Cerevisiae Extracts Research Articles

Thermal and storage stability of Saccharomyces cerevisiae (baker's yeast) allergens

The effect of storage and high temperatures on the stability of Saccharomyces cerevisiae allergens was studied by immunoblotting. Saccharomyces cerevisiae allergic serum pool and 125I- and galactosidase-labelled anti-IgE were used in the assays. Freeze-dried extracts were reconstituted with saline and with 50% glycerol and then stored at room (+20 degrees C) and refrigerator temperature (+6 degrees C) for different time periods. The stability was better in 50% glycerol at +6 degrees C than at room temperature without glycerol. However, after 1 month, two of the most important allergens of S. cerevisiae, the 48 and 32 kDa protein allergens, lost their IgE-binding capacity even in the extracts stored with 50% glycerol at +6 degrees C. The 45 kDa allergen was, on the other hand, quite stable after storage for 9 months at +6 degrees C. Although the beneficial effect of 50% glycerol was clear, storage at +6 degrees C, even with 50% glycerol should not exceed 1 month for S. cerevisiae extracts. Two commercially available S. cerevisiae extracts in solution with valid expiry dates were also analysed. They had only little allergenic potency, while a freeze-dried extract stored for 8 years showed good allergenic potency. Heating S. cerevisiae extracts resulted in precipitation, the precipitated fraction contained almost all the specific proteins as judged by electrophoresis and IgE detection. The supernatant fraction contained only a few allergens.

The 5′-leader sequence of tobacco mosaic virus RNA mediates initiation-factor-4E-independent, but still initiation-factor-4A-dependent translation in yeast extracts

Messenger RNAs encoding chloramphenicol acetyltransferase (CAT) with or without the 5′-leader sequence of tobacco mosaic virus (TMV) RNA were synthesized in vitro and translated in Saccharomyces cerevisiae extracts dependent on eukaryotic initiation factors eIF-4E or eIF-4A. The 5′-leader sequence of TMV RNA renders translation of CAT mRNA eIF-4E-independent but still 4A-dependent.

Molecular tools for inactivating a yeast enzyme in vivo.

As part of an effort to develop a new means of inducibly inactivating cellular proteins in vivo, three monoclonal antibodies which neutralize yeast alcohol dehydrogenase (ADH) activity were isolated and characterized with respect to criteria important for the inactivation strategy. The significance of these criteria is considered, and a general means of generating appropriate antibodies is suggested. All three antibodies described here were specific for ADH I; they did not recognize the closely related isozyme ADH II in a plate-binding assay and did not immunoprecipitate molecules other than ADH from a Saccharomyces cerevisiae extract. Neutralization occurred in a yeast extract and, for two antibodies, was blocked by high concentrations of the coenzyme NAD+. This finding suggests that the antibodies may block enzyme activity by stabilizing an inactive form of ADH lacking bound NAD+. These results provide a foundation for the use of these antibodies to inactivate ADH in vivo.

Molecular tools for inactivating a yeast enzyme in vivo.

As part of an effort to develop a new means of inducibly inactivating cellular proteins in vivo, three monoclonal antibodies which neutralize yeast alcohol dehydrogenase (ADH) activity were isolated and characterized with respect to criteria important for the inactivation strategy. The significance of these criteria is considered, and a general means of generating appropriate antibodies is suggested. All three antibodies described here were specific for ADH I; they did not recognize the closely related isozyme ADH II in a plate-binding assay and did not immunoprecipitate molecules other than ADH from a Saccharomyces cerevisiae extract. Neutralization occurred in a yeast extract and, for two antibodies, was blocked by high concentrations of the coenzyme NAD+. This finding suggests that the antibodies may block enzyme activity by stabilizing an inactive form of ADH lacking bound NAD+. These results provide a foundation for the use of these antibodies to inactivate ADH in vivo.

Structure-activity relationships of sparsomycin: modification at the hydroxyl group

Ten analogues of the antibiotic sparsomycin were prepared and evaluated in several in vitro tests. Nine of them carry a modification at the hydroxymethylene group of the molecules, two have a disulfide bond instead of the S(O)-CH 2-S moiety at the sulfur-containing side chain of the molecules. While the presence of the S-S group decreases the activity of the analogues in all the tests performed, the modification at the OH group has no deleterious effects on the activity when a polyphenylalanine synthesis assay is used in an Escherichia coli extract. The same modifications, however, diminish drastically the activity of the analogues when tested in a similar Saccharomyces cerevisiae extract. A polymerization system in the archaebacterium Halobacterium halobium extract behaves like the eukaryotic preparations. A discrepancy is also found between the results of the polymerization tests and those of the ‘puromycin reaction’ which is also less sensitive to the modified sparsomycin analogues. The results of cell growth inhibition tests in bacteria as well as in eukaryotic organisms agree only partially with the in vitro data.

Evidence for nonregulatory trehalase activity inDictyostelium discoideum

An in vitro activation treatment, stimulatory to the regulatory cytoplasmic trehalase ofSaccharomyces cerevisiae, had no effect on the lysosomal trehalase ofDictyostelium discoideum. Concentrations of cAMP that produced a 19 to 22-fold increase in trehalase activity inS. cerevisiae extracts did not stimulate trehalase activity inD. discoideum extracts.. cGMP and 5′-AMP were also not effective in activating the enzyme.Dictyostelium discoideum trehalase exhibits characteristics typical of nonregulatory trehalases, in agreement with its lysosomal localization. The data are consistent with the hypothesis that changes in compartmentation regulate trehalose mobilization inD. discoideum.

Assembly of a tRNA splicing complex: evidence for concerted excision and joining steps in splicing in vitro.

