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.

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.

scholarly journals The ubiquitous octamer-binding protein(s) is sufficient for transcription of immunoglobulin genes.

1990 ◽  
Vol 10 (3) ◽  
pp. 982-990 ◽  
Author(s):  
D G Johnson ◽  
L Carayannopoulos ◽  
J D Capra ◽  
P W Tucker ◽  
J H Hanke
Keyword(s):  
B Cell ◽  
Protein S ◽  
Cell Types ◽  
In Vitro Transcription ◽  
Immunoglobulin Gene ◽  
Immunoglobulin Genes ◽  
Specific Expression ◽  
Specific Regulation

All immunoglobulin genes contain a conserved octanucleotide promoter element, ATGCAAAT, which has been shown to be required for their normal B-cell-specific transcription. Proteins that bind this octamer have been purified, and cDNAs encoding octamer-binding proteins have been cloned. Some of these proteins (referred to as OTF-2) are lymphoid specific, whereas at least one other, and possibly more (referred to as OTF-1), is found ubiquitously in all cell types. The exact role of these different proteins in directing the tissue-specific expression of immunoglobulin genes is unclear. We have identified two human pre-B-cell lines that contain extremely low levels of OTF-2 yet still express high levels of steady-state immunoglobulin heavy-chain mRNA in vivo and efficiently transcribe an immunoglobulin gene in vitro. Addition of a highly enriched preparation of OTF-1 made from one of these pre-B cells or from HeLa cells specifically stimulated in vitro transcription of an immunoglobulin gene. Furthermore, OFT-1 appeared to have approximately the same transactivation ability as OTF-2 when normalized for binding activity. These results suggest that OTF-1, without OTF-2, is sufficient for transcription of immunoglobulin genes and that OTF-2 alone is not responsible for the B-cell-specific regulation of immunoglobulin gene expression.

The ubiquitous octamer-binding protein(s) is sufficient for transcription of immunoglobulin genes

1990 ◽  
Vol 10 (3) ◽  
pp. 982-990
Author(s):  
D G Johnson ◽  
L Carayannopoulos ◽  
J D Capra ◽  
P W Tucker ◽  
J H Hanke
Keyword(s):  
B Cell ◽  
Protein S ◽  
Cell Types ◽  
In Vitro Transcription ◽  
Immunoglobulin Gene ◽  
Immunoglobulin Genes ◽  
Specific Expression ◽  
Specific Regulation

All immunoglobulin genes contain a conserved octanucleotide promoter element, ATGCAAAT, which has been shown to be required for their normal B-cell-specific transcription. Proteins that bind this octamer have been purified, and cDNAs encoding octamer-binding proteins have been cloned. Some of these proteins (referred to as OTF-2) are lymphoid specific, whereas at least one other, and possibly more (referred to as OTF-1), is found ubiquitously in all cell types. The exact role of these different proteins in directing the tissue-specific expression of immunoglobulin genes is unclear. We have identified two human pre-B-cell lines that contain extremely low levels of OTF-2 yet still express high levels of steady-state immunoglobulin heavy-chain mRNA in vivo and efficiently transcribe an immunoglobulin gene in vitro. Addition of a highly enriched preparation of OTF-1 made from one of these pre-B cells or from HeLa cells specifically stimulated in vitro transcription of an immunoglobulin gene. Furthermore, OFT-1 appeared to have approximately the same transactivation ability as OTF-2 when normalized for binding activity. These results suggest that OTF-1, without OTF-2, is sufficient for transcription of immunoglobulin genes and that OTF-2 alone is not responsible for the B-cell-specific regulation of immunoglobulin gene expression.

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.

A 40-kilodalton protein binds specifically to an upstream sequence element essential for muscle-specific transcription of the human myoglobin promoter

1992 ◽  
Vol 12 (11) ◽  
pp. 5024-5032
Author(s):  
R Bassel-Duby ◽  
M D Hernandez ◽  
M A Gonzalez ◽  
J K Krueger ◽  
R S Williams
Keyword(s):  
Mammalian Cells ◽  
Mutational Analysis ◽  
Transcriptional Start Site ◽  
Upstream Sequence ◽  
Specific Expression ◽  
Sequence Element ◽  
Promoter Function ◽  
Human Myoglobin

To define transcriptional control elements responsible for muscle-specific expression of the human myoglobin gene, we performed mutational analysis of upstream sequences (nucleotide positions -373 to +7 relative to the transcriptional start site) linked to a firefly luciferase gene. Transient expression assays in avian and mammalian cells indicated that a CCCACCCCC (CCAC box) sequence (-223 to -204) is necessary for muscle-specific transcription directed either by the native myoglobin promoter or by a heterologous minimal promoter linked to the myoglobin upstream enhancer region. A putative MEF2-like site (-160 to -169) was likewise necessary for full transcriptional activity in myotubes. Mutations within either of two CANNTG (E-box) motifs (-176 to -148) had only minimal effects on promoter function. We identified and partially purified from nuclear extracts a 40-kDa protein (CBF40) that binds specifically to oligonucleotides containing the CCAC box sequence. A mutation of the CCAC box that disrupted promoter function in vivo also impaired binding of CBF40 in vitro. These data suggest that cooperative interactions between CBF40 and other factors including MEF-2 are required for expression of the human myoglobin gene in skeletal muscle.

scholarly journals A 40-kilodalton protein binds specifically to an upstream sequence element essential for muscle-specific transcription of the human myoglobin promoter.

