A nuclear mutant of Chlamydomonas that exhibits increased sensitivity to UV irradiation,reduced recombination of nuclear genes,and altered transmission of chloroplast genes

Summary Meiotic progeny of Chlamydomonas reinhardtii normally receive chloroplast genomes only from the mt ⁺ parent. However, exceptional zygotes, which transmit the chloroplast genomes of both parents or, more rarely, only those of the mt ^- parent, arise at a low frequency. Mutations at the mt ⁺-linked mat-3 locus were found previously to elevate the transmission of chloroplast genomes from the mt-parent, resulting in a much higher than normal frequency of exceptional zygotes. In this paper we demonstrate that an ultraviolet-sensitive nuclear mutation mapping at the uvsE1 locus, which is unlinked to mating type, also promotes chloroplast genome transmission from the mt ^- parent. This mutant, which was previously shown to reduce recombination of nuclear genes in meiosis, acts synergistically which the mat3-3 mutation to produce an extremely high frequency of exceptional zygotes. Through the use of restriction fragment length polymorphisms existing in the chloroplast genomes of C. reinhardtii and the interfertile strain C. smithii, we show that chloroplast DNA fragments from the mt ^- parent normally begin to disappear shortly after zygote formation. However, this process appears to be blocked totally in the absence of wild-type uvsE1 and mat-3 gene products. Our findings are consistent with the hypothesis that both gene products contribute to the mechanism responsible for uniparental inheritance of the chloroplast genome from the mt ⁺ parent.     

Hyper-recombination and mutator effects of the mms9-1, mms13-1, and mms21-1 mutations in Saccharomyces cerevisiae

Summary Spontaneous mitotic intragenic and intergenic recombination at various sites is enhanced 10 to 100 fold in the methyl methanesulfonate (MMS)-sensitive mutants mms9-1, mms13-1, and mms21-1 of Saccharomyces cerevisiae. All three mutants show elevated rates of spontaneous mutation. Sporulation is reduced in diploids homozygous for any of the three mutations, and a deficiency in meiotic recombination and meiotic chromosome segregation is observed. Pleiotropic effects on cell viability, growth rate, and radiation sensitivity, in combination with the alterations in recombination and mutagenesis displayed by mutant strains, suggest that the MMS9, MMS13, and MMS21 genes play important roles in DNA replication and/or genetic recombination.     

Glucosamine-resistant mutations in yeast affecting the glucose repression sensitivity of electron transport enzymes

Summary We have isolated two mutant strains, GSA^r-4 and GSA^r-5, which are able to grow on lactate in the presence of D-glucosamine. The glucosamine-resistant phenotype results from the cooperative effects of mutations in three loci, GAR1, GAR2 and GAR3. Both glucosamine resistant mutant strains were doubly mutant at gar1 gar2 (GSA^r-4) or gar1 gar3 (GSA^r-5). The mutants were also shown to exhibit glucose repression insensitive synthesis of NADH-cytochrome c reductase and cytochrome c oxidase. Glucose-repressible synthesis of the following enzymes was seen: succinic dehydrogenase, succinic: cytochrome c reductase, maltase (PNPGase), galactokinase, -galactokinase. The glucose-repression insensitivity segregates in association with the glucosamine resistance.     

Ribosomal control of translational fidelity in Podospora anserina: A suppressor and an antisuppressor affecting the paromomycin-induced misreading in vitro

Summary The hypersensitivity to paromomycin dis played by the su _1–60 suppressor, which was thought to be a ribosomal ambiguity mutation, was used to isolate a new antisuppressor, AS ₇₁. This antisuppressor is not specific for ribosomal suppressors, since it is able to act on putative tRNA-type suppressors. Compared to the wild-type, the AS _7-1 mutation produces an increased resistance to paromomycin. In addition, it was found to stop the sporulation process. In the polyuridylic acid-dependant assay, the paromomycin induced misreading was shown to be higher on su _1–60 ribosomes and lower on ribosomes from AS _7–1 mutant and AS _7S-1-su _1–60 double mutant than on wild-type ribosomes. These data confirm that su _1–60 and AS _7-1 mutants are ribosomal mutants involved in the control of translational fidelity.     

In-vitro recombination in rad and rnc mutants of Saccharomyces cerevisiae

Summary Extracts of S. cerevisiae cells can catalyze homologous recombination between plasmids in vitro. Extracts prepared from rad50, rad52 or rad54 disruption mutants all have reduced recombinational activity compared to wild-type. The rad52 and rad54 extracts are more impaired in the recombination of plasmids containing double-strand breaks than of intact plasmids, whereas rad50 extracts are deficient equally for both types of substrate. The nuclease RhoNuc (previously designated yNucR), encoded by the RNC1 (previously designated NUC2) gene and regulated by the RAD52 gene, is not required for recombination when one substrate is single-stranded but is essential for the majority of recombination events when both substrates are double-stranded. Furthermore, elimination of this nuclease restores recombination in rad52 extracts to levels comparable to those in wild-type extracts.     

