scholarly journals RNA polymerase II interacts with the promoter region of the noninduced hsp70 gene in Drosophila melanogaster cells.

1986 ◽  
Vol 6 (11) ◽  
pp. 3984-3989 ◽  
Author(s):  
D S Gilmour ◽  
J T Lis
Keyword(s):  
Drosophila Melanogaster ◽  
Heat Shock ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Promoter Region ◽  
High Energy ◽  
Heat Shock Gene ◽  
Cross Linking ◽  
Hsp70 Gene

By using a protein-DNA cross-linking method (D. S. Gilmour and J. T. Lis, Mol. Cell. Biol. 5:2009-2018, 1985), we examined the in vivo distribution of RNA polymerase II on the hsp70 heat shock gene in Drosophila melanogaster Schneider line 2 cells. In heat shock-induced cells, a high level of RNA polymerase II was detected on the entire gene, while in noninduced cells, the RNA polymerase II was confined to the 5' end of the hsp70 gene, predominantly between nucleotides -12 and +65 relative to the start of transcription. This association of RNA polymerase II was apparent whether the cross-linking was performed by a 10-min UV irradiation of chilled cells with mercury vapor lamps or by a 40-microsecond irradiation of cells with a high-energy xenon flash lamp. We hypothesize that RNA polymerase II has access to, and a high affinity for, the promoter region of this gene before induction, and this poised RNA polymerase II may be critical in the mechanism of transcription activation.

RNA polymerase II interacts with the promoter region of the noninduced hsp70 gene in Drosophila melanogaster cells

1986 ◽  
Vol 6 (11) ◽  
pp. 3984-3989
Author(s):  
D S Gilmour ◽  
J T Lis
Keyword(s):  
Drosophila Melanogaster ◽  
Heat Shock ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Promoter Region ◽  
High Energy ◽  
Heat Shock Gene ◽  
Cross Linking ◽  
Hsp70 Gene

By using a protein-DNA cross-linking method (D. S. Gilmour and J. T. Lis, Mol. Cell. Biol. 5:2009-2018, 1985), we examined the in vivo distribution of RNA polymerase II on the hsp70 heat shock gene in Drosophila melanogaster Schneider line 2 cells. In heat shock-induced cells, a high level of RNA polymerase II was detected on the entire gene, while in noninduced cells, the RNA polymerase II was confined to the 5' end of the hsp70 gene, predominantly between nucleotides -12 and +65 relative to the start of transcription. This association of RNA polymerase II was apparent whether the cross-linking was performed by a 10-min UV irradiation of chilled cells with mercury vapor lamps or by a 40-microsecond irradiation of cells with a high-energy xenon flash lamp. We hypothesize that RNA polymerase II has access to, and a high affinity for, the promoter region of this gene before induction, and this poised RNA polymerase II may be critical in the mechanism of transcription activation.

In vivo interactions of RNA polymerase II with genes of Drosophila melanogaster

1985 ◽  
Vol 5 (8) ◽  
pp. 2009-2018
Author(s):  
D S Gilmour ◽  
J T Lis
Keyword(s):  
Gene Expression ◽  
Drosophila Melanogaster ◽  
Heat Shock ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Protein Characterization ◽  
Heat Shock Gene ◽  
Transcriptional Level ◽  
Intact Cells

We describe a method for examining the in vivo distribution of a protein on specific eucaryotic DNA sequences. In this method, proteins are cross-linked to DNA in intact cells, and the protein-DNA adducts are isolated by immunoprecipitation with antiserum against the protein. Characterization of the DNA cross-linked to the precipitated protein identifies the sequences with which the protein is associated in vivo. Here, we applied these methods to detect RNA polymerase II-DNA interactions in heat-shocked and untreated Drosophila melanogaster Schneider line 2 cells. The level of RNA polymerase II associated with several heat shock genes increased dramatically in response to heat shock, whereas the level associated with the copia genes decreased, indicating that both induction of heat shock gene expression and repression of the copia gene expression by heat shock occur at the transcriptional level. Low levels of RNA polymerase II were present on DNA outside of the transcription units, and for at least two genes, hsp83 and hsp26, RNA polymerase II initiated binding near the transcription start site. Moreover, for hsp70, the density of RNA polymerase II on sequences downstream of the polyadenylate addition site was much lower than that observed on the gene internal sequences. Examination of the amount of specific restriction fragments cross-linked to RNA polymerase II provides a means of detecting RNA polymerase II on individual members of multigene families. This analysis shows that RNA polymerase II is associated with only one of the two cytoplasmic actin genes.

scholarly journals In vivo interactions of RNA polymerase II with genes of Drosophila melanogaster.

