High level expression of isocitrate lyase gene of n-alkane-utilizing yeast Candida tropicalis in Sacchromyces cerevisiae

The genomic DNA of peroxisomal isocitrate lyase (ICL) isolated from an n-alkane-assimilating yeast, Candida tropicalis, was truncated to utilize the original open reading frame under the control of the GAL7 promoter and was expressed in Saccharomyces cerevisiae. The recombinant ICL was synthesized as a functionally active enzyme with a specific activity similar to the enzyme purified from C. tropicalis, and was accounted for approximately 30% of the total extractable proteins in the yeast cells. This recombinant enzyme was easily purified to homogeneity. N-Terminal amino acid sequence, molecular masses of native form and subunit, amino acid composition, peptide maps, and kinetic parameters of the recombinant ICL were essentially the same as those of ICL purified from C. tropicalis. From these facts, S. cerevisiae was suggested to be an excellent microorganism to highly express the genes encoding peroxisomal proteins of C. tropicalis.Abbreviations ICL isocitrate lyase - SDS-PAGE sodium dodecylsulfate-polyacrylamide gel electrophoresis     

The Drosophila TRPA channel, Painless, regulates sexual receptivity in virgin females

Transient receptor potential (TRP) channels play crucial roles in sensory perception. Expression of the Drosophila painless ( pain ) gene, a homolog of the mammalian TRPA1/ANKTM1 gene, in the peripheral nervous system is required for avoidance behavior of noxious heat or wasabi. In this study, we report a novel role of the Pain TRP channel expressed in the nervous system in the sexual receptivity in Drosophila virgin females. Compared with wild-type females, pain mutant females copulated with wild-type males significantly earlier. Wild-type males showed comparable courtship latency and courtship index toward wild-type and pain mutant females. Therefore, the early copulation observed in wild-type male and pain mutant female pairs is the result of enhanced sexual receptivity in pain mutant females. Involvement of pain in enhanced female sexual receptivity was confirmed by rescue experiments in which expression of a pain transgene in a pain mutant background restored the female sexual receptivity to the wild-type level. Targeted expression of pain RNA interference (RNAi) in putative cholinergic or GABAergic neurons phenocopied the mutant phenotype of pain females. However, target expression of pain RNAi in dopaminergic neurons did not affect female sexual receptivity. In addition, conditional suppression of neurotransmission in putative GABAergic neurons resulted in a similar enhanced sexual receptivity. Our results suggest that Pain TRP channels expressed in cholinergic and/or GABAergic neurons are involved in female sexual receptivity.     

Neuroblast ablation in Drosophila P[GAL4] lines reveals origins of olfactory interneurons

Hydroxyurea (HU) treatment of early first instar larvae in Drosophila was previously shown to ablate a single dividing lateral neuroblast (LNb) in the brain. Early larval HU application to P[GAL4] strains that label specific neuron types enabled us to identify the origins of the two major classes of interneurons in the olfactory system. HU treatment resulted in the loss of antennal lobe local interneurons and of a subset of relay interneurons (RI), elements usually projecting to the calyx and the lateral protocerebrum (LPR). Other RI were resistant to HU and still projected to the LPR. However, they formed no collaterals in the calyx region (which was also ablated), suggesting that their survival does not depend on targets in the calyx. Hence, the ablated interneurons were derived from the LNb, whereas the HU-resistant elements originated from neuroblasts which begin to divide later in larval life. Developmental GAL4 expression patterns suggested that differentiated RI are present at the larval stage already and may be retained through metamorphosis. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 443–456, 1997     

GAL4 enhancer traps that can be used to drive gene expression in developing Drosophila spermatocytes

The Drosophila testis has proven to be a valuable model organ for investigation of germline stem cell (GSC) maintenance and differentiation as well as elucidation of the genetic programs that regulate differentiation of daughter spermatogonia. Development of germ cell specific GAL4 driver transgenes has facilitated investigation of gene function in GSCs and spermatogonia but specific GAL4 tools are not available for analysis of postmitotic spermatogonial differentiation into spermatocytes. We have screened publically available pGT1 strains, a GAL4‐encoding gene trap collection, to identify lines that can drive gene expression in late spermatogonia and early spermatocytes. While we were unable to identify any germline‐specific drivers, we did identify an insertion in the chiffon locus, which drove expression specifically in early spermatocytes within the germline along with the somatic cyst cells of the testis. genesis 50:914–920, 2012. © 2012 Wiley Periodicals, Inc.     

Galactose induction in yeast involves association of Gal80p with Gal1p or Gal3p

Gal1p carries out two functions in the galactose pathway of yeast. It activates Gal4p by interacting with Gal80p – a function that can also served by Gal3p – and it catalyzes the formation of galactose-1-phosphate. Recently, we and others have presented biochemical evidence for complex formation between Gal1p and Gal80p. Here, we extend these data and present genetic evidence for an interaction between Gal1p and Gal80p in vivo, using a two-hybrid assay. Interaction between Gal1p and Gal80p depends on the presence of galactose, but not on the catalytic activity of Gal1p. A new class of Kluyveromyces lactis mutants was isolated, designated Klgal1-m, which have lost the derepressing activity but retain galactokinase activity, indicating that the two Gal1p activities are functionally independent. The KlGal1-m proteins are defective in their ability to interact with Gal80p in a two-hybrid assay. The locations of gal1-m mutations identify putative interaction sites in Gal1p and Gal80p. A dominant mutation, KlGAL1-d, leads to a high level of constitutive expression of genes of the galactose pathway. The behavior of chimeric proteins consisting of Gal3p and KlGal1p sequences indicates that both the N-terminal and C-terminal halves of KlGal1p are involved in specific interaction with KlGal80p. Received: 12 November 1998 / Accepted: 18 December 1998     

Conditional modification of behavior in Drosophila by targeted expression of a temperature‐sensitive shibire allele in defined neurons

Behavior is a manifestation of temporally and spatially defined neuronal activities. To understand how behavior is controlled by the nervous system, it is important to identify the neuronal substrates responsible for these activities, and to elucidate how they are integrated into a functional circuit. I introduce a novel and general method to conditionally perturb anatomically defined neurons in intact Drosophila. In this method, a temperature‐sensitive allele of shibire (shi^ts1) is overexpressed in neuronal subsets using the GAL4/UAS system. Because the shi gene product is essential for synaptic vesicle recycling, and shi^ts1 is semidominant, a simple temperature shift should lead to fast and reversible effects on synaptic transmission of shi^ts1 expressing neurons. When shi^ts1 expression was directed to cholinergic neurons, adult flies showed a dramatic response to the restrictive temperature, becoming motionless within 2 min at 30°C. This temperature‐induced paralysis was reversible. After being shifted back to the permissive temperature, they readily regained their activity and started to walk in 1 min. When shi^ts1 was expressed in photoreceptor cells, adults and larvae exhibited temperature‐dependent blindness. These observations show that the GAL4/UAS system can be used to express shi^ts1 in a specific subset of neurons to cause temperature‐dependent changes in behavior. Because this method allows perturbation of the neuronal activities rapidly and reversibly in a spatially and temporally restricted manner, it will be useful to study the functional significance of particular neuronal subsets in the behavior of intact animals. © 2001 John Wiley & Sons, Inc. J Neurobiol 47: 81–92, 2001