[Architecture of the X chromosome, expression of LIM kinase 1, and recombination in the agnostic mutants of Drosophila: a model of human Williams syndrome]. / Osobennosti arkhitektury X-khromosomy, ékspressii LIM kinazy 1 i rekombinatsii u mutantov drozofily lokusa agnostic: model' sindroma uil'iamsa cheloveka.

As the Human Genome and Drosophila Genome Projects were completed, it became clear that functions of human disease-associated genes may be elucidated by studying the phenotypic expression of mutations affecting their structural or functional homologs in Drosophila. Genomic diseases were identified as a new class of human disorders. Their cause is recombination, which takes place at gene-flanking duplicons to generate chromosome aberrations such as deletions, duplications, inversions, and translocations. The resulting imbalance of the dosage of developmentally important genes arises at a frequency of 10(-3) (higher than the mutation rate of individual genes) and leads to syndromes with multiple manifestations, including cognitive defects. Genomic DNA fragments were cloned from the Drosophila melanogaster agnostic locus, whose mutations impair learning ability and memory. As a result, the locus was exactly localized in X-chromosome region 11A containing the LIM kinase 1 (LIMK1) gene (CG1848), which is conserved among many species. Hemizygosity for the LIMK1 gene, which is caused by recombination at neighboring extended repeats, underlies cognitive disorders in human Williams syndrome. LIMK1 is a component of the integrin signaling cascade, which regulates the functions of the actin cytoskeleton, synaptogenesis, and morphogenesis in the developing brain. Immunofluorescence analysis revealed LIMK1 in all subdomains of the central complex and the visual system of Drosophila melanogaster. Like in the human genome, the D. melanogaster region is flanked by numerous repeats, which were detected by molecular genetic methods and analysis of ectopic chromosome pairing. The repeats determined a higher rate of spontaneous and induced recombination. including unequal crossing over, in the agnostic gene region. Hence, the agnostic locus was considered as the first D. melanogaster model suitable for studying the genetic defect associated with Williams syndrome in human.

Molecular cloning and expression of two hexamerin cDNAs from the mosquito, Aedes aegypti.

Fourth-instar larvae of Aedes aegypti synthesize two types of hexamerins, Hexamerin-1 (AaHex-1) and Hexamerin-2 (AaHex-2), whose subunits are distinguished by different methionine and aromatic amino acid contents. In early female pupae only the methionine-rich AaHex-1gamma subunit accumulates to two-fold higher levels than in males. To investigate the relationship between hexamerin structure and the roles of Hex-1 and Hex-2 during mosquito development and reproduction, we have cloned and sequenced cDNAs encoding the AaHex-2alpha, -2beta and AaHex-1gamma subunits. Comparison with other insect hexamerins revealed that the Aedes Hex-1 and Hex-2 proteins belong, respectively, to the two hexamerin subfamilies previously defined for brachyceran Diptera. Probes specific for the Hex-2alpha and Hex-1gamma transcripts showed that expression of both genes follows the same developmental timetable. However, greater Hex-1gamma mRNA accumulation may contribute to the higher levels of Hex-1 gamma protein in early female pupae.

Molecular cloning of cDNAs for two pro-phenol oxidase subunits from the malaria vector, Anopheles gambiae.

Phenol oxidase exists in insect hemolymph as a zymogen, pro-phenol oxidase (pro-PO), which is activated by specific proteolysis in response to infection or wounding. Phenol oxidase catalyses the synthesis of quinones that polymerize to form melanin deposits, which encapsulate parasites and help to seal wounds. Antibodies to pro-PO from Manduca sexta bound to 76, 72, and 71 kDa polypeptide bands from hemolymph of Anopheles gambiae larvae. This antiserum was used to screen a cDNA library from A. gambiae fourth-instar larvae. Full-length clones were isolated for two different pro-POs, designated A. gambiae proPO-p1 and proPO-p2, which are 67% identical in nucleotide sequence and 66% identical in deduced amino acid sequence. The A. gambiae pro-PO sequences are more similar to pro-PO from Drosophila melanogaster than to lepidopteran or crustacean pro-PO sequences in the GenBank database. Like the other arthropod pro-POs, the A. gambiae pro-PO sequences lack a signal peptide and have two conserved regions predicted to bind two copper atoms in the active site of the enzyme. The availability of these pro-PO cDNAs should be useful in examining the biochemical differences between A. gambiae strains that are refractory or susceptible to Plasmodium infection, and differ in their ability to encapsulate the parasites.

