Differential expression and isozymic composition of sn-glycerol-3-phosphate dehydrogenase in tissues from variant lines of Drosophila melanogaster.

The tissue-specific expression and isozymic composition of Drosophila sn-glycerol-3-phosphate dehydrogenase (GPDH) (EC 1.1.1.8) have been determined for a high-activity control line and two variant lines that alter either the temporal or systemic expression of GPDH through a reduction in rates of polypeptide synthesis. The temporal variant exhibits a reduction in enzyme levels in all larval tissues and in the adult abdomen, while levels of activity in the adult thorax are equal to the control line. Isozymic analyses of these tissues demonstrate that it is the GPDH-3 species that is reduced in a temporal and tissue-specific manner. In contrast, the systemic variant demonstrates a uniform reduction of all isozymic species in each tissue and developmental stage. Analyses of the tissues of F1 hybrid offspring of each variant line and appropriately marked electrophoretic variants demonstrate that the tissue-specific effects observed are due to cis-acting elements that are tightly linked to the structural gene.

Gene duplication and concerted evolution of the GPDH locus in natural populations of Drosophila melanogaster.

The sn-glycerol-3-phosphate dehydrogenase (GPDH, EC 1, 1, 1, 8) locus of Drosophila melanogaster is polymorphic with respect to the number of tandemly duplicated genes in natural populations. The duplicated genes were cloned and the nucleotide sequences were determined. The duplication deletes both the first and second exons and has a size of 4500 b.p. The fact that there is no sequence variation at the junction point of the duplicated units among strains suggests a single origin for the duplication event. Comparison of the nucleotide sequences among the duplicates indicates that the frequent transfer of genetic information occurs from one to the other of the duplicates on the same chromosome either by gene conversion or by unequal crossing over. Because the GPDH duplication is partial and therefore a kind of pseudogene, the observed polymorphism of the number of tandemly duplicated GPDH genes appears to have been driven mainly by random genetic drift.

Sequence, structure and evolution of the gene coding for sn-glycerol-3-phosphate dehydrogenase in Drosophila melanogaster.

We present the complete nucleotide and deduced amino acid sequence for the gene encoding Drosophila sn-glycerol-3-phosphate dehydrogenase. A transcription unit of 5kb was identified which is composed of eight protein encoding exons. Three classes of transcripts were shown to differ only in the 3'-end and to code for three protein isoforms each with a different C-terminal amino acid sequence. Each transcript is shown to arise through the differential expression of three isotype-specific exons at the 3'-end of the gene by a developmentally regulated process of 3'-end formation and alternate splicing pathways of the pre-mRNA. In contrast, the 5'-end of the gene is simple in structure and each mRNA is transcribed from the same promoter sequence. A comparison of the organization of the Drosophila and murine genes and the primary amino acid sequence between a total of four species indicates that the GPDH gene-enzyme system is highly conserved and is evolving slowly.

The genetics of catalase in Drosophila melanogaster: isolation and characterization of acatalasemic mutants.

Activated oxygen species have been demonstrated to be the important agents in oxygen toxicity by disrupting the structural and functional integrity of cells through lipid peroxidation events, DNA damage and protein inactivation. The biological consequences of free radical damage have long been hypothesized to be a causal agent in many aging-related diseases. Catalase (H2O2:H2O2 oxidoreductase; EC 1.15.1.1) is one of several enzymes involved in the scavenging of oxygen free radicals and free radical derivatives. The structural gene for catalase in Drosophila melanogaster has been localized to region 75D1-76A on chromosome 3L by dosage responses to segmental aneuploidy. This study reports the isolation of a stable deficiency, Df(3L)CatDH104(75C1-2;75F1), that uncovers the catalase locus and the subsequent isolation of six acatalasemic mutants. All catalase mutants are viable under standard culture conditions and recessive lethal mutations within the 75Cl-F1 interval have been shown not to affect catalase activity. Two catalase mutations are amorphic while four are hypomorphic alleles of the Cat+ locus. The lack of intergenic complementation between the six catalase mutations strongly suggests that there is only one functional gene in Drosophila. One acatalesemic mutation was mapped to position 3-47.0 which resides within the catalase dosage sensitive region. While complete loss of catalase activity confers a severe viability effect, residual levels are sufficient to restore viability to wild type levels. These results suggest a threshold effect for viability and offer an explanation for the general lack of phenotypic effects associated with the known mammalian acatalasemics.

