DNA damage and heat shock dually regulate genes in Saccharomyces cerevisiae

1986 ◽  
Vol 6 (1) ◽  
pp. 90-96
Author(s):  
T McClanahan ◽  
K McEntee

Two Saccharomyces cerevisiae genes isolated in a differential hybridization screening for DNA damage regulation (DDR genes) were also transcriptionally regulated by heat shock treatment. A 0.45-kilobase transcript homologous to the DDRA2 gene and a 1.25-kilobase transcript homologous to the DDR48 gene accumulated after exposure of cells to 4-nitroquinoline-1-oxide (NQO; 1 to 1.5 microgram/ml) or brief heat shock (20 min at 37 degrees C). The DDRA2 transcript, which was undetectable in untreated cells, was induced to high levels by these treatments, and the DDR48 transcript increased more than 10-fold as demonstrated by Northern hybridization analysis. Two findings argue that dual regulation of stress-responsive genes is not common in S. cerevisiae. First, two members of the heat shock-inducible hsp70 family of S. cerevisiae, YG100 and YG102, were not induced by exposure to NQO. Second, at least one other DNA-damage-inducible gene, DIN1, was not regulated by heat shock treatment. We examined the structure of the induced RNA homologous to DDRA2 after heat shock and NQO treatments by S1 nuclease protection experiments. Our results demonstrated that the DDRA2 transcript initiates equally frequently at two sites separated by 5 base pairs. Both transcriptional start sites were utilized when cells were exposed to either NQO or heat shock treatment. These results indicate that DDRA2 and DDR48 are members of a unique dually regulated stress-responsive family of genes in S. cerevisiae.

1986 ◽  
Vol 6 (1) ◽  
pp. 90-96 ◽  
Author(s):  
T McClanahan ◽  
K McEntee

Two Saccharomyces cerevisiae genes isolated in a differential hybridization screening for DNA damage regulation (DDR genes) were also transcriptionally regulated by heat shock treatment. A 0.45-kilobase transcript homologous to the DDRA2 gene and a 1.25-kilobase transcript homologous to the DDR48 gene accumulated after exposure of cells to 4-nitroquinoline-1-oxide (NQO; 1 to 1.5 microgram/ml) or brief heat shock (20 min at 37 degrees C). The DDRA2 transcript, which was undetectable in untreated cells, was induced to high levels by these treatments, and the DDR48 transcript increased more than 10-fold as demonstrated by Northern hybridization analysis. Two findings argue that dual regulation of stress-responsive genes is not common in S. cerevisiae. First, two members of the heat shock-inducible hsp70 family of S. cerevisiae, YG100 and YG102, were not induced by exposure to NQO. Second, at least one other DNA-damage-inducible gene, DIN1, was not regulated by heat shock treatment. We examined the structure of the induced RNA homologous to DDRA2 after heat shock and NQO treatments by S1 nuclease protection experiments. Our results demonstrated that the DDRA2 transcript initiates equally frequently at two sites separated by 5 base pairs. Both transcriptional start sites were utilized when cells were exposed to either NQO or heat shock treatment. These results indicate that DDRA2 and DDR48 are members of a unique dually regulated stress-responsive family of genes in S. cerevisiae.


1984 ◽  
Vol 4 (4) ◽  
pp. 591-598
Author(s):  
J Cappello ◽  
C Zuker ◽  
H F Lodish

The Dictyostelium genome contains 40 copies of a 4.7-kilobase repetitive and apparently transposable DNA sequence (DIRS-1) and about 250 smaller elements that appear to be deletions or rearrangements of DIRS-1. Transcripts of these sequences are induced during differentiation and also by heat shock treatment of growing cells. We showed that one such cloned element, pB41.6 (2.5 kilobases) contains a nucleotide sequence identical to the Drosophila consensus heat shock promotor. To test whether this sequence might indeed control the expression of DIRS-1-related RNAs, we have cloned this genomic segment into yeast cells. In yeast cells, 41.6 directs synthesis of a 1.7-kilobase RNA that is induced at least 10-fold by heat shock. Transcription initiates at about 124 bases 3' of the putative promotor sequence and terminates within the 41.6 insert. A 381-base-pair subclone that contains the putative promotor sequence is sufficient to induce the heat shock response of 41.6 in yeast cells.