Splicing of tRNA precursors in Saccharomyces cerevisiae extracts proceeds in two steps; excision of the intervening sequence and ligation of the tRNA halves. The ability to resolve these two steps and the distinct physical properties of the endonuclease and ligase suggested that the splicing steps may not be concerted and that these two enzymes may act independently in vivo. A ligase competition assay was developed to examine whether the excision and ligation steps in tRNA splicing in vitro are concerted or independent. The ability of either yeast ligase or T4 ligase plus kinase to join the tRNA halves produced by endonuclease and the distinct structures of the reaction products provided the basis for the competition assay. In control reactions, joining of isolated tRNA halves formed by preincubation with endonuclease was measured. The ratio of yeast to T4 reaction products in these control assays reflected the ratio of the enzyme activities, as would be expected if each has equal access to the substrate. In splicing competition assays, endonuclease and pre-tRNA were added to ligase mixtures, and joining of the halves that were formed was measured. In these assays the products were predominantly those of the yeast ligase even when the T4 enzymes were present in excess. These results demonstrate preferential access of yeast ligase to the endonuclease products and provide evidence for the assembly of a functional tRNA splicing complex in vitro. This observation has important implications for the organization of the splicing components and of the gene expression pathway in vivo.

Assembly of a tRNA splicing complex: evidence for concerted excision and joining steps in splicing in vitro.

Splicing of tRNA precursors in Saccharomyces cerevisiae extracts proceeds in two steps; excision of the intervening sequence and ligation of the tRNA halves. The ability to resolve these two steps and the distinct physical properties of the endonuclease and ligase suggested that the splicing steps may not be concerted and that these two enzymes may act independently in vivo. A ligase competition assay was developed to examine whether the excision and ligation steps in tRNA splicing in vitro are concerted or independent. The ability of either yeast ligase or T4 ligase plus kinase to join the tRNA halves produced by endonuclease and the distinct structures of the reaction products provided the basis for the competition assay. In control reactions, joining of isolated tRNA halves formed by preincubation with endonuclease was measured. The ratio of yeast to T4 reaction products in these control assays reflected the ratio of the enzyme activities, as would be expected if each has equal access to the substrate. In splicing competition assays, endonuclease and pre-tRNA were added to ligase mixtures, and joining of the halves that were formed was measured. In these assays the products were predominantly those of the yeast ligase even when the T4 enzymes were present in excess. These results demonstrate preferential access of yeast ligase to the endonuclease products and provide evidence for the assembly of a functional tRNA splicing complex in vitro. This observation has important implications for the organization of the splicing components and of the gene expression pathway in vivo.

Mutations preventing expression of sup3 tRNASer nonsense suppressors of Schizosaccharomyces pombe.

Suppression of nonsense codons in Schizosaccharomyces pombe by sup3-e tRNASerUGA or sup3-i tRNASerUAA is reduced or abolished by mutations within the suppressor locus. Twenty-five suppressor-inactive sup3-e genes and thirteen mutant sup3-i genes were isolated from S. pombe genomic clone banks by colony hybridization. Sequence analysis of these revertant alleles corroborates genetic evidence for mutational hotspots within the sup3 tRNA gene. Fifteen types of point mutations or insertions were found. Many of these replace bases which are highly or completely conserved in eucaryotic tRNA genes. Transcription of the altered sup3 genes in a Saccharomyces cerevisiae extract enabled the identification of mutations which affect the rate of 5'-end maturation or splicing of the tRNA precursors or both. A total of seven mutations were found which alter transcriptional efficiencies. Of these, five are located outside the internal transcription control regions.

Mutations preventing expression of sup3 tRNASer nonsense suppressors of Schizosaccharomyces pombe.

Suppression of nonsense codons in Schizosaccharomyces pombe by sup3-e tRNASerUGA or sup3-i tRNASerUAA is reduced or abolished by mutations within the suppressor locus. Twenty-five suppressor-inactive sup3-e genes and thirteen mutant sup3-i genes were isolated from S. pombe genomic clone banks by colony hybridization. Sequence analysis of these revertant alleles corroborates genetic evidence for mutational hotspots within the sup3 tRNA gene. Fifteen types of point mutations or insertions were found. Many of these replace bases which are highly or completely conserved in eucaryotic tRNA genes. Transcription of the altered sup3 genes in a Saccharomyces cerevisiae extract enabled the identification of mutations which affect the rate of 5'-end maturation or splicing of the tRNA precursors or both. A total of seven mutations were found which alter transcriptional efficiencies. Of these, five are located outside the internal transcription control regions.

Transcription of a Drosophila tRNAArg gene in yeast extract: 5'-flanking sequence dependence for transcription in a heterologous system.

The Drosophila tRNA gene encoded on pArg is efficiently transcribed in extracts of Saccharomyces cerevisiae, but the efficiency is 5'-flanking sequence dependent: deletion to between positions -21 and -17 (relative to position +1 of the mature coding sequence) reduces transcription to a very low level. This demonstrates that requirement for wild-type 5'-flanking sequence exists in the case of a heterologous combination of a tRNA gene and transcription extract. Expression of pArg in vivo in S. cerevisiae is also dependent on the wild-type 5'-flanking sequence, but only with deletion to between -17 and -11 is the steady-state level of pArg transcripts reduced to near zero. The 5'-flanking sequence requirement in S. cerevisiae extract is similar to that found in Drosophila Kc cell extract. However, transcription kinetics distinguish S. cerevisiae extract from that of Drosophila Kc cells. tRNA genes added to S. cerevisiae extract exhibit a lag phase before initiation of active transcription, but this lag is much shorter and much less temperature dependent than is the lag phase in Drosophila Kc cell extract.