Drosophila sn-glycerol-3-phosphate dehydrogenase isozymes are generated by alternate pathways of RNA processing resulting in different carboxyl-terminal amino acid sequences.

Glycerol-3-phosphate dehydrogenase (GPDH, Ec 1.1.1.8) in Drosophila melanogaster consists of a family of three isozymes designated as GPDH-1, 2, and 3 which exhibit a unique temporal and tissue-specific pattern of expression. While each isozyme is encoded by the same structural gene, they differ by the amino acid sequence at the COOH-terminal end, with GPDH-3 having the sequence Asn-His-Pro-Glu-His-Met-COOH and with GPDH-1 extended by the three amino acid sequence Glu-Asn-Leu-COOH. We have isolated both genomic and cDNA clones in order to examine the structure of the 3'-end of this gene and its transcriptional products. This analysis has demonstrated three classes of transcripts, each differing in the 3'-untranslated region and coding for an enzyme with a different COOH-terminal amino acid sequence. Each transcript is shown to arise through the differential expression of three isotype-specific exons at the 3'-end of the gene. We propose a model where the expression of each isotype-specific transcript is controlled through a developmentally regulated process of 3'-end formation and alternate splicing pathways of the pre-mRNA. Furthermore, since each transcript and its cognant isozyme is tissue-specific in expression, this model suggests a role for tissue-specific trans-acting factors in these processing events.

Isolation of a cDNA clone for murine catalase and analysis of an acatalasemic mutant.

We have investigated the genetic control of murine catalase expression by analyzing catalase transcription and translation products from the tissues of control (Csa) and acatalasemic (Csb) mouse strains. Csb animals possess nearly normal catalase enzyme activity levels in liver, while displaying approximately 20 and 1% of normal activity levels in kidney and red blood cells, respectively. Immunoblot analyses of catalase in these tissues have revealed reduced levels of immunologically reactive catalase protein in Csb kidney and red blood cells, paralleling the reduction of catalase enzyme activity in these tissues. In order to determine the molecular basis for Csb acatalasemia, we have isolated a cDNA clone for murine catalase and have used this probe to analyze Csa and Csb genomic DNA and catalase mRNA. These studies have revealed: 1) no restriction fragment length polymorphisms between Csa and Csb genomic DNAs; 2) no differences in the levels of Csa and Csb catalase mRNA within a single tissue; and 3) no differences in the sizes of Csa and Csb catalase mRNAs. These observations suggest that the genetic defect that produces the tissue-specific reduction of catalase expression in Csb mice is not due to a marked rearrangement of DNA within the Csb catalase structural gene. Furthermore, the Csb mutation does not act at the level of gene transcription or mRNA stability, but rather at the level of mRNA translation and/or catalase protein turnover.

Isolation of a genomic clone for Drosophila sn-glycerol-3-phosphate dehydrogenase using synthetic oligonucleotides.

A genomic clone containing Drosophila sn-glycerol-3-phosphate dehydrogenase sequences has been isolated using a mixture of synthetic tridecanucleotides as a hybridization probe. The clone as well as the synthetic probe mixture was found to hybridize to an abundant poly(A)+ RNA of 1700 bases. A partial DNA sequence obtained for a 40-amino acid region containing the oligonucleotide hybridization site was found to agree with the known Drosophila protein sequence data for this region of the protein. In situ hybridization of this clone to the polytene chromosomes of wild type flies and flies bearing chromosomal aberrations that delimit the Gpdh+ locus have allowed us to decisively place the gene in the distal region of 26A on the left arm of the second chromosome.