1985 ◽  
Vol 5 (8) ◽  
pp. 2009-2018 ◽  
Author(s):  
D S Gilmour ◽  
J T Lis
Keyword(s):  
Gene Expression ◽  
Drosophila Melanogaster ◽  
Heat Shock ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Protein Characterization ◽  
Heat Shock Gene ◽  
Transcriptional Level ◽  
Intact Cells

We describe a method for examining the in vivo distribution of a protein on specific eucaryotic DNA sequences. In this method, proteins are cross-linked to DNA in intact cells, and the protein-DNA adducts are isolated by immunoprecipitation with antiserum against the protein. Characterization of the DNA cross-linked to the precipitated protein identifies the sequences with which the protein is associated in vivo. Here, we applied these methods to detect RNA polymerase II-DNA interactions in heat-shocked and untreated Drosophila melanogaster Schneider line 2 cells. The level of RNA polymerase II associated with several heat shock genes increased dramatically in response to heat shock, whereas the level associated with the copia genes decreased, indicating that both induction of heat shock gene expression and repression of the copia gene expression by heat shock occur at the transcriptional level. Low levels of RNA polymerase II were present on DNA outside of the transcription units, and for at least two genes, hsp83 and hsp26, RNA polymerase II initiated binding near the transcription start site. Moreover, for hsp70, the density of RNA polymerase II on sequences downstream of the polyadenylate addition site was much lower than that observed on the gene internal sequences. Examination of the amount of specific restriction fragments cross-linked to RNA polymerase II provides a means of detecting RNA polymerase II on individual members of multigene families. This analysis shows that RNA polymerase II is associated with only one of the two cytoplasmic actin genes.

scholarly journals Rapid changes in Drosophila transcription after an instantaneous heat shock.

1993 ◽  
Vol 13 (6) ◽  
pp. 3456-3463 ◽  
Author(s):  
T O'Brien ◽  
J T Lis
Keyword(s):  
Heat Shock ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Uv Light ◽  
Hsp70 Gene ◽  
Rapid Induction ◽  
Rapid Changes ◽  
Rapid Activation ◽  
Kinetics Of

Heat shock rapidly activates expression of some genes and represses others. The kinetics of changes in RNA polymerase distribution on heat shock-modulated genes provides a framework for evaluating the mechanisms of activation and repression of transcription. Here, using two methods, we examined the changes in RNA polymerase II association on a set of Drosophila genes at 30-s intervals following an instantaneous heat shock. In the first method, Drosophila Schneider line 2 cells were quickly frozen to halt transcription, and polymerase distribution was analyzed by a nuclear run-on assay. RNA polymerase transcription at the 5' end of the hsp70 gene could be detected within 30 to 60 s of induction, and by 120 s the first wave of polymerase could already be detected near the 3' end of the gene. A similar rapid induction was found for the small heat shock genes (hsp22, hsp23, hsp26, and hsp27). In contrast to this rapid activation, transcription of the histone H1 gene was found to be rapidly repressed, with transcription reduced by approximately 90% within 300 s of heat shock. Similar results were obtained by an in vivo UV cross-linking assay. In this second method, cell samples removed at 30-s intervals were irradiated with 40-microseconds bursts of UV light from a Xenon flash lamp, and the distribution of polymerase was examined by precipitating UV cross-linked protein-DNA complexes with an antibody to RNA polymerase II. Both approaches also showed the in vivo rate of movement of the first wave of RNA polymerase through the hsp70 gene to be approximately 1.2 kb/min.

