scholarly journals Saccharomyces cerevisiae HSP70 heat shock elements are functionally distinct.

1993 ◽  
Vol 13 (9) ◽  
pp. 5637-5646 ◽  
Author(s):  
M R Young ◽  
E A Craig
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heat Shock ◽  
Point Mutations ◽  
Growth Conditions ◽  
Mobility Shift ◽  
Basal Transcription ◽  
Basal Activity ◽  
Flanking Sequences ◽  
Gel Mobility Shift ◽  
Ability To Drive

The Saccharomyces cerevisiae HSP70 gene SSA1 has multiple heat shock elements (HSEs). To determine the significance of each of these sequences for expression of SSA1, we analyzed expression from a set of promoters containing point mutations in each of the HSEs, individually and in pairwise combinations. Of the three HSE-like sequences, two (HSE2 and HSE3) were active promoter elements; only one, HSE2, was active under basal growth conditions. Either HSE2 or HSE3 alone was able to drive SSA1 transcription at near-normal rates after heat shock. Both HSE2 and HSE3 were capable of driving basal transcription when placed in the context of the CYC1 promoter. Previous analysis had identified an upstream repressing sequence overlapping HSE2 that repressed basal transcription driven by HSE2. Our analysis showed that basal transcription driven by HSE3 was repressed both by the distant upstream repressing sequence and by closer flanking sequences. The ability to drive basal transcription is not inherent in all natural HSEs, since the HSEs from the heat-inducible SSA3 and SSA4 genes showed no basal activity when placed in the CYC1 vector. Gel mobility shift experiments showed that the same population of heat shock transcription factor molecules bound to HSEs capable of driving basal activity and to HSEs having very low or undetectable basal activity.

scholarly journals Saccharomyces cerevisiae HSP70 heat shock elements are functionally distinct

1993 ◽  
Vol 13 (9) ◽  
pp. 5637-5646
Author(s):  
M R Young ◽  
E A Craig
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heat Shock ◽  
Point Mutations ◽  
Growth Conditions ◽  
Mobility Shift ◽  
Basal Transcription ◽  
Basal Activity ◽  
Flanking Sequences ◽  
Gel Mobility Shift ◽  
Ability To Drive

The Saccharomyces cerevisiae HSP70 gene SSA1 has multiple heat shock elements (HSEs). To determine the significance of each of these sequences for expression of SSA1, we analyzed expression from a set of promoters containing point mutations in each of the HSEs, individually and in pairwise combinations. Of the three HSE-like sequences, two (HSE2 and HSE3) were active promoter elements; only one, HSE2, was active under basal growth conditions. Either HSE2 or HSE3 alone was able to drive SSA1 transcription at near-normal rates after heat shock. Both HSE2 and HSE3 were capable of driving basal transcription when placed in the context of the CYC1 promoter. Previous analysis had identified an upstream repressing sequence overlapping HSE2 that repressed basal transcription driven by HSE2. Our analysis showed that basal transcription driven by HSE3 was repressed both by the distant upstream repressing sequence and by closer flanking sequences. The ability to drive basal transcription is not inherent in all natural HSEs, since the HSEs from the heat-inducible SSA3 and SSA4 genes showed no basal activity when placed in the CYC1 vector. Gel mobility shift experiments showed that the same population of heat shock transcription factor molecules bound to HSEs capable of driving basal activity and to HSEs having very low or undetectable basal activity.

scholarly journals A bipartite operator interacts with a heat shock element to mediate early meiotic induction of Saccharomyces cerevisiae HSP82.

1995 ◽  
Vol 15 (12) ◽  
pp. 6754-6769 ◽  
Author(s):  
C Szent-Gyorgyi
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heat Shock ◽  
Point Mutations ◽  
Early Step ◽  
Heat Shock Element ◽  
Cis Acting ◽  
Major Mode ◽  
Dna Elements ◽  
Insight Into ◽  
Activation Sequence

Although key genetic regulators of early meiotic transcription in Saccharomyces cerevisiae have been well characterized, the activation of meiotic genes is still poorly understood in terms of cis-acting DNA elements and their associated factors. I report here that induction of HSP82 is regulated by the early meiotic IME1-IME2 transcriptional cascade. Vegetative repression and meiotic induction depend on interactions of the promoter-proximal heat shock element (HSE) with a nearby bipartite repression element, composed of the ubiquitous early meiotic motif, URS1 (upstream repression sequence 1), and a novel ancillary repression element. The ancillary repression element is required for efficient vegetative repression, is spatially separable from URS1, and continues to facilitate repression during sporulation. In contrast, URS1 also functions as a vegetative repression element but is converted early in meiosis into an HSE-dependent activation element. An early step in this transformation may be the antagonism of URS1-mediated repression by IME1. The HSE also nonspecifically supports a second major mode of meiotic activation that does not require URS1 but does require expression of IME2 and concurrent starvation. Interestingly, increased rather than decreased URS1-mediated vegetative transcription can be artificially achieved by introducing rare point mutations into URS1 or by deleting the UME6 gene. These lesions offer insight into mechanisms of URS-dependent repression and activation. Experiments suggest that URS1-bound factors functionally modulate heat shock factor during vegetative transcription and early meiotic induction but not during heat shock. The loss of repression and activation observed when the IME2 activation element, T4C, is substituted for the HSE suggests specific requirements for URS1-upstream activation sequence interactions.