Regional brain variations of cytochrome oxidase activity and motor coordination in Lurcher mutant mice

Lurcher mutant mice are characterized by massive degeneration of cerebellar Purkinje cells and granule cells and by deficits in motor coordination. Regional brain variations of cytochrome oxidase (CO) activity were analyzed to identify those brain regions with abnormal metabolic activity as a secondary consequence of the cerebellar atrophy and to establish the relationship between CO activity and motor deficits. Lurcher mutants had higher CO activity in all three cerebellar deep nuclei than normal littermate controls of the same background strain. Higher CO activity was also found in Lurcher mutants in brain regions directly connected to the cerebellum, such as the lateral vestibular nucleus, the cochlear nucleus, the red nucleus, the ventrolateral thalamus, the dorsal raphe, the interpeduncular nucleus, and the inferior colliculus. By contrast, there was a sharp decrease in CO activity in the inferior olive. As for brain regions not directly connected to the cerebellum, higher CO activity was observed in the trigeminal motor nucleus and the CA1 molecular layer of the hippocampus, which highlights probable transsynaptic alterations as a secondary consequence of cerebellar atrophy. A positive correlation between CO activity in the red nucleus and latencies before falling in two motor-coordination tests indicates that a compensatory increase of metabolic activity in a cerebellar efferent region is associated with improved behavior. Received: 15 September 1997 / Accepted: 3 March 1998     

On the pharmacological phenocopying of memory mutations inDrosophila: Alkylxanthines accelerate memory decay

Theophylline and 3-isobutyl-1-methylxanthine, two cyclic nucleotide phosphodiesterase inhibitors, when fed to wild-typeDrosophila adults, cause the rapid decay of learning index after training in a shock-odor learning paradigm. The drugs practically do not affect the olfactory acuity of flies, hence they influence the learning/memory process itself. The time courses of memory decay resemble those of the memory mutantsrutabaga andamnesiac and, to a lesser extent,dunce ² anddunce ^M11. Theophylline further deteriorates the learning performance ofdunce ^M11. Biochemical characterization of the inhibition of the two major phosphodiesterase isoenzymes inDrosophila by theophylline predicts only a slight inhibition of these enzymesin vivo, in accordance with the unchanged level of cAMP in wild-type fly heads during drug feeding. 8-Phenyltheophylline, an adenosine receptor antagonist in mammals, slightly retards memory decay in the wild-type. It is suggested that alkylxanthines induce memory decay inDrosophila by interfering with cAMP dynamics at more than one point of its metabolism.This work was supported by Grants OTKA and OKKFT Tt to P.F.     

The mitochondrial genome of the fission yeast Schizosaccharomyces pombe

Summary The three mutator strains ana ^r-8, ana ^r-14, and diu ^r-301 were shown to produce respiratory deficient mutants at different rates. The frequency of respiratory deficient mutants in a culture could be increased by adding ethidium bromide. According to their cytochrome spectra and enzymatic activities they form three classes, namely mutants defective in cytochrome oxidase, in cytochrome b, and in both cytochromes. By restriction enzyme analysis of mitochondrial DNA from about 100 mutants, 22 deletion mutants were identified. The deletions, ranging from 50 to 1,500 base pairs were physically mapped. Deletions were localized in the genes coding for subunit 1 of cytochrome oxidase with its two introns, within the cytochrome b gene and its intron, and within the genes for subunits 2 and 3 of cytochrome oxidase. In several cases, where the physical mapping yielded ambiguous results, pairwise genetic crosses ruled out an overlap between two neighbouring deletions.Using these mitochondrial deletion mutants as tester strains, it was shown that only tetrad analysis and chemical haploidization, but not mitotic segregation analysis, allows a decision between chromosomal and mitochondrial inheritance of respiratory deficiency in Schizosaccharomyces pombe. Abbreviations. MtDNA = mitochondrial DNA; S. pombe = Schizosaccharomyces pombe; cox1, cox2, and cox3 refer to the mt genes coding for the three subunits of cytochrome oxidase; ATPase 6 (oli2), ATPase 8 (aapl in Saccharomyces cerevisiae, urf a61 in HeLa) and ATPase 9 (olil) refer to the three respective subunits of ATP synthase; cob is thegene for apocytochrome b; urf a is the single intergenic unassigned reading frame in S. pombe; 1 rRNA and s rRNA refer to the large and small ribosomal RNA, respectively. Mut^– is a cytoplasmic mutator (the corresponding wild type allele is mut⁺). Mit^– are mitochondrially inherited respiratory deficient mutants with mitochondrial protein synthesis; RC = respiratory competent, RD = respiratory deficient.     

Cloning and expression of surface antigens from occult chronic hepatitis B virus infections and their recognition by commercial detection assays

Occult hepatitis B virus (HBV) infections show little or no serological markers of viral infection, including the absence of hepatitis B surface antigen (HBsAg) which is the main marker of ongoing HBV infection. Such infections can be important in the context of blood and/or organ donations. To study whether mutations contribute to HBsAg seronegativity, S gene sequences from such patients were amplified and cloned. Sequencing revealed 12 clones from seven different patients which contained potentially important mutations. The sequences were subcloned into an expression vector and mutant HBsAgs were expressed in cell culture. The capacity of three HBsAg detection assays to recognise the mutant HBsAgs was studied. Three categories were found: mutant HBsAgs that are not recognised by the assays, those that are recognised as well as wild-type (WT) antigen and an intermediate category where detection of the mutant HBsAgs is reduced with respect to WT. Most of the isolates fall into the second category. Mutations can therefore contribute to HBsAg seronegativity in occult HBV infections, but in most cases the explanation is probably the low level of viral replication.