A genetic method for generating Drosophila eyes composed exclusively of mitotic clones of a single genotype.
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The genetic analysis of a gene at a late developmental stage can be impeded if the gene is required at an earlier developmental stage. The construction of mosaic animals, particularly in Drosophila, has been a means to overcome this obstacle. However, the phenotypic analysis of mitotic clones is often complicated because standard methods for generating mitotic clones render mosaic tissues that are a composite of both mutant and phenotypically normal cells. We describe here a genetic method (called EGUF/hid) that uses both the GAL4/UAS and FLP/FRT systems to overcome this limitation for the Drosophila eye by producing genetically mosaic flies that are otherwise heterozygous but in which the eye is composed exclusively of cells homozygous for one of the five major chromosome arms. These eyes are nearly wild type in size, morphology, and physiology. Applications of this genetic method include phenotypic analysis of existing mutations and F(1) genetic screens to identify as yet unknown genes involved in the biology of the fly eye. We illustrate the utility of the method by applying it to lethal mutations in the synaptic transmission genes synaptotagmin and syntaxin. (+info)
Loss of Daxx, a promiscuously interacting protein, results in extensive apoptosis in early mouse development.
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The mammalian Daxx gene has been identified in a diverse set of yeast interaction trap experiments. Although a facilitating role for Daxx in Fas-induced apoptosis has been suggested, Daxx's physiologic function remains unknown. To elucidate the in vivo role of Daxx, we have generated Daxx-deficient mice. Surprisingly, rather than a hyperproliferative disorder expected from the loss of a pro-apoptotic gene, mutation of Daxx results in extensive apoptosis and embryonic lethality. These findings argue against a role for Daxx in promoting Fas-induced cell death and suggest that Daxx either directly or indirectly suppresses apoptosis in the early embryo. (+info)
Specialization and targeting of B-type cyclins.
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The B-type cyclins of S. cerevisiae are diversified with respect to time of expression during the cell cycle as well as biological function. We replaced the early-expressed CLB5 coding sequence with the late-expressed CLB2 coding sequence, at the CLB5 locus. CLB5::CLB2 exhibited almost no rescue of clb5-specific replication defects, although it could rescue clb1 clb2 lethality, and in synchronized cells Clb2p-associated kinase activity from CLB5::CLB2 rose early in the cell cycle, similar to that of Clb5p. Mutagenesis of a potential substrate-targeting domain of CLB5 reduced biological activity without reducing Clb5p-associated kinase activity. Thus, Clb5p may have targeting domains required for CLB5-specific biological activity. (+info)
Molecular characterization of the 5' control region and of two lethal alleles affecting the hsp60 gene in Drosophila melanogaster.
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The chaperonins are evolutionarily conserved essential cellular proteins that help folding newly synthesized or translocated proteins, spending ATP. We present here the molecular analysis of the hsp60 gene promoter region and of two Drosophila hsp60 ethyl methane sulfonate embryonic lethal alleles that have an identical phenotype. No heat shock element sequences were found in the 5' region, supporting previous data (Kozlova, T. et al., 1997) which suggests that mitochondrial Drosophila melanogaster HSP60.1 is not heat inducible. By sequencing the lethal allele's entire open reading frame (ORF), we found a C-T transition in the hsp60F409 allele that produces a serine to leucine change, apparently distorting the protein equatorial domain structure. No changes were found in the hsp60G93 ORF. However, an analysis of the heterogeneous nuclear RNA levels showed a reduction of the hsp60 transcript in hsp60G93 flies as compared to the wild-type. These data suggest that although the defects in the hsp60 gene produced by these alleles are at different levels, both behave as null mutations. (+info)
Evidence for a conserved system for iron metabolism in the mitochondria of Saccharomyces cerevisiae.
