The chicken ubiquitin gene contains a heat shock promoter and expresses an unstable mRNA in heat-shocked cells

1986 ◽  
Vol 6 (12) ◽  
pp. 4602-4610
Author(s):  
U Bond ◽  
M J Schlesinger
Keyword(s):  
Heat Shock ◽  
Chicken Embryo ◽  
Actinomycin D ◽  
Genomic Library ◽  
Protein Coding ◽  
Noncoding Regions ◽  
Genomic Location ◽  
Embryo Fibroblasts ◽  
The Stability ◽  
Chicken Embryo Fibroblasts

A chicken genomic library was screened to obtain genomic clones for ubiquitin genes. Two genes that differ in their genomic location and organization were identified. One gene, designated Ub I, contains four copies of the protein-coding sequence arranged in tandem, while the second gene, Ub II, contains three. The origin of the two major mRNAs that are induced after heat shock in chicken embryo fibroblasts was determined by generating DNA probes from the 5'-and 3'-noncoding regions of the two genes. Both mRNAs are transcribed from Ub I, the larger being the unspliced precursor of the smaller. A 674-base-pair intron was located within the 5'-noncoding region of Ub I. The second gene, Ub II, does not appear to code for an RNA species in normal or heat-shocked chicken embryo fibroblasts. The expression of ubiquitin mRNA during heat shock and recovery was examined. Addition of actinomycin D before heat shock completely abolished the response of ubiquitin mRNA to the stress. Analysis of the stability of the mRNA during recovery revealed that the mRNA accumulated during the heat shock is rapidly degraded with a half-life of approximately 1.5 h, suggesting a specialized but transient role for ubiquitin during heat shock.

scholarly journals The chicken ubiquitin gene contains a heat shock promoter and expresses an unstable mRNA in heat-shocked cells.

1986 ◽  
Vol 6 (12) ◽  
pp. 4602-4610 ◽  
Author(s):  
U Bond ◽  
M J Schlesinger
Keyword(s):  
Heat Shock ◽  
Chicken Embryo ◽  
Actinomycin D ◽  
Genomic Library ◽  
Protein Coding ◽  
Noncoding Regions ◽  
Genomic Location ◽  
Embryo Fibroblasts ◽  
The Stability ◽  
Chicken Embryo Fibroblasts

A chicken genomic library was screened to obtain genomic clones for ubiquitin genes. Two genes that differ in their genomic location and organization were identified. One gene, designated Ub I, contains four copies of the protein-coding sequence arranged in tandem, while the second gene, Ub II, contains three. The origin of the two major mRNAs that are induced after heat shock in chicken embryo fibroblasts was determined by generating DNA probes from the 5'-and 3'-noncoding regions of the two genes. Both mRNAs are transcribed from Ub I, the larger being the unspliced precursor of the smaller. A 674-base-pair intron was located within the 5'-noncoding region of Ub I. The second gene, Ub II, does not appear to code for an RNA species in normal or heat-shocked chicken embryo fibroblasts. The expression of ubiquitin mRNA during heat shock and recovery was examined. Addition of actinomycin D before heat shock completely abolished the response of ubiquitin mRNA to the stress. Analysis of the stability of the mRNA during recovery revealed that the mRNA accumulated during the heat shock is rapidly degraded with a half-life of approximately 1.5 h, suggesting a specialized but transient role for ubiquitin during heat shock.

Ubiquitin is a heat shock protein in chicken embryo fibroblasts

1985 ◽  
Vol 5 (5) ◽  
pp. 949-956
Author(s):  
U Bond ◽  
M J Schlesinger
Keyword(s):  
Heat Shock ◽  
Chicken Embryo ◽  
Recombinant Dna ◽  
Protein Product ◽  
Amino Acid Sequences ◽  
In Vitro Translation ◽  
Bovine Ubiquitin ◽  
Embryo Fibroblasts ◽  
Chicken Embryo Fibroblasts

