scholarly journals Functional steps in transcription initiation and reinitiation from the major late promoter in a HeLa nuclear extract.

1987 ◽  
Vol 262 (8) ◽  
pp. 3452-3461 ◽  
Author(s):  
D.K. Hawley ◽  
R.G. Roeder
Keyword(s):  
Transcription Initiation ◽  
Nuclear Extract ◽  
Late Promoter ◽  
Hela Nuclear Extract ◽  
Major Late Promoter

Downstream sequences affect transcription initiation from the adenovirus major late promoter

1986 ◽  
Vol 6 (7) ◽  
pp. 2684-2694 ◽  
Author(s):  
S L Mansour ◽  
T Grodzicker ◽  
R Tjian
Keyword(s):  
Transcription Initiation ◽  
Simian Virus 40 ◽  
T Antigen ◽  
Control Element ◽  
Coding Region ◽  
Late Promoter ◽  
Sv40 T Antigen ◽  
Transient Expression Assay ◽  
Adenovirus Major Late Promoter ◽  
Major Late Promoter

We analyzed a set of adenovirus-simian virus 40 (SV40) hybrids in which the SV40 T antigen coding sequences are inserted downstream from the adenovirus major late promoter within the first, second, and third segments of the tripartite leader. In infected cells, these viruses give rise to a matched set of hybrid SV40 mRNAs that differ only in the number of tripartite leader segments attached to the complete SV40 T antigen coding region. We found that the number of tripartite leader segments present at the 5' end of the hybrid SV40 mRNAs had little effect on the efficiency of T antigen translation. Surprisingly, insertion of SV40 sequences within the first leader segment, at +33 relative to the start of transcription, significantly reduced the frequency of transcription initiation from the major late promoter. The 3' boundary of this downstream transcriptional control element was mapped between +33 and +190 by showing that insertion of SV40 sequences within the intron after the first leader segment at +190 had very little effect on transcription initiation from the late promoter. A transient expression assay was used to show that the effect of downstream sequences on transcription initiation from the major late promoter is dependent on a trans-acting factor encoded or induced by adenovirus.

Mutations in the adenovirus major late promoter: effects on viability and transcription during infection

1987 ◽  
Vol 7 (3) ◽  
pp. 1091-1100
Author(s):  
L J Brunet ◽  
L E Babiss ◽  
C S Young ◽  
D R Mills
Keyword(s):  
Transcription Initiation ◽  
Experimental System ◽  
Transcription Unit ◽  
Tata Box ◽  
Productive Infection ◽  
Late Promoter ◽  
Lethal Mutant ◽  
Adenovirus Major Late Promoter ◽  
Specific Nucleotide Sequence ◽  
Major Late Promoter

We developed an experimental system to examine the effects of mutations in the adenovirus major late promoter in its correct genomic location during a productive infection. A virus was constructed whose genome could be digested to give a rightward terminal DNA fragment extending from the XhoI site at 22.9 map units, which can be ligated or recombined with plasmid DNA containing adenovirus sequences extending from 0 to 22.9 or 26.5 map units, respectively. Mutations were made by bisulfite mutagenesis in the region between base pairs -52 and -12 with respect to the cap site at +1 and transferred to the appropriate plasmids for viral reconstruction. Of 19 mutant plasmid sequences containing single or multiple G-to-A transitions, 14 could be placed in the viral genome with no apparent change in phenotype. These mutant sequences included those which contained four transitions in the string of G residues immediately downstream of the TATA box. There were no alterations in rates of transcription from the major late promoter, sites of transcription initiation, or steady-state levels of late mRNAs. All of the five mutant sequences which could not be placed in virus contained multiple transitions both up- and downstream of the TATA box. Two of these apparently lethal mutant sequences were used in promoter fusion experiments to test their ability to promote transcription of rabbit beta-globin sequences placed in the dispensable E1 region of the virus. Both sequences showed diminished ability compared with wild-type sequences to promote transcription in this context. Comparisons between these two sequences and the viable mutant sequences suggest a role for the string of G residues located between -38 and -33 in promoting transcription from the major late promoter. The data as a whole also demonstrate that the specific nucleotide sequence of this region of the major late promoter, which overlaps transcription elements of the divergent IVa2 transcription unit and coding sequences of the adenovirus DNA polymerase, is not rigidly constrained but can mutate extensively without loss of these several functions.

