scholarly journals c-myc can induce expression of G0/G1 transition genes.

1988 ◽  
Vol 8 (8) ◽  
pp. 3080-3087 ◽  
Author(s):  
C W Schweinfest ◽  
S Fujiwara ◽  
L F Lau ◽  
T S Papas
Keyword(s):  
Heat Shock ◽  
Cdna Clones ◽  
Nuclear Fraction ◽  
Mrna Levels ◽  
Protein Species ◽  
3T3 Cells ◽  
Hsp70 Promoter ◽  
Myc Oncogene ◽  
Serum Stimulation ◽  
Specific Induction

The human c-myc oncogene was linked to the heat shock-inducible Drosophila hsp70 promoter and used to stably transfect mouse BALB/c 3T3 cells. Heat shock of the transfectants at 42 degrees C followed by recovery at 37 degrees C resulted in the appearance of the human c-myc protein which was appropriately localized to the nuclear fraction. Two-dimensional analysis of the proteins of density-arrested cells which had been heat shock treated revealed the induction of eight protein species and the repression of five protein species. All of the induced and repressed proteins were nonabundant. cDNA clones corresponding to genes induced during the G0/G1 transition were used as probes to assay for c-myc inducibility of these genes. Two anonymous sequences previously identified as serum inducible (3CH77 and 3CH92) were induced when c-myc was expressed. In response to serum stimulation, 3CH77 and 3CH92 were expressed before c-myc mRNA levels increased. However, in response to specific induction of c-myc by heat shock of serum arrested cells, 3CH77 and 3CH92 mRNA levels increased after the rise in c-myc mRNA. Therefore, we hypothesize that abnormal expression of c-myc can induce genes involved in the proliferative response.

c-myc can induce expression of G0/G1 transition genes

1988 ◽  
Vol 8 (8) ◽  
pp. 3080-3087
Author(s):  
C W Schweinfest ◽  
S Fujiwara ◽  
L F Lau ◽  
T S Papas
Keyword(s):  
Heat Shock ◽  
Cdna Clones ◽  
Nuclear Fraction ◽  
Mrna Levels ◽  
Protein Species ◽  
3T3 Cells ◽  
Hsp70 Promoter ◽  
Myc Oncogene ◽  
Serum Stimulation ◽  
Specific Induction

The human c-myc oncogene was linked to the heat shock-inducible Drosophila hsp70 promoter and used to stably transfect mouse BALB/c 3T3 cells. Heat shock of the transfectants at 42 degrees C followed by recovery at 37 degrees C resulted in the appearance of the human c-myc protein which was appropriately localized to the nuclear fraction. Two-dimensional analysis of the proteins of density-arrested cells which had been heat shock treated revealed the induction of eight protein species and the repression of five protein species. All of the induced and repressed proteins were nonabundant. cDNA clones corresponding to genes induced during the G0/G1 transition were used as probes to assay for c-myc inducibility of these genes. Two anonymous sequences previously identified as serum inducible (3CH77 and 3CH92) were induced when c-myc was expressed. In response to serum stimulation, 3CH77 and 3CH92 were expressed before c-myc mRNA levels increased. However, in response to specific induction of c-myc by heat shock of serum arrested cells, 3CH77 and 3CH92 mRNA levels increased after the rise in c-myc mRNA. Therefore, we hypothesize that abnormal expression of c-myc can induce genes involved in the proliferative response.

Control of proliferin gene expression in serum-stimulated mouse cells

1987 ◽  
Vol 7 (6) ◽  
pp. 2080-2086
Author(s):  
D I Linzer ◽  
E L Wilder
Keyword(s):  
Protein Synthesis ◽  
S Phase ◽  
Mrna Levels ◽  
3T3 Cells ◽  
Serum Stimulation ◽  
Posttranscriptional Processing ◽  
A Cell ◽  
Mouse Cells ◽  
Stimulation Of

The serum-inducible expression of proliferin genes in BALB/c 3T3 cells was found to be dependent on both protein synthesis and an extended presence of serum in the medium. Even though no mature proliferin mRNA was detected in serum-starved cells, transcription of the proliferin genes occurred in these resting-cell cultures, indicating that posttranscriptional events may be important for regulating proliferin mRNA levels. These results suggest that protein synthesis after serum stimulation of quiescent mouse fibroblasts is required for posttranscriptional processing or stabilization of proliferin RNA. Proliferin RNA levels were found to be heterogeneous among serum-stimulated cells analyzed by in situ hybridization. This heterogeneity is probably due to asynchrony in the population and may point to a correlation between the time of proliferin expression and the time of entry of a cell into S phase.

scholarly journals Control of proliferin gene expression in serum-stimulated mouse cells.