Clones containing heat-inducible mRNA sequences were selected from a cDNA library prepared from polyadenylated RNA isolated from heat-shocked chicken embryo fibroblasts. One recombinant DNA clone, designated clone 7, hybridized to a 1.2-kilobase RNA that was present in normal cells and increased fivefold during heat shock. Clone 7 also hybridized to an RNA species of 1.7 kilobases that was present exclusively in heat-shocked cells. In vitro translation of mRNA hybrid selected from clone 7 produced a protein product with a molecular weight of approximately 8,000. Increased synthesis of a protein of similar size was detected in chicken embryo fibroblasts after heat shock. DNA sequence analysis of clone 7 indicated its protein product has amino acid sequences identical to bovine ubiquitin. In addition, clone 7 contains tandem copies of the ubiquitin sequences contiguous to each other with no untranslated sequences between them. We discuss some possible roles for ubiquitin in the heat shock response.

scholarly journals The dynamic state of heat shock proteins in chicken embryo fibroblasts.

1986 ◽  
Vol 103 (4) ◽  
pp. 1495-1507 ◽  
Author(s):  
N C Collier ◽  
M J Schlesinger
Keyword(s):  
Heat Shock ◽  
Heat Shock Proteins ◽  
Intermediate Filament ◽  
Chicken Embryo ◽  
Dynamic State ◽  
Hsp 70 ◽  
Embryo Fibroblasts ◽  
Shock Proteins ◽  
Filament Network ◽  
Chicken Embryo Fibroblasts

Subcellular fractionation and immunofluorescence microscopy have been used to study the intracellular distributions of the major heat shock proteins, hsp 89, hsp 70, and hsp 24, in chicken embryo fibroblasts stressed by heat shock, allowed to recover and then restressed. Hsp 89 was localized primarily to the cytoplasm except during the restress when a portion of this protein concentrated in the nuclear region. Under all conditions, hsp 89 was readily extracted from cells by detergent. During stress and restress, significant amounts of hsp 70 moved to the nucleus and became resistant to detergent extraction. Some of this hsp 70 was released from the insoluble form in an ATP-dependent reaction. Hsp 24 was confined to the cytoplasm and, during restress, aggregated to detergent-insoluble perinuclear phase-dense granules. These granules dissociated during recovery and hsp 24 could be solubilized by detergent. The nuclear hsps reappeared in the cytoplasm in cells allowed to recover at normal temperatures. Sodium arsenite also induces hsps and their distributions were similar to that observed after a heat shock, except for hsp 89, which remained cytoplasmic. We also examined by immunofluorescence the cytoskeletal systems of chicken embryo fibroblasts subjected to heat shock and found no gross morphological changes in cytoplasmic microfilaments or microtubules. However, the intermediate filament network was very sensitive and collapsed around the nucleus very shortly after a heat shock. The normal intermediate filament morphology reformed when cells were allowed to recover from the stress. Inclusion of actinomycin D during the heat shock--a condition that prevents synthesis of the hsps--did not affect the intermediate filament collapse, but recovery of the normal morphology did not occur. We suggest that an hsp(s) may aid in the formation of the intermediate filament network after stress.

scholarly journals HSP47: a tissue-specific, transformation-sensitive, collagen-binding heat shock protein of chicken embryo fibroblasts.

1991 ◽  
Vol 11 (8) ◽  
pp. 4036-4044 ◽  
Author(s):  
K Hirayoshi ◽  
H Kudo ◽  
H Takechi ◽  
A Nakai ◽  
A Iwamatsu ◽  
...  
Keyword(s):  
Amino Acid ◽  
Heat Shock ◽  
Amino Acid Sequence ◽  
Heat Shock Protein ◽  
Chicken Embryo ◽  
Noncoding Region ◽  
Isolation And Characterization ◽  
Embryo Fibroblasts ◽  
Retention Signal ◽  
Chicken Embryo Fibroblasts

We report the isolation and characterization of a cDNA clone encoding HSP47, a transformation-sensitive heat shock protein that binds to collagen. A cDNA library was prepared from total RNA isolated from heat-shocked chicken embryo fibroblasts and screened by using oligonucleotide mixtures prepared on the basis of the N-terminal amino acid sequence of biochemically purified HSP47. The cDNA insert contained 3,278 bp, which encoded a 15-amino-acid signal peptide and a mature protein coding region consisting of 390 amino acid residues; it also included part of the 5' noncoding region and a long 3' noncoding region. The deduced amino acid sequence revealed an RDEL sequence at the C terminus, which is a variant of the KDEL retention signal for retention of proteins in the endoplasmic reticulum. Northern (RNA) blot analyses and nuclear run-on assays established that the induction of HSP47 by heat shock and its suppression after transformation of chicken embryo fibroblasts by Rous sarcoma virus are regulated at the transcriptional level. A homology search revealed that this protein belongs to the serpin family, the superfamily of plasma serine protease inhibitors. Although structurally homologous to the serpins, HSP47 lacks the active site thought to be essential for the inhibition of proteases and does not appear to bind to intracellular proteases. HSP47 is the first heat shock protein found to be a member of the serpin superfamily. Conversely, it is the first serpin family member that is not secreted from cells, which could be explained by acquisition of the RDEL retention signal during evolution.

