scholarly journals Specific protein binding to the simian virus 40 enhancer in vitro.

1986 ◽  
Vol 6 (6) ◽  
pp. 2098-2105 ◽  
Author(s):  
A G Wildeman ◽  
M Zenke ◽  
C Schatz ◽  
M Wintzerith ◽  
T Grundström ◽  
...  
Keyword(s):  
Point Mutations ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Specific Protein ◽  
Dnase I ◽  
Wild Type ◽  
Dnase I Footprinting ◽  
Nuclear Extracts

HeLa cell nuclear extracts and wild-type or mutated simian virus 40 enhancer DNA were used in DNase I footprinting experiments to study the interaction of putative trans-acting factors with the multiple enhancer motifs. We show that these nuclear extracts contain proteins that bind to these motifs. Because point mutations which are detrimental to the activity of a particular enhancer motif in vivo specifically prevent protection of that motif against DNase I digestion in vivo, we suggest that the bound proteins correspond to trans-acting factors involved in enhancement of transcription. Using mutants in which the two domains A and B of the simian virus 40 enhancer are either separated by insertion of DNA fragments or inverted with respect to their natural orientation, we also demonstrate that the trans-acting factors bind independently to the two domains.

Specific protein binding to the simian virus 40 enhancer in vitro

1986 ◽  
Vol 6 (6) ◽  
pp. 2098-2105
Author(s):  
A G Wildeman ◽  
M Zenke ◽  
C Schatz ◽  
M Wintzerith ◽  
T Grundström ◽  
...  
Keyword(s):  
Point Mutations ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Specific Protein ◽  
Dnase I ◽  
Wild Type ◽  
Dnase I Footprinting ◽  
Nuclear Extracts

HeLa cell nuclear extracts and wild-type or mutated simian virus 40 enhancer DNA were used in DNase I footprinting experiments to study the interaction of putative trans-acting factors with the multiple enhancer motifs. We show that these nuclear extracts contain proteins that bind to these motifs. Because point mutations which are detrimental to the activity of a particular enhancer motif in vivo specifically prevent protection of that motif against DNase I digestion in vivo, we suggest that the bound proteins correspond to trans-acting factors involved in enhancement of transcription. Using mutants in which the two domains A and B of the simian virus 40 enhancer are either separated by insertion of DNA fragments or inverted with respect to their natural orientation, we also demonstrate that the trans-acting factors bind independently to the two domains.

Determination of the DNA-binding sequences of ARGR proteins to arginine anabolic and catabolic promoters

1991 ◽  
Vol 11 (5) ◽  
pp. 2852-2863
Author(s):  
F Messenguy ◽  
E Dubois ◽  
C Boonchird
Keyword(s):  
Dna Binding ◽  
Protein Complex ◽  
Point Mutations ◽  
Dnase I ◽  
Gel Retardation Assay ◽  
Dnase I Footprinting ◽  
Catabolic Genes

ARGRI, ARGRII, and ARGRIII proteins regulate the expression of arginine anabolic and catabolic genes. The integrity of these three proteins is required to observe the formation of a DNA-protein complex with the different promoters of arginine coregulated genes. A study of deletions and point mutations created in the 5' noncoding region of ARG3, ARG5,6, CAR1, and CAR2 genes shows that at least two regions, called BoxA and BoxB, are required for proper regulation of these genes by arginine and ARGR proteins. By gel retardation assay and DNase I footprinting analysis, we have determined precisely the target of the ARGR proteins. Sequences in and around BoxA are necessary for ARGR binding to these four promoters in vitro, whereas sequences in and around BoxB are clearly protected against DNase I digestion only for CAR1. Sequences present at BoxA and BoxB are well conserved among the four promoters. Moreover, pairing can occur between sequences at BoxA and BoxB which could lead to the creation of secondary structures in ARG3, ARG5,6, CAR1, and CAR2 promoters, favoring the binding of ARGR proteins in vivo.

Functional analysis of the role of the A + T-rich region and upstream flanking sequences in simian virus 40 DNA replication

1986 ◽  
Vol 6 (12) ◽  
pp. 4570-4577
Author(s):  
R Gerard ◽  
Y Gluzman
Keyword(s):  
Dna Replication ◽  
Point Mutations ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Rich Region ◽  
Viral Dnas ◽  
Origin Function

One boundary of the minimal origin of replication of simian virus 40 DNA lies within the A + T-rich region. Deletion of only a few bases into the adenine-thymine (AT) stretch results in a DNA template which is defective for replication both in vivo and in vitro (B. Stillman, R. D. Gerard, R. A. Guggenheimer, and Y. Gluzman, EMBO J. 4:2933-2939, 1985). In the present study, such deletion mutations have been reconstructed into a simian virus 40 genome containing an intact early promoter-enhancer region. The resulting mutants synthesized wild-type levels of T antigen, but were defective for replication and would not form plaques on CV-1 monkey cells. Replication-competent phenotypic revertants were selected after transfection of large quantities of the replication-defective viral DNAs into CV-1 cells. DNA sequence analysis showed that most of these revertants contained insertions or point mutations which partially regenerate the length of the AT stretch. These genotypic alterations were shown to be responsible for the revertant phenotype by replication analysis in vivo of subcloned revertant origin fragments. In general, our results emphasize the importance of the AT region to simian virus 40 origin function. However, one revertant retained the altered AT region but deleted six nucleotides upstream. Experiments using this mutant indicate that the 21-base-pair repeats identified as part of the early transcriptional promoter may compensate for defects in simian virus 40 DNA replication in vivo caused by mutations in the A + T-rich region when positioned at an appropriate distance from the core origin.

scholarly journals Simian virus 40 major late promoter: a novel tripartite structure that includes intragenic sequences.

