Sex-, stage- and tissue-specific regulation by a mosquito hexamerin promoter.

A portion of the 5'-flanking region of the female-specific hexamerin gene, Hex-1.2, from the mosquito Ochlerotatus atropalpus was used to drive expression of the luciferase reporter gene in Drosophila melanogaster. The proximal 0.7 kb of 5'-flanking DNA were sufficient to partially repress reporter gene activity in males and to drive tissue- and stage-specific expression comparable with that of the endogenous O. atropalpus Hex-1.2 gene. The Drosophila doublesex transcription factor (DSX), expressed in Escherichia coli, bound putative DSX sites of the Hex-1.2 gene differentially in vitro. Blocking expression of the female isoform of the Doublesex transcription factor in transgenic female flies resulted in reduction of luciferase expression to levels comparable with those in males, suggesting that Doublesex could contribute to regulation of female-specific expression of the O. atropalpus Hex-1.2 gene.

Lack of association between polymorphism of the human cyclic GMP-dependent protein kinase gene and obesity.

OBJECTIVE: To investigate whether genetic variation in the cyclic GMP-dependent protein kinase gene (PRKG1) is associated with obesity. METHODS: The study included 143 individuals from New York City area, NY, USA. The subjects were sampled on the basis of body mass index (BMI): obese (BMI ranging from 33.8 to 89.5 kg/m(2)), and nonobese (BMI ranging from 16.0 to 29.4 kg/m(2)). The association between C2276T polymorphism in PRKG1 gene and obesity was tested using linear regression analysis. RESULTS: BMI levels were predicted by linear regression models adjusted for demographic factors. An analysis was performed twice: in individuals of all ethnic backgrounds and in European-Americans only. In both cases, genotype did not have a significant effect. CONCLUSION: We found no evidence that the C2276T polymorphism in the PKRG1 gene is associated with obesity.

Single-nucleotide polymorphisms in the C-reactive protein (CRP) gene promoter that affect transcription factor binding, alter transcriptional activity, and associate with differences in baseline serum CRP level.

To investigate whether functional polymorphisms exist in the C-reactive protein (CRP) gene, i.e., ones that contribute directly to differences in baseline CRP among individuals, we sequenced a 1,156-nucleotide-long stretch of the CRP gene promoter in 287 ostensibly healthy people. We identified two single-nucleotide polymorphisms (SNPs), a bi-allelic one at nucleotide -409 (G-->A), and a tri-allelic one at -390 (C-->T-->A), both resident within the hexameric core of transcription factor binding E-box elements. Electrophoretic mobility shift assays confirmed that the SNP within the sequence (-412)CACGTG(-407) (E-box 1) modulates transcription factor binding, and that the one within (-394)CACTTG(-389) (E-box 2) supports transcription factor binding only when the -390 T allele is present. The commonest of four E-box 1/E-box 2 haplotypes (-409G/-390T) identified in the population supported highest promoter activity in luciferase reporter assays, and the rarest one (-409A/-390T) supported the least. Importantly, serum CRP in people with these haplotypes reproduced this rank order, i.e., people with the -409G/-390T haplotype had the highest baseline serum CRP (mean +/- SEM 10.9 +/- 2.25 microg/ml) and people with the -409A/-390T haplotype had the lowest (5.01 +/- 1.56 microg/ml). Furthermore, haplotype-associated differences in baseline CRP were not due to differences in age, sex, or race, and were still apparent in people with no history of smoking. At least two other SNPs in the CRP promoter lie within E-box elements (-198 C-->T, E-box 4, and -861 T-->C, E-box 3), indicating that not only is the quality of E-box sites in CRP a major determinant of baseline CRP level, but also that the number of E-boxes may be important. These data confirm that the CRP promoter does encode functional polymorphisms, which should be considered when baseline CRP is being used as an indicator of clinical outcome. Ultimately, development of genetic tests to screen for CRP expression variants could allow categorization of healthy people into groups at high versus low future risk of inflammatory disease.

Female-specific expression of a hexamerin gene in larvae of an autogenous mosquito.

Fourth-instar larvae of the autogenous mosquito, Aedes atropalpus, synthesize three hexamerins or hexameric storage proteins which are distinguished by different methionine and aromatic amino-acid contents. One protein, Hexamerin-1.2 (AatHex-1.2) is only found in female larvae and pupae. In order to investigate the molecular basis for this sex-specific accumulation, we have cloned and sequenced the cDNA encoding AatHex-1.2 and isolated and sequenced over 1 kb of the 5' flanking region of the AatHex-1.2 gene. The AatHex-1.2 transcript encodes a 81.6-kDa hexamerin subunit which contains 19.8% phenylalanine, tyrosine and tryptophan and 8.6% methionine residues. The single-copy AatHex-1.2 gene consists of three exons and two small introns located at its 5' end. A 2.3-kb AatHex-1.2 mRNA accumulates only in female larvae and pupae and is expressed at very low levels in adult female mosquitoes. The temporal expression profile of this transcript is typical of other mosquito hexamerin genes, with rapid disappearance of the mRNA shortly after pupation. Hence this is the first observation of exclusively female-specific gene activity during preadult development of an insect. In the 5' flanking region of the AatHex-1.2 gene, we identified putative binding sites for transcription factors, such as GATA, C/EBP and Doublesex, typically involved in fat body- and female-specific gene activity in Diptera. These findings suggest that mechanisms for sex-specific transcription in the fat body may be well conserved between flies and mosquitoes.

