintains two phylogenetically distinct type-III secretion systems encoded by chromosomal symbiosis regions designated SSR-1 and SSR-2. Although both symbiosis regions are closely related to extant pathogenicity islands with similar gene inventories, SSR-2 has undergone novel degenerative adaptations in the transition to mutualism. Notably, SSR-2 lacks homologs of genes found in SSR-1 that encode secreted effector proteins known to facilitate the host cell cytoskeletal rearrangements necessary for bacterial entry and uptake into eukaryotic cells. Also, as a result of relaxed selection, SSR-2 has undergone inactivation of genes encoding components of the type-III secretion system needle substructure. In the current study, we used quantitative PCR to determine the expression profiles of ysaV (SSR-1) and invA (SSR-2) transcripts when S. glossinidius infects an insect cell line, and we used an invasion assay to characterize the phenotype of an S. glossinidius mutant that lacks the ability to produce an OrgA protein that is required for function of the SSR-2 secretome. Whereas SSR-1 is required for bacterial invasion of host cells and ysaV is expressed when bacteria contact host cells, SSR-2 is required for bacterial proliferation after entry, and invA is only expressed in the intracellular stage of infection. These results demonstrate that degenerative genetic adaptations in SSR-2 have promoted functional diversification of the Sodalis SSR-2 type-III secretion system.

Animals↗

Birth-and-death evolution of the Cecropin multigene family in Drosophila.

Cecropins are insect antibacterial peptides that are part of the insect humoral immune response and could, therefore, be potential targets of natural selection. In Drosophila, the Cec genes constitute a multigene family whose members are arranged in tandem. The complete Cec family was isolated in two obscura group species: D. subobscura and D. pseudoobscura. The chromosomal regions encompassing the Cec genes were subsequently sequenced and mapped by in situ hybridization. In D. pseudoobscura, as in species of the D. melanogaster complex and in D. virilis, the Cec genes constitute a single cluster with five genes. In D. subobscura, unlike in the rest of the species, the eight members of the family are split into two clusters located in different parts of the same chromosome. Remarkable differences in levels of divergence were observed between copies in both species. The genomic organization and the phylogenetic relationships among members of this family in the genus Drosophila indicate (i) that the presence of two clusters is the derived state of the family, (ii) repeated gene duplication within species, (iii) nonfunctionalization and loss of some Cec copies, and (iv) the presence of both highly divergent and highly similar copies within species. These features are better explained by the birth-and-death model of molecular evolution, which posits that the high instability of the Cec multigene family in Drosophila is simply determined by the duplication rate and the subsequent loss of duplicated loci.

Amino Acid Sequence↗