1987 ◽  
Vol 7 (6) ◽  
pp. 2080-2086 ◽  
Author(s):  
D I Linzer ◽  
E L Wilder
Keyword(s):  
Protein Synthesis ◽  
S Phase ◽  
Mrna Levels ◽  
3T3 Cells ◽  
Serum Stimulation ◽  
Posttranscriptional Processing ◽  
A Cell ◽  
Mouse Cells ◽  
Stimulation Of

The serum-inducible expression of proliferin genes in BALB/c 3T3 cells was found to be dependent on both protein synthesis and an extended presence of serum in the medium. Even though no mature proliferin mRNA was detected in serum-starved cells, transcription of the proliferin genes occurred in these resting-cell cultures, indicating that posttranscriptional events may be important for regulating proliferin mRNA levels. These results suggest that protein synthesis after serum stimulation of quiescent mouse fibroblasts is required for posttranscriptional processing or stabilization of proliferin RNA. Proliferin RNA levels were found to be heterogeneous among serum-stimulated cells analyzed by in situ hybridization. This heterogeneity is probably due to asynchrony in the population and may point to a correlation between the time of proliferin expression and the time of entry of a cell into S phase.

Isolation of mutations that act in trans to alter expression from a yeast hsp70 promoter

1988 ◽  
Vol 8 (8) ◽  
pp. 3423-3431
Author(s):  
R C Findly ◽  
H Alavi ◽  
T Platt
Keyword(s):  
Heat Shock ◽  
Fusion Gene ◽  
Fusion Transcript ◽  
Mrna Levels ◽  
Hsp70 Promoter ◽  
Transcript Stability ◽  
In Trans ◽  
Selection For ◽  
Rna Blots ◽  
Beta Galactosidase

Transcription of SSA1 (formerly YG100), a member of the hsp70 gene family in Saccharomyces cerevisiae, increases dramatically upon heat shock. An expression vector in which the promoter of SSA1 is fused to the Escherichia coli galactokinase gene (galK) was constructed and transformed into a galactokinase-deficient yeast strain. The transformants grew on galactose at 23 degrees C, but increased expression of the SSA1-galK fusion gene inhibited growth of cells on galactose at 37 degrees C. Selection for survivors under nonpermissive conditions yielded a class of mutants, termed HSR (for heat shock regulation), which showed reduced levels of expression of the hsp70-galK gene fusion as determined by measurement of galactokinase activity. Similar effects on beta-galactosidase activity were obtained when an SSA1-lacZ fusion vector was introduced into the mutants, suggesting action in trans through the SSA1 promoter. Analysis of Northern (RNA) blots demonstrated that the reduction in expression was a result of decreased mRNA levels for the fusion gene. In addition, mRNA levels of the endogenous SSA1 gene are reduced in an HSR mutant. Genetic analysis has shown that these mutations act in trans and affect both transcription from the SSA1 promoter and turnover of the fusion transcript. These are the first trans-acting mutations known to affect directly the transcriptional regulation and transcript stability of heat shock genes in eucaryotes.

scholarly journals Isolation of mutations that act in trans to alter expression from a yeast hsp70 promoter.

1988 ◽  
Vol 8 (8) ◽  
pp. 3423-3431 ◽  
Author(s):  
R C Findly ◽  
H Alavi ◽  
T Platt
Keyword(s):  
Heat Shock ◽  
Fusion Gene ◽  
Fusion Transcript ◽  
Mrna Levels ◽  
Hsp70 Promoter ◽  
Transcript Stability ◽  
In Trans ◽  
Selection For ◽  
Rna Blots ◽  
Beta Galactosidase

Transcription of SSA1 (formerly YG100), a member of the hsp70 gene family in Saccharomyces cerevisiae, increases dramatically upon heat shock. An expression vector in which the promoter of SSA1 is fused to the Escherichia coli galactokinase gene (galK) was constructed and transformed into a galactokinase-deficient yeast strain. The transformants grew on galactose at 23 degrees C, but increased expression of the SSA1-galK fusion gene inhibited growth of cells on galactose at 37 degrees C. Selection for survivors under nonpermissive conditions yielded a class of mutants, termed HSR (for heat shock regulation), which showed reduced levels of expression of the hsp70-galK gene fusion as determined by measurement of galactokinase activity. Similar effects on beta-galactosidase activity were obtained when an SSA1-lacZ fusion vector was introduced into the mutants, suggesting action in trans through the SSA1 promoter. Analysis of Northern (RNA) blots demonstrated that the reduction in expression was a result of decreased mRNA levels for the fusion gene. In addition, mRNA levels of the endogenous SSA1 gene are reduced in an HSR mutant. Genetic analysis has shown that these mutations act in trans and affect both transcription from the SSA1 promoter and turnover of the fusion transcript. These are the first trans-acting mutations known to affect directly the transcriptional regulation and transcript stability of heat shock genes in eucaryotes.

