The peroxiredoxin gene family in Drosophila melanogaster.

Five peroxiredoxin genes have been identified in Drosophila melanogaster on the basis of a genome-wide search. Three of the genes (DPx-4156, DPx-4783, and DPx-5037) fall into the 2-Cys subgroup, while the other two (DPx-2540 and DPx-6005) belong to the 1-Cys subgroup. Using cDNAs, all five were expressed in E. coli and the purified recombinant proteins were shown to reduce H(2)O(2) in the presence of dithiothreitol. The three 2-Cys Prx were also shown to be active in the thioredoxin system and were, consequently, classified as thioredoxin peroxidases. Antisera raised against the DPx-4783 recombinant protein crossreacted with all family members and recognized protein species of the predicted sizes (22-27 kD). All five family members, when individually overexpressed in Drosophila S2 cells, conferred some resistance to H(2)O(2) treatment, as measured by cell viability. Functional diversification of the Drosophila peroxiredoxin family members was suggested by two lines of evidence: (i) the patterns of mRNA accumulation varied for the different genes during development and (ii) recombinant proteins fused to an epitope tag and overexpressed in Drosophila cells, differed in subcellular localizations--three proteins occurred in the cytosol, one was localized to the mitochondria, and one was found to be secreted.

Catalase expression in Drosophila melanogaster is responsive to ecdysone and exhibits both transcriptional and post-transcriptional regulation.

In the present study, we have examined catalase protein and mRNA levels and the factors that may regulate catalase expression in Drosophila melanogaster during development. Both mRNA and protein changes are in general accord with variations in ecdysteroid titer during development. Differences in mRNA and protein accumulation profiles, particularly in embryos and young adults, suggest that catalase may be regulated at both transcriptional and post-transcriptional levels. It was possible to induce catalase expression by administering exogenous 20-hydroxyecdysone (Ec) in culture at certain stages of development (usually at time points corresponding to previously observed hormone and catalase peaks). Experiments with exogenous administration of Ec, cycloheximide, and actinomycin D suggest a complex interplay of factors affecting catalase expression. In cultured third instar larvae, superinduction of catalase occurred in the presence of both Ec and cycloheximide. If ecdysteroid production was suppressed prior to antibiotic treatment by temperature upshift of the conditional mutant dre4(e55), superinduction occurred mostly at the protein level. In cultured adult abdomens, we observed induction by Ec and superinduction in the presence of hormone and translation or transcription inhibitors. Unlike what was observed in larvae, superinduction of catalase protein was dramatically more pronounced in control flies.

Overexpression of Mn-containing superoxide dismutase in transgenic Drosophila melanogaster.

The general objective of this study was to examine the role of mitochondria in the aging process. Two alternative hypotheses were tested: (i) that overexpression of Mn superoxide dismutase (Mn SOD) in the mitochondria of Drosophila melanogaster would slow the accrual of oxidative damage and prolong survival or (ii) that there is an evolved optimum level of superoxide anion radical, such that overexpression of Mn SOD would have deleterious or neutral effects. Microinjection and mobilization of a transgene, which contained a 9-kb genomic sequence encoding Mn SOD, produced 15 experimental lines overexpressing Mn SOD by 5-116% relative to the parental y w strain. Comparisons between these lines and control lines containing inserted vector sequences alone indicated that the mean longevity of the experimental lines was decreased by 4-5% relative to controls. There were no compensatory changes in the metabolic rate, level of physical activity, or the levels of other antioxidants, namely Cu-Zn SOD, catalase, and glutathione. There were no differences between groups in rates of mitochondrial hydrogen peroxide release, protein oxidative damage, or resistance to 100% oxygen or starvation conditions. The experimental lines had a marginally increased resistance to moderate heat stress. These results are consistent with the existence of an optimum level of Mn SOD activity which minimizes oxidative stress. The naturally evolved level of Mn SOD activity in Drosophila appears to be near the optimum required under normal conditions, although the optimum may be shifted to a higher level under more stressful conditions.

CuZn-SOD promoter-driven expression in the Drosophila central nervous system.

The aim of this study was to ascertain the status of CuZn superoxide dismutase (CuZn-SOD) expression in the central nervous system of Drosophila melanogaster. Immunoblot analysis of dissected tissue extracts revealed low levels of the CuZn-SOD protein in adult brains relative to other adult and larval tissues. To explore further this observation, three different reporter constructs containing different elements of the CuZn-SOD promoter domain were used for the generation of transgenic flies. A high level of reporter gene expression occurred during the second wave of neurogenesis (third instar and early pupal stages) in scattered, proliferating neuroblasts (NBs) and in proliferation centers of the optic lobe. In mature, postmitotic neurons, this expression was lower relative to other tissues. In adult flies, at all ages examined, there was little if any detectable reporter gene expression in cells of the central nervous system. These results suggest that one of the key components of the antioxidant defenses, CuZn-SOD, is quite low in postmitotic neural tissue, rendering it particularly susceptible to oxidative damage during aging.

Molecular organization of the glutathione reductase gene in Drosophila melanogaster.

