Transcription of a trout protamine gene in vitro: the effects of alteration of promoters

1984 ◽  
Vol 62 (5) ◽  
pp. 291-300 ◽  
Author(s):  
Jacek M. Jankowski ◽  
Gordon H. Dixon
Keyword(s):  
Rna Polymerase Ii ◽  
Dna Sequences ◽  
Tata Box ◽  
Base Pairs ◽  
S1 Nuclease ◽  
Promoter Sequences ◽  
Transcription System ◽  
Nuclease Digestion ◽  
Simplex Virus

An in vitro approach has been used to study trout protamine gene expression using various recombinant plasmids containing trout protamine genes as templates in the HeLa cell lysate transcription system. The specific RNA transcript which is protected against S1 nuclease digestion by hybridization to the protamine gene sequence is α-amanitin sensitive (1 μg/mL), showing that RNA polymerase II is involved. The sizes of transcripts from templates linearized with Bam HI, Rsa I, and Hpa II (all downstream from the putative TATA box) are consistent with those predicted from the known sequence of the protamine gene. Digestion at an Alu I site only 14 base pairs (bp) upstream from TATA box has no effect on the accuracy of transcription in vitro; however, cutting at an Ava II site 9 bp downstream from the TATA box (reading from the first T) abolishes transcription. Chimeric plasmids, in which a herpes simplex virus (HSV-1) thymidine kinase (tk) promoter is tandemly inserted upstream from the trout protamine DNA sequences or as a replacement of the natural protamine promoter, were constructed. Use of these plasmids allowed an examination in a single assay of eight different putative promoter sequences (TATAAAA, TATAAA, TACAAA, TATATA, TATTTAA, CATATTA, TATATTAT, and TATTTAT) that are localized in either the protamine or the tk genes. The canonical TATAAAA promoter (the natural protamine promoter) was the strongest one and, in its presence, none of the others were used significantly for transcription. However, when this promoter was removed the weaker promoters were able to promote transcription.

DNA sequences which influence the selection and efficient utilisation of transcriptional start sites of a eukaryotic gene by RNA polymerase II in vitro and in vivo

1986 ◽  
Vol 6 (11) ◽  
pp. 937-944
Author(s):  
Balazs J. Kovacs ◽  
Peter H. W. Butterworth
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Dna Sequences ◽  
Mammalian Cells ◽  
Tata Box ◽  
Flanking Sequence ◽  
Eukaryotic Gene ◽  
Transcriptional Start Sites

Experiments are described which probe the relationship between three sequence elements which make up the eukaryotic RNA polymerase II promoter. A cloned eukaryotic gene, from which the TATA-box and 400 base pairs of Y-flanking sequence has been deleted, is still transcriptionally active in vivo (following its transfection into cultured mammalian cells) and in vitro. Deletion has appropriately positioned a cluster of five TATA box-like sequences upstream from multiple potential cap sites. Which cap sites are actually used can be predicted from the DNA sequence of TATA box-like sequences and their spatial relationship with respect to possible transcriptional start sites, although there appears to be some difference in cap site utilisation in vitro and in vivo. Data suggest that deletion has also removed “upstream” sequences which affect promoter function.

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 DNA sequences required for specific and efficient initiation of transcription at the polyoma virus early promoter.

1982 ◽  
Vol 2 (7) ◽  
pp. 737-751 ◽  
Author(s):  
P Jat ◽  
U Novak ◽  
A Cowie ◽  
C Tyndall ◽  
R Kamen
Keyword(s):  
Dna Sequences ◽  
In Vitro Transcription ◽  
Polyoma Virus ◽  
Tata Box ◽  
Deletion Mutants ◽  
Base Pairs ◽  
Transcription Analysis ◽  
In Vivo Expression

The 5'-flanking DNA sequences involved in the specific and efficient transcription of the polyoma virus early region have been investigated. Sequence requirements for efficient in vivo expression differed from those in vitro. Deletion of DNA located between 200 and 400 base pairs before the principal cap sites severely inhibited in vivo expression as measured by transformation ability, but did not affect in vitro transcription. Viable deletion mutants which lack the principal cap sites and the "TATA" box were very poor templates for in vitro transcription. Analysis of other deletion mutants in vitro demonstrated that no specific sequences more than 46 base pairs before the cap sites were important. Removal of the TATA box reduced in vitro transcriptional efficiency but did not alter the initiation sites. The synthesis of transcripts with abnormal 5' termini did not occur in vitro until sequence between the TATA box and the normal cap sites was also deleted. We further observed a nonspecific requirement for 90 to 100 base pairs of DNA 5' to the cap site for optimal transcription of DNA fragments in vitro.

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.