scholarly journals Induced expression of a Drosophila hsp70 promoter-fusion transgene is reduced after repeated heat shocks

1992 ◽  
Vol 59 (3) ◽  
pp. 183-188 ◽  
Author(s):  
S. M. N. Hunt ◽  
M. R. Wilkins ◽  
H. W. Stokes ◽  
G. E. Daggard ◽  
R. Frankham
Keyword(s):  
Heat Shock ◽  
Antisense Rna ◽  
Heat Shock Protein 70 ◽  
Fusion Gene ◽  
Mrna Levels ◽  
Hsp70 Promoter ◽  
Expression Studies ◽  
Promoter Fusion ◽  
Heat Shocks

SummaryLevels of transcripts produced by a heat shock protein 70 (hsp70)-antisense white transgene in Drosophila were measured after single and multiple heat shocks to determine whether the hsp70 promoter could produce sustained high levels of transgene transcripts. A single heat shock resulted in typical highly inducible levels of RNA, but the amount of antisense RNA was substantially reduced after multiple heat shocks. Endogenous hsp70 mRNA levels were also less abundant after multiple heat shocks as compared to a single heat shock. The hsp70 promoter is unsuitable for use in fusion gene constructs for long term expression studies where repeated heat shocks are required.

Complexity of the early genetic response to growth factors in mouse fibroblasts

1988 ◽  
Vol 8 (5) ◽  
pp. 2140-2148
Author(s):  
J M Almendral ◽  
D Sommer ◽  
H Macdonald-Bravo ◽  
J Burckhardt ◽  
J Perera ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Mrna Levels ◽  
Genetic Response ◽  
3T3 Cells ◽  
Recombinant Phage ◽  
Cross Hybridization ◽  
Serum Stimulation ◽  
Cell Proliferation And Differentiation ◽  
Proliferation And Differentiation ◽  
Nih 3T3

Genes whose expression is growth factor regulated are likely to be important components in the mechanisms controlling cell proliferation and differentiation. With the aim of identifying some of those genes, a lambda cDNA library was prepared with poly(A)+ RNA from quiescent NIH 3T3 cells stimulated with serum for 4 h in the presence of cycloheximide. Differential screening of approximately 200,000 recombinant phage plaques revealed 2,540 clones that cross hybridized preferentially with [32P]cDNA derived from RNA of stimulated cells rather than with cDNA derived from nonstimulated cells. Cross hybridization of these clones identified 82 independent sequences, including c-fos and c-myc. Seventy-one clones were further studied. Analysis of the changes in transcription and mRNA levels after serum stimulation demonstrated that the kinetics and extent of the induction vary dramatically between the different genes. Cycloheximide in all cases superinduced the mRNA levels by two mechanisms, inhibiting the shutoff of transcription and prolonging the half-lives of the mRNAs. Our results showed that induction of proliferation is accompanied by the onset of a complex genetic program.

scholarly journals Complexity of the early genetic response to growth factors in mouse fibroblasts.

1988 ◽  
Vol 8 (5) ◽  
pp. 2140-2148 ◽  
Author(s):  
J M Almendral ◽  
D Sommer ◽  
H Macdonald-Bravo ◽  
J Burckhardt ◽  
J Perera ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Mrna Levels ◽  
Genetic Response ◽  
3T3 Cells ◽  
Recombinant Phage ◽  
Cross Hybridization ◽  
Serum Stimulation ◽  
Cell Proliferation And Differentiation ◽  
Proliferation And Differentiation ◽  
Nih 3T3

Genes whose expression is growth factor regulated are likely to be important components in the mechanisms controlling cell proliferation and differentiation. With the aim of identifying some of those genes, a lambda cDNA library was prepared with poly(A)+ RNA from quiescent NIH 3T3 cells stimulated with serum for 4 h in the presence of cycloheximide. Differential screening of approximately 200,000 recombinant phage plaques revealed 2,540 clones that cross hybridized preferentially with [32P]cDNA derived from RNA of stimulated cells rather than with cDNA derived from nonstimulated cells. Cross hybridization of these clones identified 82 independent sequences, including c-fos and c-myc. Seventy-one clones were further studied. Analysis of the changes in transcription and mRNA levels after serum stimulation demonstrated that the kinetics and extent of the induction vary dramatically between the different genes. Cycloheximide in all cases superinduced the mRNA levels by two mechanisms, inhibiting the shutoff of transcription and prolonging the half-lives of the mRNAs. Our results showed that induction of proliferation is accompanied by the onset of a complex genetic program.

scholarly journals Cell-cycle-specific and serum-dependent expression of gamma-actin mRNA in Swiss mouse 3T3 cells.

