An actidione resistantCandida tropicalis from custard apple juice

An actidione resistant yeast,Candida tropicalis, was isolated from fermenting custard apple juice. Though a slight inhibition of growth was observed on the first day with 5000 ppm of actidione, growth was equal to control after one week. Sorbic acid at 500 ppm and above inhibited the growth of this yeast while sodium benzoate and potassium metabisulphite were unable to suppress the growth even at 1000 ppm. Fermentation and assimilation of different carbon sources were delayed in the presence of 1000 ppm of actidione suggesting the disruption of protein synthesis by actidione. Contribution no. 985.     

The effect of actidione on the regeneration of yeast protoplasts

InSaccharomyces cerevisiae, actidione (cycloheximide) at a concentration of 10 μg/ml inhibits protein synthesis, cell multiplication and regeneration of protoplasts to normal cells. Resistance of cells to actidione is not determined by properties of the cell wall. The effect of actidione is rather cytostatic than cytocidal. The ability of protoplasts to regenerate is irreversibly blocked after more than 2 h incubation with actidione.     

The role of the functional sites of the merlin tumor suppressor in <Emphasis Type="Italic">Drosophila Spermatogenesis</Emphasis>

The Merlin gene of Drosophila is homologous to the human Neurofibromatosis 2 (NF2) gene, an important regulator of proliferation and endocytosis of cell receptors. It was earlier shown that the Thr⁵⁵⁹ residue of the Drosophila Merlin protein was homologous to Ser⁵¹⁸ of the human protein (which was already known to undergo phosphorylation); hence, it was assumed that Thr⁵⁵⁹ of Drosophila also was a substrate of phosphorylation. The mutant Merlin proteins MerT^559D (an analog of the phosphorylated form) and MerT^559A (a nonphosphorylated form) were constructed and tested, under the conditions of ectopic expression, for the ability to correct the spermatogenesis defects induced by the Mer4 mutation. The mutant form MerT^559D was demonstrated to restore the abnormal nebenkern phenotype induced by this mutation, whereas the MerT^559A substituted form did not restore this phenotype. Ectopic expression o the wild-type Merlin protein, MerT^559A mutant form, and mycMer^345–635 truncated protein in a normal genotype resulted in the abnormal nebenkern phenotype, whereas this phenotype was not observed in the case of ectopic expression of the Mer^T559D analog of the phosphorylated form. Ectopic expression of the mycMer³, mycMer^ΔBB, and mycMer^1–379 truncate variants led to disturbance of meiotic cytokinesis.     

Manipulating the aggregation activity of human prion-like proteins

Considerable advances in understanding the protein features favoring prion formation in yeast have facilitated the development of effective yeast prion prediction algorithms. Here we discuss a recent study in which we systematically explored the utility of the yeast prion prediction algorithm PAPA for designing mutations to modulate the aggregation activity of the human prion-like protein hnRNPA2B1. Mutations in hnRNPA2B1 cause multisystem proteinopathy in humans, and accelerate aggregation of the protein in vitro. Additionally, mutant hnRNPA2B1 forms cytoplasmic inclusions when expressed in Drosophila, and the mutant prion-like domain can substitute for a portion of a yeast prion domain in supporting prion activity in yeast. PAPA was quite successful at predicting the effects of PrLD mutations on prion activity in yeast and on in vitro aggregation propensity. Additionally, PAPA successfully predicted the effects of most, but not all, mutations in the PrLD of the hnRNPA2B1 protein when expressed in Drosophila. These results suggest that PAPA is quite effective at predicting the effects of mutations on intrinsic aggregation propensity, but that intracellular factors can influence aggregation and prion-like activity in vivo. A more complete understanding of these intracellular factors may inform the next generation of prion prediction algorithms.     

Evolution of the<Emphasis Type="Italic"> Drosophila broad</Emphasis> locus: the<Emphasis Type="Italic"> Manduca sexta broad</Emphasis> Z4 isoform has biological activity in<Emphasis Type="Italic"> Drosophila</Emphasis>

The Drosophila melanogaster broad locus is essential for normal metamorphic development. Broad encodes three genetically distinct functions (rbp, br, and 2Bc) and a family of four zinc-finger DNA-binding proteins (Z1-Z4). The Z1, Z2, and Z3 protein isoforms are primarily associated with the rbp, br, and 2Bc genetic functions respectively. The Z4 protein isoform also provides some rbp genetic function, however an essential function for the Z4 isoform in metamorphosis has not been identified. To determine the degree of conservation of Z4 function between the tobacco hornworm Manduca sexta and Drosophila we generated transgenic Drosophila expressing the Manduca broad Z4 isoform and used this transgene to rescue rbp mutant lethality during Drosophila metamorphosis. We find that the Manduca Z4 protein has significant biological activity in Drosophila with respect to rescue of rbp-associated lethality. There was also some overlap in effects on cuticle gene expression between the Manduca Z4 and Drosophila Z1 isoforms that was not shared with the Drosophila Z4 isoform. Our findings show that Z4 function has been conserved over the 260-million-year period since the divergence of Diptera and Lepidoptera, and are consistent with the hypothesis that the Drosophila Z4 and Manduca Z4 isoforms have essential roles in metamorphosis.Edited by M. Akam     

