scholarly journals Transcription reinitiation rate: a special role for the TATA box.

1997 ◽  
Vol 17 (7) ◽  
pp. 3809-3816 ◽  
Author(s):  
D Yean ◽  
J Gralla
Keyword(s):  
Point Mutations ◽  
In Vitro Assays ◽  
Special Role ◽  
Promoter Elements ◽  
Tata Element ◽  
Transcription Reinitiation ◽  
Active Transcription ◽  
Transcription Complexes ◽  
Transcriptional Induction

Promoters need to specify both the timing of transcriptional induction and the amount of transcript synthesized. In order to explore each of these effects separately, in vitro assays for the level of active preinitiation complex formation and for the rate of continuous RNA production were done. The effects were found to be influenced differently by different promoter elements. A consensus TATA element had a very strong effect on the rate of continuous RNA production, whereas two types of activators were important primarily in forming active transcription preinitiation complexes. Consensus TATA promoters exhibited high rates of continuous transcription; they assembled active preinitiation transcription complexes slowly but then produced transcripts continuously at an approximately fivefold-higher rate. Initiator-containing TATA-less promoters produced continuous transcripts slowly. Point mutations in the TATA element led to lower levels of transcription by reducing the number of preinitiation complexes and amplifying this reduction by lowering the apparent reinitiation rate. The results allow understanding of the sequence diversity of promoter elements in terms of specifying separate controls over the sensitivity of gene induction and over the strength of the induced promoter.

Domains of the rat rDNA promoter must be aligned stereospecifically

1992 ◽  
Vol 12 (3) ◽  
pp. 1266-1275
Author(s):  
W Q Xie ◽  
L I Rothblum
Keyword(s):  
Protein Complexes ◽  
Point Mutations ◽  
In Vitro Transcription ◽  
Promoter Elements ◽  
Repeat Distance ◽  
Deletion Mutations ◽  
The Core ◽  
In Vivo Experiments

Efficient transcription from the rat rDNA promoter results from an undefined interaction between the core (CPE) and upstream (UPE) promoter elements or the protein complexes which form on them. These interactions were demonstrated by the behavior of promoters that contained either linker-scanning or deletion mutations of the UPE in combination with point mutations of the CPE (bidomain mutants). In vivo transcription experiments using point mutations within the CPE (G----A mutation at either -16 or -7) demonstrated that the CPE may in fact consist of two domains. Whereas both of these mutants were rescued by the addition of UBF to in vitro transcription reactions, the CPE mutant -7A/G was inactive in vivo. Experiments with these bidomain mutants demonstrated that the UPE was required for the rescue of the CPE mutants. We also examined the hypothesis that this interaction might require a stereospecific alignment of the promoter elements. Our results indicate that the promoter consists of several domains with differing responses to mutations that alter the distance between, or within, the promoter elements. For example, the insertion or deletion of half-multiples of the helical repeat distance between -167 and -147 had no significant effect on transcription. On the other hand, some sites were sensitive to deletions of any size but not to insertions of up to 20 bp. The analyses of two sites yielded results suggesting that they lay between domains of the promoter that must be on the same side of the DNA helix for promoter activity. The first of these sites mapped between -106 and -95.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals Silk gland-specific tRNA(Ala) genes interact more weakly than constitutive tRNA(Ala) genes with silkworm TFIIIB and polymerase III fractions.

1994 ◽  
Vol 14 (3) ◽  
pp. 1806-1814 ◽  
Author(s):  
H S Sullivan ◽  
L S Young ◽  
C N White ◽  
K U Sprague
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Silk Gland ◽  
Flanking Sequence ◽  
Transcription Machinery ◽  
Polymerase Iii ◽  
Active Transcription ◽  
The Difference ◽  
Transcription Complexes

