scholarly journals Efficient transcription of a Caenorhabditis elegans heat shock gene pair in mouse fibroblasts is dependent on multiple promoter elements which can function bidirectionally.

1986 ◽  
Vol 6 (9) ◽  
pp. 3134-3143 ◽  
Author(s):  
R J Kay ◽  
R J Boissy ◽  
R H Russnak ◽  
E P Candido
Keyword(s):  
Caenorhabditis Elegans ◽  
Heat Shock ◽  
Gene Pair ◽  
Mammalian Cells ◽  
Heat Shock Gene ◽  
Promoter Strength ◽  
Wild Type ◽  
Kinase Gene ◽  
Mouse Fibroblasts ◽  
Bidirectional Transcription

A divergently transcribed pair of Caenorhabditis elegans hsp16 genes was introduced into mouse fibroblasts by stable transfection with vectors containing bovine papillomavirus plasmid maintenance sequences and a selectable gene. The hsp16 genes were transcriptionally inactive in the mouse cells under normal growth conditions and were strongly induced by heat shock or arsenite. In a cell line with 12 copies of the gene pair, there were estimated to be more than 10,000 hsp16 transcripts in each cell after 2 h of heat shock treatment. The hsp16 transcript levels were more than 100 times higher than those of a gene with a herpes simplex virus thymidine kinase gene promoter carried on the same vector. A single heat shock promoter element (HSE) could activate bidirectional transcription of the two hsp16 genes when placed between the two TATA elements, but the transcriptional efficiency was reduced 10-fold relative to that of the wild-type gene pair. Four overlapping HSEs positioned between the two TATA elements resulted in inducible bidirectional transcription at greater than wild-type levels. The number of HSEs can therefore be a major determinant of the promoter strength of heat-inducible genes in mammalian cells. Partial disruption of an alternating purine-pyrimidine sequence between the two hsp16 genes had no significant effect on their transcriptional activity.

Efficient transcription of a Caenorhabditis elegans heat shock gene pair in mouse fibroblasts is dependent on multiple promoter elements which can function bidirectionally

1986 ◽  
Vol 6 (9) ◽  
pp. 3134-3143
Author(s):  
R J Kay ◽  
R J Boissy ◽  
R H Russnak ◽  
E P Candido
Keyword(s):  
Caenorhabditis Elegans ◽  
Heat Shock ◽  
Gene Pair ◽  
Mammalian Cells ◽  
Heat Shock Gene ◽  
Promoter Strength ◽  
Wild Type ◽  
Kinase Gene ◽  
Mouse Fibroblasts ◽  
Bidirectional Transcription

A divergently transcribed pair of Caenorhabditis elegans hsp16 genes was introduced into mouse fibroblasts by stable transfection with vectors containing bovine papillomavirus plasmid maintenance sequences and a selectable gene. The hsp16 genes were transcriptionally inactive in the mouse cells under normal growth conditions and were strongly induced by heat shock or arsenite. In a cell line with 12 copies of the gene pair, there were estimated to be more than 10,000 hsp16 transcripts in each cell after 2 h of heat shock treatment. The hsp16 transcript levels were more than 100 times higher than those of a gene with a herpes simplex virus thymidine kinase gene promoter carried on the same vector. A single heat shock promoter element (HSE) could activate bidirectional transcription of the two hsp16 genes when placed between the two TATA elements, but the transcriptional efficiency was reduced 10-fold relative to that of the wild-type gene pair. Four overlapping HSEs positioned between the two TATA elements resulted in inducible bidirectional transcription at greater than wild-type levels. The number of HSEs can therefore be a major determinant of the promoter strength of heat-inducible genes in mammalian cells. Partial disruption of an alternating purine-pyrimidine sequence between the two hsp16 genes had no significant effect on their transcriptional activity.

Transcriptional activation and subsequent control of the human heat shock gene during adenovirus infection

1983 ◽  
Vol 3 (11) ◽  
pp. 2058-2065 ◽  
Author(s):  
H T Kao ◽  
J R Nevins
Keyword(s):  
Heat Shock ◽  
Transcriptional Activation ◽  
Peak Level ◽  
Heat Shock Gene ◽  
Adenovirus Infection ◽  
Wild Type ◽  
Rapid Turnover ◽  
Transformed Cell Line ◽  
Shock Gene ◽  
E1a Gene

