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At least 577 records · Page 32Linked to original sources
PubMed1994

Construction of a versatile promoter analysis vector and its use for analysis of the Serratia marcescens aspartase promoter region.

A new versatile promoter analysis vector, pLGlacZ7, which contains a multiple cloning site and the lacZ structural gene in a low-copy-number plasmid pLG339, has been constructed. This plasmid, which can be stably maintained in Escherichia coli and Serratia marcescens, is useful for analysis of gene expression using lacZ gene fusions. The multiple cloning site of pLGlacZ7 is convenient for the insertion or deletion of promoter DNA fragments, the latter by using exonuclease III. The promoter of the S. marcescens aspA gene encoding aspartase was analyzed using plasmid pLGlacZ7. The S. marcescens aspA gene is composed of 1434 nucleotides and codes for a protein with a Mr of 52,543 whose predicted amino acid sequence was very similar to that of the E. coli aspA gene product. Two functional regions that may participate in the transcription of the S. marcescens aspA gene were found in the promoter region by using lacZ gene fusions in pLGlacZ7.

Amino Acid Sequence↗
PubMed2000

Dox-dependent SIVmac with tetracycline-inducible promoter in the U3 promoter region.

An attenuated live vaccine is a candidate in developing vaccines against human immunodeficiency virus type 1 (HIV-1). The study using macaques and simian immunodeficiency virus (SIVmac) showed an attenuated virus to be more effective than any other vaccine candidate. However, development of a safer vaccine is required for clinical application. In this study, we constructed pSIVmac Delta nef with tetracycline inducible promoter (pTet) and tried to control viral expression in a drug-dependent manner. Promoter/enhancer motifs in the U3 region of the long terminal repeats (LTRs) were serially deleted and replaced with pTet. In mutant LTRs, which lack NF-kappaB and Sp1 binding sites, TATA box motifs, and the 5' half of the U3 region, promoter activity was stringently controlled by doxycycline (Dox). Their activities were similar to or higher than that of wild-type LTR in the presence of Dox, based on the transient chloramphenicol acetyltransferase reporter assay. Three of these mutant LTRs were introduced into the pSIVmac239 Delta nef genome. Viral protein from these viruses was efficiently expressed in a Dox-dependent manner after transfection to a HeLa cell, which expresses reverse tetracycline transactivator (rtTA). The 2-LTR-form viral DNA of these viruses could be detected in M8166 cells that had been infected with supernatants from the transfected rtTA HeLa cell. These results suggest that pSIVmac Delta nef containing mutant LTRs can proceed through one viral replication cycle consisting of transcription, formation of viral particles, infection to cells, and reverse transcription. Although continuous replication of these Dox-dependent viruses requires a supply of rtTA as a constituent for the pTet-On viral genome, the successful replacement of the original promoter with a drug-dependent promoter suggests a new possibility for developing a safer attenuated live virus.

Doxycycline↗
PubMed1995

Analysis of the lupin Nodulin-45 promoter: conserved regulatory sequences are important for promoter activity.

The promoter from the Lupinus angustifolius late nodulin gene, Nodulin-45, has been analysed to identify cis-elements and trans-acting factors. Various regions of the Nodulin-45 promoter, fused to the luciferase reporter gene, were introduced into Lotus roots using an Agrobacterium rhizogenes, transformation procedure. The transgenic roots were then nodulated. The promoter region A (-172 to +13, relative to the transcription start site) was capable of directing low-level expression of the reporter gene and in a nodule-enhanced manner when compared to roots. The addition of region C (-676 to -345) resulted in a significant increase in the expression within the nodule, whilst a low level of root expression was maintained. The C region, which confers this high-level nodule expression, contains the nodule consensus motifs AAAGAT and CTCTT. When region C was ligated to a minimal promoter element from the unrelated asparaginase gene rather than the Nodulin-45 A region, nodule-enhanced expression was still apparent, but at a much lower level. Mutation of the AAAGAT element in this construct resulted in a further significant decrease of expression. Gel retardation assays revealed that a factor from lupin nodule nuclear extracts interacted with two sequences of the C region. The binding of the factor to both of these regions could be removed by the addition of an oligonucleotide containing the AT-rich binding site for the soybean factor NAT2. This suggests that the lupin factor identified here is a NAT2 homologue. No factor binding was observed to the AAAGAT or CTCTT elements present in the C region.

Base Sequence↗
PubMed1995

Deletion analysis of the Brassica napus cruciferin gene cru 1 promoter in transformed tobacco: promoter activity during early and late stages of embryogenesis is influenced by cis-acting elements in partially separate regions.

To define sequences in the cruciferin gene cru1 promoter of importance for expression, tobacco (Nicotina tabacum L.) plants were transformed with constructs in which the cru1 promoter, in front of the intact cru1 structural gene, was truncated at -1216, -974, -736, -515, -306, -46 and -17 bp relative to the cap-site. Cru1 expression in tobacco seeds was studied by Northern analysis, Western analysis and in-situ hybridizations. Comparisons of the Northern analysis of RNA from tobacco seeds harvested at 18 d after pollination with the Western analysis of protein from mature seeds showed that the regions between -974 to -736 and -306 to -46 were important for the expression of cru1 at an early developmental stage, whereas the regions -736 to -515 and -515 to -306 were important for expression throughout embryogenesis. By investigating the mRNA levels in transgenic seeds at different stages of development, indications were obtained that the two latter regions exerted their effects during the later stages. The in-situ hybridization showed that cru1 mRNA was distributed in parenchyma cells throughout the embryo in seeds expressing constructs -974 and -736. Constructs -515 and -306 showed an expression restricted to the axis or axis and parts of the cotyledons. Sequence comparisons of the cru1 promoter with other storage-protein gene promoters, identified several motifs implicated in gene regulation.(ABSTRACT TRUNCATED AT 250 WORDS)

Allergens↗
PubMed1995

A method for easy isolation of promoter fragments from promoter-probe libraries of filamentous fungi.

