Sulfite sensitivity and sulfite oxidase activity in Drosophila melanogaster.

The relationship between sulfite oxidase (SO) and sulfite sensitivity in Drosophila melanogaster is addressed. Significant improvements to the SO assay have provided an investigative tool which can be applied to further studies of this molybdoenzyme. Using the second-instar larval stage of D, melanogaster, we have shown a direct relationship between measured levels of sulfite oxidase activity and the organism's ability to withstand a sulfite challenge. Implementation of a sulfite-testing procedure confirmed the documented instability of sulfite in solution and may explain some of the conflicting results reported in the SO literature. Results of the tungstate-addition experiments confirm that Drosophila SO is a molybdoenzyme and its activity was shown to be governed by three of the four loci known to affect more than one molybdoenzyme. The ability of D. melanogaster to withstand the application of exogenous sulfites is shown to be dependent on sulfite oxidase activity.

The isolation and characterization of mutant alleles at a new X-linked locus, mex, affecting NADP(+)-dependent enzymes in Drosophila melanogaster.

The isolation and characterization of mutant alleles in a regulatory gene affecting NADP(+)-dependent enzymes are described. The locus, mex, is at position 26.5 +/- 0.74 on the X chromosome of Drosophila melanogaster. The newly isolated mutant allele, mex1, is recessive to either the mex allele found in Oregon-R wild-type individuals or that found in the cm v parental stock in which the new mutants were induced. The mex1 mutant allele is associated with statistically significant decreases in malic enzyme (ME) specific activity and ME specific immunologically cross-reacting material (ME-CRM) in newly emerged adult males. During this same developmental stage in males, the NADP(+)-dependent isocitrate dehydrogenase specific activity increases to statistically significant levels. Females of the mex1 mutant strain show statistically significant elevated levels of the pentose phosphate shunt enzymes, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. Isoelectric focusing and thermolability comparisons of the active ME from mutant and control organisms indicate that the enzyme is the same. Developmental profiles of mex1 and control strains indicate that this mutant allele differentially modulates the levels of ME enzymatic activity and ME-CRM during development.

The expression of sequences similar to the human c-erb-A oncogene are regulated in a tissue and stage specific manner in Drosophila melanogaster.

The gene encoding the human thyroid hormone receptor, c-erb-A, is hypothesized to be a member of a superfamily of genes, which originated from a primordial receptor encoding gene. To trace the molecular evolutionary background of this gene, we initiated the search for it in the genome of Drosophila melanogaster by using low stringency hybridization analysis. We report here the presence of more than one gene in Drosophila with varying degrees of sequence similarity. By RNA blot hybridization analyses utilizing both wild type and a temperature sensitive ecdysone deficient mutant strain, ecd1, with a human c-erb-A probe, we show that a single high molecular weight RNA of 6.8kb in size is recognized by human c-erb-A, is regulated in a stage and tissue specific manner, and is also inducible in organisms of the ecd1 mutant strain in the presence of exogeneous ecdysone.

The aldox-2 locus of Drosophila melanogaster also affects sulfite oxidase and molybdenum metabolism.

Mutation at the aldox-2 locus in Drosophila melanogaster affects the specific activities of four molybdoenzymes differentially during development. Sulfite oxidase activity is normal during late larval and pupal stages but is reduced during early adult stages in aldox-2 organisms. There was complete concordance among the effects of aldox-2 on sulfite oxidase, aldehyde oxidase, xanthine dehydrogenase, and pyridoxal oxidase, when 38 stocks were analyzed which were derived from single recombination events between c and px, markers which flank aldox-2. Several different biochemical analyses indicate that the active molybdoenzymes present in the aldox-2 strain are normal with respect to size, shape, pH-activity profile, Km, and molecular weight. Significant differences were found between the aldox-2 strain and the OR control strain in their responses to dietary Na2MoO4 and Na2WO4. The mutant strain is much more resistant to the effects of dietary Na2WO4 and much more responsive to the administration of Na2MoO4 than the OR control strain when these effects are quantitated by measurements of molybdoenzyme specific activities. This evidence suggests that the aldox-2+ gene product has a molybdenum binding site which can also bind tungsten and that this site is altered in the mutant strain. The hypothesis presented explains the observed effects of the aldox-2 mutation and relates them to the other mutations reported in this gene-enzyme system.

Induction of alcohol dehydrogenase by ethanol in Drosophila melanogaster.

When Drosophila melanogaster larvae were fed a defined fat-free, low sucrose medium, alcohol dehydrogenase (ADH) was increased to a higher activity with a moderate, nontoxic level of ethanol (2.5% vol/vol) within 5 h. Ethanol-stimulated increases in ADH activity and cross-reacting material in late third-instar larvae were paralleled by increases in the larval ADH mRNA as indicated by dot blot analysis. Northern blot observations indicated that both adult and larval ADH messages were increased by dietary ethanol. The increased levels of the ADH mRNA transcribed from the proximal and distal promoters of ethanol-fed larvae argue that the induction is a consequence of elevated levels of mRNA, not a result of changes in enzyme stability or synthesis. To determine whether the induction is of nutritional significance to larvae, the rate of flux from ethanol to lipid was estimated in control larvae and larvae that were pre-fed ethanol. Flux changes occurred; the rate of incorporation of [14C]ethanol into body lipid showed a strong association with larval ADH activity. Because the induced increase in larval ADH activity did not extend into the adult stage and attempts to stimulate ADH activity by exposing adults to ethanol were unsuccessful, the modulation of ADH activity by dietary ethanol may be a mechanism by which larvae utilize environmental ethanol as a resource, especially when free sugar levels are low. In addition, ADH in larvae is postulated to perform a second, nonethanol function that expedites the conversion of sugars to lipid when habitats are low in fats, low in ethanol and high in sugars.

