Sequence and evolution of the Drosophila pseudoobscura glycerol-3-phosphate dehydrogenase locus

The Gpdh genomic region has been cloned and sequenced in Drosophila pseudoobscura. A total of 6.8 kb of sequence was obtained, encompassing all eight exons of the gene. The exons have been aligned with the sequence from D. melanogaster, and the rates of synonymous and nonsynonymous substitution have been compared to those of other genes sequenced in these two species. Gpdh has the lowest rate of nonsynonymous substitution yet seen in genes sequenced in both D. pseudoobscura and D. melanogaster. No insertion/deletion events were observed, and the overall architecture of the gene (i.e., intron sites, etc.) is conserved. An interesting amino acid reversal was noted between the D. melanogaster Fast allele and the D. pseudoobscura gene.     

Changes in gene expression during post-eclosional development in the olfactory system of Drosophila melanogaster

We have found that the expression of some genes in Drosophila melanogaster changes during the life of the adult fly. These changes can be illustrated by the use of enhancer trap lines which mark the expression of particular genes in the adult fly. Although the fly is considered able to perform most necessary adult functions within the first 72 h after eclosion from the pupal case, we find changes in expression over the first 10 days of life in the antennae of several of the genes we have examined. Some genes change by increasing from an initially low level of expression of the marked gene, while other lines, which we have termed 'late-onset' genes, show no expression of the marked gene until 4-5 days following eclosion. In contrast, some genes decrease their expression during the first 10 days of life. The changes in gene expression seen over the first 10 days of the fly's adult life provides molecular evidence of the many maturational changes occurring during the early life of the adult fly.     

Maternal Nanos regulates zygotic gene expression in germline progenitors of Drosophila melanogaster

Maternal Nanos (Nos) protein is required for germline development in Drosophila embryos. Here we show that Nos regulates zygotic gene expression in the germline progenitors, or pole cells. In order to probe the gene expression in pole cells, we screened ten enhancer-trap lines which showed beta-gal expression in pole cells. All of these enhancer-trap markers were fully activated in pole cells after their migration to the embryonic gonads. In the pole cells lacking Nos, the expression of nine out of ten enhancer-trap markers was affected. Among nine markers, five (Type-A) were prematurely expressed in the pole cells during the course of their migration. The expression of other four markers (Type-B) initiated correctly after pole-cell migration, but their expression was significantly reduced. Thus, we conclude that the maternal Nos plays a dual role in zygotic gene regulation in pole cells: to define the stages of expression for Type-A markers, and to enhance expression for Type-B markers. Contrary to our results, "Heller and Steinmann-Zwicky (1998)" have recently reported that no premature expression of Type-A markers occurs in the pole cells of embryos derived from nos mutant females. This discrepancy is due to the difference in the nos mutant alleles used for these analyses. We used the much stronger allele, nosBN.     

Gene-enzyme alcohol dehydrogenase system and adaptability in Drosophila melanogaster

As follows from the experiments on the genotypic related populations of Drosophila melanogaster with different frequency of ADH allozymes selection for postponement of ageing and resistance to hypothermia lead to saturation of population with S-allozyme ADH, and genotypic adaptation to ethanol-to increasing of frequency of F-allozyme. It is supposed that genotypic adaptation is realized by selection of specimens with the most favourable alleles of genes. At the same time ontogenetic adaptation is accompanied by biochemical modification of existing allozymes.     

Divergent and conserved features in the spatial expression of the Drosophila pseudoobscura esterase-5B gene and the esterase-6 gene of Drosophila melanogaster

The regulatory regions of homologous genes encoding esterase 6 (Est-6) of Drosophila melanogaster and esterase 5B (Est-5B) of Drosophila pseudoobscura show very little similarity. We have undertaken a comparative study of the pattern of expression directed by the Est-5B and Est-6 5'-flanking DNA to attempt to reveal conserved elements regulating tissue-specific expression in adults. Esterase regulatory sequences were linked to a lacZ reporter gene and transformed into D. melanogaster embryos. Est-5B, 5' upstream elements, give rise to a beta-galactosidase expression pattern that coincides with the wild-type expression of Est-5B in D. pseudoobscura. The expression patterns of the Est-5B/lacZ construct are different from those of a fusion gene containing the upstream region of Est-6. Common sites of expression for both kinds of constructs are the third segment of antenna, the maxillary palps, and salivary glands. In vitro deletion mutagenesis has shown that the two genes have a different organization of regulatory elements controlling expression in both the third segment of antenna and maxillary palps. The results suggest that the conservation of the expression pattern in genes that evolved from a common ancestor may not be accompanied by preservation of the corresponding cis-regulatory elements.     