1984 ◽  
Vol 4 (4) ◽  
pp. 591-598 ◽  
Author(s):  
J Cappello ◽  
C Zuker ◽  
H F Lodish

The Dictyostelium genome contains 40 copies of a 4.7-kilobase repetitive and apparently transposable DNA sequence (DIRS-1) and about 250 smaller elements that appear to be deletions or rearrangements of DIRS-1. Transcripts of these sequences are induced during differentiation and also by heat shock treatment of growing cells. We showed that one such cloned element, pB41.6 (2.5 kilobases) contains a nucleotide sequence identical to the Drosophila consensus heat shock promotor. To test whether this sequence might indeed control the expression of DIRS-1-related RNAs, we have cloned this genomic segment into yeast cells. In yeast cells, 41.6 directs synthesis of a 1.7-kilobase RNA that is induced at least 10-fold by heat shock. Transcription initiates at about 124 bases 3' of the putative promotor sequence and terminates within the 41.6 insert. A 381-base-pair subclone that contains the putative promotor sequence is sufficient to induce the heat shock response of 41.6 in yeast cells.


1996 ◽  
Vol 82 (2) ◽  
pp. 187-190 ◽  
Author(s):  
Toshiro Omori ◽  
Kiyoshi Ogawa ◽  
Yasufumi Umemoto ◽  
Kenji Yuki ◽  
Yasuhiro Kajihara ◽  
...  

1997 ◽  
Vol 83 (1) ◽  
pp. 64-69 ◽  
Author(s):  
Toshiro Omori ◽  
Yasufumi Umemoto ◽  
Kiyoshi Ogawa ◽  
Yasuhiro Kajiwara ◽  
Masahiko Shimoda ◽  
...  

Zygote ◽  
2021 ◽  
pp. 1-5
Author(s):  
Nivaldo Ferreira do Nascimento ◽  
Rafaela Manchin Bertolini ◽  
Lucia Soares Lopez ◽  
Laura Satiko Okada Nakaghi ◽  
Paulo Sérgio Monzani ◽  
...  

Summary Triploidization plays an important role in aquaculture and surrogate technologies. In this study, we induced triploidy in the matrinxã fish (Brycon amazonicus) using a heat-shock technique. Embryos at 2 min post fertilization (mpf) were heat shocked at 38°C, 40°C, or 42°C for 2 min. Untreated, intact embryos were used as a control. Survival rates during early development were monitored and ploidy status was confirmed using flow cytometry and nuclear diameter analysis of erythrocytes. The hatching rate reduced with heat-shock treatment, and heat-shock treatments at 42°C resulted in no hatching events. Optimal results were obtained at 40°C with 95% of larvae exhibiting triploidy. Therefore, we report that heat-shock treatments of embryos (2 mpf) at 40°C for 2 min is an effective way to induce triploid individuals in B. amazonicus.


1988 ◽  
Vol 106 (4) ◽  
pp. 1105-1116 ◽  
Author(s):  
L A Mizzen ◽  
W J Welch

Exposure of mammalian cells to a nonlethal heat-shock treatment, followed by a recovery period at 37 degrees C, results in increased cell survival after a subsequent and otherwise lethal heat-shock treatment. Here we characterize this phenomenon, termed acquired thermotolerance, at the level of translation. In a number of different mammalian cell lines given a severe 45 degrees C/30-min shock and then returned to 37 degrees C, protein synthesis was completely inhibited for as long as 5 h. Upon resumption of translational activity, there was a marked induction of heat-shock (or stress) protein synthesis, which continued for several hours. In contrast, cells first made thermotolerant (by a pretreatment consisting of a 43 degrees C/1.5-h shock and further recovery at 37 degrees C) and then presented with the 45 degrees C/30-min shock exhibited considerably less translational inhibition and an overall reduction in the amount of subsequent stress protein synthesis. The acquisition and duration of such "translational tolerance" was correlated with the expression, accumulation, and relative half-lives of the major stress proteins of 72 and 73 kD. Other agents that induce the synthesis of the stress proteins, such as sodium arsenite, similarly resulted in the acquisition of translational tolerance. The probable role of the stress proteins in the acquisition of translational tolerance was further indicated by the inability of the amino acid analogue, L-azetidine 2-carboxylic acid, an inducer of nonfunctional stress proteins, to render cells translationally tolerant. If, however, analogue-treated cells were allowed to recover in normal medium, and hence produce functional stress proteins, full translational tolerance was observed. Finally, we present data indicating that the 72- and 73-kD stress proteins, in contrast to the other major stress proteins (of 110, 90, and 28 kD), are subject to strict regulation in the stressed cell. Quantitation of 72- and 73-kD synthesis after heat-shock treatment under a number of conditions revealed that "titration" of 72/73-kD synthesis in response to stress may represent a mechanism by which the cell monitors its local growth environment.