Genetic and developmental characterization of the aldox-2 locus of Drosophila melanogaster.

The aldox-2 locus in Drosophila melanogaster has been shown to affect differentially three molybdoenzymes, aldehyde oxidase, pyridoxal oxidase, and xanthine dehydrogenase. These effects are most obvious at times surrounding the pupal-adult boundary, when the normal organism accumulates large amounts of these enzymes in their active form. This locus has been more precisely mapped genetically to 2-82.9 +/- 2.1, with complete concordance between the effects of all recombinant chromosomes on all three enzymes. The cytogenetic location has also been determined to be between 52E and 54E8, with the likelihood that it lies within the region 54B1-54E8. The aldox-2 mutant allele has no visible phenotype and is completely recessive for enzyme effects at all stages tested. Segmental duplication of this region, including the aldox-2+ allele, has no apparent effect on the visible phenotype or the enzymatic activity. The mutant aldox-2 allele has no effect on the developmental expression of two unrelated enzymes, 6-phosphogluconate dehydrogenase and NADP+-dependent isocitrate dehydrogenase. The effects of this locus on aldehyde oxidase, xanthine dehydrogenase, and pyridoxal oxidase suggest that this locus may code for a product involved in the synthesis of the molybdenum cofactor common to these enzymes.

Analysis of Aldoxn alleles isolated from natural populations of Drosophila melanogaster.

Aldox "null" alleles which were isolated from natural populations in Great Britain and North Carolina were analyzed for complementation. No complementation was observed between any combinations of "null" alleles for aldehyde oxidase (AO) specific activity in late third-instar larvae and newly emerged adults. AO immunologically cross-reacting material (AO-CRM) was quantitated in all homozygous stocks at both developmental stages as well as all allelic combinations in newly emerged adults. When the adult organism contains only Aldoxn alleles, the polypeptides are not immunologically recognizable or may be rapidly degraded. Larvae and adults have different abilities to degrade mutationally altered enzymatically inactive AO polypeptide or synthesize them differentially. This is indicated by easily measurable AO-CRM levels in late third-instar larvae of Aldoxn homozygotes, while newly emerged adult Aldoxn homozygotes have very little, if any, AO-CRM. Newly emerged adult heterozygotes of Aldoxn/Aldox+ do have increased AO-CRM, indicating that the Aldoxn alleles can code for a polypeptide which can be "rescued" if Aldox+ gene product is present. Heterozygotes containing an Aldox+ allele with a deficiency for the Aldox region produce 74.2% of the AO-CRM found in Aldox+ homozygotes. This may indicate the presence of trans-acting factors which serve to activate gene expression in a system in which each gene copy is not maximally expressed.

Cytogenetic localization of a putative regulatory gene affecting an NADP+-dependent enzyme in Drosophila melanogaster.

The cytogenetic localization of a putative regulatory locus affecting an NADP+-dependent enzyme is described. This locus, Mex, is in region 8D10-12 to 9A1-2 on the X chromosome of Drosophila melanogaster. Hyperploidy for this region is associated with significant increases in NADP+-dependent malic enzyme specific activity and specific immunologically cross-reacting material. This is the first report of a specific locus, unlinked to the locus coding for the major polypeptide of the enzyme, which affects an NADP+-dependent enzyme in Drosophila melanogaster.

Comparative properties of three forms of glucose-6-phosphate dehydrogenase in Drosophila melanogaster.

Three alleles of the Zw locus of Drosophila melanogaster--ZwA, ZwB, and Zwlol--apparently code for dimeric, tetrameric, and monomeric forms of glucose-6-phosphate dehydrogenase (G6PD), respectively. The three forms of G6PD are characterized by different apparent Km values for glucose-6-phosphate but similar apparent Km values for NAPD+. When high concentrations of NAPD+ were added to enzyme preparations, the ZwA and Zwlol forms of G6PD assumed tetrameric and dimeric properties, respectively. Although Zwlol adults exhibit little G6PD activity, they maintain levels of G6PD-antigen comparable to those in ZwA and ZwB adults. Thus the low level of G6PD activity in Zwlol individuals cannot be explained as the consequence of lack of synthesis of the G6PD subunit.

Characterization of a low-activity allele of NADP+-dependent isocitrate dehydrogenase from Drosophila melanogaster.