scholarly journals Ubiquitin is a heat shock protein in chicken embryo fibroblasts.

1985 ◽  
Vol 5 (5) ◽  
pp. 949-956 ◽  
Author(s):  
U Bond ◽  
M J Schlesinger
Keyword(s):  
Heat Shock ◽  
Chicken Embryo ◽  
Recombinant Dna ◽  
Protein Product ◽  
Amino Acid Sequences ◽  
In Vitro Translation ◽  
Bovine Ubiquitin ◽  
Embryo Fibroblasts ◽  
Chicken Embryo Fibroblasts

Clones containing heat-inducible mRNA sequences were selected from a cDNA library prepared from polyadenylated RNA isolated from heat-shocked chicken embryo fibroblasts. One recombinant DNA clone, designated clone 7, hybridized to a 1.2-kilobase RNA that was present in normal cells and increased fivefold during heat shock. Clone 7 also hybridized to an RNA species of 1.7 kilobases that was present exclusively in heat-shocked cells. In vitro translation of mRNA hybrid selected from clone 7 produced a protein product with a molecular weight of approximately 8,000. Increased synthesis of a protein of similar size was detected in chicken embryo fibroblasts after heat shock. DNA sequence analysis of clone 7 indicated its protein product has amino acid sequences identical to bovine ubiquitin. In addition, clone 7 contains tandem copies of the ubiquitin sequences contiguous to each other with no untranslated sequences between them. We discuss some possible roles for ubiquitin in the heat shock response.

HSP47: a tissue-specific, transformation-sensitive, collagen-binding heat shock protein of chicken embryo fibroblasts

1991 ◽  
Vol 11 (8) ◽  
pp. 4036-4044
Author(s):  
K Hirayoshi ◽  
H Kudo ◽  
H Takechi ◽  
A Nakai ◽  
A Iwamatsu ◽  
...  
Keyword(s):  
Amino Acid ◽  
Heat Shock ◽  
Amino Acid Sequence ◽  
Heat Shock Protein ◽  
Chicken Embryo ◽  
Noncoding Region ◽  
Isolation And Characterization ◽  
Embryo Fibroblasts ◽  
Retention Signal ◽  
Chicken Embryo Fibroblasts

We report the isolation and characterization of a cDNA clone encoding HSP47, a transformation-sensitive heat shock protein that binds to collagen. A cDNA library was prepared from total RNA isolated from heat-shocked chicken embryo fibroblasts and screened by using oligonucleotide mixtures prepared on the basis of the N-terminal amino acid sequence of biochemically purified HSP47. The cDNA insert contained 3,278 bp, which encoded a 15-amino-acid signal peptide and a mature protein coding region consisting of 390 amino acid residues; it also included part of the 5' noncoding region and a long 3' noncoding region. The deduced amino acid sequence revealed an RDEL sequence at the C terminus, which is a variant of the KDEL retention signal for retention of proteins in the endoplasmic reticulum. Northern (RNA) blot analyses and nuclear run-on assays established that the induction of HSP47 by heat shock and its suppression after transformation of chicken embryo fibroblasts by Rous sarcoma virus are regulated at the transcriptional level. A homology search revealed that this protein belongs to the serpin family, the superfamily of plasma serine protease inhibitors. Although structurally homologous to the serpins, HSP47 lacks the active site thought to be essential for the inhibition of proteases and does not appear to bind to intracellular proteases. HSP47 is the first heat shock protein found to be a member of the serpin superfamily. Conversely, it is the first serpin family member that is not secreted from cells, which could be explained by acquisition of the RDEL retention signal during evolution.

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