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nifU of nitrogen-fixing bacteria is involved in the synthesis of the Fe-S cluster of nitrogenase. In a synthetic lethal screen with the mitochondrial heat shock protein (HSP)70, SSQ1, we identified a gene of Saccharomyces cerevisiae, NFU1, which encodes a protein with sequence identity to the C-terminal domain of NifU. Two other yeast genes were found to encode proteins related to the N-terminal domain of bacterial NifU. They have been designated ISU1 and ISU2. Isu1, Isu2, and Nfu1 are located in the mitochondrial matrix. ISU genes of yeast carry out an essential function, because a Deltaisu1Deltaisu2 strain is inviable. Growth of Deltanfu1Delta isu1 cells is significantly compromised, allowing assessment of the physiological roles of Nfu and Isu proteins. Mitochondria from Deltanfu1Deltaisu1 cells have decreased activity of several respiratory enzymes that contain Fe-S clusters. As a result, Deltanfu1Deltaisu1 cells grow poorly on carbon sources requiring respiration. Deltanfu1Deltaisu1 cells also accumulate abnormally high levels of iron in their mitochondria, similar to Deltassq1 cells, indicating a role for these proteins in iron metabolism. We suggest that NFU1 and ISU1 gene products play a role in iron homeostasis, perhaps in assembly, insertion, and/or repair of mitochondrial Fe-S clusters. The conservation of these protein domains in many organisms suggests that this role has been conserved throughout evolution. (+info)
A direct screen identifies new flight muscle mutants on the Drosophila second chromosome.
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An ethyl methanesulfonate mutagenesis of Drosophila melanogaster was undertaken, and >3000 mutagenized second chromosomes were generated. More than 800 homozygous viable lines were established, and adults were screened directly under polarized light for muscle defects. A total of 16 mutant strains in which the indirect flight muscles were reduced in volume or disorganized or were otherwise abnormal were identified. These fell into seven recessive and one semidominant complementation groups. Five of these eight complementation groups, including the semidominant mutation, have been mapped using chromosomal deficiencies and meiotic recombination. Two complementation groups mapped close to the Myosin heavy chain gene, but they are shown to be in different loci. Developmental analysis of three mutations showed that two of these are involved in the early stages of adult myogenesis while the other showed late defects. This is the first report of results from a systematic and direct screen for recessive flight muscle defects. This mutant screen identifies genes affecting the flight muscles, which are distinct from those identified when screening for flightlessness. (+info)
The catecholamines up (Catsup) protein of Drosophila melanogaster functions as a negative regulator of tyrosine hydroxylase activity.
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We report the genetic, phenotypic, and biochemical analyses of Catecholamines up (Catsup), a gene that encodes a negative regulator of tyrosine hydroxylase (TH) activity. Mutations within this locus are semidominant lethals of variable penetrance that result in three broad, overlapping effective lethal phases (ELPs), indicating that the Catsup gene product is essential throughout development. Mutants from each ELP exhibit either cuticle defects or catecholamine-related abnormalities, such as melanotic salivary glands or pseudotumors. Additionally, Catsup mutants have significantly elevated TH activity that may arise from a post-translational modification of the enzyme. The hyperactivation of TH in Catsup mutants results in abnormally high levels of catecholamines, which can account for the lethality, visible phenotypes, and female sterility observed in these mutants. We propose that Catsup is a component of a novel system that downregulates TH activity, making Catsup the fourth locus found within the Dopa decarboxylase (Ddc) gene cluster that functions in catecholamine metabolism. (+info)
In vivo dominant lethal effect of pyrimethamine in male mouse germ cells.
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Pyrimethamine is used for treatment of malaria and toxoplasmosis. The embryotoxicity and clastogenicity of pyrimethamine is known and our aim was to investigate its dominant lethal effect in vivo. For this purpose, we used three groups of Swiss-albino male mice and a control group. We injected males with doses of 16, 32 or 64 mg/kg pyrimethamine and housed them with 10 females/male for each mating interval. Females were sacrificed and their uteri were evaluated for dominant lethality. As a result of this study we found that pyrimethamine induced dominant lethal mutations in the third, fourth and sixth weeks at the 64 mg/kg dose level, without the effect being dose-dependent. We conclude that pyrimethamine is a suspected germ cell mutagen. (+info)