Sex-Specific Effects of Cis-Regulatory Variants in Drosophila melanogaster

Sexual dimorphism at the level of gene expression is common and well documented, but much less is known about how different cis-regulatory alleles interact with the different trans-regulatory environments present in males and females. Here we show that sex-specific effects of cis-regulatory variants are common in Drosophila.     

Sex Differences in Drosophila melanogaster Heterochromatin Are Regulated by Non-Sex Specific Factors

The eukaryotic genome is assembled into distinct types of chromatin. Gene-rich euchromatin has active chromatin marks, while heterochromatin is gene-poor and enriched for silencing marks. In spite of this, genes native to heterochromatic regions are dependent on their normal environment for full expression. Expression of genes in autosomal heterochromatin is reduced in male flies mutated for the noncoding roX RNAs, but not in females. roX mutations also disrupt silencing of reporter genes in male, but not female, heterochromatin, revealing a sex difference in heterochromatin. We adopted a genetic approach to determine how this difference is regulated, and found no evidence that known X chromosome counting elements, or the sex determination pathway that these control, are involved. This suggested that the sex chromosome karyotype regulates autosomal heterochromatin by a different mechanism. To address this, candidate genes that regulate chromosome organization were examined. In XX flies mutation of Topoisomerase II (Top2), a gene involved in chromatin organization and homolog pairing, made heterochromatic silencing dependent on roX, and thus male-like. Interestingly, Top2 also binds to a large block of pericentromeric satellite repeats (359 bp repeats) that are unique to the X chromosome. Deletion of X heterochromatin also makes autosomal heterochromatin in XX flies dependent on roX and enhances the effect of Top2 mutations, suggesting a combinatorial action. We postulate that Top2 and X heterochromatin in Drosophila comprise a novel karyotype-sensing pathway that determines the sensitivity of autosomal heterochromatin to loss of roX RNA.     

Biochemical properties of esterase 6 in Drosophila melanogaster

Biochemical properties of esterase 6 in Drosophila melanogaster were investigated using partially purified preparations from three genotypes, 1/1, 1/2, and 2/2. The molecular weight of the enzyme is estimated to be about 90,000, and treatment with sodium dodecylsulfate cleaves the enzyme into four units with a molecular weight of about 22,000. The activity toward 28 naturally occurring esters was assayed and shown to vary considerably with substrate, the 1/1 preparation having in general higher activity than 1/2 and 2/2, which were very similar. Heat sensitivity, the effect of metal ions, and the effects of the presence or absence of an end product were also studied. The differences demonstrated between allozymes would allow considerable scope, under appropriate conditions, for differential selection to operate between genotypes.Supported in part by an SRC Research Studentship (N.D.D.).     

Enzyme polymorphism in Drosophila melanogaster populations in Iraq

Electrophoretic studies of the degree and pattern of polymorphism at two third-chromosome loci, esterase-6 (Est-6) and phosphoglucomutase (PGM), were carried out in three Drosophila melanogaster populations collected from different localities in Iraq: Mosul, Tuwaitha, and Basrah. The results show that only the Tuwaitha population was polymorphic for both loci; the other two populations were polymorphic for Est-6 and monomorphic for PGM. The allele frequency changes at both loci were followed for 20 generations in an experimental cage derived from the Tuwaitha population; it was found that there is a deviation from Hardy-Weinberg equilibrium at both loci toward the homozygote.     

A Luminal Glycoprotein Drives Dose-Dependent Diameter Expansion of the Drosophila melanogaster Hindgut Tube

An important step in epithelial organ development is size maturation of the organ lumen to attain correct dimensions. Here we show that the regulated expression of Tenectin (Tnc) is critical to shape the Drosophila melanogaster hindgut tube. Tnc is a secreted protein that fills the embryonic hindgut lumen during tube diameter expansion. Inside the lumen, Tnc contributes to detectable O-Glycans and forms a dense striated matrix. Loss of tnc causes a narrow hindgut tube, while Tnc over-expression drives tube dilation in a dose-dependent manner. Cellular analyses show that luminal accumulation of Tnc causes an increase in inner and outer tube diameter, and cell flattening within the tube wall, similar to the effects of a hydrostatic pressure in other systems. When Tnc expression is induced only in cells at one side of the tube wall, Tnc fills the lumen and equally affects all cells at the lumen perimeter, arguing that Tnc acts non-cell-autonomously. Moreover, when Tnc expression is directed to a segment of a tube, its luminal accumulation is restricted to this segment and affects the surrounding cells to promote a corresponding local diameter expansion. These findings suggest that deposition of Tnc into the lumen might contribute to expansion of the lumen volume, and thereby to stretching of the tube wall. Consistent with such an idea, ectopic expression of Tnc in different developing epithelial tubes is sufficient to cause dilation, while epidermal Tnc expression has no effect on morphology. Together, the results show that epithelial tube diameter can be modelled by regulating the levels and pattern of expression of a single luminal glycoprotein.     