Constitutive and silk gland-specific tRNA(Ala) genes from silkworms have very different transcriptional properties in vitro. Typically, the constitutive type, which encodes tRNA(AlaC), directs transcription much more efficiently than does the silk gland-specific type, which encodes tRNA(AlaSG). We think that the inefficiency of the tRNA(AlaCG) gene underlies its capacity to be turned off in non-silk gland cells. An economical model is that the tRNA(AlaSG) promoter interacts poorly, relative to the tRNA(AlaC) promoter, with one or more components of the basal transcription machinery. As a consequence, the tRNA(AlaSG) gene directs the formation of fewer transcription complexes or of complexes with reduced cycling ability. Here we show that the difference in the number of active transcription complexes accounts for the difference in tRNA(AlaC) and tRNA(AlaSG) transcription rates. To determine whether a particular component of the silkworm transcription machinery is responsible for reduced complex formation on the tRNA(AlaSG) gene, we measured competition by templates for defined fractions of this machinery. We find that the tRNA(AlaSG) gene is greatly impaired, in comparison with the tRNA(AlaC) gene, in competition for either TFIIIB or RNA polymerase III. Competition for each of these fractions is also strongly influenced by the nature of the 5' flanking sequence, the promoter element responsible for the distinctive transcriptional properties of tRNA(AlaSG) and tRNA(AlaC) genes. These results suggest that differential interaction with TFIIIB or RNA polymerase III is a critical functional distinction between these genes.

scholarly journals Reversal of Mefloquine and Quinine Resistance in Plasmodium falciparum with NP30

2003 ◽  
Vol 47 (8) ◽  
pp. 2393-2396 ◽  
Author(s):  
Michelle Ciach ◽  
Kathleen Zong ◽  
Kevin C. Kain ◽  
Ian Crandall
Keyword(s):  
Plasmodium Falciparum ◽  
Multidrug Resistance ◽  
Genetic Analysis ◽  
Resistance Genes ◽  
Mammalian Cells ◽  
Point Mutations ◽  
In Vitro Assays ◽  
Drug Efflux ◽  
P Glycoprotein

ABSTRACT Quinoline resistance in malaria is frequently compared with P-glycoprotein-mediated multidrug resistance (mdr) in mammalian cells. We have previously reported that nonylphenolethoxylates, such as NP30, are potential Plasmodium falciparum P-glycoprotein substrates and drug efflux inhibitors. We used in vitro assays to compare the ability of verapamil and NP30 to sensitize two parasite isolates to four quinolines: chloroquine (CQ), mefloquine (MF), quinine (QN), and quinidine (QD). NP30 was able to sensitize (reversal, >80%) P. falciparum to MF, QN, QD, and, to a lesser extent, CQ. The presence of 2 μM verapamil had no effect on mefloquine resistance; however, the presence of verapamil modulated the activities of QN and QD in a manner parallel to that observed for CQ. Genetic analysis of putative quinoline resistance genes did not suggest an association between known point mutations in pfcrt and pfmdr1 and NP30 sensitization activity. We conclude that the sensitization action of NP30 is distinct both phenotypically and genotypically from that of verapamil.

scholarly journals Effect of the Attenuating Deletion and of Sequence Alterations Evolving In Vivo on Simian Immunodeficiency Virus C8-Nef Function

1999 ◽  
Vol 73 (4) ◽  
pp. 2790-2797 ◽  
Author(s):  
Silke Carl ◽  
A. John Iafrate ◽  
Jacek Skowronski ◽  
Christiane Stahl-Hennig ◽  
Frank Kirchhoff
Keyword(s):  
Viral Replication ◽  
Simian Immunodeficiency Virus ◽  
Point Mutations ◽  
Residual Activity ◽  
Intermediate Phenotype ◽  
Cell Surface Expression ◽  
In Vitro Assays ◽  
Immunodeficiency Virus

ABSTRACT The simian immunodeficiency virus macC8 (SIVmacC8) variant has been used in a European Community Concerted Action project to study the efficacy and safety of live attenuated SIV vaccines in a large number of macaques. The attenuating deletion in the SIVmacC8nef-long terminal repeat region encompasses only 12 bp and is “repaired” in a subset of infected animals. It is unknown whether C8-Nef retains some activity. Since it seems important to use only well-characterized deletion mutants in live attenuated vaccine studies, we analyzed the relevance of the deletion, and the duplications and point mutations selected in infected macaques for Nef function in vitro. The deletion, affecting amino acids 143 to 146 (DMYL), resulted in a dramatic decrease in Nef stability and function. The initial 12-bp duplication resulted in efficient Nef expression and an intermediate phenotype in infectivity assays, but it did not significantly restore the ability of Nef to stimulate viral replication and to downmodulate CD4 and class I major histocompatibility complex cell surface expression. The additional substitutions however, which subsequently evolved in vivo, gradually restored these Nef functions. It was noteworthy that coinfection experiments in the T-lymphoid 221 cell line revealed that even SIVmac nef variants carrying the original 12-bp deletion readily outgrew an otherwise isogenic virus containing a 182-bp deletion in the nef gene. Thus, although C8-Nef is unstable and severely impaired in in vitro assays, it maintains some residual activity to stimulate viral replication.

