In Vitro Transcription: Whole-Cell Extract

1989 ◽  
pp. 653-666
Author(s):  
JAMES L. MANLEY ◽  
ANDREW FIRE ◽  
MARK SAMUELS ◽  
PHILLIP A. SHARP
Keyword(s):  
In Vitro Transcription ◽  
Cell Extract ◽  
Whole Cell ◽  
Whole Cell Extract

[35] In Vitro transcription: Whole-cell extract

1983 ◽  
pp. 568-582 ◽  
Author(s):  
James L. Manley ◽  
Andrew Fire ◽  
Mark Samuels ◽  
Phillip A. Sharp
Keyword(s):  
In Vitro Transcription ◽  
Cell Extract ◽  
Whole Cell ◽  
Whole Cell Extract

Transcriptional activation in an improved whole-cell extract from Saccharomyces cerevisiae

1991 ◽  
Vol 11 (9) ◽  
pp. 4555-4560 ◽  
Author(s):  
M Woontner ◽  
P A Wade ◽  
J Bonner ◽  
J A Jaehning
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
In Vitro Transcription ◽  
Cell Extract ◽  
Whole Cell ◽  
Transcription System ◽  
Upstream Activation Sequence ◽  
Activation Sequence

We report an improved in vitro transcription system for Saccharomyces cerevisiae. Small changes in assay and whole-cell extraction procedures increase selective initiation by RNA polymerase II up to 60-fold over previous conditions (M. Woontner and J. A. Jaehning, J. Biol. Chem. 265:8979-8982, 1990), to levels comparable to those obtained with nuclear extracts. We have found that the simultaneous use of distinguishable templates with and without an upstream activation sequence is critical to the measurement of apparent activation. Transcription from any template was very sensitive to the concentrations of template and nontemplate DNA, extract, and activator (GAL4/VP16). Alterations in reaction conditions led to proportionately greater changes from a template lacking an upstream activation sequence; thus, the apparent ratio of activation is largely dependent on the level of basal transcription. Using optimal conditions for activation, we have also demonstrated activation by a bona fide yeast activator, heat shock transcription factor.

scholarly journals The block to transcription elongation at the minute virus of mice attenuator is regulated by cellular elongation factors.

1991 ◽  
Vol 11 (7) ◽  
pp. 3515-3521 ◽  
Author(s):  
A Krauskopf ◽  
E Bengal ◽  
Y Aloni
Keyword(s):  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Cell Extract ◽  
Elongation Factors ◽  
Minute Virus Of Mice ◽  
Whole Cell ◽  
Minute Virus ◽  
Whole Cell Extract

We have previously reported that both in vivo and in vitro, RNA polymerase II pauses or prematurely terminates transcription at a specific attenuation site located 142 to 147 nucleotides downstream from the P4 promoter of minute virus of mice (MVM). In this report, we show that an in vitro block to transcription elongation in HeLa whole-cell extract occurs at elevated KCl concentrations (0.2 to 1.5 M) but not at the standard KCl concentration (50 mM). Briefly initiated transcription complexes, devoid of dissociated elongation factors by passage through a Sephacryl S-1000 column at 0.3 M KCl, were allowed to elongate the briefly initiated nascent RNA, and a block to transcription elongation at the attenuation site was observed independently of the KCl concentration at the time of elongation. Moreover, the block to elongation was overcome by the addition, during elongation, to the column of purified complexes of whole-cell extract from EA cells but not from MVM-infected EA cells or HeLa cells. The general transcription factors IIF and IIX were also shown to alleviate this block to transcription elongation. On the basis of these results, we suggest that the block to elongation at the MVM attenuation site observed late in MVM infection results, at least in part, from the inactivation of the general transcription elongation factors.

scholarly journals Transcriptional activation in an improved whole-cell extract from Saccharomyces cerevisiae.

