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S Lindquist

Publications and source records attributed to S Lindquist.

At least 109 records · Page 6Linked to original sources
PubMed1988

The Drosophila hsp70 message is rapidly degraded at normal temperatures and stabilized by heat shock.

When heat-shocked Drosophila cells are returned to normal temperatures, heat-shock protein (HSP) synthesis is repressed and normal protein synthesis is restored. The repression of HSP70 synthesis is accompanied by the selective degradation of its mRNA. We have engineered cells to produce a modified hsp70 mRNA that behaves exactly as the wild-type message. That is, it is stable during heat shock but degraded during recovery when protein synthesis returns to normal. When this message, placed under the control of the metallothionein promoter, is induced at normal temperatures it is rapidly degraded, with a half life of 15-30 min. Apparently, the hsp70 message is inherently unstable. During heat-shock, degradation of the message is suspended; during recovery degradation is restored.

Animals↗
PubMed1988

Characterization of an HSP70 Cognate Gene Family in Arabidopsis.

Analysis of the polypeptide composition of extracts from heat-shocked leaves of Arabidopsis indicated the presence of at least 12 HSP70-related polypeptides, most of which were constitutively expressed. In vitro translation of mRNA from heat-shocked and control leaves indicated that the amount of mRNA encoding four HSP70 polypeptides was increased strongly by heat-shock. Three Arabidopsis genes which exhibit homology to a Drosophila HSP70 gene were cloned. Two of the three genes are arranged in direct orientation approximately 1.5 kilobases apart. The third gene is not closely linked to the other two. Nucleotide sequence analysis of the 5' regions of the two linked genes revealed that both contain a TATA box, the CAAT motif, and several short sequences which are homologous to the Drosophila heat-shock consensus sequence. The deduced partial amino acid sequence of the open reading frames were 79 and 72% homologous to the corresponding regions of the Drosophila HSP70-cognate and HSP70 sequences, respectively. As with the two maize HSP70 genes which have been characterized, and the Drosophila HSP70-cognate genes, the Arabidopsis genes contained a putative intron in the codon specifying amino acid 72. Analysis of mRNA levels with gene-specific oligonucleotide probes indicated that two of the genes were not expressed or were expressed at very low levels in leaves during normal growth or after heat-shock, whereas the other gene was constitutively expressed. By analogy with the results of similar studies of other organisms, it appears that the three cloned genes are members of a small family which are most closely related to the HSP70-cognate genes found in other species.

Journal Article↗
PubMed1987

Inactivation of the ampD gene causes semiconstitutive overproduction of the inducible Citrobacter freundii beta-lactamase.

In Citrobacter freundii and Enterobacter cloacae, synthesis of AmpC beta-lactamase is inducible by the addition of beta-lactams to the growth medium. Spontaneous mutants that constitutively overproduce the enzyme occur at a high frequency. When the C. freundii ampC beta-lactamase gene is cloned into Escherichia coli together with the regulatory gene ampR, beta-lactamase expression from the clone is inducible. Spontaneous cefotaxime-resistant mutants were selected from an E. coli strain carrying the cloned C. freundii ampC and ampR genes on a plasmid. Virtually all isolates had chromosomal mutations leading to semiconstitutive overproduction of beta-lactamase. The mutation ampD2 in one such mutant was caused by an IS1 insertion into the hitherto unknown ampD gene, located between nadC and aroP at minute 2.4 on the E. coli chromosome. The wild-type ampD allele cloned on a plasmid could fully trans-complement beta-lactamase-overproducing mutants of both E. coli and C. freundii, restoring the wild-type phenotype of highly inducible enzyme synthesis. This indicates that these E. coli and C. freundii mutants have their lesions in ampD. We hypothesize that induction of beta-lactamase synthesis is caused by blocking of the AmpD function by the beta-lactam inducer and that this leads directly or indirectly to an AmpR-mediated stimulation of ampC expression.

Ampicillin↗
PubMed1986

Hsp26 is not required for growth at high temperatures, nor for thermotolerance, spore development, or germination.

Hsp26 is one of the major heat shock proteins of eukaryotic cells. It is also strongly induced at particular times during development at normal temperatures. We have isolated the unique gene for this protein from the yeast Saccharomyces cerevisiae, and we have used it to create disruption and deletion mutations. Surprisingly, the mutations have no detectable effect on the following characteristics: growth rates at various temperatures, in fermentative or in respiratory metabolism, in rich or in minimal media; the acquisition of thermotolerance in log phase or in stationary phase cells; resistance to ethanol; spore development; thermoresistance during sporulation; spore germination; thermoresistance of mature or germinating spores; or survival after long-term storage in stationary phase or as spores.

Chromosome Deletion↗
PubMed1986

Subcellular differentiation in sporulating yeast cells.