Silk gland-specific tRNA(Ala) genes interact more weakly than constitutive tRNA(Ala) genes with silkworm TFIIIB and polymerase III fractions

1994 ◽  
Vol 14 (3) ◽  
pp. 1806-1814
Author(s):  
H S Sullivan ◽  
L S Young ◽  
C N White ◽  
K U Sprague
Keyword(s):  
Rna Polymerase ◽  
Rna Polymerase Iii ◽  
Silk Gland ◽  
Flanking Sequence ◽  
Transcription Machinery ◽  
Polymerase Iii ◽  
Active Transcription ◽  
The Difference ◽  
Transcription Complexes

Constitutive and silk gland-specific tRNA(Ala) genes from silkworms have very different transcriptional properties in vitro. Typically, the constitutive type, which encodes tRNA(AlaC), directs transcription much more efficiently than does the silk gland-specific type, which encodes tRNA(AlaSG). We think that the inefficiency of the tRNA(AlaCG) gene underlies its capacity to be turned off in non-silk gland cells. An economical model is that the tRNA(AlaSG) promoter interacts poorly, relative to the tRNA(AlaC) promoter, with one or more components of the basal transcription machinery. As a consequence, the tRNA(AlaSG) gene directs the formation of fewer transcription complexes or of complexes with reduced cycling ability. Here we show that the difference in the number of active transcription complexes accounts for the difference in tRNA(AlaC) and tRNA(AlaSG) transcription rates. To determine whether a particular component of the silkworm transcription machinery is responsible for reduced complex formation on the tRNA(AlaSG) gene, we measured competition by templates for defined fractions of this machinery. We find that the tRNA(AlaSG) gene is greatly impaired, in comparison with the tRNA(AlaC) gene, in competition for either TFIIIB or RNA polymerase III. Competition for each of these fractions is also strongly influenced by the nature of the 5' flanking sequence, the promoter element responsible for the distinctive transcriptional properties of tRNA(AlaSG) and tRNA(AlaC) genes. These results suggest that differential interaction with TFIIIB or RNA polymerase III is a critical functional distinction between these genes.

scholarly journals Point Mutations and Cytochrome P450 Can Contribute to Resistance to ACCase-Inhibiting Herbicides in Three Phalaris Species

Plants ◽  
2021 ◽  
Vol 10 (8) ◽  
pp. 1703
Author(s):  
José G. Vázquez-García ◽  
Joel Torra ◽  
Candelario Palma-Bautista ◽  
Ricardo Alcántara-de la Cruz ◽  
Rafael De Prado
Keyword(s):  
Point Mutations ◽  
Resistance Mechanisms ◽  
Target Site ◽  
In Vitro Assays ◽  
Cross Resistance ◽  
Northern Iran ◽  
Target Site Resistance ◽  
Acetyl Coenzyme A Carboxylase ◽  
Selection Of

Species of Phalaris have historically been controlled by acetyl-coenzyme A carboxylase (ACCase)-inhibiting herbicides; however, overreliance on herbicides with this mechanism of action has resulted in the selection of resistant biotypes. The resistance to ACCase-inhibiting herbicides was characterized in Phalaris brachystachys, Phalaris minor, and Phalaris paradoxa samples collected from winter wheat fields in northern Iran. Three resistant (R) biotypes, one of each Phalaris species, presented high cross-resistance levels to diclofop-methyl, cycloxydim, and pinoxaden, which belong to the chemical families of aryloxyphenoxypropionates (FOPs), cyclohexanediones (DIMs), and phenylpyrazolines (DENs), respectively. The metabolism of 14C-diclofop-methyl contributed to the resistance of the P. brachystachys R biotype, while no evidence of herbicide metabolism was found in P. minor or P. paradoxa. ACCase in vitro assays showed that the target sites were very sensitive to FOP, DIM, and DEN herbicides in the S biotypes of the three species, while the R Phalaris spp. biotypes presented different levels of resistance to these herbicides. ACCase gene sequencing confirmed that cross-resistance in Phalaris species was conferred by specific point mutations. Resistance in the P. brachystachys R biotype was due to target site and non-target-site resistance mechanisms, while in P. minor and P. paradoxa, only an altered target site was found.