1988 ◽  
Vol 8 (5) ◽  
pp. 2021-2033 ◽  
Author(s):  
D E Ayer ◽  
W S Dynan
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Point Mutations ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Transcription Unit ◽  
Tata Box ◽  
Base Pairs

Unlike most genes transcribed by RNA polymerase II, the simian virus 40 late transcription unit does not have a TATA box. To determine what sequences are required for initiation at the major late mRNA cap site of simian virus 40, clustered point mutations were constructed and tested for transcriptional activity in vitro and in vivo. Three promoter elements were defined. The first is centered 31 base pairs upstream of the cap site in a position normally reserved for a TATA box. The second is at the cap site. The third occupies a novel position centered 28 base pairs downstream of the cap site within a protein-coding sequence. The ability of RNA polymerase II to recognize this promoter suggests that there is greater variation in promoter architecture than had been believed previously.

scholarly journals Functional analysis of the role of the A + T-rich region and upstream flanking sequences in simian virus 40 DNA replication.

1986 ◽  
Vol 6 (12) ◽  
pp. 4570-4577 ◽  
Author(s):  
R Gerard ◽  
Y Gluzman
Keyword(s):  
Dna Replication ◽  
Point Mutations ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
T Antigen ◽  
Rich Region ◽  
Viral Dnas ◽  
Origin Function

One boundary of the minimal origin of replication of simian virus 40 DNA lies within the A + T-rich region. Deletion of only a few bases into the adenine-thymine (AT) stretch results in a DNA template which is defective for replication both in vivo and in vitro (B. Stillman, R. D. Gerard, R. A. Guggenheimer, and Y. Gluzman, EMBO J. 4:2933-2939, 1985). In the present study, such deletion mutations have been reconstructed into a simian virus 40 genome containing an intact early promoter-enhancer region. The resulting mutants synthesized wild-type levels of T antigen, but were defective for replication and would not form plaques on CV-1 monkey cells. Replication-competent phenotypic revertants were selected after transfection of large quantities of the replication-defective viral DNAs into CV-1 cells. DNA sequence analysis showed that most of these revertants contained insertions or point mutations which partially regenerate the length of the AT stretch. These genotypic alterations were shown to be responsible for the revertant phenotype by replication analysis in vivo of subcloned revertant origin fragments. In general, our results emphasize the importance of the AT region to simian virus 40 origin function. However, one revertant retained the altered AT region but deleted six nucleotides upstream. Experiments using this mutant indicate that the 21-base-pair repeats identified as part of the early transcriptional promoter may compensate for defects in simian virus 40 DNA replication in vivo caused by mutations in the A + T-rich region when positioned at an appropriate distance from the core origin.

Simian virus 40 major late promoter: a novel tripartite structure that includes intragenic sequences

1988 ◽  
Vol 8 (5) ◽  
pp. 2021-2033
Author(s):  
D E Ayer ◽  
W S Dynan
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Point Mutations ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
Transcription Unit ◽  
Tata Box ◽  
Base Pairs

Unlike most genes transcribed by RNA polymerase II, the simian virus 40 late transcription unit does not have a TATA box. To determine what sequences are required for initiation at the major late mRNA cap site of simian virus 40, clustered point mutations were constructed and tested for transcriptional activity in vitro and in vivo. Three promoter elements were defined. The first is centered 31 base pairs upstream of the cap site in a position normally reserved for a TATA box. The second is at the cap site. The third occupies a novel position centered 28 base pairs downstream of the cap site within a protein-coding sequence. The ability of RNA polymerase II to recognize this promoter suggests that there is greater variation in promoter architecture than had been believed previously.

scholarly journals Determination of the DNA-binding sequences of ARGR proteins to arginine anabolic and catabolic promoters.