We have previously described the induction of two sets of sporulation-specific mRNAs in Saccharomyces cerevisiae. Herein we correlate the appearance of these RNAs with the major morphogenic events of sporulation, and we analyze the spatial distribution of the RNAs within the ascus. Several observations suggest that the first set of messages is involved in spore wall synthesis. In fractionation experiments, these mRNAs are detected in the ascal cytoplasm but not in developing spores, indicating that the proteinaceous component of the spore wall is synthesized from the external compartment. The second set of messages is induced later in the course of spore maturation. These mRNAs accumulate within the spores and, unlike the first set of mRNAs, are retained in mature asci until the early stages of germination. We conclude that the development of ascospores proceeds through the differentiation of functionally distinct subcellular compartments.

Cell Compartmentation↗
PubMed1986

RNA splicing is interrupted by heat shock and is rescued by heat shock protein synthesis.

The transcripts of most eukaryotic genes contain intervening sequences and must be spliced to yield functional messenger RNA. We report that a brief severe heat shock blocks the processing of intervening sequences in Drosophila cells and that this block persists for at least 2 hr after cells are returned to normal temperatures. If a mild heat shock, which induces the synthesis of heat shock proteins, is administered prior to the severe heat shock, processing occurs under otherwise restrictive conditions. When heat shock protein synthesis is inhibited, this protection is not observed. We suggest that the disruption of intron processing contributes to heat-induced lethality and developmental abnormalities and that one function of the heat shock proteins is to protect processing from heat-induced disruption.

Animals↗
PubMed1986

An ancient developmental induction: heat-shock proteins induced in sporulation and oogenesis.

Every eukaryotic and prokaryotic organism tested to date synthesizes a small number of heat-shock proteins in response to heat and other forms of stress. A particular pattern of heat-shock gene expression was observed during ascospore development in Saccharomyces: heat-shock proteins hsp26 and hsp84 were strongly induced nor inducible by heat shock. Instead, two proteins related to hsp70 were induced. A strikingly similar pattern of expression occurs during oogenesis in Drosophila, suggesting that it may be one of the earliest developmental pathways to evolve in eukaryotic cells.

Drosophila↗
PubMed1986

Inhibition of heat shock protein synthesis by heat-inducible antisense RNA.

We show that antisense RNAs transcribed from genes that are stably integrated into the genome can be used to inhibit the expression of an endogenous cellular gene. Drosophila tissue culture cells were stably transformed with a gene encoding a heat-inducible RNA complementary to the message for hsp26, one of the small heat shock proteins. These cells produced much less hsp26 after heat shock than did untransformed cells. The inhibition was highly specific: expression of the closely related heat shock proteins hsp22, hsp23, and hsp28 was unaffected. By varying the copy number of the antisense gene, the degree of inhibition was varied over a broad range. Reducing the rate of hsp26 synthesis did not appear to affect the synthesis of any other protein during either heat shock or recovery.

Animals↗
PubMed1986

Induction of chromosomal beta-lactamase expression in enterobacteria.

Enterobacteria and Pseudomonas express evolutionarily related chromosomally encoded beta-lactamases. These enzymes have a high affinity for modern cephalosporins, and beta-lactamase overproduction is the most important factor in the development of resistance against such drugs. In some species the beta-lactamase is produced constitutively at a low level, and mutations to high expression occur only infrequently. In other species beta-lactamase synthesis can be induced by beta-lactams, and mutation to constitutive overexpression of the enzyme is a frequent event. We discuss the current knowledge concerning the genetic basis of the two different modes of beta-lactamase regulation, as well as the mechanisms through which a high level of beta-lactamase synthesis is reached.

Base Sequence↗
PubMed1986

Chromosomal beta-lactam resistance in enterobacteria.

Most enterobacterial species carry a chromosomal ampC beta-lactamase gene. In Escherichia coli and Shigella, expression from ampC is non-inducible and the beta-lactamase is synthesized at low levels. Mutations leading to increased beta-lactamase synthesis occur rather infrequently, making resistance to modern cephalosporins a rare event in these species. In other enterobacteria and Pseudomonas, ampC beta-lactamase synthesis is induced by beta-lactams. In Enterobacter cloacae, Citrobacter freundii and probably also in other species with inducible beta-lactamase expression, ampC is regulated by at least two genes, ampR and ampD. Mutations affecting ampR abolish beta-lactamase inducibility, and mutants devoid of ampR, produce ampC beta-lactamase at low constitutive levels. Mutations in ampD lead to constitutive overproduction of inducible beta-lactamase if an intact ampR protein is present in the cell. The latter type of mutations occur at a high frequency and result in clinical resistance to several third-generation cephalosporins.

Anti-Bacterial Agents↗
PubMed1985

The preferential translation of Drosophila hsp70 mRNA requires sequences in the untranslated leader.