The J gene of ?X174: Isolation and characterization of a J gene mutant

Summary The J gene protein of bacteriophage X174 is a component of the mature phage. The previous lack of J gene mutants has prevented an in vivo analysis of J protein functions. A X174 mutant was constructed by inserting an 11 nucleotide sequence into the J gene. This mutant, designated insJ, was viable only in the presence of a wild-type J gene carried on a plasmid that could provide J protein. An analysis of DNA synthesis during insJ mutant infection under non-permissive conditions confirmed that the J protein is not required for viral DNA synthesis.     

Role of AWD/Nucleoside Diphosphate Kinase in Drosophila Development

The abnormal wing discs gene of Drosophila encodes a soluble protein with nucleosidediphosphate kinase activity. This enzymic activity is necessary for the biological function ofthe abnormal wing discs gene product. Complete loss of function, i.e., null, mutations causelethality after the larval stage. Most larval organs in such null mutant larvae appear to benormal, but the imaginal discs are small and incapable of normal differentiation.Killer-of-prune is a neomorphic mutation in the abnormal wing discs gene. It causes dominant lethalityin larvae that lack prune gene activity. The Killer-of-prune mutant protein may have alteredsubstrate specificity. Null mutant larvae have a low level of nucleoside diphosphate kinaseactivity. This suggests that there may be additional Drosophila genes that encode proteinswith nucleoside dipthosphate kinase activity. Candidate genes have been found in theDrosophila genome.     

The role of carcinine in signaling at the Drosophila photoreceptor synapse

The Drosophila melanogaster photoreceptor cell has long served as a model system for researchers focusing on how animal sensory neurons receive information from their surroundings and translate this information into chemical and electrical messages. Electroretinograph (ERG) analysis of Drosophila mutants has helped to elucidate some of the genes involved in the visual transduction pathway downstream of the photoreceptor cell, and it is now clear that photoreceptor cell signaling is dependent upon the proper release and recycling of the neurotransmitter histamine. While the neurotransmitter transporters responsible for clearing histamine, and its metabolite carcinine, from the synaptic cleft have remained unknown, a strong candidate for a transporter of either substrate is the uncharacterized inebriated protein. The inebriated gene (ine) encodes a putative neurotransmitter transporter that has been localized to photoreceptor cells in Drosophila and mutations in ine result in an abnormal ERG phenotype in Drosophila. Loss-of-function mutations in ebony, a gene required for the synthesis of carcinine in Drosophila, suppress components of the mutant ine ERG phenotype, while loss-of-function mutations in tan, a gene necessary for the hydrolysis of carcinine in Drosophila, have no effect on the ERG phenotype in ine mutants. We also show that by feeding wild-type flies carcinine, we can duplicate components of mutant ine ERGs. Finally, we demonstrate that treatment with H₃ receptor agonists or inverse agonists rescue several components of the mutant ine ERG phenotype. Here, we provide pharmacological and genetic epistatic evidence that ine encodes a carcinine neurotransmitter transporter. We also speculate that the oscillations observed in mutant ine ERG traces are the result of the aberrant activity of a putative H₃ receptor.     

Apurinic endonuclease activity from wild-type and repair-deficient mei-9 Drosophila ovaries.

Summary An endonuclease which acts on apurinic (AP) sites in DNA was partially purified from Drosophila ovaries. The enzyme present in the female germ line has a molecular weight of 63 000 daltons, is Mg⁺⁺ dependent, and produces a site upon cleaving depurinated DNA that supports DNA repair synthesis. Although the same characteristics are shared by the enzyme present in the excision-deficient mutant mei-9, specific activity for the AP endonuclease is reduced 98% when compared with that found for its wild-type counterpart. Moreover, cross-reactivity toward an antibody that recognizes the wild-type AP endonuclease protein is reduced roughly 90% for partially purified preparations from mei-9. Mixing experiments between extracts of mei-9 and wild type suggest that the mei-9 structural gene somehow alters or influences the levels of the AP endonuclease protein, but in view of the complex phenotype of this mutant the endonuclease is probably not the product of the gene itself.     