Allele-specific population structure of Drosophila melanogaster alcohol dehydrogenase at the molecular level

The history of the Drosophila melanogaster alcohol dehydrogenase (ADH) Fast/Slow polymorphism was studied by recording molecular variation and inversion polymorphism in 233 chromosomes from European and African populations. Silent molecular variation in the Slow allele was very different between standard chromosomes and chromosomes bearing the In(2L)t inversion. Within populations, inverted Slow haplotypes were more variable than standard Slow haplotypes. Between populations, geographical structure was almost nonexistent for inverted Slow haplotypes and highly significant for standard Slow. All Fast haplotypes occurred on standard chromosomes. They showed little variation within and between populations. They were highly significantly closer to standard Slow haplotypes from Europe. These results suggest that the current range of Fast and In(2L)t Slow haplotypes is recent and that an older genetic differentiation between populations was followed by allele-specific gene flow.     

Purification and biochemical properties of allelic forms of cytoplasmic glycerol-3-phosphate dehydrogenase from Drosophila virilis

Three homozygous allelic forms (alpha GPDHf, alpha GPDHm and alpha GPDHs) of NAD+-dependent glycerol-3-phosphate dehydrogenase (sn-glycerol-3-phosphate:NAD+ 2-oxidoreductase, EC 1.1.1.8) of Drosophila virilis were purified to homogeneity and their biochemical properties were compared. Although these three forms were mutually distinguishable by electrophoresis, no significant differences were found with respect to pH optima for both forward and reverse reactions (pH 6.0--6.5 for dihydroxyacetone phosphate reduction; pH 10.0--10.5 for glycerol 3-phosphate oxidation), native and subunit molecular weights (65 000 for native form; 35 000--37 000 for subunit) and Michaelis constants for NADH, glycerol 3-phosphate and NAD+ (5.3--6.0 microM for NADH; 1.8--1.9 mM for glycerol 3-phosphate; 100--110 microM for NAD+). Significant differences among three forms were observed in thermostability at 35 degrees C and inhibition by excess of dihydroxyacetone phosphate. The alpha GPDHf form was found to be most thermolabile and the alpha GPDHs form most susceptible to the inhibition.     

Inhibition of glyceraldehyde-3-phosphate dehydrogenase in post-ischaemic myocardium

OBJECTIVE: Myocardial reperfusion following brief period of ischaemic is associated with prolonged, reversible periods of metabolic dysfunction. As the glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is inhibited in vitro by reactive oxygen species, we hypothesized that production of reactive oxygen species during reperfusion would lead to inhibition of GAPDH in post-ischaemic myocardium. METHODS: Anaesthetized closed-chest-dogs were subjected to 20 min balloon occlusion of the left anterior descending coronary artery. Biopsy samples were taken after 3 and 24 h of reperfusion, to determine the activity of GAPDH and the concentrations of glycolytic intermediates in post-ischaemic and remote, non-ischaemic territories. RESULTS: A significant reduction in GAPDH activity was observed in post-ischaemic relative to remote tissue after 3 h reperfusion (4.8 +/- 0.5 vs. 2.9 +/- 0.2 mumol/min/mg protein; P < 0.01). Western blotting revealed no reduction in the levels of GAPDH protein. Analysis of enzyme kinetics showed the loss of activity to be associated with decreased Vmax (5.9 +/- 0.5 vs. 3.2 +/- 0.2 mumol/min/mg protein; P < 0.01) with no significant change in the Km for glyceraldehyde-3-phosphate (GAP). Incubation of the inhibited enzyme under both mild and strong reducing conditions failed to reactivate the enzyme. The acute reduction in enzyme activity in post-ischaemic tissue was accompanied by regional differences in glycolytic intermediates, notably a twofold accumulation of GAP (P < 0.05), and a reduction in the glucose metabolic rate (GMR) determined by positron emission tomography and [18F]2-fluorodeoxyglucose. By 24 h reperfusion, no regional differences in GAPDH activity, reaction Vmax or Km, GAP concentrations or GMR were detectable. CONCLUSIONS: These results suggest that inhibition of GAPDH activity may represent an important point at which glycolysis is limited during reperfusion, and further, that the mechanisms of enzyme inhibition do not involve simple oxidation or S-thiolation of critical active site thiol groups.     

Inactivation of glyceraldehyde-3-phosphate dehydrogenase by ferrylmyoglobin

This article describes the development and psychometric evaluation of self-report measurement instruments in (nursing) research. The aim of this study is to gain more insight into, and understanding of the use of such instruments. To be more specific, this paper deals with: (1) What is a self-report measurement instrument?; (2) How to develop such an instrument?; (3) What is its psychometric quality in terms of validity and reliability?; (4) How to analyze an instrument statistically?; (5) Which are the pros en cons of a self-report measurement instrument in general?; and (6) Where do we find examples of good measurement instruments? These six questions will be answered with the help of several practical research examples. The article concludes with a few suggestions for literature concerning existing measurement instruments and their psychometric qualities.     

