Yeast resistance to polyene antibiotics. VI. Isolation and biochemical analysis of strains of Saccharomyces cerevisiae yeasts with mutations in the 2 nystatin-resistance genes

The comparative analysis of sterol content in the yeast Saccharomyces cerevisiae strains singly or doubly defective in nystatin resistance genes was carried out. The strains with two mutations in NYS genes were shown to accumulate the sterol mixture, similar to that of the parental singly defective mutant. This type of gene interaction allows to define the main biochemical order of reaction in ergosterol synthesis: methylation in C24 (NYS1), delta 8----delta 7 isomerization (NYS2), C5 (6) and C22 (23) desaturation (NYS3 and NYSX).     

Resistance of yeasts to polyene antibiotics

The strains of Saccharomyces cerevisiae yeast with mutations in two genes NYS3 NYS4 were obtained by tetrad analysis. Sterol fraction of these mutants contains two sterols: ergosta-7-en-3 beta-ol (fungisterol) and ergosta-7,24-dien-3 beta-ol (episterol). The findings allowed to testify the sequence of the ergosterol biosynthesis reactions. Dehydrogenization of the sterol nucleus in C5(6) which is controlled by gene NYS3 occurs simultaneously with the introduction of double bond in C22(23) site of the side chain regulated by gene NYS4.     

Synthesis of Drosophila melanogaster alcohol dehydrogenase in yeast

Expression systems for the heterologous expression of Drosophila melanogaster alcohol dehydrogenase (ADH) in Saccharomyces cerevisiae have been designed, analyzed and compared. Four different yeast/Escherichia coli shuttle vectors were constructed and used to transform four different yeast strains. Expression was detectable in ADH- yeast strains, from either a constitutive promoter, yeast ADH1 promoter (ADCp), or a regulated promoter, yeast GALp. The highest amount of D. melanogaster ADH was obtained from a multicopy plasmid with the D. melanogaster Adh gene expressed constitutively under the control of yeast ADCp promoter. The D. melanogaster enzyme was produced in cell extracts, as assessed by Coomassie blue staining and Western blotting after polyacrylamide-gel electrophoresis and it was fully active and able to complement the yeast ADH deficiency. Results show that D. melanogaster ADH subunits synthesized in yeast are able to assemble into functional dimeric forms. The synthesized D. melanogaster ADH represents up to 3.5% of the total extracted yeast protein.     

Review of the mutagenicity/genotoxicity of butylated hydroxytoluene.

Butylated hydroxytoluene (BHT) is an effective, widely used, low cost antioxidant. A host of studies examining the potential of BHT to cause point mutations have been published. They include in vitro studies on various bacterial species and strains and on various types of mammalian cell lines as well as in vivo studies on Drosophila melanogaster, silk worms and also the mouse specific locus test (involving long-term exposure). Together these studies convincingly show the absence of a potential for BHT to cause point mutations. A great number of studies on many cell types and species have also been carried out to examine the potential of BHT to cause chromosome aberrations. In vitro studies have been published using plant cells and the WI-38, CHL, CHO, and V79 mammalian cell lines. In vivo studies have been carried out on somatic and/or germ cells of Drosophila melanogaster, rats and mice. Nearly all studies, especially those using validated test systems, indicate that BHT lacks clastogenic potential. In vitro studies on bacterial, yeast and various mammalian cell lines including DON, CHO, CHL cells and primary hepatocytes demonstrate the absence of interactions with or damage to DNA. Taking all the existing data into account, the weight of evidence suggests that BHT does not represent a relevant mutagenic/genotoxic risk to man.     

Drosophila ARSs contain the yeast ARS consensus sequence and a replication enhancer.

A number of restriction fragments that function as autonomously replicating sequences (ARSs) in yeast have been isolated from Drosophila melanogaster DNA. The behaviour in yeast of plasmids containing Drosophila ARS elements was studied and compared to that exhibited by the archetypal yeast ARS-1 plasmid. ARS functions were localised by subcloning and BAL-31 deletion analysis. These studies demonstrated the structural and functional complexity of Drosophila ARSs. Each Drosophila ARS element has at least two domains, one essential for replication (the replication sequence, RS) and a second (the replication enhancer, RE) which is essential for maximum function of the RS. The RS of three Drosophila ARSs was shown to contain a sequence identical to an 11 bp yeast ARS consensus sequence (5' A/T TTTATPuTTT A/T 3'). These observations lend support to the hypothesis that heterologous ARS elements may be of biological significance.     

