Meiotic mapping of the nuclear determinant of cell lysis of the osmotic dependent Saccharomyces cerevisiae mutant VY1160

Summary A recessive nuclear mutation, sorb ^-, which determines the ability for lysis of the osmotic dependent Saccharomyces cerevisiae mutant VY1160 has been mapped on the right arm of chromosome I. sorb ^- is not centromere linked and is approximately 31 recombination units from ade1.     

Low ribonuclease activity in cellular lysates of osmotic sensitive Saccharomyces cerevisiae mutants.

Summary Cellular lysates with very low total ribonuclease activities are obtained by lysis of Saccharomyces cerevisiae VY1160 osmotic sensitive mutant cells in 1% sorbitol solution. These lysates could be used for isolation of intact polysomes and messenger RNA molecules, or for studying of specific ribonucleases.     

Decreased synthesis of alkali-soluble glucan in a cell-wall mutant of Saccharomyces cerevisiae

In vivo studies and quantitative measurements of glucans provide evidence for a decreased rate of synthesis and a lower amount of alkali-soluble glucan in cells of the osmotically fragile VY1160 mutant of the yeast Saccharomyces cerevisiae. Combined genetic and biochemical analysis shows that the srb1 mutation is responsible for the reduction of alkali-soluble glucan. Data on beta(1----3) glucan synthase activity did not indicate the participation of the enzyme in the in vivo synthesis of alkali-soluble glucan and suggest the existence of other glucan synthases in Saccharomyces cerevisiae.     

Evidence for an early regulatory function in phage P22

Summary A temperature sensitive mutant of P22 phage (ts X) was isolated and studied.This mutant seems to have a basic regulatory function: it is defective in an early function like the typical DNA^- mutant ts 12.1; it is unable to direct the phage DNA synthesis and does not lyse infected or induced cells.Unlike ts 12.1, the mutation ts X seems to involve a gene product necessary for the expression of any vegetative function, since no phage protein synthesis, no alteration of host DNA synthesis, and no cell killing can be observed under non-permissive conditions.The possible functional similarity between the N-cistron of the phage and the present X-cistron in P22 is discussed.     

Mitochondrial ribosomal RNA genes of yeast: Their mutations and a common nuclear suppressor

Summary Due to the absence of repetition of the rRNA genes in S. cerevisiae mitochondria, isolation of ribosomal mutants at the level of the rRNA genes is relatively easy in this system. We describe here a novel thermosensitive mutation, ts1297, localized by rho^- deletion mapping in (or very close to) the sequence corresponding to the small ribosomal RNA (15S) gene. Defective mutations of the small rRNA have not been reported so far.In the mutant, the amount of 15S rRNA and of the small ribosomal subunit, 37S, is reduced. The quantity of the large ribosomal RNA (21S), directly extracted from mitochondria, appears normal. However, the large ribosomal subunit, 50S, seems to be fragile and could be recovered only in the presence of Ca²⁺ in place of Mg²⁺. The 50S particles seem to be completely degraded under normal conditions of extraction with Mg²⁺.The thermosensitive phenotype of the ts1297 mutant is suppressed by a nuclear mutation SU₁₀₁. The SU₁₀₁ mutation had been originally isolated as a suppressor of another mitochondrial mutation, ts902, which is located within the 21S rRNA gene.These results suggest that the mitochondrial mutations ts1297 and ts902 are both involved in the interaction of the large and small ribosomal subunits.     