1992 ◽  
Vol 12 (11) ◽  
pp. 5024-5032 ◽  
Author(s):  
R Bassel-Duby ◽  
M D Hernandez ◽  
M A Gonzalez ◽  
J K Krueger ◽  
R S Williams
Keyword(s):  
Mammalian Cells ◽  
Mutational Analysis ◽  
Transcriptional Start Site ◽  
Upstream Sequence ◽  
Specific Expression ◽  
Sequence Element ◽  
Promoter Function ◽  
Human Myoglobin

To define transcriptional control elements responsible for muscle-specific expression of the human myoglobin gene, we performed mutational analysis of upstream sequences (nucleotide positions -373 to +7 relative to the transcriptional start site) linked to a firefly luciferase gene. Transient expression assays in avian and mammalian cells indicated that a CCCACCCCC (CCAC box) sequence (-223 to -204) is necessary for muscle-specific transcription directed either by the native myoglobin promoter or by a heterologous minimal promoter linked to the myoglobin upstream enhancer region. A putative MEF2-like site (-160 to -169) was likewise necessary for full transcriptional activity in myotubes. Mutations within either of two CANNTG (E-box) motifs (-176 to -148) had only minimal effects on promoter function. We identified and partially purified from nuclear extracts a 40-kDa protein (CBF40) that binds specifically to oligonucleotides containing the CCAC box sequence. A mutation of the CCAC box that disrupted promoter function in vivo also impaired binding of CBF40 in vitro. These data suggest that cooperative interactions between CBF40 and other factors including MEF-2 are required for expression of the human myoglobin gene in skeletal muscle.

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 dyskerin ribonucleoprotein complex as an OCT4/SOX2 coactivator in embryonic stem cells

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Yick W Fong ◽  
Jaclyn J Ho ◽  
Carla Inouye ◽  
Robert Tjian
Keyword(s):  
Stem Cell ◽  
Es Cells ◽  
Embryonic Stem ◽  
In Vitro Transcription ◽  
Specific Gene ◽  
Transcriptional Level ◽  
Ips Cell ◽  
Ribonucleoprotein Complex ◽  
Transcription System

Acquisition of pluripotency is driven largely at the transcriptional level by activators OCT4, SOX2, and NANOG that must in turn cooperate with diverse coactivators to execute stem cell-specific gene expression programs. Using a biochemically defined in vitro transcription system that mediates OCT4/SOX2 and coactivator-dependent transcription of the Nanog gene, we report the purification and identification of the dyskerin (DKC1) ribonucleoprotein complex as an OCT4/SOX2 coactivator whose activity appears to be modulated by a subset of associated small nucleolar RNAs (snoRNAs). The DKC1 complex occupies enhancers and regulates the expression of key pluripotency genes critical for self-renewal in embryonic stem (ES) cells. Depletion of DKC1 in fibroblasts significantly decreased the efficiency of induced pluripotent stem (iPS) cell generation. This study thus reveals an unanticipated transcriptional role of the DKC1 complex in stem cell maintenance and somatic cell reprogramming.

scholarly journals Concentration dependence of transcriptional transactivation in inducible E1A-containing human cells.

1988 ◽  
Vol 8 (11) ◽  
pp. 4799-4807 ◽  
Author(s):  
L J Brunet ◽  
A J Berk
Keyword(s):  
Adenovirus Type ◽  
In Vitro Transcription ◽  
Mrna Levels ◽  
Adenovirus E1a ◽  
Transcription System ◽  
Nuclear Extracts ◽  
Tumor Virus ◽  
E1a Gene ◽  
High Level

The adenovirus E1A proteins are essential for the normal temporal activation of transcription from every other adenoviral early promoter. High-level E1A expression in the absence of viral infection would facilitate biochemical studies of E1A-mediated transactivation. Toward this end, we introduced the adenovirus type 2 E1A gene under the control of the murine mammary tumor virus promoter into HeLa cells. Uninduced cells expressed little or no detectable E1A mRNA. Upon induction, mRNA levels accumulated to about 50% of the level observed in 293 cells. The level of E1A expression in these cells could be controlled by varying the concentration of the inducing glucocorticoid. Under these conditions of varying E1A concentrations, it was observed that activation of the E2, E3, and E4 promoters of H5dl312 initiated at the same E1A concentration and that transcription from each promoter increased as the E1A concentration increased. These results indicate that E1A-mediated transactivation is proportional to the concentration of E1A protein. E1A-dependent transcriptional stimulation of the E4 promoter was reproduced in an in vitro transcription system, demonstrating that expression of only the E1A proteins was sufficient to increase the transcriptional activity of nuclear extracts.

Sign in / Sign up

Export Citation Format

Share Document