1988 ◽  
Vol 8 (6) ◽  
pp. 2288-2294 ◽  
Author(s):  
A S Masibay ◽  
P K Qasba ◽  
D N Sengupta ◽  
G P Damewood ◽  
T Sreevalsan
Keyword(s):  
Growth Factor ◽  
Calcium Ionophore ◽  
Swiss Mouse ◽  
Cdna Clones ◽  
Ionophore A23187 ◽  
Resting Cells ◽  
Mrna Levels ◽  
3T3 Cells ◽  
Specific Transcript ◽  
Actin Mrna

We isolated cDNA clones that represent genes whose expression is enhanced when resting Swiss mouse 3T3 cells are stimulated to proliferate with serum. Two clones (designated pME1 and pMR6) were analyzed further. A partial sequence analysis of the pME1 insert DNA indicated that it contained a 104-base-pair stretch with extensive homology to the 3' untranslated region of gamma actin. Similar analysis of the insert DNA from the pMR6 clone indicated that it did not correspond to any previously reported gene sequence. We used the pME1 clone as a probe to determine the level of gamma actin-specific transcript in 3T3 cells under a variety of conditions. The level of gamma actin-specific mRNA began to increase in resting cells upon serum stimulation and reached a peak at 6 h. Thereafter its level declined, and by 24 h it was hardly detectable. In contrast, pMR6-specific transcript was detectable in resting cells but remained elevated even at 24 h poststimulation. The level of gamma-actin mRNA was elevated in resting cells by 12-O-tetradecanoylphorbol-13-acetate, calcium ionophore A23187, and bombesin and to a lesser extent by cholera toxin, fibroblast-derived growth factor, and dibutyryl cyclic AMP. However, insulin, vasopressin, or epidermal growth factor failed to enhance gamma-actin mRNA levels in resting cells. Inhibitors of transcription diminished the induction of gamma-actin mRNA. Gamma-actin gene was superinduced in serum-stimulated cells by cycloheximide, an inhibitor of translation. Analysis of proteins from serum-stimulated cells by two-dimensional gel electrophoresis indicated that enhanced transcription of gamma-actin mRNA resulted in a concomitant increase in the corresponding actin protein. The possible role of gamma actin, a component of the cytoskeleton, in the regulation of cell growth is discussed.

Cell-cycle-specific and serum-dependent expression of gamma-actin mRNA in Swiss mouse 3T3 cells

1988 ◽  
Vol 8 (6) ◽  
pp. 2288-2294
Author(s):  
A S Masibay ◽  
P K Qasba ◽  
D N Sengupta ◽  
G P Damewood ◽  
T Sreevalsan
Keyword(s):  
Growth Factor ◽  
Calcium Ionophore ◽  
Swiss Mouse ◽  
Cdna Clones ◽  
Ionophore A23187 ◽  
Resting Cells ◽  
Mrna Levels ◽  
3T3 Cells ◽  
Specific Transcript ◽  
Actin Mrna

We isolated cDNA clones that represent genes whose expression is enhanced when resting Swiss mouse 3T3 cells are stimulated to proliferate with serum. Two clones (designated pME1 and pMR6) were analyzed further. A partial sequence analysis of the pME1 insert DNA indicated that it contained a 104-base-pair stretch with extensive homology to the 3' untranslated region of gamma actin. Similar analysis of the insert DNA from the pMR6 clone indicated that it did not correspond to any previously reported gene sequence. We used the pME1 clone as a probe to determine the level of gamma actin-specific transcript in 3T3 cells under a variety of conditions. The level of gamma actin-specific mRNA began to increase in resting cells upon serum stimulation and reached a peak at 6 h. Thereafter its level declined, and by 24 h it was hardly detectable. In contrast, pMR6-specific transcript was detectable in resting cells but remained elevated even at 24 h poststimulation. The level of gamma-actin mRNA was elevated in resting cells by 12-O-tetradecanoylphorbol-13-acetate, calcium ionophore A23187, and bombesin and to a lesser extent by cholera toxin, fibroblast-derived growth factor, and dibutyryl cyclic AMP. However, insulin, vasopressin, or epidermal growth factor failed to enhance gamma-actin mRNA levels in resting cells. Inhibitors of transcription diminished the induction of gamma-actin mRNA. Gamma-actin gene was superinduced in serum-stimulated cells by cycloheximide, an inhibitor of translation. Analysis of proteins from serum-stimulated cells by two-dimensional gel electrophoresis indicated that enhanced transcription of gamma-actin mRNA resulted in a concomitant increase in the corresponding actin protein. The possible role of gamma actin, a component of the cytoskeleton, in the regulation of cell growth is discussed.

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