Transcription initiation complexes and upstream activation with RNA polymerase II lacking the C-terminal domain of the largest subunit

1990 ◽  
Vol 10 (10) ◽  
pp. 5562-5564
Author(s):  
S Buratowski ◽  
P A Sharp
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Adenovirus Type ◽  
Late Promoter ◽  
Terminal Domain ◽  
Late Transcription ◽  
Major Late Promoter

RNA polymerase II assembles with other factors on the adenovirus type 2 major late promoter to generate pairs of transcription initiation complexes resolvable by nondenaturing gel electrophoresis. The pairing of the complexes is caused by the presence or absence of the C-terminal domain of the largest subunit. This domain is not required for transcription stimulation by the major late transcription factor in vitro.

scholarly journals Downstream sequences affect transcription initiation from the adenovirus major late promoter.

1986 ◽  
Vol 6 (7) ◽  
pp. 2684-2694 ◽  
Author(s):  
S L Mansour ◽  
T Grodzicker ◽  
R Tjian
Keyword(s):  
Transcription Initiation ◽  
Simian Virus 40 ◽  
T Antigen ◽  
Control Element ◽  
Coding Region ◽  
Late Promoter ◽  
Sv40 T Antigen ◽  
Transient Expression Assay ◽  
Adenovirus Major Late Promoter ◽  
Major Late Promoter

We analyzed a set of adenovirus-simian virus 40 (SV40) hybrids in which the SV40 T antigen coding sequences are inserted downstream from the adenovirus major late promoter within the first, second, and third segments of the tripartite leader. In infected cells, these viruses give rise to a matched set of hybrid SV40 mRNAs that differ only in the number of tripartite leader segments attached to the complete SV40 T antigen coding region. We found that the number of tripartite leader segments present at the 5' end of the hybrid SV40 mRNAs had little effect on the efficiency of T antigen translation. Surprisingly, insertion of SV40 sequences within the first leader segment, at +33 relative to the start of transcription, significantly reduced the frequency of transcription initiation from the major late promoter. The 3' boundary of this downstream transcriptional control element was mapped between +33 and +190 by showing that insertion of SV40 sequences within the intron after the first leader segment at +190 had very little effect on transcription initiation from the late promoter. A transient expression assay was used to show that the effect of downstream sequences on transcription initiation from the major late promoter is dependent on a trans-acting factor encoded or induced by adenovirus.

scholarly journals Construction of a modular dihydrofolate reductase cDNA gene: analysis of signals utilized for efficient expression.

1982 ◽  
Vol 2 (11) ◽  
pp. 1304-1319 ◽  
Author(s):  
R J Kaufman ◽  
P A Sharp
Keyword(s):  
Dihydrofolate Reductase ◽  
Splice Site ◽  
Transcription Initiation ◽  
Simian Virus 40 ◽  
Variable Region ◽  
Polyadenylation Signal ◽  
Immunoglobulin Gene ◽  
Late Promoter ◽  
Adenovirus Major Late Promoter ◽  
Major Late Promoter