scholarly journals GENE-04. THE ONCOGENIC FUNCTIONS OF YAP1-GENE FUSIONS CAN BE INHIBITED BY DISRUPTION OF YAP1-TEAD INTERACTION

2019 ◽  
Vol 21 (Supplement_6) ◽  
pp. vi98-vi98
Author(s):  
Frank Szulzewsky ◽  
Pia Hoellerbauer ◽  
Hua-Jun Wu ◽  
P J Cimino ◽  
Franziska Michor ◽  
...  
Keyword(s):  
Hippo Pathway ◽  
Point Mutations ◽  
Tumor Formation ◽  
In Vitro Assays ◽  
Gene Fusions ◽  
Genetic Ablation ◽  
Molecular Features ◽  
Cancer Types

Abstract Supratentorial ependymoma can be sub-stratified into clinically relevant subtypes characterized by distinct molecular features. The subtype defined by high YAP1 activity harbored two distinct YAP1 gene fusions, YAP1-MAMLD1 and YAP1-FAM118B. In addition, YAP1 gene fusions have been detected in several other cancer types, including Epithelioid Hemangioendothelioma and Endocervical Adenocarcinoma. YAP1 is a key transcriptional co-activator and proto-oncogene that is negatively regulated by the Hippo pathway. Here, we show that both YAP1-MAMLD1 and YAP1-FAM118B, as well as additional YAP1 fusion genes found in other cancer types, are potent oncogenic drivers that cause tumor formation in the brain and the hindlimb in mice upon overexpression by somatic cell gene transfer. Using different in vitro assays, including Luciferase, RNA-, and ChIP Seq, we show that both the N-terminal YAP1 part and the C-terminal fusion partners exert activity. We can show that the YAP1 activity still relies on the binding to TEAD transcription factors, whereas the C terminal activity does not. Furthermore, the different fusion proteins have become independent from negative Hippo pathway signaling by constitutive nuclear localization and protection from degradation. In addition, by introducing point mutations and truncations to block the YAP1 and the MAMLD1 function we can show that the activity of both halves contributes to the oncogenic function of YAP1-MAMLD1. Using in vitro and in vivo assays we can show that pharmacological and genetic ablation of YAP-TEAD interaction diminishes the oncogenic potential of the fusions, indicating that this might be a potential therapeutic approach for these tumors in the future.

scholarly journals Intragenic activating and repressing elements control transcription from the adenovirus IVa2 initiator.

1994 ◽  
Vol 14 (1) ◽  
pp. 676-685 ◽  
Author(s):  
H Chen ◽  
R Vinnakota ◽  
S J Flint
Keyword(s):  
Regulatory Element ◽  
Point Mutations ◽  
Adenovirus Type ◽  
Mobility Shift ◽  
Negative Regulatory Element ◽  
Promoter Sequences ◽  
Tata Element ◽  
Tata Sequence ◽  
Promoter Mutations

The downstream stimulatory segment of the adenovirus type 2 IVa promoter includes a TA-rich sequence that binds recombinant TATA-binding proteins (TBP) in vitro. We now demonstrate that when placed upstream of the IVa2, initiator, this TA-rich sequence operated as a TATA element but exhibited significantly lower transcriptional and TBP-binding activities than did the TATA box of the adenovirus major late (ML) promoter. In sharp contrast, changing the IVa2 TA-rich sequence in its natural, intragenic context to the ML TATA sequence increased the activity of the IVa2 promoter only slightly. In view of this discrepancy, we examined the effects of single, double, and clustered point mutations in the downstream sequence on the activity of a minimal IVa2 promoter. Mutations between positions +21 and +29 inhibited IVa2 transcription, in some cases to the very low level directed by the IVa2 initiator alone. By contrast, substitutions within the TA-rich sequence increased the efficiency of IVa2 transcription. These results indicated that the downstream, TA-rich sequence does not function as an intragenic TFIID-binding site but rather is included within a negative regulatory element. Electrophoretic mobility shift and methylation interference assays using wild-type and mutated, intragenic promoter sequences identified a HeLa cell component whose binding to the sequence +11 to +27 correlated with repression of IVa2 transcription, suggesting that a negative regulatory element is superimposed upon the intragenic sequence required for efficient transcription from the IVa2 initiator.

scholarly journals Domains of the rat rDNA promoter must be aligned stereospecifically.