A novel simple satellite DNA is colocalized with the Stalker retrotransposon in Drosophila melanogaster heterochromatin.

In the T(1:2)dor(var7) multibreak rearrangement the distal 1A-2B segment of the X chromosome of Drosophila melanogaster is juxtaposed to an inverted portion of the heterochromatin of chromosome 2. Analysis of mitotic chromosomes by a series of banding techniques has permitted us precisely to locate the heterochromatic breakpoint of this translocation in the h42 region of 2R. Cloning and sequencing of the eu-heterochromatic junction revealed that the translocated 1A-2B fragment is joined to (AACAC)n repeats, which represent a previously undescribed satellite DNA in D. melanogaster. These repeated sequences have been estimated to account for about 1 Mb of the D. melanogaster genome. The repeats are located mainly in the Y chromosome and in the heterochromatin of the right arm of chromosome 2 (2Rh), where they are colocalized with the Stalker retrotransposon.

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.

The evolution of hexamerins and the phylogeny of insects.

The evolutionary relationships among arthropod hemocyanins and insect hexamerins were investigated. A multiple sequence alignment of 12 hemocyanin and 31 hexamerin subunits was constructed and used for studying sequence conservation and protein phylogeny. Although hexamerins and hemocyanins belong to a highly divergent protein superfamily and only 18 amino acid positions are identical in all the sequences, the core structures of the three protein domains are well conserved. Under the assumption of maximum parsimony, a phylogenetic tree was obtained that matches perfectly the assumed phylogeny of the insect orders. An interesting common clade of the hymenopteran and coleopteran hexamerins was observed. In most insect orders, several paralogous hexamerin subclasses were identified that diversified after the splitting of the major insect orders. The dipteran arylphorin/LSP-1-like hexamerins were subject to closer examination, demonstrating hexamerin gene amplification and gene loss in the brachyceran Diptera. The hexamerin receptors, which belong to the hexamerin/hemocyanin superfamily, diverged early in insect evolution, before the radiation of the winged insects. After the elimination of some rapidly or slowly evolving sequences, a linearized phylogenetic tree of the hexamerins was constructed under the assumption of a molecular clock. The inferred time scale of hexamerin evolution, which dates back to the Carboniferous, agrees with the available paleontological data and reveals some previously unknown divergence times among and within the insect orders.

Genes of succinyl-CoA ligase from Saccharomyces cerevisiae.

Succinyl-CoA ligase (succinyl-CoA synthetase) catalyzes the nucleotide-dependent conversion of succinyl-CoA to succinate. This enzyme functions in the tricarboxylic acid (TCA) cycle and is also involved in ketone-body breakdown in animals. The enzyme is composed of alpha and beta subunits that are required for catalytic activity. Two genes, LSC1 (YOR142W) and LSC2 (YGR244C), with high similarity to succinyl-CoA ligase subunits from other species were isolated from Saccharomyces cerevisiae. The expression of these genes was repressed by growth on glucose and was induced threefold to sixfold during growth on nonfermentable carbon sources. The LSC genes were deleted singly and in combination. Unlike other yeast strains with defects in TCA cycle genes, strains lacking either or both LSC genes were able to grow with acetate as a carbon source. However, growth on glycerol or pyruvate was impaired. An antiserum against both subunits of the Escherichia coli enzyme was capable of recognizing the yeast succinyl-CoA ligase alpha subunit, and this band was absent in delta lsc1 deletion strains. Succinyl-CoA ligase activity was absent in mitochondria isolated from strains deleted for one or both LSC genes, but activity was restored by the presence of the appropriate LSC gene on a plasmid. The yeast succinyl-CoA ligase was shown to utilize ATP but not GTP for succinyl-CoA synthesis.

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.

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.

[An analysis of the DNA sequence in the region of the breakpoint of the T(1;2)dor(var7) translocation inducing the mosaic-type position effect in Drosophila melanogaster]. / Analiz posledovatel'nosti DNK v raione tochki razryva translokatsii T(1;2)dor(var7), vyzyvaiushchei éffekt polozheniia mozaichnogo tipa u Drosophila melanogaster.

The sites of location of (AC/GT) minisatellite sequence were detected in 2B region of X-chromosome of Drosophila melanogaster. The distribution of (AC/GT) repeats coincided with the location of one-half of the mapped breakpoints of known rearrangements within this region. Analysis of sequences neighbouring the breakpoints of the rearrangement T(1;2)dor(var7) demonstrated that the breaks between two sequences of (AC/GT) repeats occurred at a distance 80 and 156 bp, respectively, but there were no (AC/GT) repeats in r combination directly. T-rich sequences 25 bp long as well as short tandem repeats were found near the breakpoints. The pentanucleotide repeat (CTGTT)10 is located at a distance of 660 bp from the breakpoint. It differs by one nucleotide from the sequence of the GTGTT satellite attached to the 1A-2B fragment of X-chromosome as a result of rearrangement.