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U Bond

Publications and source records attributed to U Bond.

18 recordsLinked to original sources
PubMed2003

Transcription profile of brewery yeast under fermentation conditions.

AIMS: Yeast strains, used in the brewing industry, experience distinctive physiological conditions. During a brewing fermentation, yeast are exposed to anaerobic conditions, high pressure, high specific gravity and low temperatures. The purpose of this study was to examine the global gene expression profile of yeast subjected to brewing stress. METHODS AND RESULTS: We have carried out a microarray analysis of a typical brewer's yeast during the course of an 8-day fermentation in 15 degrees P wort. We used the probes derived from Saccharomyces cerevisiae genomic DNA on the chip and RNA isolated from three stages of brewing. This analysis shows a high level of expression of genes involved in fatty acid and ergosterol biosynthesis early in fermentation. Furthermore, genes involved in respiration and mitochondrial protein synthesis also show higher levels of expression. CONCLUSIONS: Surprisingly, we observed a complete repression of many stress response genes and genes involved in protein synthesis throughout the 8-day period compared with that at the start of fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: This microarray data set provides an analysis of gene expression under brewing fermentation conditions. The data provide an insight into the various metabolic processes altered or activated by brewing conditions of growth. This study leads to future experiments whereby selective alterations in brewing conditions could be introduced to take advantage of the changing transcript profile to improve the quality of the brew.

Anaerobiosis↗
PubMed2000

Dynamic changes in small nuclear ribonucleoproteins of heat-stressed and thermotolerant HeLa cells.

Living organisms when subjected to various forms of environmental stress mount a physiological response to survive the long- and short-term ill-effects of the stress. The stress response may involve selective shut down of non-essential metabolic activities and the repair of macromolecular damage resulting from the stress. Messenger RNA splicing in cultured HeLa cells is one of the processes inhibited by heat stress. Splicing is protected from such inhibition in stress-preconditioned cells that have acquired a tolerant state characterised by increased cell survival and resistance to other environmental stresses. Stress tolerant cells have heat shock proteins (HSPs) that had been induced by the preconditioning process. To examine the biochemical changes induced by stress in the splicing apparatus, we analysed the small nuclear ribonucleoprotein (snRNP) particles associated with spliceosomes in normal, stressed, and stress tolerant cells. We show that (a) the spliceosomal component U4/U5/U6 snRNP particle is disassembled by heat stress into intermediates of splicing assembly, (b) prior induction of stress tolerance protects the structural and functional integrity of snRNPs if cells are subsequently exposed to a severe stress and (c) a novel 65 kDa protein is associated with small nuclear ribonucleoprotein particles in stress tolerant cells.

HeLa Cells↗
PubMed2000

The stress response is repressed during fermentation in brewery strains of yeast.

Yeast cells encounter a variety of environmental stresses during brewing and must respond to ensure cell survival. Cells can respond to stress by inducing a Heat Shock Response in which heat shock proteins (Hsps) are synthesized. In laboratory strains of Saccharomyces cerevisiae, the heat shock protein, Hsp104, plays a major role in the acquisition of tolerance to a variety of stresses such as heat, ethanol and sodium arsenite, and as such acts as an excellent stress indicator. The induction of Hsp104 in bottom-and top-fermenting brewery strains was examined when grown under laboratory and industrial fermentation conditions, and it was found that each brewing strain exhibits its own unique pattern of Hsp104 expression. During industrial fermentations, brewery strains are capable of mounting a stress response at the early stages of fermentation. However, as the fermentation proceeds, the response is repressed. The results suggest that conditions experienced in industrial brewing prevent the activation of the stress response. This study increases our understanding of alterations in gene expression patterns during the brewing process, and yields information that will aid in the definition of best practice in yeast management.

Aerobiosis↗
PubMed1999

Reassembly and protection of small nuclear ribonucleoprotein particles by heat shock proteins in yeast cells.