DNA sequences required for specific and efficient initiation of transcription at the polyoma virus early promoter

1982 ◽  
Vol 2 (7) ◽  
pp. 737-751
Author(s):  
P Jat ◽  
U Novak ◽  
A Cowie ◽  
C Tyndall ◽  
R Kamen
Keyword(s):  
Dna Sequences ◽  
In Vitro Transcription ◽  
Polyoma Virus ◽  
Tata Box ◽  
Deletion Mutants ◽  
Base Pairs ◽  
Transcription Analysis ◽  
In Vivo Expression

The 5'-flanking DNA sequences involved in the specific and efficient transcription of the polyoma virus early region have been investigated. Sequence requirements for efficient in vivo expression differed from those in vitro. Deletion of DNA located between 200 and 400 base pairs before the principal cap sites severely inhibited in vivo expression as measured by transformation ability, but did not affect in vitro transcription. Viable deletion mutants which lack the principal cap sites and the "TATA" box were very poor templates for in vitro transcription. Analysis of other deletion mutants in vitro demonstrated that no specific sequences more than 46 base pairs before the cap sites were important. Removal of the TATA box reduced in vitro transcriptional efficiency but did not alter the initiation sites. The synthesis of transcripts with abnormal 5' termini did not occur in vitro until sequence between the TATA box and the normal cap sites was also deleted. We further observed a nonspecific requirement for 90 to 100 base pairs of DNA 5' to the cap site for optimal transcription of DNA fragments in vitro.

scholarly journals Activation of yeast polymerase II transcription by herpesvirus VP16 and GAL4 derivatives in vitro.

1989 ◽  
Vol 9 (11) ◽  
pp. 4746-4749 ◽  
Author(s):  
D I Chasman ◽  
J Leatherwood ◽  
M Carey ◽  
M Ptashne ◽  
R D Kornberg
Keyword(s):  
Rna Polymerase Ii ◽  
Fusion Proteins ◽  
In Vitro System ◽  
Transcription System ◽  
Natural Mechanism ◽  
Transcription In Vitro ◽  
Rna Polymerase Ii Transcription ◽  
Fold Stimulation

Fusion proteins known to activate transcription in vivo were tested for the ability to stimulate transcription in vitro in a recently developed Saccharomyces cerevisiae RNA polymerase II transcription system. One fusion protein, whose activation domain was derived from the herpesvirus transcriptional activator VP16, gave more than 100-fold stimulation in the in vitro system. The order of effects of the various proteins was the same for transcription in vitro and in vivo, suggesting that the natural mechanism of activation is preserved in vitro.

scholarly journals In vitro analysis of a transcription termination site for RNA polymerase II.

1990 ◽  
Vol 10 (11) ◽  
pp. 5782-5795 ◽  
Author(s):  
D K Wiest ◽  
D K Hawley
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Dna Sequences ◽  
Nuclear Extract ◽  
Transcription Unit ◽  
Dependent Manner ◽  
Wide Range ◽  
Vitro Analysis

Transcription from the adenovirus major late (ML) promoter has previously been shown to pause or terminate prematurely in vivo and in vitro at a site within the first intron of the major late transcription unit. We are studying the mechanism of elongation arrest at this site in vitro to define the DNA sequences and proteins that determine the elongation behavior of RNA polymerase II. Our assay system consists of a nuclear extract prepared from cultured human cells. With standard reaction conditions, termination is not observed downstream of the ML promoter. However, in the presence of Sarkosyl, up to 80% of the transcripts terminate 186 nucleotides downstream of the start site. Using this assay, we showed that the DNA sequences required to promote maximal levels of termination downstream of the ML promoter reside within a 65-base-pair region and function in an orientation-dependent manner. To test whether elongation complexes from the ML promoter were functionally homogeneous, we determined the termination efficiency at each of two termination sites placed in tandem. We found that the behavior of the elongation complexes was different at these sites, with termination being greater at the downstream site over a wide range of Sarkosyl concentrations. This result ruled out a model in which the polymerases that read through the first site were stably modified to antiterminate. We also demonstrated that the ability of the elongation complexes to respond to the ML termination site was promoter specific, as the site did not function efficiently downstream of a heterologous promoter. Taken together, the results presented here are not consistent with the simplest class of models that have been proposed previously for the mechanism of Sarkosyl-induced termination.

scholarly journals Regulation of protamine gene expression in an in vitro homologous system.

1996 ◽  
Vol 43 (2) ◽  
pp. 369-377 ◽  
Author(s):  
J M Jankowski ◽  
P D Cannon ◽  
F Van der Hoorn ◽  
L D Wasilewska ◽  
N C Wong ◽  
...  
Keyword(s):  
Gene Expression ◽  
Regulatory Elements ◽  
In Vitro Transcription ◽  
Tata Box ◽  
Transcription System ◽  
Proposed Model ◽  
Binding Factor ◽  
Gc Box ◽  
Responsive Element