We have characterized biochemical effects of IdhGB1 in Drosophila melanogaster. This is a "null"-activity allele for NADP+-dependent isocitrate dehydrogenase (NADP-IDH) isolated from a natural population. The homozygous mutant strain has 5% of the NADP-IDH specific activity found in controls and less than 24% of the immunologically cross-reacting material (CRM). This mutation maps to 27.2 on the third chromosome, to the right of h. The biochemical phenotype of this mutant strain includes a coordinate reduction in malic enzyme (ME) specific activity and CRM and an increase in specific activity for the pentose-phosphate shunt enzymes, 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase. The Km values for purified NADP-IDH are not different from those found for the purified control enzyme for NADP+ or isocitrate. It is suggested that this allele may represent a cis-acting control mutation for one of at least two loci involved in the production of NADP-IDH in D. melanogaster.

Dosage compensation in Drosophila: NADP-enzyme activities and cross-reacting material.

The relationships between gene dosage, enzyme activities and CRM levels have been determined for G6PD and 6PGD. Enzyme activities and CRM levels were directly proportional and increased in genotypes carrying duplications of the respective structural genes. When a duplication consisting of the distal 45% of the X chromosome was used to duplicate Pgd+, 6PGD activity and CRM increased and G6PD activity decreased. When the proximal 55% of the X chromosome was duplicated, G6PD activity and CRM increased whereas 6PGD activity and CRM levels decreased. These observations support the model of dosage compensation of X-linked genes that invokes an autosomal activator in limited concentrations for which X-linked loci compete. The distal 45% of the X chromosome, when duplicated, caused a significant increase in NADP-malic enzyme activity and CRM levels, as if a structural gene for NADP-ME is sex-linked.

The developmental analysis of aldehyde oxidase activity in cin allelic heterozygotes of Drosophila melanogaster.

Aldehyde oxidase (AO) activity has been determined at 11 stages during the development of selected cin allelic homo-, hemi- and heterozygotes in Drosophila melanogaster. The AO activity levels found during development were completely consistent with the levels previously reported for adults, less than 24 h of age (Bentley and Williamson, 1979b). All of the cin homo- and hemizygotes tested exhibited no significant levels of AO activity at any of the 11 stages during development. All cin allelic heterozygotes, which were defined as complementing in adults, less then 24h of age, displayed similar levels of complementation at all stages tested. Conversely, all cin allelic heterozygotes which were defined as noncomplementing in adults, less than 24h of age, were found to lack measurable AO activity at all developmental stages tested.

The control of aldehyde oxidase and xanthine dehydrogenase activities and CRM levels by the mal locus in Drosophila melanogaster.

The effects of five new mal alleles on aldehyde oxidase (AO) and xanthine dehydrogenase (XDH) activities and CRM levels in Drosophila melanogaster are described. These alleles were isolated by taking full advantage of the pleiotropic phenotype exhibited by all previously described mal alleles and represent at least three unique examples of mal function. Al least one of these alleles is a representative of a new complementation group. Two other alleles exhibit a wild-type eye color in homozygous stock and one of these is "leaky", exhibiting some 50% of the XDH activity normally found in Oregon-R control flies and some 12% of the AO activity. CRM and activity levels have been quantitated for both enzymes in all allelic heterozygotes. XDH-CRM levels vary only slightly around wild-type levels while AO-CRM levels appear much more sensitive to mutational alterations.

The effects of molybate, tungstate and lxd on aldehyde oxidase and xanthine dehydrogenase in Drosophila melanogaster.

The effects of dietary sodium molybdate and sodium tungstate on eye color and aldehyde oxidase and xanthine dehydrogenase activities have been determined in Drosophila melanogaster. Dietary sodium tungstate administration has been used as a screening procedure to identify two new lxd alleles. Tungstate administration results in increased frequencies of "brown-eyed" flies in lxd stocks and a coordinate decrease in AO and XDH activities in all genotypes tested. The two new lxd alleles affect AO and XDH in a qualitatively but not quantitatively similar fashion to the original lxd allele. AO and XDH activity and AO-CRM levels appear much more sensitive to mutational perturbations of this gene-enzyme than do XDH-CRM levels in the genotypes tested.

A new mutant affecting aldehyde oxidase in Drosophila melanogaster.

A new locus, Aldox-2, which affects the activity and heat stability of aldehyde oxidase in D. melanogaster is described. The Aldox-2 locus is localized to map position 86 on chromosome 2, between c and px. Aldehyde oxidase activity in aldox-2 homozygotes is approximately 25--30% that of the Oregon-R wild-type control strain. The enzyme from the mutant stock is much more heat labile than is the enzyme from the wild-type strain. Both the activity and heat phenotypes are completely recessive.

The effects of lao on aldehyde, oxidase activity and cross-reacting-material in Drosophila melanogaster.

In Drosophila melanogaster aldehyde oxidase occurs in at least two forms that can be separated electrophoretically. The mutant allele lao (low aldehyde oxidase activity) causes a deficiency of the major form of this enzyme. Immunoelectrophoretic analyses suggest that lao homozygotes produce aldehyde oxidase cross-reacting-material in nearly wild-type levels. Although aldehyde oxidase from the mutant stock is heat labile. properties such as Km and pH optima are not different from the normal enzyme.