Dihydrofolate reductase (DHFR) modular genes have been constructed with segments containing the adenovirus major late promoter, a 3' splice site from a variable region immunoglobulin gene, a DHFR cDNA, and portions of the simian virus 40 (SV40) genome. DNA-mediated transfer of these genes transformed Chinese hamster ovary DHFR- cells to the DHFR+ phenotype. Transformants contained one to several copies of the transfected DNA integrated into the host genome. Clones subjected to growth in increasing concentrations of methotrexate eventually gave rise to lines containing several hundred copies of the transforming DNA. Analysis of the DHFR mRNA produced in amplified lines indicated the following. (i) All clones utilize the adenovirus major late promoter for transcription initiation. (ii) A hybrid intron formed by the 5' splice site of the adenovirus major late leader and a 3' splice site from a variable-region immunoglobulin gene is properly excised. (iii) The mRNA is not efficiently polyadenylated at sequences in the 3' end of the DHFR cDNA but rather uses polyadenylation signals downstream from the DHFR cDNA. Three independent clones produce a DHFR mRNA containing SV40 or pBR322 and SV40 sequences, and the RNA is polyadenylated at the SV40 late polyadenylation site. Another clone has recombined into cellular DNA and apparently uses a cellular sequence for polyadenylation. Introduction of a segment containing the SV40 early polyadenylation signal into the 3' end of the DHFR cDNA gene generated a recombinant capable of transforming cells to the DHFR+ phenotype with at least a 10-fold increase in efficiency, demonstrating the necessity for an efficient polyadenylation signal. Attachment of a DNA segment containing the transcription enhancer (72-base pair repeat) of SV40 further increased the biological activity of the modular DHFR gene 50- to 100-fold.

scholarly journals Functional Analysis of the CAAT Box in the Major Late Promoter of the Subgroup C Human Adenoviruses

1998 ◽  
Vol 72 (4) ◽  
pp. 3213-3220 ◽  
Author(s):  
Byeongwoon Song ◽  
C. S. H. Young
Keyword(s):  
Binding Site ◽  
Transcription Initiation ◽  
Mutational Analysis ◽  
Wild Type ◽  
Late Promoter ◽  
Phenotypic Effect ◽  
Partial Restoration ◽  
Wild Type Phenotype ◽  
Major Late Promoter

ABSTRACT Comparisons among sequences predicted to encode the major late promoter (MLP) of adenoviruses from a wide variety of host species show that an inverted CAAT box is among the most highly conserved transcription elements found in the putative MLPs. The high degree of conservation suggests that the CAAT box plays an important role in the function of the MLP in vivo, an idea supported by a previous mutational analysis of the core CCAAT sequence. To address the importance of the CAAT box, in terms both of quantitative levels of transcription and of specificity, a further set of mutations was created and examined in the context of the viral genome. One mutation, CAAT5, contains individual changes at five positions, four of which correspond to invariant residues in a CAAT box consensus derived either by computer analysis or empirically. The CAAT5 mutation had no discernible phenotype by itself but when coupled with the previously described USF0 mutation, which disrupts binding of the upstream stimulating factor (USF) but is otherwise phenotypically silent, gave rise to virus with a severe replication deficiency. Nuclear run-on assays showed that transcription initiation at the mutant MLP was significantly reduced compared with that of the wild type or the virus containing CAAT5 alone. Replication of the double mutant was lower than that of the previously described USF0::CCCAT virus, suggesting that the additional mutations in the CAAT box had further lowered the binding of transcription factor CP1 (also called CBF, NF-Y). Replacement of the CAAT box by an ATF binding site or an OCT1 binding site had no phenotypic effect in an otherwise wild-type background, but replacement in a USF0::CCCAT background led to only partial restoration of the wild-type phenotype. The failure to restore the functional redundancy normally exhibited by the CAAT box and the proximal upstream activating element is consistent with the idea that in the adenovirus MLP the CAAT box is preferred over others as the distal transcriptional element.

scholarly journals Mutations in the adenovirus major late promoter: effects on viability and transcription during infection.