Expression of the pspA gene stimulates efficient protein export in Escherichia coli

Expression of several mutant forms of outer membrane protein PhoE of Escherichia coli, which are disturbed in normal biogenesis, resulted in high expression of a 26kDa protein. This 26kDa protein fractionated as a peripherally bound inner membrane protein. It appeared to be identical to a previously identified protein (PspA = phage shock protein A) of unknown function that is induced upon infection of E. coli with filamentous phages. PspA was not expressed upon synthesis of mutant PhoE proteins in a secB mutant, nor upon expression of a PhoE mutant that lacks the signal sequence, suggesting that entrance into the export pathway of prePhoE is essential for induction. PspA synthesis was also induced under other conditions that are known to block the export apparatus, i.e. in secA, secD and secF mutants when grown at their non-permissive temperature or upon induction of the synthesis of MalE-LacZ or LamB-LacZ hybrid proteins. The inducing conditions for PspA synthesis suggested a rote for this protein in export. In vivo pulse-chase experiments showed that the translocation of (mutant) prePhoE and of the precursors of other exported proteins was retarded in a pspA mutant strain. Also, in in vitro translocation assays, a role for PspA in protein transport could be demonstrated.     

Overexpression of wild-type and mutant NDP kinase in Dictyostelium discoideum

Nucleoside diphosphate (NDP) kinase has a central role in the synthesis of (deoxy-)trinucleotides. In addition, mutations in the gene encoding NDP kinase have been shown to have important consequences for Drosophila development and mammalian tumorogenesis. We have overexpressed, in Dictyostelium discoideum, a genomic clone encoding the enzyme NDP kinase. The concomitant increase in the levels of RNA and enzyme activity identifies a 5′ non-coding genomic region of 0.9 kb as being the complete promoter region. Overexpression of wild-type NDP kinase has no effect on development. This is also true for an inactive mutant H122C that does not have a dominant inhibitor effect. Overexpression of the P105G mutant NDP kinase, which is known to be affected in its stability in vitro, only leads to a small increase in total NDP-kinase activity. Thermal and chemical denaturation experiments demonstrate the formation of hexameric hybrids between wild-type and mutant monomers.     

Selektion Actidion-resistenter Mutanten bei Neurospora crassa sowie ihre genetische und biochemische Analyse

Summary Conidia of an actidione-sensitive wildtype strain of Neurospora crassa were irradiated with UV-light. They were then plated into nutrient-agar, either with or without actidione. The latter plates were incubated for several hours, before nutrient agar containing actidone was layered onto the plates. Colonies formed in both sets of plates were isolated as actidione-resistent. They were studied further by genetic and biochemical means.Pre-incubation of the irradiated conidia before subjecting them to the action of actidione increased the mutant yield considerably, as compared to immediate plating with the drug. E.g. a 13 hours pre-incubation gave ca. 100 times more resistent colonies than were obtained without pre-incubation (Fig. 2). Their resistent phenotype was stable on vegetative propagation.17 mutants were mapped by crossing them with suitable tester-strains. Of them, 14 were found to belong to linkage group I, the remaining to linkage group V. The mutants are, therefore, considered as characterizing resp. genes act-1 and -2 of Hsu (1963). Act-1 and -2 mutants were crossed with suitable auxotrophic strains to obtain auxotrophic, actidione-resistent isolates. These were combined on minimal medium with auxotrophic, actodione-sensitive strains of the same mating type. Conidia of the arising heterokaryotic mycelia were tested on minimal medium with and without actidione. In these tests resistence of act-1 and -2 mutants was found to be dominant over the sensitivity of the wildtype. However, an analysis of nuclear ratios in the conidial populations by differential plating does not exclude incomplete dominance of act-1.Incorporation of 14-C-leucine into protein of conidia of the wildtype was strongly inhibited by 1 actidione/ml. Resistence in two mutants, representing the two separate genes, was accompanied by a marked decrease of this inhibition. No significant differences in the amount of inhibition were found between the two mutants. It is suggested that cytoplasmic ribosomes may be the cellular components influenced by actidione. In the case of the mutant cells the actidione is no longer effective in this capacity, possibly because of changes in the ribosomal proteins.     

