James B. Courtright

Intracellular localization and properties of glycerokinase and glycerophosphate dehydrogenase in Neurospora crassa

Abstract Glycerokinase and glycerol-3-phosphate dehydrogenase activities have been examined in cell extracts obtained from Neurospora crassa after growth in media containing glycerol. The glycerokinase is located in the cytosol and has been partially purified by ion exchange and gel-filtration chromatography. The molecular weight of the enzyme has been estimated by sucrose density centrifugation to be approximately 120,000. No effect of either fructose-1,6-bisphosphate or other sugar phosphates on enzyme activity was observed. The G3P dehydrogenase activity in cell extracts is apparently catalyzed by a flavin-linked enzyme as no dependence for either NAD+ or NADP+ could be demonstrated. The enzyme is located primarily in the mitochondria and is not removed from mitochondrial membranes by treatment with digitonin. Separation of digitonin-treated mitochondria on discontinuous sucrose gradients indicated that the enzyme is located on the mitochondrial inner membrane. The synthesis of both enzymes is under some form of catabolite repression since increased specific activities could only be observed in cells grown on acetate, but not glucose, sucrose, or xylose.

Tissue-specific and substrate-specific detection of aldehyde and pyridoxal oxidase in larval and imaginal tissues of Drosophila melanogaster

The substrate specificities of aldehyde and pyridoxal oxidases in Drosophila melanogaster have been determined with a variety of aliphatic and aromatic aldehydes. This analysis has led to the discovery that 2,4,5-trimethoxy-benzaldehyde is a specific substrate for pyridoxal oxidase, as based on the histochemical distribution of oxidase activity, the absence of enzymatic activity in the lpo1strains, and the dosage dependence on the number of lpo+genes present. The tissue-specific localization of aldehyde oxidase (AO) and pyridoxal oxidase (PO) in the larval and adult structures showed that AO was present in all the major internal organs of the larvae and adults, including brain, imaginal discs, Malpighian tubules, digestive system, and reproductive structures. Pyridoxal oxidase is present in many of the same structures which possess AO, but is missing from the cardia, crop, imaginal discs, ovarian follicle cells, paragonia, pericardial cells, and wreath cells. The only structure which possesses PO but lacks AO is the larval salivary gland. These histochemical differences in AO and PO distribution were also confirmed by enzymatic analysis of the activities present in homogenates of ovaries, paragonia, and salivary glands. The general pattern of enzyme expression appears to be established during embryogenesis and maintained throughout the life of the individual.

Drosophila Gene-Enzyme Systems

Publisher Summary The genetic analysis of morphological mutants of drosophila in the past years has led to a nearly complete catalog of specific genetic sites that control many features of the adult phenotype. An approach to understanding drosophila gene action has relied on studying mutations of genes for specific enzymes that affect subtle secondary characteristics of the enzyme, have little or no effect on enzymatic properties in vivo , and do not impair the viability of the mutant organism. Several different gene-enzyme systems in drosophila have been described and genetically characterized, thereby adding substantially to the understanding of genetic expression in eukaryotic cells. The chapter focuses on the specific properties of individual gene-enzyme systems that have been amenable to analysis and hold promise for additional investigation, and discusses the genetic nomenclature, specific gene-enzyme systems, dosage compensation of gene activity, genetic organization, genetic regulation of enzyme formation, and such. Emphasis on enzyme polymorphisms is given with the view that the results of these comparisons can often provide information on possible interactions of gene systems and products.

Ultrastructural changes induced by juvenile hormone analogue in oöcyte membranes of apterous4Drosophila melanogaster

Abstract Egg chambers from apterous 4 (ap 4 ) , a female sterile mutant of Drosophila melanogaster , show none of the microvilli or pinocytotic vesicles which are a prominent feature of the membrane of the wild-type vitellogenic oocyte. The studies reported here show that a juvenile hormone analogue (ZR515) stimulates formation of microvilli and pinocytotic vesicles in oocytes of ap 4 flies. Within 12 hr after topical application of ZR515 to homozygous ap 4 females the oocyte membranes exhibit extensive microvilli and pinocytotic activity. The follicle-cell surface adjoining the oocyte also shows some changes. In vitro studies in which ap 4 ovaries were incubated in Schneiders Drosophila tissue-culture medium in the presence of ZR515 with or without female haemolymph, or in the absence of ZR515, showed that the analogue acts alone directly on the ovary to cause formation of microvilli and pinocytotic vesicles on the oocyte membrane.