A cDNA copy of the major human heat shock mRNA was cloned. The clone is complementary to the mRNA encoding the major 70-kilodalton heat shock protein as shown by hybrid arrest translation. We utilized the cloned DNA to measure induction of the gene during adenovirus infection. The mRNA increases in abundance approximately 100-fold during a wild-type adenovirus infection but does not increase more than 2-fold during an infection in which there is no E1A gene function [high multiplicity of infection of an E1A (-) mutant]. Furthermore, by measuring transcription in isolated nuclei, we found that the induction was transcriptional and was mediated by the E1A gene product. The induction was not maintained, however. After a peak level was obtained, transcription returned to preinfection levels. This decline was also reflected in the cytoplasmic mRNA abundance indicating a rapid turnover of the heat shock mRNA. This rapid turnover of the heat shock mRNA appears to be induced by the viral infection since the heat shock mRNA was found to be stable when synthesized in an adenovirus-transformed cell line.

scholarly journals Electrophysiological Measures of Aging Pharynx Function in C. elegans Reveal Enhanced Organ Functionality in Older, Long-lived Mutants

2017 ◽  
Vol 74 (8) ◽  
pp. 1173-1179 ◽  
Author(s):  
Joshua Coulter Russell ◽  
Nikolay Burnaevskiy ◽  
Bridget Ma ◽  
Miguel Arenas Mailig ◽  
Franklin Faust ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Heat Shock ◽  
Life Span ◽  
Insulin Signaling ◽  
Model System ◽  
Wild Type ◽  
Organ Function ◽  
C Elegans ◽  
Age Related ◽  
The Relationship

Abstract The function of the pharynx, an organ in the model system Caenorhabditis elegans, has been correlated with life span and motility (another measure of health) since 1980. In this study, in order to further understand the relationship between organ function and life span, we measured the age-related decline of the pharynx using an electrophysiological approach. We measured and analyzed electropharyngeograms (EPG) of wild type animals, short-lived hsf-1 mutants, and long-lived animals with genetically decreased insulin signaling or increased heat shock pathway signaling; we recorded a total of 2,478 EPGs from 1,374 individuals. As expected, the long-lived daf-2(e1370) and hsf-1OE(uthIs235) animals maintained pharynx function relatively closer to the youthful state during aging, whereas the hsf-1(sy441) and wild type animals’ pharynx function deviated significantly further from the youthful state at advanced age. Measures of the amount of variation in organ function can act as biomarkers of youthful physiology as well. Intriguingly, the long-lived animals had greater variation in the duration of pharynx contraction at older ages.

scholarly journals Temporal and spatial expression patterns of the small heat shock (hsp16) genes in transgenic Caenorhabditis elegans.

1992 ◽  
Vol 3 (2) ◽  
pp. 221-233 ◽  
Author(s):  
E G Stringham ◽  
D K Dixon ◽  
D Jones ◽  
E P Candido
Keyword(s):  
Caenorhabditis Elegans ◽  
Heat Shock ◽  
Gene Pair ◽  
Expression Patterns ◽  
Developmentally Regulated ◽  
Noncoding Sequences ◽  
C Elegans ◽  
Gene Pairs ◽  
Temporal And Spatial ◽  
Beta Galactosidase

The expression of the hsp16 gene family in Caenorhabditis elegans has been examined by introducing hsp16-lacZ fusions into the nematode by transformation. Transcription of the hsp16-lacZ transgenes was totally heat-shock dependent and resulted in the rapid synthesis of detectable levels of beta-galactosidase. Although the two hsp16 gene pairs of C. elegans are highly similar within both their coding and noncoding sequences, quantitative and qualitative differences in the spatial pattern of expression between gene pairs were observed. The hsp16-48 promoter was shown to direct greater expression of beta-galactosidase in muscle and hypodermis, whereas the hsp16-41 promoter was more efficient in intestine and pharyngeal tissue. Transgenes that eliminated one promoter from a gene pair were expressed at reduced levels, particularly in postembryonic stages, suggesting that the heat shock elements in the intergenic region of an hsp16 gene pair may act cooperatively to achieve high levels of expression of both genes. Although the hsp16 gene pairs are never constitutively expressed, their heat inducibility is developmentally restricted; they are not heat inducible during gametogenesis or early embryogenesis. The hsp16 genes represent the first fully inducible system in C. elegans to be characterized in detail at the molecular level, and the promoters of these genes should find wide applicability in studies of tissue- and developmentally regulated genes in this experimental organism.

scholarly journals The Heat Shock Protein YbeY Is Required for Optimal Activity of the 30S Ribosomal Subunit