CD8+ T Cell Cross-Reactivity Profiles and HIV-1 Immune Escape towards an HLA-B35-Restricted Immunodominant Nef Epitope

Antigen cross-reactivity is an inbuilt feature of the T cell compartment. However, little is known about the flexibility of T cell recognition in the context of genetically variable pathogens such as HIV-1. In this study, we used a combinatorial library containing 24 billion octamer peptides to characterize the cross-reactivity profiles of CD8⁺ T cells specific for the immunodominant HIV-1 subtype B Nef epitope VY8 (VPLRPMTY) presented by HLA-B^*35∶01. In conjunction, we examined naturally occurring antigenic variations within the VY8 epitope. Sequence analysis of plasma viral RNA isolated from 336 HIV-1-infected individuals revealed variability at position (P) 3 and P8 of VY8; Phe at P8, but not Val at P3, was identified as an HLA-B^*35∶01-associated polymorphism. VY8-specific T cells generated from several different HIV-1-infected patients showed unique and clonotype-dependent cross-reactivity footprints. Nonetheless, all T cells recognized both the index Leu and mutant Val at P3 equally well. In contrast, competitive titration assays revealed that the Tyr to Phe substitution at P8 reduced T cell recognition by 50–130 fold despite intact peptide binding to HLA-B^*35∶01. These findings explain the preferential selection of Phe at the C-terminus of VY8 in HLA-B^*35∶01⁺ individuals and demonstrate that HIV-1 can exploit the limitations of T cell recognition in vivo.     

Clonal analysis of two mutations in the large subunit of RNA polymerase II of Drosophila

Summary Two mutations in the gene, RpII215, were analyzed to determine their effects on cell differentiation and proliferation. The mutations differ in that one, RpII215 ^ts(ts), only displays a conditional recessive lethality, while the other, RpII215 ^Ubl (Ubl), is a recessive lethal mutation that also displays a dominant mutant phenotype similar to that caused by the mutation Ultrabithorax (Ubx). Ubl causes a partial transformation of the haltere into a wing; however, this transformation is more complete in flies carrying both Ubl and Ubx. The present study shows that patches of Ubl/- tissue in gynandromorphs are morphologically normal. Cuticle that has lost the wild-type copy of the RpII215 locus fails to show a haltere to wing transformation, nor does it show the synergistic enhancement of Ubx by Ubl. We conclude that an interaction between the two RpII215 alleles, Ubl and RpII215 ⁺, is responsible for the mutant phenotype. Gynandromorphs carrying the ts allele, when raised at permissive temperature, display larger patches of ts/- cuticle than expected, possibly indicating that the proliferation of ts/+ cells is reduced. This might result from an antagonistic interaction between different RpII215 alleles. Classical negative complementation does not appear to be the cause of the antagonistic interaction described above, as only one RpII215 subunit is thought to be present in an active multimeric polymerase enzyme. We have therefore coined the term negative heterosis to describe the aforementioned interactions.We also observed that the effects of mutationally altered RNA polymerase II on somatic cells are different from its effects on germ cells. Mutant somatic cells (either Ubl/- or ts/-, the latter shifted to restrictive temperature) reduce cell proliferation, but otherwise do not appear to disrupt cell differentiation. However, mutant germ cells often differentiate into morphologically abnormal oocytes.     

Different composition of the cell wall polysaccharides in Saccharomyces cerevisiae S288 and in an osmosis sensitive mutant]

Determination of the polysaccharide contents and structural studies on the mannan by acetolysis and permethylation analysis shows an altered polysaccharide biosynthesis of the osmotic-sensitive mutant VY 1160 of Saccharomyces cerevisiae S 288. The mutant contains more glucan, less mannan, and less alkali-soluble glycogen. Its mannan is characterized by more short side chains and less long side chains. Its main chain is 1 leads to 6-linked, but its side chains consist of more 1 leads to 3- than 1 leads to 2-linked mannose units.     

Zymomonas mobilis mutants blocked in fructose utilization

Summary A mutant ofZymomonas mobilis deficient in the utilization of fructose for growth and ethanol formation was shown to lack fructokinase activity. When grown in media which contained glucose+fructose or sucrose, both the mutant and wild type produced sorbitol in amounts up to 60 g·l^-1, depending on the initial concentrations of sugars. Sorbitol formation was accompanied by an accumulation of acetaldehyde, gluconate, and acetoin. A ferricyanide-dependent sorbitol dehydrogenase could be localized in the cell membrane; it thus resembles the sorbitol dehydrogenase ofGluconobacter suboxydans. Neither a NAD(P)H dependent reduction of fructose nor a NAD(P) dependent dehydrogenation of sorbitol could be detected in cell-free extracts. The use of fructose-negative mutants ofZ. mobilis for the enrichment of fructose in glucose+fructose mixtures is discussed.     