Genetic instability and transposition of mobile element mdg4 in a mutator strain of Drosophila melanogaster

Laboratory mutator strain of Drosophila melanogaster is characterized by increased (up to 10(-3)-10(-4) frequency of spontaneous mutability. Mutations appear in premeiotic stages of gametes development. The majority of mutations were unstable (high frequencies of reversions, appearance of new mutations at the same and other loci, replicating instability). Localization of mobile elements mdg1, mdg2, mdg3, mdg4, copia and P element in X chromosomes of mutator individuals and its mutations y, ct, sbt was studied by hybridization in situ. In all strains P element was absent. The distribution of mdg1, mdg2, mdg3 and copia was identical in mutator strains and its derivatives, but distribution of mdg4 was different. The essential heterogeneity in localization of mdg4 and increased (up to 30-40) copy number in the mutator strain individuals was observed. The ability of single element mdg4 to autonomous transpositions was thus shown.     

Chromosome aberrations induced by gamma radiation in the somatic cells of a radiosensitive mutant line of Drosophila melanogaster

Chromosome aberrations induced by gamma-rays in ganglia cells of Drosophila melanogaster larvae have been studied. Two strains of Drosophila were used: radiosensitive mutant rad (2) 201G1 and normal strain. It has been shown that the frequency of cells with chromosome aberrations in radiosensitive larvae is much more than in normal larvae after gamma-irradiation. The ratio of chromosome and chromatid deletions number to the number of exchange type aberrations is the same for both strains. The kinetics of chromosome aberrations induced in rad-larvae is similar to the normal one. The conclusion has been made that the realization of rad (2) 201G1 mutation takes place on the cell level.     

The pharmacological flexibility of the insect voltage gated sodium channel: toxicity of AaIT to knockdown resistant (kdr) flies.

AaIT is an insect selective neurotoxic polypeptide shown to affect insect neuronal sodium conductance by binding to excitable sodium channels. In the present study the paralytic potency of AaIT to wild type and various mutant strains of houseflies (Musca domestica) and fruitflies (Drosophila melanogaster) was examined and it has been shown that: On the basis of body weight when compared to published data on Sarcophaga falculata blowflies, the Musca and Drosophila flies reveal at least two orders of magnitude decreased susceptibility to the AaIT. When compared to wild type flies the toxicity of AaIT is greatly altered in knockdown resistant fly strains which are mutated in their para gene encoding the voltage gated sodium channel. Several strains, with genetically mapped para mutations conferring pyrethroid resistance, exhibited opposing response to AaIT. The para ts2 Drosophila strain, with a point of mutation in domain I of the para gene conferring a 6-fold resistance to deltamethrin also showed about 15-fold tolerance to AaIT. On the other hand the Musca kdr and super-kdr flies, with a single or a double point mutation, respectively in domain II of the para gene, are about 9- and 14-fold more susceptible to AaIT, respectively. The above data are interpreted in terms of the pharmacological diversity and flexibility ("allosteric coupling") of voltage gated sodium channels and their implications for the management of pesticide resistance are discussed.     

The acetylcholinesterase gene and organophosphorus resistance in the Australian sheep blowfly, Lucilia cuprina