Temporal variation for the expression of catalase in Drosophila melanogaster: correlations between rates of enzyme synthesis and levels of translatable catalase-messenger RNA.

Two variants that alter the temporal expression of catalase have been isolated from a set of third chromosome substitution lines. Each variant has been mapped to a cytogenetic interval flanked by the visible markers st (3-44.0) and cu (3-50.0) at a map position of 47.0, which is within or near the interval 75D-76A previously identified as containing the catalase structural gene on the bases of dosage responses to segmental aneuploidy. Each variant operates by modulating the rate of enzyme synthesis and the level of translatable catalase-mRNA.

RNA directed synthesis of catalytically active Drosophila sn-glycerol-3-phosphate dehydrogenase in Xenopus oocytes.

Xenopus laevis oocytes injected with poly(A)+ RNA isolated from Drosophila melanogaster direct the synthesis of catalytically active glycerol-3-phosphate dehydrogenase (NAD+) (EC 1.1.1.8). The de novo synthesized enzyme reflects the electrophoretic properties appropriate to the stock of flies from which the injected RNA was isolated and is electrophoretically distinct from endogenous Xenopus activity. Immunoprecipitation of 35S-labeled translation products has demonstrated two immunologically related proteins with molecular masses of 32- and 34kDa which are encoded by two separate mRNA molecules. The 32-kDa protein is identical in size and charge properties to the protein purified from the fly and possesses the catalytic activity observed in the Xenopus translational assay. Poly(A)+ RNA isolated from a strain of flies bearing a CRM- null mutation at the GPDH locus does not contain translatable RNA for the 32-kDa protein. These results suggest that the two immunologically related proteins are the translational products of two separate transcripts derived from either two related loci or from differential transcription and/or processing of the same genetic locus.

Genetic variation affecting the expression of catalase in Drosophila melanogaster: correlations with rates of enzyme synthesis and degradation.

Both second and third chromosome substitution lines isolated from natural populations of Drosophila melanogaster affect the expression of catalase (EC 1.11.1.6) at both the larval and adult stages of development. In each case, the level of catalase activity is strongly related to the level of catalase-specific cross-reacting material. Turnover studies employing the catalase inhibitor 3-amino-1,2,4-triazole were conducted on a selected number of lines. Although the variation in steady state levels of catalase protein was highly significant among lines, variation in intracellular degradation rate was not. These results suggest that the different steady state levels observed among lines largely reflect different rates of catalase synthesis.

Purification and characterization of the naturally occurring allelic variants of sn-glycerol-3-phosphate dehydrogenase in Drosophila melanogaster.

The naturally occurring electrophoretic variants of sn-glycerol-3-phosphate dehydrogenase and a heterodimeric form of the enzyme resulting from a genetic cross of two variant strains of Drosophila were purified to homogeneity by a combination of DEAE-cellulose chromatography and 8-(6-aminohexyl)-amino-ATP-Sepharose affinity chromatography. Each purified protein was compared with respect to a number of physicochemical and kinetic properties. All forms of the enzyme were found to be similar, except for pI differences associated with the electrophoretic variation observed.

Naturally occurring genetic variation affecting the expression of sn-glycerol-3-phosphate dehydrogenase in Drosophila melanogaster.

Genetic variation among second and third chromosomes from natural populations of Drosophila melanogaster affects the activity level of sn-glycerol-3-phosphate dehydrogenase (EC 1.1.1.8; GPDH) at both the larval and the adult stages. The genetic effects, represented by differences among chromosome substitution lines with coisogenic backgrounds, are very repeatable over time and are generally substantially larger than environmental and measurement error effects. Neither the GPDH allozyme, the geographic origin, nor the karyotype of the chromosome contributes significantly to GPDH activity variation. The strong relationship between GPDH activity level and GPDH-specific CRM level, as well as our failure to find any thermostability variation among the lines, indicates that most, if not all, of the activity variation is due to variation in the steady-state quantity of enzyme rather than in its catalytic properties. The lack of a strong relationship between adult and larval activity levels suggests the importance of stage- or isozyme-specific effects.