2010 ◽  
Vol 192 (18) ◽  
pp. 4592-4596 ◽  
Author(s):  
Aviram Rasouly ◽  
Chen Davidovich ◽  
Eliora Z. Ron
Keyword(s):  
Heat Shock ◽  
Ribosomal Subunit ◽  
Heat Shock Gene ◽  
Wild Type ◽  
Lower Efficiency ◽  
Ribosomal Subunits ◽  
Translation Machinery ◽  
Shock Gene

ABSTRACT The highly conserved bacterial ybeY gene is a heat shock gene whose function is not fully understood. Previously, we showed that the YbeY protein is involved in protein synthesis, as Escherichia coli mutants with ybeY deleted exhibit severe translational defects in vivo. Here we show that the in vitro activity of the translation machinery of ybeY deletion mutants is significantly lower than that of the wild type. We also show that the lower efficiency of the translation machinery is due to impaired 30S small ribosomal subunits.

scholarly journals Transcriptional activation and subsequent control of the human heat shock gene during adenovirus infection.

1983 ◽  
Vol 3 (11) ◽  
pp. 2058-2065 ◽  
Author(s):  
H T Kao ◽  
J R Nevins
Keyword(s):  
Heat Shock ◽  
Transcriptional Activation ◽  
Peak Level ◽  
Heat Shock Gene ◽  
Adenovirus Infection ◽  
Wild Type ◽  
Rapid Turnover ◽  
Transformed Cell Line ◽  
Shock Gene ◽  
E1a Gene

A cDNA copy of the major human heat shock mRNA was cloned. The clone is complementary to the mRNA encoding the major 70-kilodalton heat shock protein as shown by hybrid arrest translation. We utilized the cloned DNA to measure induction of the gene during adenovirus infection. The mRNA increases in abundance approximately 100-fold during a wild-type adenovirus infection but does not increase more than 2-fold during an infection in which there is no E1A gene function [high multiplicity of infection of an E1A (-) mutant]. Furthermore, by measuring transcription in isolated nuclei, we found that the induction was transcriptional and was mediated by the E1A gene product. The induction was not maintained, however. After a peak level was obtained, transcription returned to preinfection levels. This decline was also reflected in the cytoplasmic mRNA abundance indicating a rapid turnover of the heat shock mRNA. This rapid turnover of the heat shock mRNA appears to be induced by the viral infection since the heat shock mRNA was found to be stable when synthesized in an adenovirus-transformed cell line.

Structure, organization, and expression of the 16-kDa heat shock gene family of Caenorhabditis elegans

Genome ◽  
1989 ◽  
Vol 31 (2) ◽  
pp. 690-697 ◽  
Author(s):  
E. P. M. Candido ◽  
D. Jones ◽  
D. K. Dixon ◽  
R. W. Graham ◽  
R. H. Russnak ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Heat Shock ◽  
Normal Growth ◽  
Growth Conditions ◽  
Amino Acid Sequences ◽  
Heat Shock Gene ◽  
Coding Region ◽  
Heat Shock Element ◽  
Hypersensitive Sites ◽  
Intergenic Regions

Exposure of the nematode Caenorhabditis elegans to a heat shock results in the induction of a number of genes not normally expressed in the animals under normal growth conditions. Among these are a family of genes encoding 16 kDa heat shock proteins (hsp16s). The major hsp16 genes have been cloned and characterized, and found to reside at two clusters in the C. elegans genome. One cluster contains two distinct genes, hsp16-1 and hsp16-48, arranged in divergent orientations separated by only 348 base pairs (bp). An identical pair, duplicated and inverted with respect to the first pair, is located 415 bp away. This cluster, located on chromosome V, therefore contains four genes as two identical pairs within less than 4 kilobases of DNA, and the pairs form the arms of a large inverted repeat. A second pair of genes, hsp16-2 and hsp16-41, constitutes a second hsp16 locus with an organization very similar to that of the hsp16-1/48 locus, except that it is not duplicated. Comparisons of the derived amino acid sequences show that hsp16-1 and hsp16-2 form a closely related pair, as do hsp16-41 and hsp16-48. These hsps show extensive sequence identity with the small hsps of Drosophila, as well as with mammalian alpha-crystallins. The coding region of each gene is interrupted by a single intron of approximately 50 bp, in a position homologous to that of the first intron in a mouse alpha-crystallin gene. The compact intergenic regions of both hsp16 loci contain a TATA element and a heat shock element (HSE) for each member of the pair, and are very similar in sequence overall. Expression studies, however, show that the level of transcripts from the hsp16-2/41 pair may be up to 14-fold higher on a per gene basis, as the level of RNA from the hsp16-1/48 pair, depending upon the induction conditions and developmental stage. This difference in message levels seems to be due to differences in the kinetics of inactivation of the genes rather than in transcription rates or rates of mRNA turnover. Distinct DNAseI hypersensitive sites are present upstream of each HSE in chromatin when the genes are inactive; these disappear and the whole intergenic region seems to become DNAse sensitive when the genes are maximally active.Key words: heat shock, 16-kDa polypeptides, gene structure, transcription, DNAseI hypersensitive sites, Caenorhabditis elegans.