Acetylcholinesterase (AChE), encoded by the Ace gene, is the primary target of organophosphorous (OP) and carbamate insecticides. Ace mutations have been identified in OP resistants strains of Drosophila melanogaster. However, in the Australian sheep blowfly, Lucilia cuprina, resistance in field and laboratory generated strains is determined by point mutations in the Rop-1 gene, which encodes a carboxylesterase, E3. To investigate the apparent bias for the Rop-1/E3 mechanism in the evolution of OP resistance in L. cuprina, we have cloned the Ace gene from this species and characterized its product. Southern hybridization indicates the existence of a single Ace gene in L. cuprina. The amino acid sequence of L. cuprina AChE shares 85.3% identity with D. melanogaster and 92.4% with Musca domestica AChE. Five point mutations in Ace associated with reduced sensitivity to OP insecticides have been previously detected in resistant strains of D. melanogaster. These residues are identical in susceptible strains of D. melanogaster and L. cuprina, although different codons are used. Each of the amino acid substitutions that confer OP resistance in D. melanogaster could also occur in L. cuprina by a single non-synonymous substitution. These data suggest that the resistance mechanism used in L. cuprina is determined by factors other than codon bias. The same point mutations, singly and in combination, were introduced into the Ace gene of L. cuprina by site-directed mutagenesis and the resulting AChE enzymes expressed using a baculovirus system to characterise their kinetic properties and interactions with OP insecticides. The K(m) of wild type AChE for acetylthiocholine (ASCh) is 23.13 microM and the point mutations change the affinity to the substrate. The turnover number of Lucilia AChE for ASCh was estimated to be 1.27x10(3) min(-1), similar to Drosophila or housefly AChE. The single amino acid replacements reduce the affinities of the AChE for OPs and give up to 8.7-fold OP insensitivity, while combined mutations give up to 35-fold insensitivity. However, other published studies indicate these same mutations yield higher levels of OP insensitivity in D. melanogaster and A. aegypti. The inhibition data indicate that the wild type form of AChE of L. cuprina is 12.4-fold less sensitive to OP inhibition than the susceptible form of E3, suggesting that the carboxylesterases may have a role in the protection of AChE via a sequestration mechanism. This provides a possible explanation for the bias towards the evolution of resistance via the Rop-1/E3 mechanism in L. cuprina.     

Stress-reactivity and juvenile hormone degradation in Drosophila melanogaster strains having stress-related mutations

Juvenile hormone (JH) degradation was studied under normal and stress conditions in young and matured females of Drosophila melanogaster strains having mutations in different genes involved in responses to stress It was shown that (1) the impairment in heat shock response elicits an alteration in stress-reactivity of the JH system; (2) the impairment JH reception causes a decrease of JH-hydrolysing activity and of stress-reactivity in young females, while in mature ones stress reactivity is completely absent; (3) the absence of octopamine results in higher JH-hydrolysis level under normal conditions and altered JH stress-reactivity; (4) the higher dopamine content elicits a dramatic decrease of JH degradation under normal conditions and of JH stress-reactivity. Thus, the impairments in any component of the Drosophila stress reaction result in changes in the reponse of JH degradation system to stress. The role of JH in the development of the insect stress reaction is discussed.     

The influence of gamma-irradiation in low doses on the rate of dominant lethal mutations in drosophila melanogaster

The dose-rate effect of acute and chronic irradiation in the dose of 0.2 Gy in Drosophila melanogaster repair (mei-41, mus209 [Russian character: see text] mus309) and free radicals detoxication (sod) mutant strains was investigated. Was shown the lack of dose rate effect on the rate of dominant lethal mutations in mei-41, mus209 and sod. However in mus309, that has defect in the main Drosophila pathway of the DNA double strand breack repair, the increase of the mutation rate after chronic irradiation was observed (inverse dose-rate effect). The obtained results suggest the main role of DNA double strand breack repair in dose-rate effect formation in Drosophila.     

Molecular analysis of chemically-induced mutations at the RpII215 locus of Drosophila melanogaster

A substantial fraction, perhaps 50% or more, of spontaneous mutations in Drosophila melanogaster have been shown by molecular analyses to be associated with the presence of a transposable element (TE) inserted into the affected gene. We are interested in the molecular structure of induced mutations in Drosophila, in particular whether TEs are also responsible for a significant proportion of chemically-induced mutations. We report here the molecular analysis of 58 mutations at the RpII215 locus induced with EMS or ENU. While we find evidence for moderately sized deletions at this locus (in 3/58, or 5% of the examined mutants), we failed to detect any mutations which were associated with an insertion event. It may be the case that induced mutations are qualitatively different from spontaneous mutations.     