scholarly journals Upstream sequences required for efficient expression of a soybean heat shock gene.

1986 ◽  
Vol 6 (2) ◽  
pp. 559-565 ◽  
Author(s):  
W B Gurley ◽  
E Czarnecka ◽  
R T Nagao ◽  
J L Key
Keyword(s):  
Heat Shock ◽  
Dna Sequences ◽  
Small Heat Shock Protein ◽  
Heat Shock Gene ◽  
Base Pairs ◽  
Basal Transcription ◽  
S1 Nuclease ◽  
Soybean Seedlings ◽  
Flanking Sequences ◽  
Shock Gene

A soybean gene (Gmhsp17.5-E) encoding a small heat shock protein was introduced into primary sunflower tumors via T-DNA-mediated transformation. RNA blot hybridizations and S1-nuclease hybrid protection studies indicated that the heat shock gene containing 3.25 kilobases of 5'-flanking sequences was strongly transcribed in a thermoinducible (40 degrees C) manner. Transcriptional induction also occurred to a lesser extent upon treatment of whole tumors with sodium arsenite and CdCl2. Basal (26 degrees C) transcription was not detected in soybean seedlings, but it was quite evident in transformed tumor tissue. A 5' deletion to -1,175 base pairs with respect to the CAP site had no effect on the levels of thermoinducible transcription, but it resulted in a large increase in basal transcription. Further removal of DNA sequences (including the TATA-distal heat shock consensus element) to -95 base pairs reduced thermoinducible transcription by 95% and also greatly decreased basal transcription. The termini of the Gmhsp17.5-E RNA in the tumor were generally the same as those present in soybean RNA, with the exception of several additional 3' termini.

U-rich sequence-binding proteins (URBPs) interacting with a 20-nucleotide U-rich sequence in the 3' untranslated region of c-fos mRNA may be involved in the first step of c-fos mRNA degradation

1992 ◽  
Vol 12 (7) ◽  
pp. 2931-2940
Author(s):  
Y You ◽  
C Y Chen ◽  
A B Shyu
Keyword(s):  
Untranslated Region ◽  
Binding Proteins ◽  
Critical Role ◽  
Point Mutations ◽  
Mrna Degradation ◽  
Binding Activity ◽  
Early Gene ◽  
Mobility Shift ◽  
Mobility Shift Assay ◽  
Gel Mobility Shift

Rapid decay of the c-fos transcript plays a critical role in controlling transforming potential of the c-fos proto-oncogene. One of the mRNA instability determinants is a 75-nucleotide AU-rich element (ARE) present in the 3' untranslated region of the c-fos transcript. It appears to control two steps in the process of c-fos mRNA degradation: removal of the poly(A) tail, which does not require the AUUUA motifs, and subsequent degradation of deadenylated mRNA, which appears to be dependent on the AUUUA motifs. In this study, we report the identification of four U-rich sequence binding proteins (URBPs) that specifically interact with a 20-nucleotide U-rich sequence within the c-fos ARE. Gel mobility shift assay and competition experiments showed that these protein factors form three specific band-shifted complexes with the c-fos ARE. Binding activity of one of the protein factors, a 37-kDa protein, is significantly affected by serum induction and by pretreatment of cells with drugs known to stabilize many of the immediate-early gene mRNAs. Combining UV cross-linking with a new approach, designated sequential RNase digestion, we were able to better determine the molecular masses of these cellular proteins. The binding sites for the four proteins were all mapped to a 20-nucleotide U-rich sequence located at the 3' half of the c-fos ARE, which contains no AUUUA pentanucleotides but stretches of uridylate residues. Single U-to-A point mutations in each of the three AUUUA motifs within the c-fos ARE have little effect on formation of the mobility-shifted complexes. Our data indicate c-fos ARE-protein interaction involves recognition of U stretches rather than recognition of the AUUUA motifs. We propose that UTBP binding may be involved in the first step, removal of the Poly(A) tail, in the c-fos ARE-mediated decay pathway.

scholarly journals In vitro studies of the binding of the ARGR proteins to the ARG5,6 promoter.