Genetics of mitochondrial ribosomes of yeast: mitochondrial lethality of a double mutant carrying two mutations of the 21S ribosomal RNA gene

Summary Among the mitochondrial conditional mutations localized in the gene coding for the 21S ribosomal RNA, one — ts 902 — produces severely reduced amounts of 21S RNA and 50S subunit. We investigated its physiological properties and found that this thermosensitive mutation was associated with highly pleiotropic effects. The mutant phenotype is associated with cell death in certain conditions, and with a massive accumulation of rho^- mutants at non-permissive temperature. Furthermore, interactions with the sites of action of erythromycin and chloramphenicol, both localized within the 21S rRNA, were detected. The mutant is hypersensitive to erythromycin and has a cis-incompatibility with the chloramphenicol-resistant mutation C ₃₂₁ ^R .Ts 902 thus appears to have a dual effect, not only at the ribosomal level but also at a cellular level.     

Use of hybridoma-resistant variant cell lines to study H-2D b/FMR-specific cytotoxic T cells

An H-2D ^b b heterozygous tumor cell line and a variant subclone bearing a mutant gene product were used to analyze the H-2D^b specificity of cytotoxic T lymphocytes (CTL) generated during a Moloney murine sarcoma virus (MSV) infection. When the mutant cells were used as targets for MSV-specific CTL, the amount of cell lysis, compared with that seen with the nonmutant parental cells, was drastically decreased. However, cells of the mutant clones remained susceptible to allogeneic CTL specific for the nonmutant H-2D^b molecule. The mutant cells also did not differ from the parent cells in their level of viral antigen expression. Biochemically the parental and mutant molecules were similar but not identical. The data indicate that minor alterations of the H-2 antigens caused by somatic mutation may prevent virus-infected cells from being recognized as targets by CTL.     

A Temperature Sensitive Suppressor Mutation of Escherichia Coli

Abstract

In an Escherichia coli K 12 strain, with a Pho ^- nonsense mutation and the suppressor gene Su-4, we have induced a mutant with a temperature dependent alkaline phosphatase negative phenotype. This phenotype can be ascribed to a mutation mapping in the Su-4 gene that makes the suppression temperature sensitive.     

A UDP-glucose derivative is required for vacuolar autophagic cell death

Autophagic cell death in Dictyostelium can be dissociated into a starvation-induced sensitization stage and a death induction stage. A UDP-glucose pyrophosphorylase (ugpB) mutant and a glycogen synthase (glcS) mutant shared the same abnormal phenotype. In vitro, upon starvation alone mutant cells showed altered contorted morphology, indicating that the mutations affected the pre-death sensitization stage. Upon induction of cell death, most of these mutant cells underwent death without vacuolization, distinct from either autophagic or necrotic cell death. Autophagy itself was not grossly altered as shown by conventional and electron microscopy. Exogenous glycogen or maltose could complement both ugpB^- and glcS^- mutations, leading back to autophagic cell death. The glcS- mutation could also be complemented by 2-deoxyglucose that cannot undergo glycolysis. In agreement with the in vitro data, upon development glcS^- stalk cells died but most were not vacuolated. We conclude that a UDP-glucose derivative (such as glycogen or maltose) plays an essential energy-independent role in autophagic cell death.     

A lethal mutation which affects the maturation of ribosomes

Summary Among a group of 31 ts^- yeast mutants screened electrophoretically for heat-sensitive synthesis of each stable RNA species, only mutant ts351 failed to accumulate 25S RNA at 36°C. Pulse-labeling experiments at 36°C showed that 35S and 27S precursor RNA and mature 18S r-RNA molecules are synthesized by ts351 cells but that 25S and 5.8S RNA species are not made and new 60S ribosomal sub-units are not assembled. The mutant is blocked at a specific point in r-RNA processing: the cutting of 27S to form 25S and 5.8S r-RNA.     