Molecular cloning and expression of a hexamerin cDNA from the malaria mosquito, Anopheles gambiae.

During the last larval instar, dipteran insects synthesize two hexamerins rich in aromatic residues, typified by the larval serum proteins 1 and 2 (LSP-1 and LSP-2) of Drosophila melanogaster. We report here the characterization of a complete cDNA sequence encoding a LSP-1-like protein from a lower dipteran insect, the malaria mosquito Anopheles gambiae. The cDNA encodes the subunit of a homohexamer, A. gambiae hexamerin-1.1 (AgHex-1.1), which is a major pupal protein but only a minor constituent of late larval hemolymph. AgHex-1.1 is moderately rich in methionine (3.9%) and particularly rich in aromatic residues (21% Phe+Tyr). Cytogenetic analysis reveals AgHex-1.1 to be encoded by a single-copy gene localized to division 22F within the proximal 2La inversion breakpoint of chromosome 2 of A. gambiae. The AgHex-1.1 transcript is first detected in fourth-instar larvae (L4) and disappears abruptly in early pupae. In situ hybridization shows accumulation of the transcript uniquely in the larval fat body. AgHex-1.1 mRNA is re-expressed in male and female adults at about 10% of the L4 level, with no effect of bloodfeeding in females. The potential roles of AgHex-1.1 in Anopheles development and reproductive maturation are discussed.

Mosquito hexamerins: characterization during larval development.

We report here the first examination of hexamerins expressed during mosquito larval development. Haemolymph proteins from fourth-instar larvae of six species representing the two major subfamilies of mosquitoes were characterized by immunoblotting using antisera to calliphorin, the major hexamerin of the blowfly. Calliphora vicina, or to LSP1 or LSP2, the two distinct hexamerins of Drosophila melanogaster. In each mosquito species the antisera demonstrated the presence of multiple abundant hexamerin polypeptides of 66-85 kDa in molecular weight. According to the subunit composition of native proteins, the larval hexamerins from both Aedes aegypti and Anopheles gambiae form heterohexamers. Furthermore, the two major Aedes hexamerin subunits (AaHex1 and AaHex2) are neither rich in aromatic amino acids nor methionine. cDNA clones encoding AaHex1 and AaHex2 were isolated and used to show that hexamerin mRNA is uniquely expressed in fourth-instar larvae of both A. aegypti and A. gambiae and disappears rapidly at the onset of pupal development.

Differential accumulation and tissue distribution of mosquito hexamerins during metamorphosis.

The pupal hexamerins were characterized for two mosquitoes representative of the culicine and anopheline families, Aedes aegypti and Anopheles gambiae. Like higher Diptera, both mosquito species express two types of hexamerins, Hex-1 and Hex-2, whose subunits are distinguished by different levels of methionine and aromatic amino acids. In A. aegypti there are two heterohexamers, AaHex-1 and AaHex-2. In A. gambiae there are two homohexamers, AgHex-1.1 and AgHex-1.2, and one heterohexamer, AgHex-2. These hexamerins are rich in aromatic residues, with 18-23% Phe + Tyr for Hex-1 subunits and 13-17% Phe + Tyr for Hex-2 subunits. In addition, both mosquito species synthesize methionine-rich Hex-1 subunits: Aedes AaHex-1 gamma (8% met) and Anopheles AgHex-1.1 (3.9% met). Aedes Hex-1 and Hex-2 proteins exhibit different, stage-specific tissue distributions: AaHex-2 is the primary hexamerin of late larval hemolymph whereas AaHex-1 is the most important non-hemolymph protein of early pupae. Although both proteins are stored in the pupal fat body, peak AaHex-1 levels are 2-fold higher. Both pupal protein levels decline rapidly between 25 and 36 h after pupation. Furthermore, AaHex-1 not only reaches peak values in female Aedes pupae later than in males, but the methionine-rich AaHex-1 gamma subunit level is specifically higher in females. These observations suggest different roles for Hex-1 and Hex-2 during mosquito development.

Overlapping Lsp-2 gene sequences target expression to both the larval and adult Drosophila fat body.