The process of mRNA splicing is sensitive to in vivo thermal inactivation, but can be protected by pretreatment of cells under conditions that induce heat-shock proteins (Hsps). This latter phenomenon is known as "splicing thermotolerance". In this article we demonstrate that the small nuclear ribonucleoprotein particles (snRNPs) are in vivo targets of thermal damage within the splicing apparatus in heat-shocked yeast cells. Following a heat shock, levels of the tri-snRNP (U4/U6.U5), free U6 snRNP, and a pre-U6 snRNP complex are dramatically reduced. In addition, we observe multiple alterations in U1, U2, U5, and U4/U6 snRNP profiles and the accumulation of precursor forms of U4- and U6-containing snRNPs. Reassembly of snRNPs following a heat shock is correlated with the recovery of mRNA splicing and requires both Hsp104 and the Ssa Hsp70 family of proteins. Furthermore, we correlate splicing thermotolerance with the protection of a subset of snRNPs by Ssa proteins but not Hsp104, and show that Hsp70 directly associates with U4- and U6-containing snRNPs in splicing thermotolerant cells. In addition, our results show that Hsp70 plays a role in snRNP assembly under normal physiological conditions.

Fungal Proteins↗
PubMed1995

Characterization of a new class of transcribed repetitive DNA sequence which also exists as a hybrid with HP1 mRNA; potential for site-specific recombination in Drosophila melanogaster.

A new class of dispersed repetitive DNA designated as vivi-sequence (VS) has been identified in Drosophila. It is relatively AT rich and is transcribed. The VS transcription is developmentally regulated and generates multiple transcripts. A hybrid transcript, designated fl-cDNA, has been identified in which a small segment of the VS is fused to the 5' end of an unrelated structural gene transcript coding for the heterochromatin protein HP1. The VS has recombination signal sequences (RSS) characteristic of vertebrate immunoglobulin genes. Such sequences are also present in the HP1 DNA. In both cases the recombination signal sequences are found close to the junction between HP1 and the VS in fl-cDNA. There is additional sequence identity both 5' and 3' of the junction between HP1 and the VS in fl-cDNA. We propose that (a) the HP1-VS composite transcript represented by the fl-cDNA may be the product of recombination between the two sequences, (b) that the process is mediated by the RSS and/or the DNA downstream of the junction between HP1 and the VS and (c) that the recombination event may lead to the inactivation of the HP1 gene in a cell and tissue specific manner.

Animals↗
PubMed1994

Point mutations in the stem-loop at the 3' end of mouse histone mRNA reduce expression by reducing the efficiency of 3' end formation.

Mammalian histone mRNAs end in a highly conserved stem-loop structure, with a six-base stem and a four-base loop. We have examined the effect of mutating the stem-loop on the expression of the histone mRNA in vivo by introducing the mutated histone genes into CHO cells by stable transfection. Point mutations have been introduced into the loop sequence and into the UA base pair at the top of the stem. Changing either the first or the third base of the conserved UYUN sequence in the loop to a purine greatly reduced expression, while changing both U's to purines abolished expression. A number of alterations in the stem sequence, including reversing the stem sequence, reversing the two base pairs at the base of the stem, or destroying the UA base pair at the top of the stem, also abolished expression. Changing the UA base pair to a CG or a UG base pair also reduced expression. The loss of expression is due to inefficient processing of the pre-mRNA, as judged by the efficiency of processing in vitro. Addition of a polyadenylation site or the wild-type histone processing signal downstream of a mutant stem-loop resulted in rescuing the processing of the mutant pre-histone mRNA. These results suggest that if the histone pre-mRNA is not rapidly processed, then it is degraded.

Animals↗
PubMed1994

The steady state levels and structure of the U7 snRNP are constant during the human cell cycle: lack of cell cycle regulation of histone mRNA 3' end formation.