The su(Hw) protein insulates expression of the Drosophila melanogaster white gene from chromosomal position-effects

Mutations in the suppressor of Hairy-wing [su(Hw)] locus reverse the phenotype of a number of tissue-specific mutations caused by insertion of a gypsy retrotransposon. The su(Hw) gene encodes a zinc finger protein which binds to a 430 bp region of gypsy shown to be both necessary and sufficient for its mutagenic effects. su(Hw) protein causes mutations by inactivation of enhancer elements only when a su(Hw) binding region is located between these regulatory sequences and a promoter. To understand the molecular basis of enhancer inactivation, we tested the effects of su(Hw) protein on expression of the mini-white gene. We find that su(Hw) protein stabilizes mini-white gene expression from chromosomal position-effects in euchromatic locations by inactivating negative and positive regulatory elements present in flanking DNA. Furthermore, the su(Hw) protein partially protects transposon insertions from the negative effects of heterochromatin. To explain our current results, we propose that su(Hw) protein alters the organization of chromatin by creating a new boundary in a pre-existing domain of higher order chromatin structure. This separates enhancers and silencers distal to the su(Hw) binding region into an independent unit of gene activity, thereby causing their inactivation.     

A study of penetrance of Drosophila melanogaster forked gene

Group I intron endonuclease I-CreI is encoded by an open reading frame contained within a self-splicing intron in the Chlamydomonas reinhardtii chloroplast 23S rRNA gene. I-CreI initiates the lateral transfer or homing of this intron by specifically recognizing and cleaving a pseudopalindromic 19-24 bp homing site in chloroplast 23S rRNA genes that lack the intron. The gene encoding this enzyme has been subcloned, and the protein product has been purified and crystallized. The crystals belong to space group P321, with unit cell dimensions a = b = 78.2 A, c = 67.4 A. The crystal unit cell is consistent with an asymmetric unit consisting of the enzyme monomer. The specific volume of this unit cell is 3.3 A3/Da. The crystals diffract to at least 3.0 A resolution after flash-cooling, when using a rotating anode x-ray source and an RAXIS image plate detector.     

Regulation of a dpp target gene in the Drosophila embryo

EnvZ is an inner membrane protein present in Escherichia coli that is important for osmosensing and required for porin gene regulation. EnvZ is phosphorylated by intracellular ATP, and EnvZ-P phosphorylates OmpR, which then binds to the porin promoters to regulate their expression. An overexpressed, truncated form of the enzyme, EnvZ115, was used to characterize the kinase reaction in vitro. Using a filter binding assay, we report the first direct measurements of the kinase activity, including the apparent affinity for ATP of 200 microM. The phosphorylation reaction is dependent on MgCl2, and the phosphoenzyme has the expected stability of a phosphohistidine; i.e., it is stable in base and less stable in acid at room temperature. The addition of OmpR and ATP to solutions containing EnvZ resulted in an OmpR-stimulated, EnvZ-dependent ATPase activity that was not vanadate-sensitive. The in vivo kinase activity of EnvZ and two mutants that were deficient in porin expression were studied using an immune complex kinase reaction. Interestingly, a mutation located in the periplasmic domain of EnvZ exhibited kinase activity that was identical to that of the wild-type enzyme, while a mutation located close to the phosphorylation site showed a significant decrease in both kinase and phosphotransferase activities. These data provide support for models of EnvZ consisting of separate sensing and kinase domains.     

Mechanism of NADH transfer between alcohol dehydrogenase and glyceraldehyde-3-phosphate dehydrogenase

To investigate the correlation between neural activity and intracellular Ca2+ ([Ca2+]i) mobilization in immature and adult brain during ischemia (hypoxia and glucose deprivation) and deprivation of glucose, hippocampal slices were prepared from 7-, 10-day-old and adult rats. Population spikes (PS) and antidromic responses (AR) were recorded in the pyramidal cell layer of the CA1 area as an index of neural function. [Ca2+]i mobilization of the stratum radiatum in the CA1 area was measured using the fluorescent dye fura-2 AM. The rise in [Ca2+]i occurred earlier in the adult animal and the decay times for the orthodromic PS and antidromic responses were shorter in the adult during ischemia. The field potentials and antidromic responses decreased substantially prior to the elevation of [Ca2+]i in both developing and adult brains. Furthermore, ATP levels decreased substantially before the elevation of [Ca2+]i during ischemia. These results suggest that neural activity and intracellular Ca2+ homeostasis in the immature rats brain are more resistant to energy failure than adult rats and that neuronal activity in the developing and adult brain is impaired initially by energy depletion during ischemia. In the immature animal, during glucose deprivation, the antidromic responses were slowly decayed or even failed to extinguish and [Ca2+]i levels were maintained for a longer period or even failed to rise in spite of the rapid loss of PS. Furthermore, ATP levels were well preserved at the time of PS loss. These results agree well with our previous reports showing that glucose plays an important role in the preservation of synaptic transmission in addition to its major function as an energy substrate.     