Structural instability of 297 element in Drosophila melanogaster

297 element Southern pattern modifications previously detected in mutation accumulation lines of Drosophila melanogaster were further investigated by in situ hybridisation, Southern blotting with different combinations of genomic digest-probe, and PCR. Only one out of the nine pattern modifications studied could be interpreted as an excision and was detectable by in situ hybridisation to polytene chromosomes. Results were consistent with most pattern modifications being small rearrangements within the body of the element. In agreement with the existence of spontaneous rearrangements of this kind is the observation that many genomic copies of element 297 are defective and these are not limited to heterochromatin. These findings have important implications for the models of transposable element (TE) number regulation as well as for the study of genome evolution. This revised version was published online in July 2006 with corrections to the Cover Date.     

Biochemical and genetic characterization of kynurenine formamidase from Drosophila melanogaster

The molecular weight forms of kynurenine formamidase were studied both genetically and biochemically. Formamidase I (native molecular weight 60,000) was purified using (NH₄)₂SO₄ and pH fractionation, DEAE-cellulose chromatography at two different pH's, hydroxylapatite chromatography, and Sephadex G-100 gel filtration. Its subunit molecular weight, as determined by SDS gel electrophoresis, is 34,000, indicating that formamidase I is a dimer. Its K _m is 1.87×10^–3 m. Its isoelectric point is pH 5.3. Its amino acid composition is reported. Formamidase II (native molecular weight 31,000) was partially purified using techniques similar to those above. Its K _m is 2.31×10^–3 m. The response of formamidase activity to change in gene dosage was measured in segmental aneuploids generated in the second, third, and X chromosomes. Two separate chromosomal regions were identified which when present in extra dosage result in an elevation of the level of formamidase activity close to that predicted for the addition of a structural gene in a two-gene system. These tentative map positions were substantiated by demonstration that addition of one of the regions, 25A–27E, causes a 50% elevation in the relative amount of formamidase II. Addition of the other region, 91B–93F, causes a similar elevation in the relative amount of formamidase I. A model of the evolutionary origin of the two forms is presented, and the significance of these results to this model is discussed.This work was supported by USPHS Grant GM-21286.     

Biochemical differences between alcohol dehydrogenases of Drosophila melanogaster.

This paper describes substrate specificities, developmental changes in activity, pH profiles, and heat stabilities of isozymes produced by four Adh genotypes in D. melanogaster. No differences are found in the substrate specificities of isozymes from the different genotypes but studies of the other three properties reveal significant differences between the isozymes. Thus relatively low activities are found among extracts of AdhF Adhn2 larvae and among extracts of AdhF AdhF adults aged 44 days. Also AdhF AdhS and AdhS AdhS extracts have relatively high activities at pH 6-5, and AdhF Adhn2 extracts have relatively low activities at pH values above 10-0. Finally, extracts of AdhF AdhF and AdhF AdhS are more stable at 40 degrees C than are those of AdhS AdhS and AdhF Adhn2.     

Reduced Enzyme Activity Following Hsp70 Overexpression in Drosophila melanogaster

Acclimation to environmental change can impose costs to organisms. One potential cost is the change in cell metabolism that follows a physiological response, e.g., high expression of heat shock proteins may alter specific activity of important enzymes. We examined the significance of this cost in a pair of Drosophila melanogaster lines transformed with additional copies of a gene that encodes the heat shock protein, Hsp70. Heat shock induces Hsp70 expression in all lines, but lines with extra copies produce much more Hsp70 than do excision control strains. The consequence of this supranormal Hsp70 expression is to reduce specific activity of both enzymes analyzed, adult alcohol dehydrogenase (ADH), which is heat sensitive, and lactate dehydrogenase, which is not. Strain differences were most pronounced under those conditions where Hsp70 expression was maximized, and not where the heat stress denatured proteins. That result supported the idea that Hsp70 expression is constrained evolutionarily by its tendency to bind nascent peptides when overabundant within the cell.     

Structural heterogeneity and genomic distribution of Drosophila melanogaster LTR-retrotransposons

Structural heterogeneity of five long terminal repeat (LTR) retrotransposon families (297, mdg 1, 412, copia, and 1731) was investigated in Drosophila melanogaster. The genomic distribution of canonical and rearranged elements was studied by comparing hybridization patterns of Southern blots on salivary glands from adult females and males with in situ hybridization on polytene chromosomes. The proportion and genomic distribution of noncanonical copies is distinctive to each family and presents constant features in the four different D. melanogaster strains studied. Most elements of families 297 and mdg 1 were noncanonical and presented large interstock and intrastock polymorphism. Noncanonical elements of these two families were mostly located in euchromatin, although not restricted to it. The elements of families 412 and copia were better conserved. The proportion of noncanonical elements was lower. The 1731 family is mainly composed of noncanonical, beta-heterochromatic elements that are highly conserved among stocks. The relation of structural polymorphism to phylogeny, transpositional activity and the role of natural selection in the maintenance of transposable elements are discussed.     