The U7 small nuclear ribonucleoprotein (snRNP) is an essential component of the endonucleolytic cleavage reaction which leads to the production of mature 3'-ends of histone premRNAs. We have examined the relative amount and the structure of the U7 snRNP, as assayed by sensitivity to micrococcal nuclease, during the cell cycle in human HeLa and WI-38 cells. Using an RNase A protection assay, we find no change in the steady state levels of U7 throughout the cell cycle. Similarly, the sensitivity of U7 to micrococcal nuclease remained unchanged in both cell types. Contact inhibited WI-38 cells, that are deemed to have left the cell cycle and entered a quiescent state, displayed similar levels of U7 to cells in S and G1 phases of the cell cycle, however, the U7 snRNA was slightly more resistant to micrococcal nuclease. Histone 3' end mRNA processing was also assayed in HeLa cell cycle phase-specific extracts. In marked contrast to previous observations in extracts prepared from the rodent cell line, C3H10T1/2, (Hoffmann and Birnstiel, 1990), we find that the 3' end processing reaction remained constant throughout the cell cycle.

Base Sequence↗
PubMed1990

Heat shock RNA levels in brain and other tissues after hyperthermia and transient ischemia.

A number of studies have demonstrated increased synthesis of heat shock proteins in brain following hyperthermia or transient ischemia. In the present experiments we have characterized the time course of heat shock RNA induction in gerbil brain after ischemia, and in several mouse tissues after hyperthermia, using probes for RNAs of the 70-kilodalton heat shock protein (hsp70) family, as well as ubiquitin. A synthetic oligonucleotide selective for inducible hsp70 sequences proved to be the most sensitive indicator of the stress response whereas a related rat cDNA detected both induced RNAs and constitutively expressed sequences that were not strongly inducible in brain. Considerable polymorphism of ubiquitin sequences was evident in the outbred mouse and gerbil strains used in these studies when probed with a chicken ubiquitin cDNA. Brief hyperthermic exposure resulted in striking induction of hsp70 and several-fold increases in ubiquitin RNAs in mouse liver and kidney peaking 3 h after return to room temperature. The oligonucleotide selective for hsp70 showed equivalent induction in brain that was more rapid and transient than observed in liver, whereas minimal induction was seen with the ubiquitin and hsp70-related cDNA probes. Transient ischemia resulted in 5- to 10-fold increases in hsp70 sequences in gerbil brain which peaked at 6 h recirculation and remained above control levels at 24 h, whereas a modest 70% increase in ubiquitin sequences was noted at 6 h. These results demonstrate significant temporal and quantitative differences in heat shock RNA expression between brain and other tissues following hyperthermia in vivo, and indicate that hsp70 provides a more sensitive index of the stress response in brain than does ubiquitin after both hyperthermia and ischemia.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acids↗
PubMed1989

Analysis of pre-rRNAs in heat-shocked HeLa cells allows identification of the upstream termination site of human polymerase I transcription.

Human rRNA precursor from normal or stressed HeLa cells were studied by S1 nuclease mapping of unlabeled RNA and by antisense RNase mapping of RNA from cells that had been labeled in vivo with [32P]PO4. Heating cells to 43 degrees C decreased the amount of newly synthesized rRNA to less than 5% of the control level and led to greater than 95% inhibition of transcription termination at a region 355 to 362 nucleotides downstream of the 3' end of 28S rRNA, with readthrough continuing into the next transcription unit. Heating of cells to 42 degrees C led to 60% inhibition of termination at this site; 50% of transcripts that extended into the nontranscribed spacer ended in a region 200 to 210 nucleotides upstream of the polymerase I (Pol I) initiation site. This is presumed to be the human upstream transcription termination site because of the absence of RNAs with a 5' end corresponding to this region, the location relative to the Pol I initiation site (which is similar to the location of upstream terminators in other species), and the fact that it is 15 to 25 nucleotides upstream of the sequence GGGTTGACC, which has an 8-of-9 base identity with the sequence 3' of the downstream termination site. Surprisingly, treatment of cells with sodium arsenite, which also leads to the induction of a stress response, did not inhibit termination. Pol I initiation was decreased to the same extent as termination, which lends support to the hypothesis that termination and initiation are coupled. Although termination was almost completely inhibited at 43 degrees C, the majority of the recently synthesized rRNAs were processed to have the correct 3' end of 28S. This finding suggests that 3'-end formation can involve an endonucleolytic cut and is not solely dependent on exonucleolytic trimming of correctly terminated rRNAs.