An in vitro transcription system from the trout testis nuclei was developed to study trout protamine gene expression. The protamine promoter contains, among others, two regulatory elements: 1) a cAMP-responsive element or CRE element (TGACGTCA) which is present in position 5' to TATA box, and 2) GC box (CCGCCC) which is present in position 3' to TATA box. The removal of the CRE-binding protein by titration (by the addition of appropriate oligonucleotides to the incubation mixture) resulted in a decrease in transcription of the protamine gene. These results were confirmed by experiments in which the pure CRE-binding factor (TPBP1) was used, as well as by those where a stimulatory effect of cAMP on protamine promoter transcription was observed. On the other hand, addition of oligonucleotides containing the GC-box sequence enhanced the protamine gene transcription indicating that the protein (Sp1 like) which binds to this sequence acts as a repressor of protamine gene expression. These results confirm the previously proposed model which suggested that the GC box played a role in negative regulation of the protamine gene expression. Involvement of some other factors in this process was also discussed.

scholarly journals Isolation of Developmentally Regulated Genes fromToxoplasma gondii by a Gene Trap with the Positive and Negative Selectable Marker Hypoxanthine-Xanthine-Guanine Phosphoribosyltransferase

1998 ◽  
Vol 18 (2) ◽  
pp. 807-814 ◽  
Author(s):  
Laura J. Knoll ◽  
John C. Boothroyd
Keyword(s):  
Antimicrobial Agents ◽  
Selectable Marker ◽  
Insertion Site ◽  
Gene Trap ◽  
Specific Gene ◽  
Mrna Levels ◽  
Developmentally Regulated ◽  
S1 Nuclease ◽  
Nuclease Digestion

ABSTRACT Within its intermediate host, Toxoplasma gondiiswitches between two forms: a rapidly replicating tachyzoite and an encysted bradyzoite. Bradyzoites persist within the host throughout its life, hidden from antimicrobial agents and the immune system. The signals that mediate switching are poorly understood. A gene trap was employed to isolate genes whose expression is up-regulated early in the switching of bradyzoites via the negative and positive selectable marker hypoxanthine-xanthine-guanine phosphoribosyltransferase (HXGPRT). T. gondii was transfected with promoterlessHXGPRT and negatively selected with 6-thioxanthine to inhibit the growth of tachyzoites expressing HXGPRT. The surviving tachyzoites were then induced for in vitro bradyzoite formation and treated with mycophenolic acid and xanthine to positively select for parasites in which the construct had integrated downstream of a bradyzoite-specific gene. Strains were checked for their ability to differentiate by using Dolichos biflorus agglutinin (a bradyzoite-specific lectin) and a monoclonal antibody against P36 (a bradyzoite-specific surface antigen). After differentiation, all gene-trapped clones had Dolichos immunofluorescence and all but one expressed P36. The sequences flanking the insertion site of this P36-negative strain were homologous to the Toxoplasmafamily of surface antigens, strongly suggesting that P36 is encoded by the disruptive gene. Genetic mapping and complementation of the P36-negative strain further indicated that the disrupted gene is P36. Reverse transcriptase PCR and S1 nuclease digestion were used to compare mRNA levels during the tachyzoite and bradyzoite stages. The presumptive P36 gene does not appear to regulate its mRNA levels between the two stages, indicating a posttranscriptional mechanism of regulation for early bradyzoite-specific genes.

Organization of the noncontiguous promoter components of adenovirus VAI RNA gene is strikingly similar to that of eucaryotic tRNA genes

1983 ◽  
Vol 3 (11) ◽  
pp. 1996-2005
Author(s):  
R A Bhat ◽  
B Metz ◽  
B Thimmappaya
Keyword(s):  
Dna Sequences ◽  
Transcriptional Control ◽  
Transcriptional Activity ◽  
Evolutionary Relationship ◽  
Trna Genes ◽  
Wild Type ◽  
Base Pairs ◽  
Internal Promoter ◽  
Cell Extracts

The intragenic transcriptional control region (internal promoter) of the adenovirus type 2 VAI RNA gene was mutated by deletion, insertion, and substitution of DNA sequences at the plasmid level. The mutant plasmids were assayed for in vitro transcriptional activity by using HeLa cell extracts. The mutant clones with substitution or insertion of DNA sequences or both between nucleotides +18 and +53 of the VAI RNA gene were all transcriptionally active, although to various extents. Substitution of unrelated DNA sequences up to +26 or between +54 and +61 abolished the transcriptional activity completely. Based on these results, the intragenic promoter sequences of the VAI RNA gene can be subdivided into two components: element A, +10 to +18; and element B, +54 to +69. The distance between the A and B components could be enlarged from its normal 35 base pairs to 75 base pairs without destroying the transcriptional activity. However, a deletion of 4 or 6 base pairs in the DNA segment separating the A and B components (segment C) reduced the transcriptional activity of the genes to less than 2% of that of the wild type. When the VAI RNA gene with its element A or B was substituted for the corresponding element A or B of the Xenopus laevis tRNAMet gene, the hybrid genes transcribed close to the level of the wild-type VAI RNA gene and about 10- to 20-fold more efficiently than the tRNAMet gene. Thus, the organization of DNA sequences in the internal promoter of the VAI RNA gene appears to be very similar to that of eucaryotic tRNA genes. This similarity suggests an evolutionary relationship of the VAI RNA gene to tRNA genes.

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