1991 ◽  
Vol 11 (9) ◽  
pp. 4555-4560 ◽  
Author(s):  
M Woontner ◽  
P A Wade ◽  
J Bonner ◽  
J A Jaehning
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
In Vitro Transcription ◽  
Cell Extract ◽  
Whole Cell ◽  
Transcription System ◽  
Upstream Activation Sequence ◽  
Activation Sequence

We report an improved in vitro transcription system for Saccharomyces cerevisiae. Small changes in assay and whole-cell extraction procedures increase selective initiation by RNA polymerase II up to 60-fold over previous conditions (M. Woontner and J. A. Jaehning, J. Biol. Chem. 265:8979-8982, 1990), to levels comparable to those obtained with nuclear extracts. We have found that the simultaneous use of distinguishable templates with and without an upstream activation sequence is critical to the measurement of apparent activation. Transcription from any template was very sensitive to the concentrations of template and nontemplate DNA, extract, and activator (GAL4/VP16). Alterations in reaction conditions led to proportionately greater changes from a template lacking an upstream activation sequence; thus, the apparent ratio of activation is largely dependent on the level of basal transcription. Using optimal conditions for activation, we have also demonstrated activation by a bona fide yeast activator, heat shock transcription factor.

scholarly journals The block to transcription elongation at the minute virus of mice attenuator is regulated by cellular elongation factors

1991 ◽  
Vol 11 (7) ◽  
pp. 3515-3521
Author(s):  
A Krauskopf ◽  
E Bengal ◽  
Y Aloni
Keyword(s):  
Rna Polymerase Ii ◽  
Transcription Elongation ◽  
Cell Extract ◽  
Elongation Factors ◽  
Minute Virus Of Mice ◽  
Whole Cell ◽  
Minute Virus ◽  
Whole Cell Extract

We have previously reported that both in vivo and in vitro, RNA polymerase II pauses or prematurely terminates transcription at a specific attenuation site located 142 to 147 nucleotides downstream from the P4 promoter of minute virus of mice (MVM). In this report, we show that an in vitro block to transcription elongation in HeLa whole-cell extract occurs at elevated KCl concentrations (0.2 to 1.5 M) but not at the standard KCl concentration (50 mM). Briefly initiated transcription complexes, devoid of dissociated elongation factors by passage through a Sephacryl S-1000 column at 0.3 M KCl, were allowed to elongate the briefly initiated nascent RNA, and a block to transcription elongation at the attenuation site was observed independently of the KCl concentration at the time of elongation. Moreover, the block to elongation was overcome by the addition, during elongation, to the column of purified complexes of whole-cell extract from EA cells but not from MVM-infected EA cells or HeLa cells. The general transcription factors IIF and IIX were also shown to alleviate this block to transcription elongation. On the basis of these results, we suggest that the block to elongation at the MVM attenuation site observed late in MVM infection results, at least in part, from the inactivation of the general transcription elongation factors.

scholarly journals Chromatin assembly in a yeast whole-cell extract

1997 ◽  
Vol 94 (17) ◽  
pp. 9034-9039 ◽  
Author(s):  
M. C. Schultz ◽  
D. J. Hockman ◽  
T. A. A. Harkness ◽  
W. I. Garinther ◽  
B. A. Altheim
Keyword(s):  
Cell Extract ◽  
Chromatin Assembly ◽  
Whole Cell ◽  
Whole Cell Extract

scholarly journals Effect of Calcium Carbide on Rat Tissue

1970 ◽  
Vol 6 (2) ◽  
pp. 93-98 ◽  
Author(s):  
Younus Patoare ◽  
Md Iqbal Hossain ◽  
Mohammad Nurul Islam ◽  
Akhtarunnessa Chowdhury ◽  
Seheli Parveen ◽  
...  
Keyword(s):  
Acid Phosphatase ◽  
Calcium Carbide ◽  
Cell Extract ◽  
Control Group ◽  
Histopathological Study ◽  
Histopathological Analysis ◽  
Whole Cell ◽  
Group I ◽  
Collecting Tubules ◽  
Whole Cell Extract

