Victoria Finnerty

Comparison of the sequences of the Aspergillus nidulans hxB and Drosophila melanogaster ma-l genes with nifS from Azotobacter vinelandii suggests a mechanism for the insertion of the terminal sulphur atom in the molybdopterin cofactor

The molybdopterin cofactor (MoCF) is required for the activity of a variety of oxidoreductases. The xanthine oxidase class of molybdoenzymes requires the MoCF to have a terminal, cyanolysable sulphur ligand. In the sulphite oxidase/nitrate reductase class, an oxygen is present in the same position. Mutations in both the ma‐l gene of Drosophila melanogaster and the hxB gene of Aspergillus nidulans result in loss of activities of all molybdoenzymes that necessitate a cyanolysable sulphur in the active centre. The ma‐l and hxB genes encode highly similar proteins containing domains common to pyridoxal phosphate‐dependent cysteine transulphurases, including the cofactor binding site and a conserved cysteine, which is the putative sulphur donor. Key similarities were found with NifS, the enzyme involved in the generation of the iron–sulphur centres in nitrogenase. These similarities suggest an analogous mechanism for the generation of the terminal molybdenum‐bound sulphur ligand. We have identified putative homologues of these genes in a variety of organisms, including humans. The human homologue is located in chromosome 18.q12.

Ribosomal RNA genes of the Anopheles gambiae species complex.

The Anopheles gambiae complex is a group of six sibling species of Afro-tropical anophelines. The two most widely distributed species Anopheles gambiae and An. arabiensis are among the principal vectors of malaria in Africa. An. melas, An. merus, and An. bwambae, are vectors of limited regional importance, whereas the highly zoophilic An. Quadri-annulatus is not believed to contribute significantly to the transmission of malaria (25, 63, 71, 72). Although some species-specific differences have been described in the distributions of morphometric criteria (15, 20), no morphological taxonomic characters have been observed that permit the unequivocal identification of the species of individual adult or larval specimens. Because the habitats of these six species overlap considerably, both on geographical and ecological scales, two or more members of the complex are often present in the same adult or larval collections.

The use of genetic complementation in the study of eukaryotic macromolecular evolution: Rate of spontaneous gene duplication at two loci ofDrosophila melanogaster

SummaryGene duplications must play an important role in the evolutionary development of living organisms. Presented here is a general scheme that uses complementary alleles to isolate gene duplications in diploid organisms. The technique was used inDrosophila melanogaster to assess the rate of spontaneous gene duplication at two loci, maroon-like and rosy. The results indicate (1) that the rate of duplication of the maroon-like locus is on the order of 2.7×10−6; (2) that the rate of duplication of the rosy locus is approximately 1.7×10−4; and (3) that duplication occurs in males, suggesting that there may actually be two modes of gene duplication inDrosophila melanogaster.

Molybdenum hydroxylases in Drosophila

SummaryThe molybdenum hydroxylases are a ubiquitous class of enzymes which contain molybdenum in association with a low molecular weight cofactor. Genetic evidence suggests that the Drosophila loci, ma-1, cin and lxd are concerned with this cofactor because mutants for any one of these loci simultaneously interrupt activity for two molybdenum hydroxylases, XDH and A0. A third enzyme activity, P0, is also absent in each of the three mutants but evidence classifying P0 as a molybdoenzyme has been lacking. This study utilizes the known tungsten sensitivity of molybdoenzymes to demonstrate directly that pyridoxal oxidase is also a molybdoenzyme. The low molecular weight molybdenum cofactor is found to be severely reduced in extracts of the lxd and cin mutants but ma-1 mutants have high levels of cofactor. A partially purified preparation of XDH crossreacting material from ma-1 was also shown to contain the molybdenum cofactor. These results, considered with data from other workers are taken to indicate that the functions of all three of the loci examined could be concerned with some aspect of cofactor biosynthesis.

Gene expression in Drosophila: post-translational modification of aldehyde oxidase and xanthine dehydrogenase.

SummaryMaroon-like homozygotes are completely deficient for xanthine dehydrogenase (XDH) and aldehyde oxidase (AO), however ma-l is not a structural locus for either enzyme. Quantitative immunoelectrophoresis of ma-l and wild type extracts suggests that the ma-l function must be post-translational. To determine whether the ma-l function involves some direct physical changes in XDH and/or AO the enyzmes were characterized with respect to temperature sensitivity and behavior in gel sieving electrophoresis. Since the XDH and AO from complementary ma-l heterozygotes is more thermolabile and different in shape from wild type XDH and AO, we conclude that ma-l is involved in a post-translational modification of these enzymes.