1992 ◽  
Vol 12 (3) ◽  
pp. 1266-1275 ◽  
Author(s):  
W Q Xie ◽  
L I Rothblum
Keyword(s):  
Protein Complexes ◽  
Point Mutations ◽  
In Vitro Transcription ◽  
Promoter Elements ◽  
Repeat Distance ◽  
Deletion Mutations ◽  
The Core ◽  
In Vivo Experiments

Efficient transcription from the rat rDNA promoter results from an undefined interaction between the core (CPE) and upstream (UPE) promoter elements or the protein complexes which form on them. These interactions were demonstrated by the behavior of promoters that contained either linker-scanning or deletion mutations of the UPE in combination with point mutations of the CPE (bidomain mutants). In vivo transcription experiments using point mutations within the CPE (G----A mutation at either -16 or -7) demonstrated that the CPE may in fact consist of two domains. Whereas both of these mutants were rescued by the addition of UBF to in vitro transcription reactions, the CPE mutant -7A/G was inactive in vivo. Experiments with these bidomain mutants demonstrated that the UPE was required for the rescue of the CPE mutants. We also examined the hypothesis that this interaction might require a stereospecific alignment of the promoter elements. Our results indicate that the promoter consists of several domains with differing responses to mutations that alter the distance between, or within, the promoter elements. For example, the insertion or deletion of half-multiples of the helical repeat distance between -167 and -147 had no significant effect on transcription. On the other hand, some sites were sensitive to deletions of any size but not to insertions of up to 20 bp. The analyses of two sites yielded results suggesting that they lay between domains of the promoter that must be on the same side of the DNA helix for promoter activity. The first of these sites mapped between -106 and -95.(ABSTRACT TRUNCATED AT 250 WORDS)

Intragenic activating and repressing elements control transcription from the adenovirus IVa2 initiator

1994 ◽  
Vol 14 (1) ◽  
pp. 676-685
Author(s):  
H Chen ◽  
R Vinnakota ◽  
S J Flint
Keyword(s):  
Regulatory Element ◽  
Point Mutations ◽  
Adenovirus Type ◽  
Mobility Shift ◽  
Negative Regulatory Element ◽  
Promoter Sequences ◽  
Tata Element ◽  
Tata Sequence ◽  
Promoter Mutations

The downstream stimulatory segment of the adenovirus type 2 IVa promoter includes a TA-rich sequence that binds recombinant TATA-binding proteins (TBP) in vitro. We now demonstrate that when placed upstream of the IVa2, initiator, this TA-rich sequence operated as a TATA element but exhibited significantly lower transcriptional and TBP-binding activities than did the TATA box of the adenovirus major late (ML) promoter. In sharp contrast, changing the IVa2 TA-rich sequence in its natural, intragenic context to the ML TATA sequence increased the activity of the IVa2 promoter only slightly. In view of this discrepancy, we examined the effects of single, double, and clustered point mutations in the downstream sequence on the activity of a minimal IVa2 promoter. Mutations between positions +21 and +29 inhibited IVa2 transcription, in some cases to the very low level directed by the IVa2 initiator alone. By contrast, substitutions within the TA-rich sequence increased the efficiency of IVa2 transcription. These results indicated that the downstream, TA-rich sequence does not function as an intragenic TFIID-binding site but rather is included within a negative regulatory element. Electrophoretic mobility shift and methylation interference assays using wild-type and mutated, intragenic promoter sequences identified a HeLa cell component whose binding to the sequence +11 to +27 correlated with repression of IVa2 transcription, suggesting that a negative regulatory element is superimposed upon the intragenic sequence required for efficient transcription from the IVa2 initiator.

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