References

Notes: When a clone of Chinese hamster ovary (CHO) cells transfected with a plasmid containing a luciferase reporter gene under the control of the human heat shock protein (hsp) 70 gene promoter was treated with cycloheximide during heat exposure at 42 and 43 degrees C for 15 to 100 minutes and then incubated at 37 degrees C after removal of cycloheximide, reporter gene expression was suppressed by the protein synthesis inhibitor only at small heat shock doses (i.e., heat shock of less than 40 minutes at 42 degrees C and 15 minutes at 43 degrees C). A similar stress dose- dependent suppression of reporter gene expression by cycloheximide was also demonstrated by treatment with sodium arsenite at 37 degrees C. However, dexamethasone-dependent reporter gene expression in a different CHO clone was not inhibited after the inducer treatment for different times in the presence of cycloheximide. In addition, synthesis of most cellular proteins (except for hsp) was not affected after heat shock treatment with cycloheximide. The results suggested that the cycloheximide inhibition of gene expression is specific to hsp gene expression induced by limited stress doses. Furthermore, a prior 42 degrees C heat shock treatment for 30 minutes induced a decreased responsiveness (tolerance) to a second 42 degrees C heat treatment for hsp gene expression, but tolerance did not develop in cells exposed to the first heat shock in the presence of cycloheximide. These results confirm previous findings that induction of hsp gene expression by stress is balanced by the severity of stress and rate of protein synthesis. They also support the proposed model of autoregulation of hsp gene expression by levels of free hsp70
Reference ID: 4948

Notes: Expression of a luciferase reporter gene by Chinese hamster ovary cells under the control of the human heat shock protein (hsp) 70 gene promoter was suppressed by incubation at 37 degrees C after treatment with cycloheximide (CHX) during 42 degrees C heat shock exposure. The CHX-induced suppression of hsp gene expression induced no development of thermotolerance. However, 42 degrees C heat shock treatment without CHX followed by CHX inhibition of protein synthesis during recovery incubation at 37 degrees C induced thermotolerance expression by inducing enhanced synthesis of hsps including hsp70 in subsequent heat challenge incubation at 43 degrees C. The results demonstrated a direct correlation between suppression (induction) of stress protein gene expression and non-expression (expression) of thermotolerance. Kinetic analysis showed that the CHX suppression of hsp gene induction was greater than the CHX inhibition of protein synthesis, and that it depended on the severity of heat stress: it decreased with increasing heat stress doses. Moreover, prior feeding of the proline analog L-azetidine 2-carboxylic acid abrogated the CHX-induced suppression of hsp gene expression. In addition, CHX treatment during heat cell-killing at 43 degrees C induced protection of cells. These results were well explained by the proposed model of negative or positive control of stress protein gene expression depending on the level of free hsp70, which may be modulated by both the rate of protein synthesis and the severity of heat stress
Reference ID: 4949

Notes: Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892-4255, USA ENGLISH Organisms respond to elevated temperatures and to chemical and physiological stresses by an increase in the synthesis of heat shock proteins. The regulation of heat shock gene expression in eukaryotes is mediated by the conserved heat shock transcription factor (HSF). HSF is present in a latent state under normal conditions; it is activated upon heat stress by induction of trimerization and high-affinity binding to DNA and by exposure of domains for transcriptional activity. Analysis of HSF cDNA clones from many species has defined structural and regulatory regions responsible for the inducible activities. The heat stress signal is thought to be transduced to HSF by changes in the physical environment, in the activity of HSF-modifying enzymes, or by changes in the intracellular level of heat shock proteins
Reference ID: 4951

Notes: Biotechnology Research Institute, National Research Council of Canada, Montreal, Quebec ENGLISH The human heat shock transcription factor (HSF) is maintained in an inactive non-DNA-binding form under nonstress conditions and acquires the ability to bind specifically to the heat shock promoter element in response to elevated temperatures or other conditions that disrupt protein structure. Here we show that constitutive overexpression of the major inducible heat shock protein, hsp70, in transfected human cells reduces the extent of HSF activation after a heat stress. HSF activation was inhibited more strongly in clones that express higher levels of hsp70. These results demonstrate that HSF activity is negatively regulated in vivo by hsp70 and suggest that the cell might sense elevated temperature as a decreased availability of hsp70. HSF activation in response to treatment with sodium arsenite or the proline analog azetidine was also depressed in hsp70-expressing cells relative to that in the nontransfected control cells. As well, the level of activated HSF decreased more rapidly in the hsp70-expressing clones when the cells were heat shocked and returned to 37 degrees C. These results suggest that hsp70 could play an active role in the conversion of HSF back to a conformation that does not bind the heat shock promoter element during the attenuation of the heat shock response
Reference ID: 4952