1987 ◽  
Vol 7 (3) ◽  
pp. 1091-1100 ◽  
Author(s):  
L J Brunet ◽  
L E Babiss ◽  
C S Young ◽  
D R Mills
Keyword(s):  
Transcription Initiation ◽  
Experimental System ◽  
Transcription Unit ◽  
Tata Box ◽  
Productive Infection ◽  
Late Promoter ◽  
Lethal Mutant ◽  
Adenovirus Major Late Promoter ◽  
Specific Nucleotide Sequence ◽  
Major Late Promoter

We developed an experimental system to examine the effects of mutations in the adenovirus major late promoter in its correct genomic location during a productive infection. A virus was constructed whose genome could be digested to give a rightward terminal DNA fragment extending from the XhoI site at 22.9 map units, which can be ligated or recombined with plasmid DNA containing adenovirus sequences extending from 0 to 22.9 or 26.5 map units, respectively. Mutations were made by bisulfite mutagenesis in the region between base pairs -52 and -12 with respect to the cap site at +1 and transferred to the appropriate plasmids for viral reconstruction. Of 19 mutant plasmid sequences containing single or multiple G-to-A transitions, 14 could be placed in the viral genome with no apparent change in phenotype. These mutant sequences included those which contained four transitions in the string of G residues immediately downstream of the TATA box. There were no alterations in rates of transcription from the major late promoter, sites of transcription initiation, or steady-state levels of late mRNAs. All of the five mutant sequences which could not be placed in virus contained multiple transitions both up- and downstream of the TATA box. Two of these apparently lethal mutant sequences were used in promoter fusion experiments to test their ability to promote transcription of rabbit beta-globin sequences placed in the dispensable E1 region of the virus. Both sequences showed diminished ability compared with wild-type sequences to promote transcription in this context. Comparisons between these two sequences and the viable mutant sequences suggest a role for the string of G residues located between -38 and -33 in promoting transcription from the major late promoter. The data as a whole also demonstrate that the specific nucleotide sequence of this region of the major late promoter, which overlaps transcription elements of the divergent IVa2 transcription unit and coding sequences of the adenovirus DNA polymerase, is not rigidly constrained but can mutate extensively without loss of these several functions.

Construction of a modular dihydrofolate reductase cDNA gene: analysis of signals utilized for efficient expression

1982 ◽  
Vol 2 (11) ◽  
pp. 1304-1319
Author(s):  
R J Kaufman ◽  
P A Sharp
Keyword(s):  
Dihydrofolate Reductase ◽  
Splice Site ◽  
Transcription Initiation ◽  
Simian Virus 40 ◽  
Variable Region ◽  
Polyadenylation Signal ◽  
Immunoglobulin Gene ◽  
Late Promoter ◽  
Adenovirus Major Late Promoter ◽  
Major Late Promoter

Dihydrofolate reductase (DHFR) modular genes have been constructed with segments containing the adenovirus major late promoter, a 3' splice site from a variable region immunoglobulin gene, a DHFR cDNA, and portions of the simian virus 40 (SV40) genome. DNA-mediated transfer of these genes transformed Chinese hamster ovary DHFR- cells to the DHFR+ phenotype. Transformants contained one to several copies of the transfected DNA integrated into the host genome. Clones subjected to growth in increasing concentrations of methotrexate eventually gave rise to lines containing several hundred copies of the transforming DNA. Analysis of the DHFR mRNA produced in amplified lines indicated the following. (i) All clones utilize the adenovirus major late promoter for transcription initiation. (ii) A hybrid intron formed by the 5' splice site of the adenovirus major late leader and a 3' splice site from a variable-region immunoglobulin gene is properly excised. (iii) The mRNA is not efficiently polyadenylated at sequences in the 3' end of the DHFR cDNA but rather uses polyadenylation signals downstream from the DHFR cDNA. Three independent clones produce a DHFR mRNA containing SV40 or pBR322 and SV40 sequences, and the RNA is polyadenylated at the SV40 late polyadenylation site. Another clone has recombined into cellular DNA and apparently uses a cellular sequence for polyadenylation. Introduction of a segment containing the SV40 early polyadenylation signal into the 3' end of the DHFR cDNA gene generated a recombinant capable of transforming cells to the DHFR+ phenotype with at least a 10-fold increase in efficiency, demonstrating the necessity for an efficient polyadenylation signal. Attachment of a DNA segment containing the transcription enhancer (72-base pair repeat) of SV40 further increased the biological activity of the modular DHFR gene 50- to 100-fold.

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