1991 ◽  
Vol 11 (5) ◽  
pp. 2852-2863 ◽  
Author(s):  
F Messenguy ◽  
E Dubois ◽  
C Boonchird
Keyword(s):  
Dna Binding ◽  
Protein Complex ◽  
Point Mutations ◽  
Dnase I ◽  
Gel Retardation Assay ◽  
Dnase I Footprinting ◽  
Catabolic Genes

ARGRI, ARGRII, and ARGRIII proteins regulate the expression of arginine anabolic and catabolic genes. The integrity of these three proteins is required to observe the formation of a DNA-protein complex with the different promoters of arginine coregulated genes. A study of deletions and point mutations created in the 5' noncoding region of ARG3, ARG5,6, CAR1, and CAR2 genes shows that at least two regions, called BoxA and BoxB, are required for proper regulation of these genes by arginine and ARGR proteins. By gel retardation assay and DNase I footprinting analysis, we have determined precisely the target of the ARGR proteins. Sequences in and around BoxA are necessary for ARGR binding to these four promoters in vitro, whereas sequences in and around BoxB are clearly protected against DNase I digestion only for CAR1. Sequences present at BoxA and BoxB are well conserved among the four promoters. Moreover, pairing can occur between sequences at BoxA and BoxB which could lead to the creation of secondary structures in ARG3, ARG5,6, CAR1, and CAR2 promoters, favoring the binding of ARGR proteins in vivo.

scholarly journals Molecular Basis of In Vivo Resistance to Sulfadoxine-Pyrimethamine in African Adult Patients Infected withPlasmodium falciparum Malaria Parasites

1998 ◽  
Vol 42 (7) ◽  
pp. 1811-1814 ◽  
Author(s):  
Leonardo K. Basco ◽  
Rachida Tahar ◽  
Pascal Ringwald
Keyword(s):  
Dihydrofolate Reductase ◽  
Point Mutations ◽  
Gene Mutations ◽  
Adult Patients ◽  
Wild Type ◽  
Dihydropteroate Synthase ◽  
Reductase Gene ◽  
Parasite Populations

ABSTRACT In vitro sulfadoxine and pyrimethamine resistance has been associated with point mutations in the dihydropteroate synthase and dihydrofolate reductase domains, respectively, but the in vivo relevance of these point mutations has not been well established. To analyze the correlation between genotype and phenotype, 10 Cameroonian adult patients were treated with sulfadoxine-pyrimethamine and followed up for 28 days. After losses to follow-up (n = 1) or elimination of DNA samples due to mixed parasite populations with pyrimethamine-sensitive and pyrimethamine-resistant profiles (n = 3), parasite genomic DNA from day 0 blood samples of six patients were analyzed by DNA sequencing. Three patients who were cured had isolates characterized by a wild-type or mutant dihydrofolate reductase gene (with one or two mutations) and a wild-type dihydropteroate synthase gene. Three other patients who failed to respond to sulfadoxine-pyrimethamine treatment carried isolates with triple dihydrofolate reductase gene mutations and either a wild-type or a mutant dihydropteroate synthase gene. Three dihydrofolate reductase gene codons (51, 59, and 108) may be reliable genetic markers that can accurately predict the clinical outcome of sulfadoxine-pyrimethamine treatment in Africa.

The AAUAAA sequence is required both for cleavage and for polyadenylation of simian virus 40 pre-mRNA in vitro

1986 ◽  
Vol 6 (7) ◽  
pp. 2317-2323
Author(s):  
D Zarkower ◽  
P Stephenson ◽  
M Sheets ◽  
M Wickens
Keyword(s):  
Hela Cell ◽  
Simian Virus 40 ◽  
Nuclear Extract ◽  
Simian Virus ◽  
Polyadenylation Site ◽  
Single Base ◽  
Cleavage And Polyadenylation ◽  
Cell Nuclear Extract

The sequence AAUAAA is found near the polyadenylation site of eucaryotic mRNAs. This sequence is required for accurate and efficient cleavage and polyadenylation of pre-mRNAs in vivo. In this study we show that synthetic simian virus 40 late pre-mRNAs are cleaved and polyadenylated in vitro in a HeLa cell nuclear extract, and that cleavage in vitro is abolished by each of four different single-base changes in AAUAAA. In this same extract, precleaved RNAs (RNAs with 3' termini at the polyadenylation site) are efficiently polyadenylated. This in vitro polyadenylation reaction also requires the AAUAAA sequence.

The number of ribosomes on simian virus 40 late 16S mRNA is determined in part by the nucleotide sequence of its leader

1984 ◽  
Vol 4 (4) ◽  
pp. 813-816
Author(s):  
A Barkan ◽  
J E Mertz
Keyword(s):  
Nucleotide Sequence ◽  
Direct Evidence ◽  
Simian Virus 40 ◽  
Simian Virus ◽  
The Body ◽  
Size Distributions ◽  
Wild Type ◽  
Leader Protein ◽  
16S Rna

The size distributions of polyribosomes containing each of three simian virus 40 late 16S mRNA species that differ in nucleotide sequence only within their leaders were determined. The two 16S RNA species with shorter leaders were incorporated into polysomes that were both larger (on average) and narrower in size distribution than was the predominant wild-type 16S RNA. Therefore, the nucleotide sequence of the leader can influence the number of ribosomes present on the body of an mRNA molecule. We propose a model in which the excision from leaders of sizeable translatable regions permits more frequent utilization of internally located translation initiation signals, thereby enabling genes encoded within the bodies of polygenic mRNAs to be translated at higher rates. In addition, the data provide the first direct evidence that VP1 can, indeed, be synthesized in vivo from the species of 16S mRNA that also encodes the 61-amino acid leader protein.

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