Hygromcyin- and paromonycin-resistant mutants of Aspergillus nidulans alter translational fidelity

Summary Mutants of Aspergillus nidulans resistant to the aminoglycoside antibiotics paromycin and hygromycin B have been isolated and their growth characteristics are described here. Most paromomycin mutants were crossresistant to hygromycin and geneticin. All the hygromycin-resistant mutants were slightly cross-resistant to geneticin. Out of the 15 mutants tested 14 had drug-resistant ribosomes in vitro and all 12 of those investigated further had reduced levels of translational misreading. Five new loci have been found-parA on linkage group I, hygA on III, hygB on IV, hygC on V, hygD on VI and parB on VIII. This increases, to at least 12, the number of translational fidelity loci in A. nidulans.Amina Sheikh is the nee Zamir     

A new selection method for isolating mutants defective in acetate utilisation inAspergillus nidulans

Propionate medium is normally toxic for the growth ofAspergillus nidulans. Spontaneous mutations relieving the toxicity to propionate, which arose on propionate medium, have been shown to be mutations in acetate metabolism. Oneacu ^– mutant is allelic withacuA (the structural gene for acetyl-CoA synthetase), another withacuB (the regulatory gene involved in the induction of enzymes concerned with acetate metabolism, including acetyl-CoA synthetase), and a third mutants,acuO, represents a newacu ^– locus that maps on likage group V.     

Chromosome instability in mutagen sensitive mutants of Neurospora

Summary Four mutagen sensitive mutants of Neurospora (mus-7, mus-9, mus-11, and mei-2) are shown to increase mitotic chromosome instability in the duplication test developed by Newmeyer. Three other mutagen-sensitive mutants (upr-1, mus-8, and mus-10) do not increase chromosome instability. Previously three mutagen-sensitive mutants (uvs-3, uvs-6, and mei-3) were also shown to increase chromosome instability. The growth of all seven mutants that increase chromosome instability, is shown here to be more sensitive to hydroxyurea than that of wild type. Hydroxyurea, a compound which inhibits the enzyme ribonucleotide diphosphate reductase, is also shown to increase chromosome instability in the absence of any mutagen-sensitive mutation. These seven mutations are known to represent seven different genes in two epistasis groups. They have been shown previously to have four other properties in common: meiotic defects and sensitivity to y-rays, methyl methane sulfonate and the amino acid histidine. Their shared properties lead to the prediction here that all have reduced or altered deoxyribonucleotide pools.     

uvsI mutants defective in UV mutagenesis define a fourth epistatic group of uvs genes in Aspergillus

Three UV-sensitive mutations of A. nidulans, uvsI, uvsJ and uvsA, were tested for epistatic relationships with members of the previously established groups, here called the UvsF, UvsC, and UvsB groups. uvsI mutants are defective for spontaneous and induced reversion of certain point mutations and differ also for other properties from previously analyzed uvs types. They are very sensitive to the killing effects of UV-light and 4-NQO (4-nitro-quinoline-N-oxide) but not to MMS (methylmethane sulfonate). When double-and singlemutant uvs strains were compared for sensitivity to these three agents, synergistic or additive effects were found for uvsI with all members of the three groups. The uvsI gene may therefore represent a fourth epistatic group, possibly involved in mutagenic repair. On the other hand, uvsJ was clearly epistatic with members of the UvsF group and fitted well into this group also by phenotype. The uvsA gene was tentatively assigned to the UvsC group. uvsA showed epistatic interactions with uvsC in all tests, and like UvsC-group mutants is UV-sensitive mainly in dividing cells. However, the uvsA mutation does not cause the defects in recombination and UV mutagenesis typical for this group.     

Phenotypic and epistatic grouping of hypo- and hyper-rec mus mutants in Aspergillus

The mutants musK to musS of Aspergillus nidulans are sensitive to methyl-methanesulfonate (MMS) and several of them are meiotic-defective and alter mitotic recombination frequencies. All were found to be cross-sensitive to 4-nitro-quinoline-N-oxide (4-NQO) but unexpectedly none of them was hypersensitive to -rays and few to UV light. Double mus;uvs mutants were constructed to test for interactions with uvs mutations of the four epistatic groups of Aspergillus, UvsF, UvsC, UvsI, and UvsB. All meiotic-defective mus mutations caused some lethal interactions, usually with uvsF. None of them showed epistasis with UvsF or UvsB group mutants and one, musO, may represent a new group. Three mus mutations that affect recombination were assigned to the UvsC group, namely musN and K, and also musL which is recombination-defective and closely resembles uvsC. While uvsC mutants are mutators and lack UV-mutagenesis, most mus mutants had no effects on mutation. Only musR, which appeared epistatic with uvsI, showed reduced UV-reversion frequencies similar to uvsI. The recombination-proficient mus mutants appeared to be epistatic with more than one group, but in several cases sensitivities were slight and overlaps insufficient to obtain corroborating results with MMS and 4-NQO.     