Larval serum protein-2 gene (Lsp-2) of Drosophila melanogaster encodes one of the major hexameric haemolymph proteins of third-instar larvae and a major component of adult serum. Regulated transcription of Lsp-2 results in high-level, ecdysone-stimulated expression throughout the larval fat body and low-level, spatially restricted expression in the adult fat cells. To localize cis-acting regulatory sequences responsible for the stage- and tissue-specific activity at Lsp-2, the expression of Lsp-2-lacZ fusion genes was studied by P element-mediated germline transformation of Drosophila. A 230 base pair larval enhancer, which includes an ecdysone response element (EcRE), specifically targets gene activity to the larval fat body. Although the adult mode of Lsp-2 expression depends on the larval enhancer, additional negative regulatory elements dictate both tissue-specificity and unique spatial restriction within the adult fat body. Implications of these findings for the identification of fat body-specific gene regulatory units in other insects are discussed.

[Nature of female sterility in mutants at the ecs locus controlling sensitivity to ecdysterone in Drosophila melanogaster]. / Issledovanie prirody steril'nosti samok u mutantov po lokusu ecs, kontroliruiushchemu chuvstvitel'nost' k ekdisteronu u Drosophila melanogaster.

Using interstrain level ovary transplantations function of ovaries was estimated in females bearing mutations at the ecs locus mapped in the 2B3-5 region of the X-chromosome of Drosophila melanogaster. Dissection of recipient flies and genetic analysis of their progeny demonstrated that the donor ovary in fertile flies was capable of normal functioning in the case of connection of the donor ovary in fertile flies with the recipient oviducts. Therefore, female-sterile mutations at the ecs locus are somatic line specific. SDS-PAGE electrophoresis revealed normal level of the yolk proteins in such females. Thus, the sterility of the ecs mutants may arise from abnormal morphogenesis of their genital disc, due to the loss of normal sensitivity to ecdysterone.

[Temperature control of the crossing-over frequency in Drosophila melanogaster. Effect of infra- and super-optimal shock temperatures in early ontogenesis on the recombination frequency]. / Temperaturnyi kontrol' chastoty krossingovera u Drosophila melanogaster. Vozdeistvie infra- i superoptimal'nykh shokovykh temperatur v rannem ontogeneze na chastotu rekombinatsii.

Effect of temperature shock treatments (0 and 37 degrees C) in early ontogenesis on recombination frequency was studied in two strains of Drosophila X1 and X2. Recombination frequency under treatment with temperature of 0 degrees C and 37 degrees C (shock treatment), as well as at 14 degrees C and 29 degrees C nonshock treatment was found to be dependent on strain genotype, the chromosomal segments under consideration, developmental stage and the age of individuals analysed. Shock treatments usually increase recombination frequency, whereas nonshock treatments lead to unstable and variable recombination frequencies. A concept of ontogenic homeostasis of recombination has been introduced. It is assumed that the effect of temperature treatments on recombination frequency is indirect--i.e. physiologically mediated.

Mitomycin C induces genomic rearrangements involving transposable elements in Drosophila melanogaster.

Mitomycin C was injected into the abdomen of male flies of the y2 sc1 waG strain of Drosophila melanogaster. They were mated with females bearing attached-X chromosomes, and the male offspring (F1) were analysed for the appearance of mutations in the X chromosome. We observed y+ and sc+ reversions induced either by excision of mdg4 (gypsy) with retention of one long terminal repeat (LTR) or by insertion of a foreign sequence into mdg4, partial reversion of the waG mutation, waG----waGd, and unstable f mutations. The overall mutation frequency was considerably higher than in control flies of the y2 sc1 waG strain. Possible mechanisms of genomic rearrangements induced by Mitomycin C, in particular the role of homologous recombination, are discussed.

[Induction of single transpositions of mobile genetic elements in Drosophila melanogaster using mitomycin C]. / Induktsiia odinochnykh transpozitsii mobil'nykh geneticheskikh élementov u Drosophila melanogaster s pomoshch'iu mitomitsina C.

Intraperitoneal injections of mitomycin C into the males from laboratory strains of Drosophila melanogaster induce several mutation events in different loci of the X-chromosome in the offspring. These mutations are caused by transposition of mobile genetic elements. The transpositions are single and are not associated with transposition explosions.