When Drosophila cells are heat shocked, the translation of normal cellular mRNAs is repressed, while mRNAs encoding the heat-shock proteins are translated at high rates. We have found that the hsp70 message is not translated at high temperatures when its leader sequence is deleted. This message is translated when the cells are allowed to recover at 25 degrees C, but the translation ceases when the cells are given a second heat shock. A message with an extra 39 bases added onto the 5' end of the leader behaves in the same way. However, if either of two conserved sequence elements in the leader is deleted, the message is still translated during heat shock. Although the specific feature responsible for the preferential translation of heat-shock messages is not yet identified, we conclude that it must reside in the 5' untranslated leader.

Amino Acid Sequence↗
PubMed1984

Expression of a Drosophila heat shock protein in mammalian cells: transient association with nucleoli after heat shock.

We transformed mouse L cells with a cloned Drosophila hsp70 gene and obtained cells with either heat-inducible or constitutively expressed copies of the gene. The distribution of hsp70 in these cells was examined by indirect immunofluorescence with monoclonal antibodies specific to the Drosophila protein. In constitutive cells, hsp70 was present in both cytoplasm and nucleus. After heat shock, the nuclear hsp70 was transiently concentrated in nucleoli, from which it had previously been excluded; the cytoplasmic hsp70 moved to a perinuclear location, a result consistent with it being associated with intermediate filaments of the cytoskeleton. The nucleolar migration took several hours and was partly inhibited by actinomycin D, but was independent of protein synthesis; it may reflect binding to newly-synthesized rRNA. Drosophila hsp70 was also expressed from replicating plasmids in monkey COS cells, and was found to be concentrated in nuclei even at low temperature. Migration to nucleoli occurred after heat shock. These results indicate that a single protein can have multiple interactions with cellular components, and form the basis for future studies of these interactions by in vitro mutagenesis and expression of the hsp70 gene.

Animals↗
PubMed1984

hsp70: nuclear concentration during environmental stress and cytoplasmic storage during recovery.

The intracellular distribution of the major Drosophila heat-shock protein hsp70 was determined by indirect immunofluorescence with monoclonal antibodies. During heat shock the protein concentrates strongly in nuclei while a small quantity remains cytoplasmic. During recovery hsp70 leaves the nuclei and becomes distributed throughout the cytoplasm. With a second heat shock it is rapidly transported back into the nucleus. Nuclear translocation depends not on the temperature per se, but on the physiological state of the cell since it also occurs after exposure to an anoxic atmosphere at normal temperatures. We also provide evidence that hsps protect cells from the toxic effects of anoxia, as well as heat, and conclude that nuclear translocation of hsp70 is related to its function in protecting the organism from both forms of environmental stress.

Animals↗
PubMed1984

Changing patterns of gene expression during sporulation in yeast.

Analysis of RNAs isolated from the yeast Saccharomyces cerevisiae reveals a dramatic series of changes in protein coding sequences during sporulation. Shortly after transfer to sporulation medium, mRNAs for certain proteins are repressed while a broad array of mRNAs for other proteins is induced. Superimposed on this general increase in transcriptional activity is the very strong induction of a particular subset of heat shock mRNAs, the same subset that is induced during the normal course of oogenesis in Drosophila. At distinct times later in sporulation, two sets of abundant mRNAs are coordinately induced. Unlike the earlier changes in the message complement, these changes are unique to sporulating cells. As asci mature, one set of sporulation-specific RNAs is selectively degraded. The second set, as well as the broad array of mRNAs induced earlier in development, is retained in a highly stable and fully translatable form.

Diploidy↗
PubMed1983

Is the major Drosophila heat shock protein present in cells that have not been heat shocked?

When eukaryotic cells are exposed to elevated temperatures they respond by vigorously synthesizing a small group of proteins called the heat shock proteins. An essential element in defining the role of these proteins is determining whether they are unique to a stressed state or are also found in healthy, rapidly growing cells at normal temperatures. To date, there have been conflicting reports concerning the major heat-induced protein of Drosophila cells, HSP 70. We report the development of monoclonal antibodies specific for this protein. These antibodies were used to assay HSP 70 in cells incubated under different culture conditions. The protein was detectable in cells maintained at normal temperatures, but only when immunological techniques were pushed to the limits of their sensitivity. To test for the possibility that these cells contain a reservoir of protein in a cryptic antigenic state (i.e., waiting posttranslational modification for use at high temperature), we treated cells with cycloheximide or actinomycin D immediately before heat shock. HSP 70 was not detected in these cells. Finally, we tested for the presence of a reservoir of inactive messages by using a high stringency hybridization of 32P-labeled cloned gene sequences to electrophoretically separated RNAs. Although HSP 70 mRNA was detectable in rapidly growing cells, it was present at less than 1/1,000th the level achieved after induction.

Animals↗