Differential rates of synthesis of glycerokinase and glycerophosphate dehydrogenase in Neurospora crassa during induction

Abstract The synthesis of the enzymes of the glycerophosphate pathway in Neurospora has been examined during exponential growth of cells on acetate as the sole carbon source. After the addition of glycerol to the media, increases in the levels of both glycerokinase and a mitochondrial glycerol-3-phosphate dehydrogenase are observed within 1 h and fully induced levels are reached within one and a half mass doublings for glycerokinase and two and a half mass doublings for glycerol-3-phosphate dehydrogenase. The increase in glycerokinase activity represents de novo synthesis of enzyme as evidenced by the absence of immunologically related protein in uninduced cell extracts. The synthesis of both glycerokinase and glycerol-3-phosphate dehydrogenase can be totally inhibited by treatment of cells with 20 μg/ml cycloheximide. During incubation with 4 mg/ml chloramphenicol, there is normal synthesis of glycerokinase but a 30–50% inhibition of mitochondrial glycerol-3-phosphate dehydrogenase synthesis. However, under these conditions, in the cytosol fraction there is a significant increase in glycerol-3-phosphate dehydrogenase specific activity, suggesting that precursors are synthesized and accumulated in the cytosol prior to incorporation into mitochondria. Upon removal of chloramphenicol, the rate of appearance of glycerol-3-phosphate dehydrogenase into the mitochondria is up to four times greater than observed in untreated controls. It is concluded that both glycerokinase and glycerol-3-phosphate dehydrogenase are synthesized on cytoplasmic ribosomes, but that final assembly of glycerol-3-phosphate dehydrogenase into mitochondria is dependent on concomitant synthesis of mitochondrial inner membrane.

Evidence for a new type of complementation among the cin, lxd and ma-l loci in Drosophila melanogaster

SummaryA phenotypic effect of the lxd locus in the expression of the cin and ma-l gene products has been described. Flies which are genotypically lxd have normal eye pigments, but maternally affected cin; lxd or ma-l; lxd flies are characterized by mutant eyes which are indistinguishable from those observed in ma-l or ry mutant strains. Furthermore, under conditions where there is only partial complementation at the ma-l locus, the presence of the lxd gene is sufficient to prevent normal eye pigmentation. The possibility that these post translational interactions of the cin, lxd, and ma-l loci may prove useful in the isolation of additional loci affecting XDH synthesis is discussed.

Requirement for specific carbon sources in the low temperature induction of glycerol kinase in Neurospora crassa

The synthesis of glycerol kinase (ATP:glycerol phosphotransferase), an inducible enzyme in Neurospora crassq, has recently been shown to occur at low temperatures during growth on sucrose media [ 1 ] The cause of this low temperature synthesis is not known, but it may be the result of unique themal dependent properties of the glycerol repressor(s) in this organism. Alternatively, the increase in activity may be the result of metabolically generated inducer(s) at depressed temperatures. Since previous work on glycerol kinase induction has indicated that at normal growth temperatures (20-35°C) glycerol is the only known effector for this system [2], these studies have been undertaken to elucidate further the mechanism involved in induction. The results indicate that the induction of GK at low temperatures is highly specific, and is dependent on the carbon source used during the low temperature incubation. It was also found that low temperatures do not cause induction of a second enzyme involved in glycerol utilization, glycerol-3phosphate (G3P) dehydrogenase (glycerol (acceptor) oxidoreductase), with normally is synthesized after growth in glycerol-containing media [2] .

A genetic engineering methodology for insect pest control: female sterilizing genes

We propose a novel method to control insect pest populations by integrating into the genome of the target species a gene fusion that serves as a dominant female sterilizing (FS) gene. Based on the developmental regulation, regulatory sequences from vitellogenin and/or chorion protein genes have been identified as suitable for construction of FS gene fusions. These promoters, which respond to tissue- and stage-specific developmental signals, can be linked to a variety of structural gene elements such as those of a scrambled chorion protein, a signal sequence-deleted neutral protease, a phospholipase, an altered tubulin or a toxin. Such a FS gene fusion, because of its tissue and developmental stage specificity, could be expected to be expressed only in adult ovaries with resulting disruption of ovarian functions. In order to introduce the FS gene into the genome of the target species we suggest placing it at a neomycin resistance or other appropriate selectable genetic marker between the termini of a Drosophila transposing P-element and transforming the germline cells in embryo. Males carrying the S gene will transmit it to their progeny which would be all female sterile. Theoretical calculations suggest that target insect populations will be reduced substantially in two to four generations if the ratio of FS males to endemic males is maintained 10:1. It is also theoretically possible to construct a FS gene which would undergo replicative transposition and thus maintain its self in the population in a high copy number thus causing continued decline in the number of pests. The current genetic engineering method allows adapting these principles to control a wide variety of insect pest species.

Induction of enzymes of the glycerophosphate pathway in leu-5 mutants of Neurospora crassa.

The inducible cytosolic glycerokinase and mitochondrial glycerol-3-phosphate2 dehydrogenase have been examined during the glycerol-specific induction in Neurospora crassa. Although both the fully induced levels and the respective rates of synthesis of these two enzymes were less than observed with wild-type cells, there were no major differences in the relative rates of induction of the glycerol-3-phosphate dehydrogenase at either permissive or restrictive temperatures. These results indicate that the processes involved in the assembly of this enzyme into the mitochondrial inner membrane are normal in a mutant lacking the mitochondrial leucyl tRNA synthetase and suggest that the functions of the mitochondrial synthetase may be replaced by those of the cytosolic leucyl tRNA synthetase.