Genetic analysis of Aspergillus niger mutants defective in benzoate-4-hydroxylase function

Summary This study was prompted by the observation that an Aspergillus niger transformant with a multicopy bphA (benzoate-4-hydroxylase gene) insert did not grow on benzoate, whereas a transformant with only one extra copy could grow. Therefore, an extensive survey has been made for other genes involved in the conversion of benzoate into 4-hydroxy-benzoate. A transformant with two copies of the bphA gene was used in part of the mutation experiments in order to avoid the isolation of many bphA mutants. Filtration enrichment was used to isolate mutants defective in the conversion of benzoate. The Bph mutants that have been isolated belong to six complementation groups. Mutants with a defected structural gene (bphA) were again predominantly found but, in addition, five other groups of mutants that could not grow on benzoate were isolated. Genetic analysis of the mutants showed that the six genes were localized in different parts of the genome. This was used as an additional proof that some mutants involved different genes. Diploids with seven copies of the bphA gene and heterozygous for one of the other bph genes were constructed. No indication has been obtained that any one of the mutant classes is responsible for the growth-limiting factor in bphA multicopy transformants. This study shows that the p-hydroxylation of benzoate is very complex, although the metabolic pathway is straight forward.     

The identification of mutations in Aspergillus nidulans that lead to increased levels of ADHII

Summary There are at least three alcohol dehydrogenases in Aspergillus nidulans. ADHII has been observed in polyacrylamide gels stained for ADH activity but, unlike ADHI and ADHIII, no physiological function has been attributed to it. This paper describes mutations that have been isolated from strains carrying a deletion in the structural gene for ADHI (alcA) and its adjacent positively-acting regulatory gene (alcR) that restore some ability to utilise ethanol as a carbon source. The mutations map at three loci, and all show elevated levels of the ADHII staining band. An assay for ADHII has been developed. The growth on ethanol has been shown to be dependent on the previously identified aldehyde dehydrogenase (structural gene, aldA). Two of the mutations, alcD and alcE, represent newly discovered mutations affecting ethanol utilisation while the third mutation is in amdA, a previously described trans-acting regulatory protein.     

Every ribosomal suppressor mutation in Aspergillus nidulans has a unique and highly pleiotropic phenotype

Summary 18 suppressors of alcR125 have been selected in Aspergillus nidulans. They have been located in genes as follows: 12 in suaA, 1 in suaB and 5 in suaC. Suppressors have been examined to see whether their phenotype is diagnostic for their genotype. Several new traits are described: conidial viability, cycloheximide resistance, fertility, suppression of niaD500, naaD501 and fWA1. These tests, added to those already in use, provide a battery of tests suitable for assigning suppressor mutations to physiological type (tRNA or ribosomal), and in one case to a specific gene since only suaA mutations suppressed fWA1. A very broad range of phenotypes was associated with suppressors such that every mutation had a unique phenotype. This indicates that the ribosomal suppressor mutations are in genes which code directly for ribosomal proteins, rather than genes which code for modifying enzymes.     

Suppressor specificity in Aspergillus nidulans

Summary Seven allele specific gene unspecific suppressors mapping at four loci have been described previously (Roberts et al. 1979). Three new suppressors mapping in suaA are characterised, and the spectrum of suppression of all the suppressors with respect to seventeen suppressible mutations in eight different genes is described. Two distinct classes of suppressor are defined. The diversity of suppression of five suaA alleles, and the temperature sensitivity of some suaA suppressor mutant combinations but not others, suggests that suppressors at this locus are acting via ribosomal protein alteration. suaC109, a mutation that results in cold-sensitivity for growth shows a similar broad spectrum of suppression. Suppressors at the suaA and suaC loci suppress mutations that have the properties of chain termination mutations as well as missense mutations. suaB111, and suaD103 and suaD108 have a very restricted range of suppression. These suppressors may be mutations in tRNA genes.     