Deletion of Siah-interacting protein gene in <Emphasis Type="Italic">Drosophila</Emphasis> causes cardiomyopathy

Drosophila is a useful model organism in which the genetics of human diseases, including recent advances in identification of the genetics of heart development and disease in the fly, can be studied. To identify novel genes that cause cardiomyopathy, we performed a deficiency screen in adult Drosophila. Using optical coherence tomography to phenotype cardiac function in awake adult Drosophila, we identified Df(1)Exel6240 as having cardiomyopathy. Using a number of strategies including customized smaller deletions, screening of mutant alleles, and transgenic rescue, we identified CG3226 as the causative gene for this deficiency. CG3226 is an uncharacterized gene in Drosophila possessing homology to the mammalian Siah-interacting protein (SIP) gene. Mammalian SIP functions as an adaptor protein involved in one of the β-catenin degradation complexes. To investigate the effects of altering β-catenin/Armadillo signaling in the adult fly, we measured heart function in flies expressing either constitutively active Armadillo or transgenic constructs that block Armadillo signaling, specifically in the heart. While, increasing Armadillo signaling in the heart did not have an effect on adult heart function, decreasing Armadillo signaling in the fly heart caused the significant reduction in heart chamber size. In summary, we show that deletion of CG3226, which has homology to mammalian SIP, causes cardiomyopathy in adult Drosophila. Alterations in Armadillo signaling during development lead to important changes in the size and function of the adult heart.     

Stable transformation and cloning mediated by <Emphasis Type="Italic">piggyBac</Emphasis> vector and RNA interference knockdown of <Emphasis Type="Italic">Drosophila</Emphasis> ovarian cell line

An in vitro study is a powerful method for elucidating gene functions in cellular and developmental events. However, until date, no reliable in vitro transformation, cloning, or knockdown system has been reported for Drosophila cells, with the exception of S2 and Kc cells. In this study, we demonstrated that the piggyBac vector stably integrates donor DNA into ovarian somatic sheets derived from follicle stem cells. The transformed ovarian somatic sheet cells were easily cloned with a new piggyBac selection vector carrying enhanced green fluorescent protein and dihydrofolate reductase genes, egfp, and dhfr, respectively, in culture media containing methotrexate, an inhibitor of DNA synthesis. Donor egfp continued to be expressed at a high level in long-term culture. Furthermore, the translation of donor egfp was inhibited by treatment with double-stranded RNA derived from the target gene. The transfection and cloning methods mediated by the piggyBac vector would thus be useful for future analyses of gene functions in OSS cells and possibly be applicable to other Drosophila cell lines.     

The vestigial locus of Drosophila melanogaster is involved in resistance to inhibitors of dTMP synthesis

The vestigal (vg) gene encodes a nuclear protein which plays a major role in the formation of the wing of Drosophila. Resistance or sensitivity to aminopterin, an inhibitor of the dihydrofolate reductase enzyme in D. melanogaster, seems to be associated with a specific alteration in vg gene function. Wild-type and vg mutant strains selected for growth on increasing concentrations of aminopterin display changes in physiological and biochemical parameters such as viability on normal and aminopterin-containing media, duration of development, wing phenotype, dihydrofolate reductase activity, and cross-resistance to fluorodeoxyuridine (FUdR) and to methotrexate. Our results indicate that the mechanisms of resistance differ in the wild-type and mutant strains. The vg ^83b27 mutant, in which the major part of intron 2 of the vg gene is deleted, is associated with a high rate of resistance to FUdR, an inhibitor of thymidylate synthetase. Moreover, vg ^83b27/vg ^BGheterozygotes, which are wild type when grown on normal medium, display a strong vg phenotype when grown on aminopterin. Our results indicate a role for the vestigial locus in mediating resistance to inhibitors of dTMP synthesis.     

Protein synthesis during flight muscle development in normal and wupB indirect flight muscles of Drosophila melanogaster

We have looked at protein synthesis in Drosophila pupae during normal and abnormal development of indirect flight muscle. Abnormal development was followed in the dominant flightless mutant wupB isolated by Hotta and Benzer (Genetic Mechanisms of Development, pp. 129–167. Academic Press, New York, 1972). The mutant muscles in adult wupB flies have abnormal morphology and disorganized myofibrils. Protein synthesis in developing muscle was followed on SDS-polyacrylamide gels. During early stages of development (55–60 h) protein synthesis patterns are similar in the mutant and the wild-type. However, at 61 h, the mutant shows a transient increase in synthesis of the 68 and 70 kDa heat shock proteins. This is followed at about 70 h by a divergence of the patterns of synthesis of other proteins seen in the mutant and wild type. These results suggest that induction of heat shock protein synthesis is an early event in abnormal morphogenesis in this mutant.     

Analysis of revertants of a ribosomal mutation in Podospora anserina: Evidence for new ribosomal mutations which confer hypersensitivity to paromomycin

This paper describes the analysis of cold-resistant revertants of a cold-sensitive mutant. Pm1-1 is a ribosomal mutation screened for its paromomycin resistance. Suppression of its cold sensitivity occurs with two kinds of external mutations localized in two different loci. One of them, PmB, is assumed to be a ribosomal gene. PmB mutations confer hypersensitivity to paromomycin in vivo as well as in vitro in a cell-free protein synthesis system.This work was supported by DGRST Grant MRM/P240 and NATO Grant 1637.