Truncation attenuates molecular chaperoning and apoptosis inhibition by p26, a small heat shock protein from Artemia franciscana

2010 ◽  
Vol 88 (6) ◽  
pp. 937-946 ◽  
Author(s):  
Yong Wu ◽  
Thomas H. MacRae
Keyword(s):  
Heat Shock ◽  
Mammalian Cells ◽  
Protein Denaturation ◽  
Maximal Activity ◽  
Artemia Franciscana ◽  
Eukaryotic Expression ◽  
Chaperone Activity ◽  
Wild Type ◽  
Transfected Cells ◽  
Apoptosis Inhibition

The small heat shock proteins (sHSPs), which prevent irreversible protein denaturation and inhibit apoptosis, consist of an amino-terminus, the canonical α-crystallin domain, and a carboxy-terminal extension. It remains difficult, however, to define sHSP structure–function relationships and with this in mind p26, an sHSP from the crustacean Artemia franciscana , was truncated by deletion mutagenesis. Wild-type p26 cDNA and three truncated variants inserted into the eukaryotic expression vector pcDNA3.1/HisC were used to generate stably transfected 293H cells. p26 shielded transfected cells against death upon exposure to heat and oxidative stress. Truncation reduced chaperone activity, with cells synthesizing the p26 α-crystallin domain being the least resistant. Wild-type p26 inhibited apoptosis in transfected cells, with protection against oxidation-generated apoptosis being more effective than that against heat-induced apoptosis. Truncation reduced p26 apoptotic inhibitory activity, with the α-crystallin domain again being the least effective. The results show that a crustacean sHSP functions effectively in mammalian cells, demonstrating interchangeability of these proteins between distantly related organisms and indicating similarities in their mechanisms of action. Moreover, maximal activity was observed for full-length p26, indicating that structural elements required for chaperone activity and apoptosis inhibition reside throughout the protein.

scholarly journals CAMKII and Calcineurin regulate the lifespan of Caenorhabditis elegans through the FOXO transcription factor DAF-16

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Li Tao ◽  
Qi Xie ◽  
Yue-He Ding ◽  
Shang-Tong Li ◽  
Shengyi Peng ◽  
...  
Keyword(s):  
Caenorhabditis Elegans ◽  
Nuclear Localization ◽  
Insulin Signaling ◽  
Mammalian Cells ◽  
Nuclear Translocation ◽  
Type Ii ◽  
Wild Type ◽  
C Elegans ◽  
Foxo Transcription Factors ◽  
Lifespan Regulation

The insulin-like signaling pathway maintains a relatively short wild-type lifespan in Caenorhabditis elegans by phosphorylating and inactivating DAF-16, the ortholog of the FOXO transcription factors of mammalian cells. DAF-16 is phosphorylated by the AKT kinases, preventing its nuclear translocation. Calcineurin (PP2B phosphatase) also limits the lifespan of C. elegans, but the mechanism through which it does so is unknown. Herein, we show that TAX-6•CNB-1 and UNC-43, the C. elegans Calcineurin and Ca2+/calmodulin-dependent kinase type II (CAMKII) orthologs, respectively, also regulate lifespan through DAF-16. Moreover, UNC-43 regulates DAF-16 in response to various stress conditions, including starvation, heat or oxidative stress, and cooperatively contributes to lifespan regulation by insulin signaling. However, unlike insulin signaling, UNC-43 phosphorylates and activates DAF-16, thus promoting its nuclear localization. The phosphorylation of DAF-16 at S286 by UNC-43 is removed by TAX-6•CNB-1, leading to DAF-16 inactivation. Mammalian FOXO3 is also regulated by CAMKIIA and Calcineurin.

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