Glutathione reductase catalyzes the conversion of the oxidized form of glutathione to regenerate reduced glutathione, which acts as a versatile intracellular reductant. The present study provides initial characterization of the glutathione reductase gene in Drosophila melanogaster and its response to experimentally induced oxidative stress. Drosophila cDNA clones were isolated, based on cross-hybridization to the Musca domestica glutathione reductase cDNA. Genomic clones were isolated by cross-hybridization with the Drosophila cDNA as hybridization probe. Northern analysis of adult Drosophila poly(A)+ RNA, utilizing the Drosophila cDNA probe, revealed a hybridization signal in the 2-kb range. The entire sequence of one cDNA was determined. In addition to a coding domain of 1431 bases, the sequence included 206 bases upstream of a putative start codon and 355 bases downstream of a putative stop codon. Based on the cDNA sequence, the 476 amino acid sequence of the Drosophila glutathione reductase gene was deduced and was found to have extensive similarities with the glutathione reductase gene from other species. Gene mapping of a 13-kb genomic fragment revealed that the glutathione reductase gene consists of at least two exons spanning approximately 5 kb. A first exon contains sequence for only the first 5 amino acids and the first base of the sixth and appears to be separated by a ca. 2.5-kb intron from the remainder of the coding region, which is confined to <2 kb. The Drosophila glutathione reductase is single copy and its cytogenetic position, as determined by in situ hybridization, is 7D-E on the X chromosome. mRNA levels of glutathione reductase, measured by RT-PCR, increased in response to exposure to 100% ambient oxygen by almost twofold and administration of paraquat by greater than threefold. Exposure of flies to hyperoxia also induced a 60% increase in the activity of glutathione reductase and augmented the concentration of total glutathione by ca. 40% following an initial drop. The present study, besides providing an initial molecular characterization of the glutathione reductase gene in Drosophila, demonstrates its dynamic involvement in response to experimentally induced oxidative stress.

[Cloning the human cytomegalovirus immediate-early gene and expression of it in Escherichia coli]. / Klonirovanie gena osnovnogo predrannego belka tsitomegalovirusa cheloveka i ékspressiia ego v kletkakh Escherichia coli.

The major immediate early gene of human cytomegalovirus has been cloned. The fragment of the immediate early protein containing 351 amino acids has been fused with cro-beta-galactosidase and expressed in Escherichia coli cells. The obtained recombinant protein reacts in immunoblotting with the antibodies in human sera from patients suffering from acute cytomegalovirus infection.

[The production of recombinant polypeptides from the herpes simplex viruses types 1 and 2]. / Poluchenie rekombinantnykh polipeptidov virusa gerpesa prostogo tipov 1 i 2.

The genes IE175, IE63, IE68, IE12, UL29 and UL19 of herpes simplex virus types 1 and 2 were cloned. Fragments of these genes were cloned into open reading frame expression vector pEX1-3. Recombinant proteins containing amino acid sequences of ICP4, ICP27, ICP22, ICP47, major DNA-binding protein and major capsid protein were expressed in E. coli cells by fusion with cro-beta-galactosidase proteins.

[Cloning and expression of certain herpes simplex virus type 1 genes in Escherichia coli]. / Klonirovanie i ékspressiia v kletkakh Escherichia coli nekotorykh genov virusa gerpesa prostogo tipa 1.

Complete genes IE12, IE63, IE68, IE175, UL19, UL29 of herpes simplex virus type I or their fragments have been cloned in Escherichia coli cells. The peptides expressed were shown to be fused with cro-beta-galactosidase proteins. The recombinant proteins containing amino acid sequences of ICP4, ICP27 and ICP47, major capsid protein, and major DNA-binding protein react in immunoblotting with the anti-HSVI serum from hyperimmune rabbit. The recombinant proteins can be used for creation of diagnosticums and other scientific and practical purposes. The immunological properties of the recombinant proteins are being investigated.

[The determination of the antigenic activity of recombinant virus-specific polypeptides from the herpes simplex virus types 1 and 2 and their use in immunoenzyme analysis]. / Opredelenie antigennoi aktivnosti rekombinantnykh virusspetsificheskikh polipeptidov virusa gerpesa prostogo tipov 1 i 2 i ispol'zovanie ikh v immunofermentnom analize.

The structural and nonstructural recombinant proteins of HSV type 1 and type 2 were expressed in E. coli by fusion with cro-beta-galactosidase proteins. These proteins containing amino acid sequences of ICP4, ICP27, ICP47, ICP22, major capsid protein and major DNA-binding proteins of HSV types 1 and 2 reacted in immunoblotting with the corresponding anti-HSV sera from hyperimmune rabbits. The nonstructural recombinant proteins can be used for enzyme immunoassay detection of HSV1 or HSV2 type-specific antibodies in sera from patients with acute HSV infection.

[Comparative study of glyceraldehyde-3-phosphate dehydrogenases isolated from rabbit skeletal muscles and baker's yeast using cationic fluorescent probes]. / Sravnitel'noe issledovanie glitseral'degid-3-fosfatdegidrogenaz, izolirovannykh iz skeletnykh myshts krolika i iz pekarskikh drozhzhei, s pomoshch'iu fluorestsentnykh zondov kationnoi prirody.

The tetrameric molecule of glyceraldehyde-3-phosphate dehydrogenase possesses the ability to bind fluorescent probes of cationic nature (auramine O and acridine orange) outside the active center. The rabbit skeletal muscle and yeast enzymes share some common features, e.g., the conformational non-equivalency of subunits; two subunits per tetramer can bind auramine O; conformational changes caused by the binding of adenyl mononucleotides and involving the microenvironment of auramine O binding sites; the ability to bind the cationic probe at pH values typical for the maximal activity of the enzyme in the reaction of glyceraldehyde 3-phosphate oxidation. The yeast and rabbit muscle enzymes are distinguished in terms of localization of the probe binding sites with respect to the active center and/or in the nature of conformational changes induced by NAD+ binding. It was demonstrated that nicotinamide mononucleotide may serve as a co-enzyme in glyceraldehyde 3-phosphate oxidation catalyzed by yeast dehydrogenase; this reaction in inhibited by AMP.