To evaluate the effects of Calcium carbide (CaC2) in biological system, an in vivo study was carried out on Long Evans rats. CaC2 was administered orally once daily for one month with specific concentrations. Group- I was considered as the control group (without CaC2), Group - II, III, IV and V were the sample groups treated with CaC2 having concentration of 1g/kg, 2g/kg, 5g/kg and 10g/kg body weight respectively. The experiment was conducted to detect any cellular and molecular level changes caused by CaC2. The histopathology and isozyme assay were performed to analyze the changes in the activities of the genes affected by the free radicals released from CaC2. The molecular analysis included different isozymes namely esterase and acid phosphatase. Polyacrylamide electrophoresis of whole cell extract of control subjects and CaC2 administered rats were performed; subsequently the gels were treated with the substrates for acid phosphatase and esterase respectively. No difference was observed in the whole cell extract band pattern between the control subject and the CaC2 administered rats, which grossly indicates that the CaC2 has no effect on the expression pattern of isozymes (acid phosphatase and esterase). Histopathological analysis of liver, heart, spleen, kidney and lungs were performed to observe any change due to the administration of CaC2. Remarkable changes were observed during the histopathological study of lungs and kidney only. The histopathological analysis of kidney showed the thickening of the lining of collecting tubules with changes in cell structure while lungs were found to be increased moderately in weight, with focal areas of consolidation that was found red-brown to red. Key words: Calcium carbide; Histopathological study; Isozymes Dhaka Univ. J. Pharm. Sci. 6(2): 93-98, 2007 (December)

Correction of the defect in initiation of DNA replication in a temperature-sensitive mutant hamster cell line by in vitro addition of extracts from normal cells

1985 ◽  
Vol 5 (4) ◽  
pp. 902-905
Author(s):  
M Narkhammar ◽  
R Hand
Keyword(s):  
Cell Line ◽  
Nuclear Extract ◽  
Cell Extract ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Wild Type ◽  
Whole Cell ◽  
Mutant Cells

ts BN-2 is a temperature-sensitive hamster cell line that is defective in DNA synthesis at the restrictive temperature. The mutant expresses its defect during in vitro replication in whole-cell lysates. Addition of a high-salt-concentration extract from wild-type BHK-21, revertant RBN-2, or CHO cells to mutant cells lysed with 0.01% Brij 58 increased the activity in the mutant three- to fourfold, so that it reached 85% of the control value, and restored replicative synthesis. The presence of extract had an insignificant effect on wild-type and revertant replication and on mutant replication at the permissive temperature. Extract prepared from mutant cells was less effective than the wild-type cell extract was. Also, the stimulatory activity was more heat labile in the mutant than in the wild-type extract. Nuclear extract was as active as whole-cell extract.

scholarly journals In vitro transcription of immunoglobulin genes in a B-cell extract: effects of enhancer and promoter sequences.

1987 ◽  
Vol 7 (5) ◽  
pp. 1989-1994 ◽  
Author(s):  
R Sen ◽  
D Baltimore
Keyword(s):  
B Cell ◽  
Dna Sequences ◽  
In Vitro Transcription ◽  
Cell Extract ◽  
Upstream Sequence ◽  
Immunoglobulin Genes ◽  
Specific Expression ◽  
Sequence Element ◽  
Transcription System

Transfection experiments have led to the identification of three DNA sequences that are responsible for the tissue-specific expression of immunoglobulin genes. As a first step toward characterizing these regulatory phenomena at the biochemical level, we report the development of an in vitro transcription system from cells of the B lymphoid lineage. In these extracts, transcription of the MOPC41 kappa promoter is correctly initiated and dependent on the presence of an upstream sequence element located between -44 and -79 base pairs from the cap site. Second, although standard in vitro transcriptions are not affected by the presence or absence of enhancer sequences, we observed that the addition of polyethylene glycol led to a B-cell extract-specific suppression of transcription from a template that carries an immunoglobulin enhancer.

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