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.

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.

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 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.

Growth-dependent expression of dihydrofolate reductase mRNA from modular cDNA genes

1983 ◽  
Vol 3 (9) ◽  
pp. 1598-1608
Author(s):  
R J Kaufman ◽  
P A Sharp
Keyword(s):  
Dihydrofolate Reductase ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Polyadenylation Signal ◽  
Ovary Cells ◽  
Dividing Cells ◽  
Major Late Promoter ◽  
The Stability

Dihydrofolate reductase (DHFR) synthesis is regulated in a growth-dependent fashion. Dividing cells synthesize DHFR at a 10-fold-higher rate than do stationary cells. To study this growth-dependent synthesis. DHFR genes have been constructed from a DHFR cDNA segment, the adenovirus major late promoter, and fragments of simian virus 40 (SV40) which provide signals for polyadenylation. These genes have been introduced into Chinese hamster ovary cells. The DHFR mRNAs produced in different transformants are identical at their 5' ends, but differ in sequences in their 3' ends as different sites are utilized for polyadenylation. Three transformants that utilize either DHFR polyadenylation signals or the SV40 late polyadenylation signal exhibit growth-dependent DHFR synthesis. The level of DHFR mRNA in growing cells is approximately 10 times that in stationary cells for these transformants. This growth-dependent DHFR mRNA production probably results from posttranscriptional events. In contrast, three transformants that utilize the SV40 early polyadenylation signal and another transformant that utilizes a cellular polyadenylation signal do not exhibit growth-dependent DHFR synthesis. In these three cell lines, the fraction of mRNAs polyadenylated at different sites in a tandem array shifts between growing and stationary cells. These results suggest that the metabolic state of the cell is important in determining either the efficiency of polyadenylation at various sites or the stability of mRNA polyadenylated at various sites.

Characterization of a mammalian ribosomal protein gene promoter

1990 ◽  
Vol 68 (6) ◽  
pp. 949-956 ◽  
Author(s):  
Peter Zahradka ◽  
Dawn E. Larson ◽  
Bruce H. Sells
Keyword(s):  
Ribosomal Protein ◽  
Rna Polymerase Ii ◽  
Dihydrofolate Reductase ◽  
Protein Gene ◽  
Gene Promoter ◽  
Ribosomal Protein Gene ◽  
Late Promoter ◽  
Promoter Elements ◽  
Adenovirus Major Late Promoter ◽  
Major Late Promoter

The presence of specific promoter elements, notably the TATA and GC boxes, has been useful for categorizing genes transcribed by RNA polymerase II. The gene for the murine ribosomal protein (r-protein) L32 lacks both of these elements, although it has GC-rich regions. The conditions required for its optimal synthesis in vitro, however, resemble the properties of promoters containing TATA (adenovirus major late promoter) rather than GC boxes (dihydrofolate reductase). To further investigate the relationship of the r-protein gene to different promoter elements, transcription competition analyses were used to distinguish the presence of common protein-binding sequences. The low levels of competition observed by either the adenovirus major late promoter or dihydrofolate reductase promoter with the r-protein gene promoter resulted from general transcription factors present in each initiation complex. Competition by factors binding to common sequence elements was not observed, indicating the r-protein L32 gene possesses elements distinct from those present in the other genes examined.Key words: ribosomal protein gene, gene promoter, cell-free transcription.

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.

scholarly journals Growth-dependent expression of dihydrofolate reductase mRNA from modular cDNA genes.

1983 ◽  
Vol 3 (9) ◽  
pp. 1598-1608 ◽  
Author(s):  
R J Kaufman ◽  
P A Sharp
Keyword(s):  
Dihydrofolate Reductase ◽  
Chinese Hamster Ovary Cells ◽  
Chinese Hamster ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Polyadenylation Signal ◽  
Ovary Cells ◽  
Dividing Cells ◽  
Major Late Promoter ◽  
The Stability

Dihydrofolate reductase (DHFR) synthesis is regulated in a growth-dependent fashion. Dividing cells synthesize DHFR at a 10-fold-higher rate than do stationary cells. To study this growth-dependent synthesis. DHFR genes have been constructed from a DHFR cDNA segment, the adenovirus major late promoter, and fragments of simian virus 40 (SV40) which provide signals for polyadenylation. These genes have been introduced into Chinese hamster ovary cells. The DHFR mRNAs produced in different transformants are identical at their 5' ends, but differ in sequences in their 3' ends as different sites are utilized for polyadenylation. Three transformants that utilize either DHFR polyadenylation signals or the SV40 late polyadenylation signal exhibit growth-dependent DHFR synthesis. The level of DHFR mRNA in growing cells is approximately 10 times that in stationary cells for these transformants. This growth-dependent DHFR mRNA production probably results from posttranscriptional events. In contrast, three transformants that utilize the SV40 early polyadenylation signal and another transformant that utilizes a cellular polyadenylation signal do not exhibit growth-dependent DHFR synthesis. In these three cell lines, the fraction of mRNAs polyadenylated at different sites in a tandem array shifts between growing and stationary cells. These results suggest that the metabolic state of the cell is important in determining either the efficiency of polyadenylation at various sites or the stability of mRNA polyadenylated at various sites.

Sign in / Sign up

Export Citation Format

Share Document