Animals↗
PubMed1988

Ubiquitin in stressed chicken embryo fibroblasts.

Ubiquitin, a small 76-amino acid protein which is highly conserved in eukaryotic cells, occurs in several forms other than the free polypeptide. Among these are protein conjugates in which ubiquitin is covalently linked in lysylpeptide bond to lysl residues of other proteins and fusion proteins in which the amino-terminal domain is the precise ubiquitin sequence. Ubiquitin plays a role in cellular proteolytic degradation and in chromatin structure and has been postulated to be involved in the induction of a set of proteins which function during the cellular response to various kinds of environmental stress. We have measured the various forms of ubiquitin in cultures of chicken embryo fibroblasts under normal growth conditions and after treatment with a thermal or chemical stress. Levels of free ubiquitin fell slightly, ubiquitin conjugate levels rose shortly after stress began, and both then increased substantially as one of the cell's ubiquitin-encoding genes was activated by stress. The level of a protein synthesized as the carboxyl-terminal domain of one ubiquitin fusion protein was unchanged by a heat stress. The most dramatic effect was seen in the rapid disappearance of the ubiquitinated form of histone H2A, one of the major ubiquitin conjugates in cells in the interphase portion of their growth cycle. A significant rise in protein turnover was detected as a result of the stress, but occurred only when cells were removed from the stress condition. These results suggest that ubiquitin plays an important role both during and after stress, but fails to support hypotheses for ubiquitin and proteolysis in the activation of stress genes.

Animals↗
PubMed1988

Heat shock but not other stress inducers leads to the disruption of a sub-set of snRNPs and inhibition of in vitro splicing in HeLa cells.

Splicing of pre-mRNA in HeLa cells exposed to various stress response inducers has been investigated. In vivo, intron-containing transcripts of the hsp27 gene accumulate in cells stressed by heat or sodium arsenite. In vitro analysis, however, reveals a differential effect of stress on splicing: nuclear extracts from cells exposed to a severe heat shock are incapable of splicing an exogenously supplied substrate while splicing is not perturbed in extracts treated with sodium arsenite, the amino acid analog canavinine or ethanol. Pretreatment of cells with a mild heat shock prior to a severe heat shock protects the splicing apparatus and allows splicing to proceed unimpeded. Analyses of the splicing defect in extracts from heat-shocked cells show that the inhibition of splicing cannot be accounted for by changes in the major RNA and protein components of small nuclear ribonucleoprotein particles (snRNPs) or in a previously described heat-labile factor that is essential for in vitro splicing. Fractionation of small nuclear ribonucleoprotein particles from heat-shock extracts by native polyacrylamide gel electrophoresis reveals dramatic changes in certain particles, most noticeably in a U4/U5/U6 snRNP complex and the U2 snRNP. Alterations in these particles are accompanied by the assembly of labeled pre-mRNA transcript into aberrant splicing complexes that differ from those formed in normal extracts.

Arsenic↗
PubMed1988

In vitro proteolytic processing of a diubiquitin and a truncated diubiquitin formed from in vitro-generated mRNAs.