The spontaneous mutation rate in Drosophila simulans

The purpose of this investigation was to determine whether there exists a correlation in Drosophila between the spontaneous mutation rate and the amount of dispersed middle repetitive (mobile) DNA sequences. The amount of these sequences is 7 times less in Drosophila simulans as compared to Drosophila melanogaster. Therefore, if a correlation exists, the spontaneous mutation rate in Drosophila simulans should be 7 times lower than that in Drosophila melanogaster. We isolated an X-chromosome inversion after X-irradiation of wild-type Drosophila simulans males, that reduced crossing-over between white and forked, two X-linked visible markers, to less than 1%. This inversion was subsequently used to determine the sex-linked recessive lethal mutation rate in Drosophila simulans males of a laboratory strain marked with white. The frequency of these lethal mutations found is not different from that observed in Drosophila melanogaster males of laboratory strains.     

Flamenco, a Gene Controlling the Gypsy Retrovirus of Drosophila Melanogaster

Gypsy is an endogenous retrovirus of Drosophila melanogaster. It is stable and does not transpose with detectable frequencies in most Drosophila strains. However, we have characterized unstable strains, known as MG, in which it transposes at high frequency. These stocks contain more copies of gypsy than usual stocks. Transposition results in mutations in several genes such as ovo and cut. They are stable and are due to gypsy insertions. Integrations into the ovo(D1) female sterile-dominant mutation result in a null allele of the gene and occurrence of fertile females. This phenomenon, known as the ovo(D1) reversion assay, can be used to quantitate gypsy activity. We have shown that the properties of MG strains result from mutation of a host gene that we called flamenco (flam). It has a strict maternal effect on gypsy mobilization: transposition occurs at high frequency only in the germ line of the progeny of females homozygous for mutations of the gene. It is located at position 65.9 (20A1-3) on the X chromosome. The mutant allele present in MG strains is essentially recessive. Flamenco seems to control the infective properties of gypsy.     

Studies on the relationship between dimethylnitrosamine-demethylase activity and dimethylnitrosamine-dependent mutagenesis in Drosophila melanogaster

The relationship between dimethylnitrosamine (DMN) demethylase activity and DMN-induced mutagenesis was investigated in Drosophila melanogaster. The activity of DMN-demethylase was at least 10-fold greater in the Hikone-R strain than in three other Drosophila strains. However, the sex-linked recessive lethal (SLRL) mutations induced by DMN in the four strains differed by less than 2-fold. Several possibilities to explain the lack of correlation between DMN-demethylase activity and DMN-induced mutations were tested and eliminated. They include: (i) the presence of inhibitors of DMN-demethylase in extracts of low-activity strains, (ii) a sex bias in the Hikone-R strain in which the enzyme activity is confined to the females, (iii) the possibility that DMN treatment induces DMN-demethylase activity in the low-activity strains and (iv) the possibility that Hikone-R has a much more efficient DNA repair system than the other strains. The results are discussed in terms of what is known about the role of DMN-demethylase in the metabolic activation of DMN in other systems.     

Minute mutations of Drosophila melanogaster change aldehyde oxidase and pyridoxal oxidase distribution patterns in imaginal wing discs

Aldehyde oxidase (AO) and pyridoxal oxidase (PO) distribution patterns were determined in the imaginal wing discs for a series of strains of Drosophila melanogaster heterozygous for different Minute mutations. The mutant severity ranged from very weak to strong. The results shown an inverse response of AO and PO to the expressivity of the Minute mutation: in weaker Minutes the extent of the AO positive area increases, whereas PO activity disappears. The results are discussed with reference to an impaired protein synthesis in Minutes.     

Genetic analysis of the contributions of individual chromosomes to the expression of mating behavior traits in Drosophila melanogaster

The model of isogenous strains with chromosome substitutions has been used to estimate the relative contributions of the X chromosome and autosomes (chromosomes 2 and 3) to the control of some mating behavior traits in Drosophila melanogaster. It has been found that the male sexual activity (SA), female sexual receptivity (SR), and copulation latency (CL) are determined by interaction between X-chromosome and autosomal genes, whereas the copulation duration (CD) is mainly controlled by the X-chromosome genes. The synthesized isogenous strains have been shown to be more similar to hybrids than to the original strains. In the offspring with hybrid genotypes, the relationships between all traits are less stable, which may be related with an increase in the heterozygosity level and changes in genetic homeostasis.