Comparative enzymatic properties of GapB-encoded erythrose-4-phosphate dehydrogenase of Escherichia coli and phosphorylating glyceraldehyde-3-phosphate dehydrogenase

GapB-encoded protein of Escherichia coli and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) share more than 40% amino acid identity. Most of the amino acids involved in the binding of cofactor and substrates to GAPDH are conserved in GapB-encoded protein. This enzyme shows an efficient non-phosphorylating erythrose-4-phosphate dehydrogenase activity (Zhao, G., Pease, A. J., Bharani, N., and Winkler, M. E. (1995) J. Bacteriol. 177, 2804-2812) but a low phosphorylating glyceraldehyde-3-phosphate dehydrogenase activity, whereas GAPDH shows a high efficient phosphorylating glyceraldehyde-3-phosphate dehydrogenase activity and a low phosphorylating erythrose-4-phosphate dehydrogenase activity. To identify the structural factors responsible for these differences, comparative kinetic and binding studies have been carried out on both GapB-encoded protein of Escherichia coli and GAPDH of Bacillus stearothermophilus. The KD constant of GapB-encoded protein for NAD is 800-fold higher than that of GAPDH. The chemical mechanism of erythrose 4-phosphate oxidation by GapB-encoded protein is shown to proceed through a two-step mechanism involving covalent intermediates with Cys-149, with rates associated to the acylation and deacylation processes of 280 s-1 and 20 s-1, respectively. No isotopic solvent effect is observed suggesting that the rate-limiting step is not hydrolysis. The rate of oxidation of glyceraldehyde 3-phosphate is 0.12 s-1 and is hydride transfer limiting, at least 2000-fold less efficient compared with that of erythrose 4-phosphate. Thus, it can be concluded that it is only the structure of the substrates that prevails in forming a ternary complex enzyme-NAD-thiohemiacetal productive (or not) for hydride transfer in the acylation step. This conclusion is reinforced by the fact that the rate of oxidation for erythrose 4-phosphate by GAPDH is 0.1 s-1 and is limited by the acylation step, whereas glyceraldehyde 3-phosphate acylation is efficient and is not rate-determining (>/=800 s-1). Substituting Asn for His-176 on GapB-encoded protein, a residue postulated to facilitate hydride transfer as a base catalyst, decreases 40-fold the kcat of glyceraldehyde 3-phosphate oxidation. This suggests that the non-efficient positioning of the C-1 atom of glyceraldehyde 3-phosphate relative to the pyridinium of the cofactor within the ternary complex is responsible for the low catalytic efficiency. No phosphorylating activity on erythrose 4-phosphate with GapB-encoded protein is observed although the Pi site is operative as proven by the oxidative phosphorylation of glyceraldehyde 3-phosphate. Thus the binding of inorganic phosphate to the Pi site likely is not productive for attacking efficiently the thioacyl intermediate formed with erythrose 4-phosphate, whereas a water molecule is an efficient nucleophile for the hydrolysis of the thioacyl intermediate. Compared with glyceraldehyde-3-phosphate dehydrogenase activity, this corresponds to an activation of the deacylation step by >/=4.5 kcal.mol-1. Altogether these results suggest subtle structural differences between the active sites of GAPDH and GapB-encoded protein that could be revealed and/or modulated by the structure of the substrate bound. This also indicates that a protein engineering approach could be used to convert a phosphorylating aldehyde dehydrogenase into an efficient non-phosphorylating one and vice versa.     

Sequence of the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Nicotiana plumbaginifolia and phylogenetic origin of the gene family

A cDNA-library has been constructed from Nicotiana plumbaginifolia seedlings, and the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GapN, EC 1.2.1.9) was isolated by plaque hybridization using the cDNA from pea as a heterologous probe. The cDNA comprises the entire GapN coding region. A putative polyadenylation signal is identified. Phylogenetic analysis based on the deduced amino acid sequences revealed that the GapN gene family represents a separate ancient branch within the aldehyde dehydrogenase superfamily. It can be shown that the GapN gene family and other distinct branches of the superfamily have its phylogenetic origin before the separation of primary life-forms. This further demonstrates that already very early in evolution, a broad diversification of the aldehyde dehydrogenases led to the formation of the superfamily.