Ubiquitin, a highly conserved protein of 76 amino acids found in all eukaryotes, is translated from mRNAs that contain either multiple, contiguous coding sequences of the protein or a single ubiquitin coding sequence fused to sequences coding for 52 or 76 amino acids. We describe here formation of monoubiquitin from in vitro translation of mRNAs containing either two complete sequences or one complete ubiquitin and 60% of a second ubiquitin. No diubiquitin precursor was found with the complete diubiquitin mRNA, but the truncated mRNA formed proteins with apparent molecular masses of 30, 24, 7, and 4 kDa. The latter two are the expected products from truncated ubiquitin mRNA. The 30-kDa protein was immunoprecipitated by anti-ubiquitin antibodies and was converted to ubiquitin and the 4-kDa form by a ubiquitin isopeptidase-like activity in wheat germ. Other data indicated that the 30-kDa protein had multiple ubiquitins, all linked by isopeptide bonds to the truncated ubiquitin. One of these was the radiolabeled translation product, which should have been linked to the truncated protein by a normal peptide bond. A model is proposed in which ubiquitin itself participates in a transpeptidase activity.

Cloning, Molecular↗
PubMed1987

Heat-shock proteins and development.

At the simplest level there is little doubt that the heat shock response is homeostatic, to protect the cell against the ravages of the environmental insult and ensure that the cell can continue its normal life after the crisis has passed.

Aging↗
PubMed1987

Ubiquitin genes.

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Amino Acid Sequence↗
PubMed1986

The chicken ubiquitin gene contains a heat shock promoter and expresses an unstable mRNA in heat-shocked cells.

A chicken genomic library was screened to obtain genomic clones for ubiquitin genes. Two genes that differ in their genomic location and organization were identified. One gene, designated Ub I, contains four copies of the protein-coding sequence arranged in tandem, while the second gene, Ub II, contains three. The origin of the two major mRNAs that are induced after heat shock in chicken embryo fibroblasts was determined by generating DNA probes from the 5'-and 3'-noncoding regions of the two genes. Both mRNAs are transcribed from Ub I, the larger being the unspliced precursor of the smaller. A 674-base-pair intron was located within the 5'-noncoding region of Ub I. The second gene, Ub II, does not appear to code for an RNA species in normal or heat-shocked chicken embryo fibroblasts. The expression of ubiquitin mRNA during heat shock and recovery was examined. Addition of actinomycin D before heat shock completely abolished the response of ubiquitin mRNA to the stress. Analysis of the stability of the mRNA during recovery revealed that the mRNA accumulated during the heat shock is rapidly degraded with a half-life of approximately 1.5 h, suggesting a specialized but transient role for ubiquitin during heat shock.

Animals↗
PubMed1985

Ubiquitin is a heat shock protein in chicken embryo fibroblasts.

Clones containing heat-inducible mRNA sequences were selected from a cDNA library prepared from polyadenylated RNA isolated from heat-shocked chicken embryo fibroblasts. One recombinant DNA clone, designated clone 7, hybridized to a 1.2-kilobase RNA that was present in normal cells and increased fivefold during heat shock. Clone 7 also hybridized to an RNA species of 1.7 kilobases that was present exclusively in heat-shocked cells. In vitro translation of mRNA hybrid selected from clone 7 produced a protein product with a molecular weight of approximately 8,000. Increased synthesis of a protein of similar size was detected in chicken embryo fibroblasts after heat shock. DNA sequence analysis of clone 7 indicated its protein product has amino acid sequences identical to bovine ubiquitin. In addition, clone 7 contains tandem copies of the ubiquitin sequences contiguous to each other with no untranslated sequences between them. We discuss some possible roles for ubiquitin in the heat shock response.

Amino Acid Sequence↗
PubMed1982

The slow kinetic transients of arylsulphatase A.

A simple model is described to account for the anomalous time course of arylsulphatase A. In the case of the ox liver and human placental enzymes the enzyme-nitrocatechol sulphate complex can, in addition to forming products, slowly break down to form an inactive species which can turn in slowly regenerate active enzyme. When the inactive form binds sulphate the rate of reactivation is enhanced, 218-fold in the case of the ox enzyme. Rat liver arylsulphatase A is refractory to reactivation by sulphate.

Animals↗