Notes: Complementation analysis at the single gene level has been carried out on 13 different, recessive, glycine-requiring mutants produced by action of mutagenic agents on the CHO-K1 Chinese hamster cell. The mutants form four different complementation classes indicating that at least four separate genes can produce a growth requirement for glycine. The four classes of mutants can be distinguished biochemically as well as genetically. Existence of requiring forms in the auxotrophs produced by treatment of the parental cell with mutagenic agents.
Reference ID: 1986

Notes: The luciferase gene from the firefly, Photinus pyralis, was used as a reporter of gene expression by light production in transfected plant cells and transgenic plants. A complementary DNA clone of the firefly licirerase gene under the control of a plant virus promoter (cauliflower mosaic virus 35S RNA promoter) was introduced into plant protoplast cells (Daucus carota) by electroporation and into plants (Nicotiana tabacum) by use of the Agrobacterium tumefaciens tumor-inducing plasmid. Extracts from electroporated cells (24 hours after the introduction of DNA) and from transgenic plants produce light when mixed with the substrates luciferin and adenosine triphosphate. Light produced by the action of luciferase was also detected in undisruped leaves or cells in culture from transgenic plants incubated in lucirerin and in whole transgenic plants "watered" with luciferin. Although light was detected in most organs in intact, transgenic plants (leaves, stems, and roots), the pattern of luminescence appeared to reflect both the organ-specific distribution of luciferase and the pathway for uptake of luciferin through the vasculature of the plant.
Reference ID: 4959

Notes: 0027-8424 ENGLISH UNITED-STATES A human 70-kDa heat shock protein (hsp70) gene segment has been isolated. The segment contains 3.15 kilobase pairs (kbp) of 5' nontranscribed sequence, an RNA leader of 119 bp, and a protein- coding region of 741 bp. The human protein sequence shows a high degree of homology to hsp70 sequences from other species. Expression experiments in Xenopus oocytes and mammalian cells indicate that a region that includes only 105 bp of 5' nontranscribed sequence contains all elements required for the efficient heat-controlled expression of the human gene. Two adjacent identical sequence elements, which are partly homologous to the Drosophila "heat shock consensus" sequence, are located 57 to 76 bp upstream from the capping site. Interestingly, the capping site itself is flanked by inverted repeat sequences JOURNAL-ARTICLE EC 3.2.1.23; 0; 0 85270444 8511 GENBANK/M11236 -------------------------------------------------- ------------------------------
Reference ID: 4955

Notes: 0270-7306 ENGLISH UNITED-STATES The nucleotide sequence of the luciferase gene from the firefly Photinus pyralis was determined from the analysis of cDNA and genomic clones. The gene contains six introns, all less than 60 bases in length. The 5' end of the luciferase mRNA was determined by both S1 nuclease analysis and primer extension. Although the luciferase cDNA clone lacked the six N-terminal codons of the open reading frame, we were able to reconstruct the equivalent of a full-length cDNA using the genomic clone as a source of the missing 5' sequence. The full-length, intronless luciferase gene was inserted into mammalian expression vectors and introduced into monkey (CV-1) cells in which enzymatically active firefly luciferase was transiently expressed. In addition, cell lines stably expressing firefly luciferase were isolated. Deleting a portion of the 5'-untranslated region of the luciferase gene removed an upstream initiation (AUG) codon and resulted in a twofold increase in the level of luciferase expression. The ability of the full-length luciferase gene to activate cryptic or enhancerless promoters was also greatly reduced or eliminated by this 5' deletion. Assaying the expression of luciferase provides a rapid and inexpensive method for monitoring promoter activity. Depending on the instrumentation employed to detect luciferase activity, we estimate this assay to be from 30- to 1,000-fold more sensitive than assaying chloramphenicol acetyltransferase expression JOURNAL-ARTICLE GM31253GMNIGMS; CA01062CANCI EC 1.13.12.-; 0; 0; 9007-49-2 87144243 8706 GENBANK/M15077 -------------------------------------------------- ------------------------------
Reference ID: 4958

Notes: Department of Biochemistry, State University of New York Health Science Center at Brooklyn 11203 0027-8424 ENGLISH UNITED-STATES We have introduced the firefly luciferase gene of Photinus pyralis into the vaccinia virus genome. This gene is expressed in a coordinate fashion during virus infection. Luminescence produced by the action of luciferase [Photinus-luciferin:oxygen 4- oxidoreductase(decarboxylating, ATP-hydrolyzing), EC 1.13.12.7] was easily detectable in infected cells in culture as well as in cells of tissues of infected mice. The limits of detection were about one infected cell in a background of a million noninfected cells. The luciferase assay was about 1000-fold more sensitive than that of beta-galactosidase. Our findings show that the luciferase assay can be conveniently used to follow viral gene expression and virus dissemination both in cell cultures and in tissues of infected animals JOURNAL-ARTICLE CA44262CANCI EC 1.13.12.- 88144497 8806
Reference ID: 4954