Mutations in the Saccharomyces Cerevisiae Type 2a Protein Phosphatase Catalytic Subunit Reveal Roles in Cell Wall Integrity, Actin Cytoskeleton Organization and Mitosis

Temperature-sensitive mutations were generated in the Saccharomyces cerevisiae PPH22 gene that, together with its homologue PPH21, encode the catalytic subunit of type 2A protein phosphatase (PP2A). At the restrictive temperature (37°), cells dependent solely on pph22(ts) alleles for PP2A function displayed a rapid arrest of proliferation. Ts(-) pph22 mutant cells underwent lysis at 37°, showing an accompanying viability loss that was suppressed by inclusion of 1 M sorbitol in the growth medium. Ts(-) pph22 mutant cells also displayed defects in bud morphogenesis and polarization of the cortical actin cytoskeleton at 37°. PP2A is therefore required for maintenance of cell integrity and polarized growth. On transfer from 24° to 37°, Ts(-) pph22 mutant cells accumulated a 2N DNA content indicating a cell cycle block before completion of mitosis. However, during prolonged incubation at 37°, many Ts(-) pph22 mutant cells progressed through an aberrant nuclear division and accumulated multiple nuclei. Ts(-) pph22 mutant cells also accumulated aberrant microtubule structures at 37°, while under semi-permissive conditions they were sensitive to the microtubule-destabilizing agent benomyl, suggesting that PP2A is required for normal microtubule function. Remarkably, the multiple defects of Ts(-) pph22 mutant cells were suppressed by a viable allele (SSD1-v1) of the polymorphic SSD1 gene.     

Genetic studies with a phosphoglucose isomerase mutant of Saccharomyces cerevisiae.

Summary A mutation pgi1 in the yeast Saccharomyces cerevisiae conferring deficiency of the glycolytic enzyme glucose 6-phosphate isomerase is characterised genetically. The mutation segregates 2⁺:2^- in tetrads from diploids heterozygous for the mutant phenotype. The mutation is semi-dominant and is located on the right arm of chromosome II in the order: tsm134-lys2-pgi1-tyr1 approximately 15 map units from tyr1. The mutation pgi1 defines the structural gene of glucose 6-phosphate isomerase and can be suppressed intragenically giving revertants that have an unstable enzyme. In one temperature-sensitive revertant no enzyme activity in excess of the mutant level could be detected although fructose 6-phosphate was converted to glucose 6-phosphate in vivo. The suppressor locus in this revertant is dominant and is unlinked to the pgi1 locus.     

Mutants of Pseudomonas aeruginosa unable to inactivate allantoinase and NADP-dependent glutamate dehydrogenase

Both allantoinase and NADP-GDH in Pseudomonas aeruginosa were inactivated when cells reached the stationary phase of growth. Mutants unable to inactivate these enzymes were isolated. Results with recombinants showed that the mutation is not located in the structural genes of these enzymes but in an independent gene involved in the inactivation.Abbreviations NADP-GDH NADP-dependent glutamate dehydrogenase - Ani^- mutant allantoinase non-inactivating mutant - GOGAT glutamate synthase     

A missense mutation P136L in the arylsulfatase A gene causes instability and loss of activity of the mutant enzyme

Metachromatic leukodystrophy is a lysosomal storage disease caused by deficiency of arylsulfatase A. Sequencing of the arylsulfatase A genes of an Ashkenazi Jewish patient suffering from the severe late infantile form of the disease revealed a point mutation in exon 2 causing proline 136 to be substituted by leucine. The patient was homozygous for this mutation. Studies on Ltk^- cells stably expressing the mutant enzyme show that the mutation causes complete loss of enzyme activity and rapid degradation in an early biosynthetic compartment.