RNA polymerase II pauses at the 5' end of the transcriptionally induced Drosophila hsp70 gene

1991 ◽  
Vol 11 (10) ◽  
pp. 5285-5290
Author(s):  
T O'Brien ◽  
J T Lis
Keyword(s):  
Drosophila Melanogaster ◽  
Heat Shock ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Intermediate Level ◽  
Hsp70 Gene ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Initiation Of Transcription ◽  
Rate Limiting

An RNA polymerase II molecule is associated with the 5' end of the Drosophila melanogaster hsp70 gene under non-heat shock conditions. This polymerase is engaged in transcription but has paused, or arrested, after synthesizing about 25 nucleotides (A. E. Rougvie and J. T. Lis, Cell 54:795-804, 1988). Resumption of elongation by this paused polymerase appears to be the rate-limiting step in hsp70 transcription in uninduced cells. Here we report results of nuclear run-on assays that measure the distribution of elongating and paused RNA polymerase molecules on the hsp70 gene in induced cells. Pausing of polymerase was detected at the 5' end of hsp70 in cells exposed to the intermediate heat shock temperatures of 27 and 30 degrees C. At 30 degrees C, each copy of hsp70 was transcribed approximately five times during the 25-min heat shock that we used. Therefore, once the hsp70 gene is induced to an intermediate level, initiation of transcription by RNA polymerase II remains more rapid than the resumption of elongation by a paused polymerase molecule.

Rapid changes in Drosophila transcription after an instantaneous heat shock

1993 ◽  
Vol 13 (6) ◽  
pp. 3456-3463
Author(s):  
T O'Brien ◽  
J T Lis
Keyword(s):  
Heat Shock ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Uv Light ◽  
Hsp70 Gene ◽  
Rapid Induction ◽  
Rapid Changes ◽  
Rapid Activation ◽  
Kinetics Of

Heat shock rapidly activates expression of some genes and represses others. The kinetics of changes in RNA polymerase distribution on heat shock-modulated genes provides a framework for evaluating the mechanisms of activation and repression of transcription. Here, using two methods, we examined the changes in RNA polymerase II association on a set of Drosophila genes at 30-s intervals following an instantaneous heat shock. In the first method, Drosophila Schneider line 2 cells were quickly frozen to halt transcription, and polymerase distribution was analyzed by a nuclear run-on assay. RNA polymerase transcription at the 5' end of the hsp70 gene could be detected within 30 to 60 s of induction, and by 120 s the first wave of polymerase could already be detected near the 3' end of the gene. A similar rapid induction was found for the small heat shock genes (hsp22, hsp23, hsp26, and hsp27). In contrast to this rapid activation, transcription of the histone H1 gene was found to be rapidly repressed, with transcription reduced by approximately 90% within 300 s of heat shock. Similar results were obtained by an in vivo UV cross-linking assay. In this second method, cell samples removed at 30-s intervals were irradiated with 40-microseconds bursts of UV light from a Xenon flash lamp, and the distribution of polymerase was examined by precipitating UV cross-linked protein-DNA complexes with an antibody to RNA polymerase II. Both approaches also showed the in vivo rate of movement of the first wave of RNA polymerase through the hsp70 gene to be approximately 1.2 kb/min.

scholarly journals RNA polymerase II pauses at the 5' end of the transcriptionally induced Drosophila hsp70 gene.

1991 ◽  
Vol 11 (10) ◽  
pp. 5285-5290 ◽  
Author(s):  
T O'Brien ◽  
J T Lis
Keyword(s):  
Drosophila Melanogaster ◽  
Heat Shock ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Intermediate Level ◽  
Hsp70 Gene ◽  
Rate Limiting Step ◽  
Limiting Step ◽  
Initiation Of Transcription ◽  
Rate Limiting