The cloning and sequencing of thealcB gene,coding for alcohol dehydrogenase II,inAspergillus nidulans

Alcohol dehydrogenase II (ADH II, structural genealcB) was purified from a strain H1035,biA1; alcE1; alc500 alcD1, which produces 100-times more ADH II activity than thealcAalcR deletion strain (alc500). Antibodies were raised against this ADH, and were used to screen a cDNA library in gt11. We have isolated the gene for an ADH which is over-expressed in H1035, and which we believe to be thealcB gene; cDNA and genomic clones were sequenced. The sequence contains three introns and encodes a protein of 367 amino acids. This protein shows a clear level of identity to a range of alcohol dehydrogenases, but is no more closely related to the ADH I and ADH III previously described inA. nidulans than to the ADHs ofS. pombe andS. cerevisiae. The significance of consensus sequences found in the 5 region of the gene is discussed in relation to the regulation of the gene.     

Structure of the Aspergillus niger pelA gene and its expression in Aspergillus niger and Aspergillus nidulans

Summary Aspergillus niger pectin lyases are encoded by a multigene family. The complete nucleotide sequence of the pectin lyase PLA-encoding gene pelA has been determined. Comparison of the deduced amino acid sequence with the deduced amino acid sequence of the other characterized pectin lyase, PLD, shows that the proteins share 69% amino acid identity. When grown on media with pectin as the sole carbon source, A. niger transformants containing multiple copies of the pelA gene show raised mRNA levels and overexpression of the gene product PLA compared with the wild-type strain. PLA was purified and characterized. In A. nidulans transformants PLA is also produced in medium containing a high concentration of glucose and no pectin.Deceased April 30, 1988     

Antisuppressor mutations reduce misreading of the genetic code in Aspergillus nidulans

Summary Antisuppressor mutations were isolated in a strain containing the omnipotent suppressor suaC109. The antisuppressors reduce the activity of translational suppressors in vivo and counteract most aspects of the pleiotropic phenotype associated with the suaC and the suaA suppressor mutations. Using an homologous system for cell-free translation, we have measured translational accuracy in two antisuppressor strains with the genotype suaC109 and either the asuB11 or the asuD14 antisuppressor mutation. Ribosomes from antisuppressor mutants have higher levels of translational accuracy than those from the suppressor strain (suaC109, asu ⁺). Mistranslation levels depended solely on the source of the sucrose-cleaned ribosomes. However, the increased accuracy associated with sucrose-cleaned ribosomes from antisuppressor strains can be nullified by salt-washing, suggesting that the component responsible can be washed off.     

Neurospora mutants sensitive both to mutagens and to histidine

Summary Previous work in this and other laboratories showed that histidine strongly inhibits growth of mutants at ten out of 20 known mutagen-sensitivity loci in Neurospora, and that nine of the histidine-sensitive mutants disturb meiosis when homozygous. These and other results suggested that histidine affects recombination or DNA repair. Current work with the histidine-sensitive mutant uvs-6 shows that it is also inhibited by several other metabolites but none of them is as effective as histidine. On minimal medium without histidine or other inhibitors, uvs-6 first grows normally, then slows drastically and begins stop-start growth. Conidia from stop-start uvs-6 mycelia produce rejuvenated cultures. The stop-start growth, UV-sensitivity, histidine-sensitivity, and recessive meiotic characters of uvs-6 segregated together in crosses, and reverted together. In tests on other mutagen-sensitive mutants, sensitivity to histidine was strongly correlated with stop-start growth and with sensitivity to other metabolites. Histidine induces premature stop-start growth in at least two mutants. Several possible explanations for the histidine-sensitivity have been excluded.     

Extremely low doses of oxytocin reduce pain sensitivity in men

The effect of extremely low doses of oxytocin (vapor) on the perception of pain (pricking of the finger) is studied on 48 healthy volunteers. Inhalation of oxytocin vapor from the standard solution in doses producing a sensation of smell lowers pain threshold by 56.5%. Inhalation of oxytocin vapor creating no sense of smell has a lower hypalgesic effect. The oxytocin-induced hypalgesia is consistent with reduction in the heart reactivity to pain. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 122, No. 11, pp. 487–489, November, 1996     

Genetic and molecular characterization of argB+ transformants of Aspergillus nidulans