1991 ◽  
Vol 11 (4) ◽  
pp. 2162-2168 ◽  
Author(s):  
E Dubois ◽  
F Messenguy
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Specific Binding ◽  
Regulatory Element ◽  
Regulatory Proteins ◽  
Arginine Metabolism ◽  
Mobility Shift ◽  
Catabolic Genes ◽  
Gel Mobility Shift ◽  
Dna Complex

ARGRI, ARGRII, and ARGRIII regulatory proteins control the expression of arginine anabolic and catabolic genes in Saccharomyces cerevisiae. We show here that they are also required in vitro to observe a protein-DNA complex with the promoter of the ARG5,6 gene. The specific binding of ARGR proteins in vitro is stimulated by arginine. Antibodies raised against a synthetic MCM1 polypeptide retard the migration of ARGR-DNA complex on gel mobility shift assays. This result suggests that MCM1 could be an additional regulatory element of arginine metabolism.

scholarly journals Opposite roles of trehalase activity in heat-shock recovery and heat-shock survival in Saccharomyces cerevisiae

1999 ◽  
Vol 343 (3) ◽  
pp. 621-626 ◽  
Author(s):  
Stefaan WERA ◽  
Ellen DE SCHRIJVER ◽  
Ilse GEYSKENS ◽  
Solomon NWAKA ◽  
Johan M. THEVELEIN
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heat Shock ◽  
Specific Activity ◽  
Point Mutations ◽  
Activity Levels ◽  
Protective Function ◽  
Trehalase Activity ◽  
Neutral Trehalase

A variety of results has been obtained consistent with activation of neutral trehalase in Saccharomyces cerevisiae through direct phosphorylation by cAMP-dependent protein kinase (PKA). A series of neutral trehalase mutant alleles, in which all evolutionarily conserved putative phosphorylation sites were changed into alanine, was tested for activation in vitro (by PKA) and in vivo (by glucose addition). None of the mutations alone affected the activation ratio, whereas all mutations combined resulted in an inactive enzyme. All mutant alleles were expressed to similar levels, as shown by Western blotting. Several of the point mutations significantly lowered the specific activity. Using this series of mutants with different activity levels we show an inverse relationship between trehalase activity and heat-shock survival during glucose-induced trehalose mobilization. This is consistent with a stress-protective function of trehalose. On the other hand, reduction of trehalase activity below a certain threshold level impaired recovery from a sublethal heat shock. This suggests that trehalose breakdown is required for efficient recovery from heat shock, and that the presence of trehalase protein alone is not sufficient for efficient heat-stress recovery.

scholarly journals Transcriptional regulation of SSA3, an HSP70 gene from Saccharomyces cerevisiae.

1990 ◽  
Vol 10 (6) ◽  
pp. 3262-3267 ◽  
Author(s):  
W R Boorstein ◽  
E A Craig
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Heat Shock ◽  
Double Mutant ◽  
Optimal Growth ◽  
Growth Conditions ◽  
Hsp70 Gene ◽  
Base Pairs ◽  
Double Mutant Strain ◽  
Low Levels ◽  
Necessary And Sufficient

The SSA3 gene of Saccharomyces cerevisiae, a member of the HSP70 multigene family, is expressed at low levels under optimal growth conditions and is dramatically induced in response to heat shock. Sequences coinciding with two overlapping heat shock elements, located 156 base pairs upstream of the transcribed region, were necessary and sufficient for regulation of heat induction. The SSA3 promoter was also activated in an ssa1ssa2 double-mutant strain. This increase in the expression of SSA3 was mediated via the same upstream activating sequences that activated transcription in response to heat shock.

scholarly journals Cellular Transcription Factors Enhance Herpes Simplex Virus Type 1 oriS-Dependent DNA Replication

1998 ◽  
Vol 72 (5) ◽  
pp. 3635-3645 ◽  
Author(s):  
Anh Tuan Nguyen-Huynh ◽  
Priscilla A. Schaffer
Keyword(s):  
Dna Replication ◽  
Binding Sites ◽  
Cellular Proteins ◽  
Consensus Binding Site ◽  
Mobility Shift ◽  
Dna Complexes ◽  
Flanking Sequences ◽  
Gel Mobility Shift ◽  
Simplex Virus