Molecular Perspectives on the Genetics of Mosquitoes

Publisher Summary This chapter presents molecular perspectives on the genetics of mosquitoes. The chapter reviews various recent molecular studies performed on mosquitoes of the genera Anopheles, Culex, and Aedes. These genera include the principal vectors of three major groups of human pathogens: malaria parasites of the genus Plasmodium, filarial worms of the genera Wuchereria and Brugia, and numerous arboviruses. Anophelines are the only mosquitoes known to transmit human malaria parasites, a group of organisms that may be responsible for more morbidity and mortality worldwide than any other human pathogen. Anophelines also transmit filarial worms as do Culex and Aedes species. The chapter also discusses how molecular techniques are used to reveal genetic differentiation, both for the identification of cryptic species and for the understanding of population structure and evolution. The chapter describes the genome organization on the basis of reassociation kinetics, molecular cloning, and genome mapping. The chapter discusses the molecular biology of specific physiological systems, including insecticide resistance, immune mechanisms, oogenesis, and salivation. The identification of endogenous mobile elements and potential transformation systems for both cell lines and embryos are also described in the chapter.

Molybdenum hydroxylases in Drosophila. III. Further characterization of the low xanthine dehydrogenase gene

The biochemical effects of several newly induced low xanthine dehydrogenase (lxd) mutations in Drosophila melanogaster were investigated. When homozygous, all lxd alleles simultaneously interrupt each of the molybdoenzyme activities to approximately the same levels: xanthine dehydrogenase, 25%; aldehyde oxidase, 12%; pyridoxal oxidase, 0%; and sulfite oxidase, 2% as compared to the wild type. In order to evaluate potentially small complementation or dosage effects, mutant stains were made coisogenic for 3R. These enzymes require a molybdenum cofactor, and lxd cofactor levels are also reduced to less than 10% of the wild type. These low levels of molybdoenzyme activities and cofactor activity are maintained throughout development from late larval to adult stages. The lxd alleles exhibit a dosage-dependent effect on molybdoenzyme activities, indicating that these mutants are leaky for wild-type function. In addition, cofactor activity is dependent upon the number of lxd+ genes present. The lxd mutation results in the production of more thermolabile XDH and AO enzyme activities, but this thermolability is not transferred with the cofactor to a reconstituted Neurospora molybdoenzyme. The lxd gene is localized to salivary region 68 A4-9, 0.1 map unit distal to the superoxide dismutase (Sod) gene.

Molybdoenzymes in Drosophila. IV. Further characterization of the cinnamon phenotype

SummaryMutations at the cin gene display drastically lowered levels of the molybdoenzymes, xanthine dehydrogenase (XDH) and aldehyde oxidase (AO), and lack pyridoxal oxidase (PO) and sulfite oxidase (SO) activities. Certain mutations at cin also display varying degrees of female sterility, which is maternally affected. Here we characterize five new cin alleles with respect to the molybdoenzyme activities as well as the molybdenum cofactor, commonly required for molybdoenzyme activity. In complementing cin heterozygotes we find that, in addition to the previously reported unusually high levels of XDH and AO activities, there are unusually elevated levels of SO activity, as well as complementation for PO activity. The levels of immunologically crossreacting material in such heterozygotes indicate that the elevated levels of molybdoenzyme activities cannot be due to increases in the number of enzyme molecules. Measurements of the level of molybdenum cofactor activity normally present in XDH, AO, PO, and SO point to the possibility that a larger fraction of the enzyme molecules are active in these heterozygotes. The possible role of SO with respect to cinnamons female sterility is also discussed.

Symposium: New Technologies for the Taxonomic Identification of Arthropods: Ribosomal DNA Probes for Identification of Member Species of the Anopheles Gambiae Complex

Many insects belong to species complexes wherein member species are morphologically indistinguishable. Yet, in numerous cases, both economic and medical exigencies require that the species of such individuals be reliably determined. The specific problem addressed here concerns the Anopheles gambiae mosquito complex which includes An. gambiae and An. arabiensis, currently the two major African malaria vectors. The present work uses a ribosomal DNA gene probe to differentiate member species of this complex. The method is shown to be extremely useful and sensitive because it can easily test just portions of a single dried adult. The rationale for using ribosomal DNA clones to provide such diagnostic probes to distinguish among other morphologically identical insect species is also discussed.

Cloning of a Eukaryotic Molybdenum Cofactor Gene

Molybdenum requiring enzymes have been identified in virtually every species. All molybdoenzymes, with the single exception of nitrogenase, require a molybdopterin cofactor for catalytic activity. Mutations leading to a simultaneous loss of all molybdoenzyme activities have been identified in organisms including, E.coli (1), higher plants such as Nicotiana and Arabidopsis (2,3), as well as Drosophila (4,5), and humans (6). Such mutations identify genes involved in the synthesis and/or activiation of the cofactor. Much of the current interest in MoCF genes stems from the crucial roles of the molybdoenzymes. One example is nitrate reductase,which is the key enzyme in nitrogen assimilation by plants. At least 90% of the total nitrogen assimilated by plants is from the mineral nitrogen, mostly in the form of nitrate (7). A mitochondrial molybdoenzyme, sulfite oxidase (SO) catalyzes the terminal step in the oxidative degradation of sulfur containing amino acids and is responsible for the detoxification of sulfite (8). Complete lack of sulfite oxidase activity leads to a buildup of toxic sulfites which (perhaps combined with a deficiency in sulphates) results in a severely debilitating human genetic defect involving abnormal neurological development.