Summary Thirty-three argB^– to argB⁺ transformants of Aspergillus nidulans have been subjected to genetic and molecular analysis. Two showed high levels of mitotic instability although it is suggested that this is a consequence of heterokaryosis rather than instability of the transformation event. Most transformants resulted from the integration of the transforming DNA in tandem with the chromosomal argB locus. The maximum number of inserted sequences was two, to generate three copies of the argB locus. The other main transformant type showed replacement of the argB^– mutation by the wild-type allele present on the transforming plasmid. Transformants were also recovered in which the transforming DNA had integrated into non-homologous chromosomal regions. Selfed or hybrid cleistothetica from all transformants, except the gene replacement types gave arginine requiring recombinants. Most transformants showed low levels of meiotic instability. Others displayed varying levels which in some cases differed between selfed and hybrid cleistotheticia. There was some correlation between meiotic instability and the nature of the transformation event. Diploid parasexual and aneuploid analysis located the integrated DNA in each transformant to chromosome III. Two transformants were isolated as heterozygous diploids. A third diploid was isolated as a stable mitotic segregant from one of the mitotically unstable transformants.     

Effect of low doses of piracetam on conditioned-response memory in rats

Effects of low doses of piracetam, a psychotropic nootropic, on the memory of rats are studied. A positive effect of the drug in a dose many times lower than the doses used routinely is demonstrated on a model of elaboration of the active avoidance reaction. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 120, N ^o 7, pp. 60–61, July, 1995 Presented by I. P. Ashmarin, Member of the Russian Academy of Medical Sciences     

Heat shock phenomena in Aspergillus nidulans

Summary Heat shock was found to induce characteristic changes in the pattern of protein synthesis in Aspergillus nidulans as analysed by SDS-polyacrylamide gel electrophoresis. Six to seven new bands were found to show increased incorporation to ³⁵S-methionine at 43 °C compared to 37 °C, the standard temperature for this organism. The heat shock response of five different strains of A. nidulans was examined. This comparative study showed that these strains (haploids and diploids) show exactly the same set of heat shock proteins.     

The Aspergillus nidulans pkcA gene is involved in polarized growth, morphogenesis and maintenance of cell wall integrity

The protein kinase C (PKC) family participates in maintaining integrity and growth of fungal cell walls. However, the precise molecular role of these proteins in the filamentous fungi remains unknown. In this work, pkcA, the gene encoding the PKC homolog in the filamentous fungus Aspergillus nidulans, was cloned and its function analyzed using a conditional alcA-PKC mutant strain. Repression of pkcA expression resulted in increased conidial swelling, decreased rates of hyphal growth, changes in the ultrastructure of the cell wall and increased sensitivity to antifungal agents. These results suggest that the protein encoded by pkcA is involved in key aspects of cell morphogenesis and cell wall integrity.     

Immuno- and phagocytosis-modulating properties of low doses of B1-aflatoxin and benzene

Benzene or benzene-dissolved B₁-aflatoxin in low doses promotes an increase of the Thy-1⁺ cell count in the bone marrow of mice and an enhancement of the thymusdependent immune response.In vitro aflatoxin and benzene are unable to induce the expression of Thy-1-antigen in bone marrow T-precursors. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 117, N ^o 2, pp. 158–160, February, 1994 Presented by S. A. Neifakh, Member of the Russian Academy of Medical Sciences     

Expression of a bacterial aspartase gene in Aspergillus nidulans: an efficient system for selecting multicopy transformants

Summary The Escherichia coli aspartase gene aspA has been expressed in the fungus Aspergillus nidulans using the powerful constitutive gpdA promoter and trpC terminator, both from A. nidulans. Multiple, but not single, copies of aspA overcome nutritional deficiencies resulting from the loss of catabolic NAD-linked glutamate dehydrogenase. They also circumvent certain nutritional deficiencies resulting from loss of the positive-acting regulatory gene product mediating nitrogen metabolite repression. Both of these cases of physiological suppression involve the aspartase-catalyzed catabolism of aspartate to ammonium plus fumarate. No physiological evidence for the opposite reaction leading to aspartate synthesis was obtained as multiple copies of aspA did not affect the phenotype resulting from the loss of anabolic NADP-linked glutamate dehydrogenase. The use of vectors containing aspA and recipients lacking NAD-linked glutamate dehydrogenase is an efficient means of selecting multicopy transformants in A. nidulans and also offers the possibility to select strains having increased aspartase levels from original transformants.