1985 ◽  
Vol 5 (1) ◽  
pp. 17-26
Author(s):  
L Naumovski ◽  
G Chu ◽  
P Berg ◽  
E C Friedberg

We determined the complete nucleotide sequence of the RAD3 gene of Saccharomyces cerevisiae. The coding region of the gene contained 2,334 base pairs that could encode a protein with a calculated molecular weight of 89,796. Analysis of RAD3 mRNA by Northern blots and by S1 nuclease mapping indicated that the transcript was approximately 2.5 kilobases and did not contain intervening sequences. Fusions between the RAD3 gene and the lac'Z gene of Escherichia coli were constructed and used to demonstrate that the RAD3 gene was not inducible by DNA damage caused by UV radiation or 4-nitroquinoline-1-oxide. Two UV-sensitive chromosomal mutant alleles of RAD3, rad3-1 and rad3-2, were rescued by gap repair of a centromeric plasmid, and their sequences were determined. The rad3-1 mutation changed a glutamic acid to lysine, and the rad3-2 mutation changed a glycine to arginine. Previous studies have shown that disruption of the RAD3 gene results in loss of an essential function and is associated with inviability of haploid cells. In the present experiments, plasmids carrying the rad3-1 and rad3-2 mutations were introduced into haploid cells containing a disrupted RAD3 gene. These plasmids expressed the essential function of RAD3 but not its DNA repair function. A 74-base-pair deletion at the 3' end of the RAD3 coding region or a fusion of this deletion to the E. coli lac'Z gene did not affect either function of RAD3.


Circulation ◽  
2000 ◽  
Vol 102 (suppl_3) ◽  
Author(s):  
Ken Suzuki ◽  
Ryszard T. Smolenski ◽  
Jay Jayakumar ◽  
Bari Murtuza ◽  
Nigel J. Brand ◽  
...  

Background —Graft survival after skeletal myoblast transplantation is affected by various pathological processes caused by environmental stress. Heat shock is known to afford protection of several aspects of cell metabolism and function. We hypothesized that prior heat shock treatment of graft cells would improve their survival after cell transplantation. Methods and Results —L6 rat skeletal myoblasts expressing β-galactosidase (β-gal) were subjected to heat shock (42°C, 1 hour). Increased expression of heat shock protein 72 was detected 24 hours later in the heat-shocked cells. After hypoxia-reoxygenation in vitro, lactate dehydrogenase leakage was significantly attenuated in the heat-shocked cells; in addition, the percentage of early apoptosis was lower in this group measured by flow cytometry with annexin V staining. For the in vivo study, 1×10 6 heat-shocked (hsCTx) or normal-cultured (CTx) myoblasts were infused into the explanted rat hearts through the coronary artery followed by heterotopic heart transplantation. β-gal activity was significantly higher in the hsCTx group after cell transplantation, with an estimated 8×10 6 surviving cells per heart in the hsCTx group and 5×10 6 cells in the CTx group on day 28. Discrete loci of grafted cells were globally observed in the myocardium of the hsCTx and CTx groups, with a higher frequency in the hsCTx group. Surviving myoblasts occasionally differentiated into myotubes and had integrated with the native cardiomyocytes. Conclusions —Heat-shocked skeletal myoblasts demonstrated improved tolerance to hypoxia-reoxygenation insult in vitro and enhanced survival when grafted into the heart. Heat shock treatment could be useful in improving graft cell survival in cell transplantation.


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