An RNA polymerase II molecule is associated with the 5' end of the Drosophila melanogaster hsp70 gene under non-heat shock conditions. This polymerase is engaged in transcription but has paused, or arrested, after synthesizing about 25 nucleotides (A. E. Rougvie and J. T. Lis, Cell 54:795-804, 1988). Resumption of elongation by this paused polymerase appears to be the rate-limiting step in hsp70 transcription in uninduced cells. Here we report results of nuclear run-on assays that measure the distribution of elongating and paused RNA polymerase molecules on the hsp70 gene in induced cells. Pausing of polymerase was detected at the 5' end of hsp70 in cells exposed to the intermediate heat shock temperatures of 27 and 30 degrees C. At 30 degrees C, each copy of hsp70 was transcribed approximately five times during the 25-min heat shock that we used. Therefore, once the hsp70 gene is induced to an intermediate level, initiation of transcription by RNA polymerase II remains more rapid than the resumption of elongation by a paused polymerase molecule.

scholarly journals Heat shock and developmental regulation of the Drosophila melanogaster hsp83 gene.

1989 ◽  
Vol 9 (4) ◽  
pp. 1746-1753 ◽  
Author(s):  
H Xiao ◽  
J T Lis
Keyword(s):  
Drosophila Melanogaster ◽  
Heat Shock ◽  
Fusion Gene ◽  
Developmental Expression ◽  
Regulatory Sequence ◽  
Mrna Levels ◽  
Hsp70 Gene ◽  
High Level ◽  
Hsp83 Gene

In contrast to the hsp70 gene, whose expression is normally at a very low level and increases by more than 2 orders of magnitude during heat shock, the hsp83 gene in Drosophila melanogaster is expressed at high levels during normal development and increases only severalfold in response to heat shock. Developmental expression of the hsp83 gene consists of a high level of tissue-general, basal expression and a very high level of expression in ovaries. We identified regions upstream of the hsp83 gene that were required for its developmental and heat shock-induced expression by assaying beta-galactosidase activity and mRNA levels in transgenic animals containing a series of 5' deletion and insertion mutations of an hsp83-lacZ fusion gene. Deletion of sequences upstream of the overlapping array of a previously defined heat shock consensus (HSC) sequence eliminated both forms of developmental expression of the hsp83 gene. As a result, the hsp83 gene with this deletion mutation was regulated like that of the hsp70 gene. Moreover, an in vivo polymer competition assay revealed that the overlapping HSC sequences of the hsp83 gene and the nonoverlapping HSC sequences of the hsp70 gene had similar affinities for the factor required for heat induction of the two heat shock genes. We discuss the functional similarity of hsp70 and hsp83 heat shock regulation in terms of a revised view of the heat shock-regulatory sequence.

scholarly journals Protein-DNA cross-linking reveals dramatic variation in RNA polymerase II density on different histone repeats of Drosophila melanogaster.

1987 ◽  
Vol 7 (9) ◽  
pp. 3341-3344 ◽  
Author(s):  
D S Gilmour ◽  
J T Lis
Keyword(s):  
Drosophila Melanogaster ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Topoisomerase I ◽  
Transcriptional Activity ◽  
Base Pairs ◽  
Nontranscribed Spacer ◽  
Short Repeat ◽  
Dramatic Variation

In Drosophila melanogaster the five histone genes are within a 5-kilobase region which is repeated 100 times at a single chromosomal site. These 5-kilobase repeats are of two distinct classes, short and long, that differ by approximately 200 base pairs of DNA in the spacer region between the H1 and H3 genes. Since the mRNA-homologous regions of the repeats are highly conserved, one cannot examine differential expression of the repeats by classical hybridization methods. In this study, we assessed their transcriptional activity by measuring in vivo the relative amounts of RNA polymerase II that were cross-linked by UV irradiation to the two different histone repeats. The RNA polymerase II density on the long repeat in Schneider line 2 cells was strikingly lower (10-fold) than the density on the short repeat. The magnitude of this difference cannot be accounted for by reduced transcription of only one or two genes of the repeat. The density of topoisomerase I, an indicator of transcriptional activity, was also much higher on the short repeat than on the long repeat of line 2 cells. In contrast, the RNA polymerase II density was slightly higher on the long repeat than on the short repeat in a second cell line, KcH. The major difference between active (KcH) and inactive (S2) long repeats resides in the H1-H3 nontranscribed spacer. This portion of the spacer may contain a component necessary for expression that can act over a moderate distance and affect multiple genes of the repeat.

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