Biochemical identification and crystal structure of kynurenine formamidase from Drosophila melanogaster

KFase (kynurenine formamidase), also known as arylformamidase and formylkynurenine formamidase, efficiently catalyses the hydrolysis of NFK (N-formyl-L-kynurenine) to kynurenine. KFase is the second enzyme in the kynurenine pathway of tryptophan metabolism. A number of intermediates formed in the kynurenine pathway are biologically active and implicated in an assortment of medical conditions, including cancer, schizophrenia and neurodegenerative diseases. Consequently, enzymes involved in the kynurenine pathway have been considered potential regulatory targets. In the present study, we report, for the first time, the biochemical characterization and crystal structures of Drosophila melanogaster KFase conjugated with an inhibitor, PMSF. The protein architecture of KFase reveals that it belongs to the α/β hydrolase fold family. The PMSF-binding information of the solved conjugated crystal structure was used to obtain a KFase and NFK complex using molecular docking. The complex is useful for understanding the catalytic mechanism of KFase. The present study provides a molecular basis for future efforts in maintaining or regulating kynurenine metabolism through the molecular and biochemical regulation of KFase.     

Biochemical studies of supernatant malate dehydrogenase allozymes in Drosophila melanogaster.

1. A biochemical comparison was made among cytoplasmic malate dehydrogenase allozymic variants from Drosophila melanogaster. Experiments were carried out on enzyme extracted from six different genotypes: three homozygotes and their respective heterozygotes. 2. The allozyme forms (MDH A, MDH B, MDH C) were indistinguishable in terms of NAD and L-malate optima, while they are distinguishable in terms of NADH and OAA saturation conditions. Activities were inhibited at concentrations greater than 0.36 and 0.40 mM NADH for BB and AA, CC, respectively, while in relation to OAA inhibition was observed at concentrations higher than 3 or 6 mM for the AA, CC and BB, respectively. 3. differences among genotypes were also observed in thermal stability: Km values for OAA, L-malate, NADH and NAD: and pG optima. 4. A simple method is presented for the separation of the cytoplasmic from the mitochondrial malate dehydrogenase.     

Structural and functional analysis of the scute locus in Drosophila melanogaster

Summary The functional expression of 12 scute alleles in homozygotes and compounds of Drosophila melanogaster at 14°, 22°, 30°C is analysed. Based on the data obtained, linear maps for bristles and mutations are built. The basic features of the maps, clustering and polarity, are invariable with respect to temperature, scute gene dosage and cross direction. In addition local dominance of the norm over bristle reduction was produced by the scute mutation; different types of complementation reactions were established for each bristle. The gene scute is treated as an operon-like system, composed of 3–4 cistrons with each controlling the formation of bristles on a particular region of the fly's body. This model argues well with the structure of maps constructed and implies a post-translational level of initial events of bristle-formation process.This paper is based on the report presented at XIV International Congress of Genetics (Moscow, August 1978)     

Biochemical Differences between Products of the Adh Locus in Drosophila

An analysis of the molecular properties of the major alcohol dehydrogenase (E.C.1.1.1.1) allozyme variants found segregating in natural populations of D. melanogaster is presented. Our results indicate: (1) ADH-S enzyme has generally lower Michaelis-Menten constants than those of ADH-F; (2) ADH-S and ADH-F enzymes display opposite interactions for both co-factor and substrate; and (3) higher levels of ADH are associated with the Adh-fast genotype. The possible adaptive significance of these findings is discussed.     

Biochemical and genetic basis of the response to 5-fluorouracil in Drosophila melanogaster

Mutant strains sensitive and resistant to the drug 5-fluorouracil (FU) have been isolated from the wild-type Pac strain of Drosophila melanogaster. The resistant strain, termed flu^r, is resistant to at least 0.0035%FU (2.7 × 10^–4 m) in the food media and exhibits cross-resistance to 5-fluorodeoxyuridine (FUdR) but not to 5-fluorouridine (FUR). The sensitive strain termed flu ^S , exhibits over 90% mortality on 0.0008% FU (6 × 10^–5 m). Genetic analysis indicates that the flu gene is located on the third chromosome, which agrees with results of previous workers. An analysis of the enzyme thymidylate synthetase from the selected sensitive and resistant strains indicates that the resistant strain enzyme possesses an elevated specific activity. Levels 4 times that of the sensitive strain were observed when the enzymes were assayed at 20 C. This increase is apparently not due to induction by FU in the food media. It is suggested that the enzyme thymidylate synthetase may be involved in the resistance process.