Genetic determination of sn-glycerol-3-phosphate dehydrogenase synthesis in Drosophila melanogaster. A cis-acting controlling element.

A variant that uniformly reduces the activity of larval and adult isozymes of sn-glycerol-3-phosphate dehydrogenase (EC 1.1.1.8) throughout development has been isolated and analyzed. This low activity line (BI 114), which bears the fast electrophoretic structural allele, has been analyzed in comparison with two high activity control lines, WGM 74 (fast electrophoretic variant) and RI09 (slow electrophoretic variant). The enzyme has been purified from each line and all three variants have similar kinetic, immunological, and physicochemical parameters, except for pI differences associated with the electrophoretic differences observed. Enzyme-specific cross-reacting material analysis corroborates the activity data indicating differential rates of enzyme accumulation between lines throughout development. Protein turnover studies indicate that the rate of intracellular degradation between lines is the same, and that the activity and enzyme-specific cross-reacting material differences observed are due to differential rates of synthesis of the enzyme. Genetic analysis indicates that the variation in the rate of glycerol phosphate dehydrogenase synthesis segregates as a single gene with additive inheritance and is closely linked to the structural gene. Zymogram analysis of F1 heterozygotes has indicated that the variant acts as a cis-regulator and affects both isozymes equally throughout development. We have therefore designated this variant as a systemic regulator controlling the rate of glycerol phosphate dehydrogenase synthesis.

Structural analysis of adult and larval isozymes of sn-glycerol-3-phosphate dehydrogenase of Drosophila melanogaster.

Compositional analysis of the soluble tryptic peptides representing about 70% of the 293 residues of sn-glycerol-3-phosphate dehydrogenase in Drosophila melanogaster reveals a single peptide difference between the sn-glycerol-3-phosphate dehydrogenase adult (GPDHF-1) and larval (GPDHF-3) isozymes. This peptide was shown to be the carboxyl terminus by sequence determination and by carboxypeptidase A digestion of the native protein. For GPDHF-1, the sequence of the COOH-terminal tryptic peptide is Asn-His-Pro-Glu-His-Met-Gln-Asn-Leu-COOH, while that of GPDHF-3 is Asn-His-Pro-Glu-His-Met-COOH.

Analyses of genetic variants of L-glycerol-3-phosphate dehydrogenase in Drosophila melanogaster by two-dimensional gel electrophoresis and immunoelectrophoresis.

A protein spot corresponding to L-glycerol-3-phosphate dehydrogenase (alpha-GPDH, E.C. 1.1.1.8, NAD+ oxidoreductase) has been identified on a two-dimensional gel (isoelectric focusing-SDS gel) containing up to 150 stained protein spots from a crude Drosophila homogenate. Preliminary identification of the alpha-GPDH in crude fly homogenates prior to electrophoresis and observing an intensity enhancement of the corresponding protein spot on the gels. When three purified electrophoretic variants (slow, fast, and ultrafast) were mixed and analyzed by two-dimensional gel electrophoresis, horizontal displacements of the three protein spots were observed. Immunoprecipitation of the enzyme prior to electrophoresis and gene mapping further confirmed the identity of the alpha GPDH protein spot. The alpha-GPDH spot can also be detected by autoradiography of a two-dimensional gel from a single fly extract, where it has been estimated to constitute 0.5-1% of the total soluble protein. Mutants which express no apparent alpha-GPDH activity were analyzed by two-dimensional gels and immunoelectrophoresis in an attempt to identify and characterize the inactive proteins. It is suggested that these techniques provide a powerful tool, for the analysis of CRM+-null activity mutants of a specific gene-enzyme system.