ABSTRACT The herpes simplex virus type 1 (HSV-1) origin of DNA replication, oriS, contains three binding sites for the viral origin binding protein (OBP) flanked by transcriptional regulatory elements of the immediate-early genes encoding ICP4 and ICP22/47. To assess the role of flanking sequences in oriS function, plasmids containing oriS and either wild-type or mutant flanking sequences were tested in transient DNA replication assays. Although the ICP4 and ICP22/47 regulatory regions were shown to enhance oriS function, most individual elements in these regions, including the VP16-responsive TAATGARAT elements, were found to be dispensable for oriS function. In contrast, two oriS core-adjacent regulatory (Oscar) elements, OscarL and OscarR, at the base of the oriS palindrome were shown to enhance oriS function significantly and additively. Specifically, mutational disruption of either element reduced oriS-dependent DNA replication by 60 to 70%, and disruption of both elements reduced replication by 90%. The properties of protein-DNA complexes formed in gel mobility shift assays using uninfected and HSV-1-infected Vero cell nuclear extracts demonstrated that both OscarL and OscarR are binding sites for cellular proteins. Whereas OscarR does not correspond to the consensus binding site of any known transcription factor, OscarL contains a consensus binding site for the transcription factor Sp1. Gel mobility shift and supershift experiments using antibodies directed against members of the Sp1 family of transcription factors demonstrated the presence of Sp1 and Sp3, but not Sp2 or Sp4, in the protein-DNA complexes formed at OscarL. The abilities of OscarL and OscarR to bind their respective cellular proteins correlated directly with the efficiency of oriS-dependent DNA replication. Cooperative interactions between the Oscar-binding factors and proteins binding to adjacent OBP binding sites were not observed. Notably, Oscar element mutations that impaired oriS-dependent DNA replication had no detectable effect on either basal or induced levels of transcription from the ICP4 and ICP22/47 promoters, as determined by RNase protection assays. The Oscar elements thus appear to provide binding sites for cellular proteins that facilitate oriS-dependent DNA replication but have no effect on transcription of oriS-flanking genes.

In vitro studies of the binding of the ARGR proteins to the ARG5,6 promoter

1991 ◽  
Vol 11 (4) ◽  
pp. 2162-2168
Author(s):  
E Dubois ◽  
F Messenguy
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Specific Binding ◽  
Regulatory Element ◽  
Regulatory Proteins ◽  
Arginine Metabolism ◽  
Mobility Shift ◽  
Catabolic Genes ◽  
Gel Mobility Shift ◽  
Dna Complex

ARGRI, ARGRII, and ARGRIII regulatory proteins control the expression of arginine anabolic and catabolic genes in Saccharomyces cerevisiae. We show here that they are also required in vitro to observe a protein-DNA complex with the promoter of the ARG5,6 gene. The specific binding of ARGR proteins in vitro is stimulated by arginine. Antibodies raised against a synthetic MCM1 polypeptide retard the migration of ARGR-DNA complex on gel mobility shift assays. This result suggests that MCM1 could be an additional regulatory element of arginine metabolism.

NF1 transcriptional factor(s) is required for basal promoter activation of the human intestinal NaPi-IIb cotransporter gene

2005 ◽  
Vol 288 (2) ◽  
pp. G175-G181 ◽  
Author(s):  
Hua Xu ◽  
Jennifer K. Uno ◽  
Michael Inouye ◽  
James F. Collins ◽  
Fayez K. Ghishan
Keyword(s):  
Gc Content ◽  
Gene Promoter ◽  
Normal Growth ◽  
Protein S ◽  
Growth Conditions ◽  
Transcriptional Factor ◽  
Minimal Promoter ◽  
Mobility Shift ◽  
Nuclear Factor 1 ◽  
Gel Mobility Shift

The human intestinal type IIb Na+-Picotransporter (hNaPi-IIb) gene promoter lacks a TATA box and has a high GC content in the 5′-flanking region. To understand the mechanism of hNaPi-IIb gene transcription, the current study was performed to characterize the minimal promoter region and transcriptional factor(s) necessary to activate gene expression in human intestinal cells (Caco-2). With the use of progressively shorter promoter constructs, a minimal promoter extending from bp −58 to +15 was identified and shown to direct high levels of hNaPi-IIb cotransporter expression in Caco-2 cells. Gel mobility shift assays (GMSAs) indicated that two regions could be bound by nuclear proteins from Caco-2 cells: region A at bp −26/−23 and region B at bp −44/−35. The introduction of mutations in region A abolished promoter activity, whereas mutations in region B had no effect. Deletion mutants of the same regions showed identical results. Furthermore, DNase I footprinting experiments confirmed the observation made by GMSAs. Additional studies, which used a specific nuclear factor 1 (NF1) antiserum, demonstrated that NF1 protein(s) binds to the minimal promoter at region A. These results indicated that the NF1 protein(s) is required to activate the basal transcription of hNaPi-IIb gene under normal growth conditions. This study has thus identified a new target gene in the small intestinal epithelium that is directly regulated by NF1 transcriptional factor(s).

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