Two genomic fragments capable of driving the expression of the hygromycin B resistance gene (hph) were isolated from the phytopathogenic ascomycete Gibberella pulicaris (anamorph Fusarium sambucinum) using a "promoter-probe library" strategy. Two libraries consisting of random, 0.5-2.0-kb fragments of genomic DNA inserted 5' of a promoterless hph gene were constructed and used for transformation of G. pulicaris. Both libraries transformed G. pulicaris at a low frequency. Transformants tolerated up to 800 micrograms/ml of hygromycin B, while untransformed colonies were inhibited completely by 50 micrograms/ml of the antibiotic. Plasmids were re-isolated from transformants by simply digesting, the genomic DNA with KpnI, which cuts once in the polylinker 5' to the insert, and transforming E. coli with the re-ligated DNA. The recovered plasmids transformed G. pulicaris with a frequency of up to 4.4 transformants/micrograms of DNA. Both promoter fragments were sequenced and found to contain TATA and CAAT boxes as well as CT-rich sequences. This method makes it possible to easily isolate many fragments with promoter activity from filamentous fungi, and should facilitate the investigation of the promoter structures necessary for the expression of fungal genes.

Base Sequence↗
PubMed1987

Characterization of a promoter up mutation in the -35 region of the promoter of the primer for ColE1 replication.

A point mutation in the -35 region of the promoter of the primer for initiation of DNA replication in the plasmid pMB1 was characterized. This base change causes a promoter up phenotype. The analysis of a second mutant obtained by site-directed mutagenesis allowed the exclusion of a role in the phenotype for the potential intrastrand secondary structure as well as for the methylation state of the DNA in the promoter region. The promoter up phenotype is concluded to be due to a change in the primary structure of the -35 element with the consequent production of a better cluster of hydrogen bond donors and acceptors for the RNA polymerase.

Base Sequence↗
PubMed1996

A ribosomal protein gene (rpl32) from tobacco chloroplast DNA is transcribed from alternative promoters: similarities in promoter region organization in plastid housekeeping genes.

Multiple transcriptional start sites have been identified in the tobacco plastid ribosomal protein gene rpl32 by RNA mapping and in vitro capping techniques. A promotor with a canonical -10 Pribnow Box (P1) produces a major transcript in leaf chloroplasts. Transcription is also driven from additional promoters in non-photosynthetic plastids from heterotrophically cultured cells (BY2 line). Among them, a second promoter located downstream (P2) generates the most prominent transcript in this type of cell. The absence of typical plastid promoter motifs upstream of this site and the higher steady-state level of the P2-derived transcript in BY2 cells suggest a distinct modulation of transcription. Mobility shift experiments also seem to indicate the existence of differences in protein-DNA binding between both kinds of plastids with respect to a DNA fragment including the sequence upstream from the P2 starting site. The structure of the rpl32 promoter region is discussed in relation to that of other plastid housekeeping genes encoding elements of the genetic machinery.

Base Sequence↗
PubMed1996

Evolution of promoter sequences: elements of a canonical promoter for prespore genes of Dictyostelium.

An attempt is made to define a minimal prespore promoter which contains all elements essential for correct regulation of expression of a prespore gene. The prespore genes of Dictyostelium are coregulated during development. Most begin transcription at the same early stage, and activity of all is restricted to prespore tissue during the later slug stage. Sequences 5' to the coding sequences of eight prespore genes were searched for all elements proposed to control transcription and for new elements. The meaningfulness of occurrences of elements and pairs of elements in prespore promoters was evaluated by comparison with frequencies of occurrences in promoters of other, nonprespore genes. These comparisons resulted in definition of a canonical prespore promoter, a stretch of about 200 nucleotides containing at least one of each of three elements. Certain limitations were found on the spacing of elements. Orientation of elements with respect to each other appeared unrestricted. All elements often occurred in multiple copies. This structure suggests that individual copies of each element are not conserved during evolution, but instead continually appear and disappear.

Animals↗
PubMed2000

Targeted neutralization of calcineurin, by expression of an inhibitor peptide under the control of a cholinergic specific promoter in PC12 cells, promotes neurite outgrowth in the presence of NGF.

We have characterized a region of the mouse vesicular acetylcholine transporter(VAChT)/choline acetyltransferase (ChAT) gene locus that serves as a cholinergic-specific promoter for the expression of both VAChT and ChAT genes, as well as a reporter gene (LacZ) in vivo. We have used this promoter to direct the expression of an inhibitor peptide, derived from the calcineurin (CalN) autoregulatory domain, to directly neutralize the function of CalN to define the role of this Ca2+/Calmodulin regulated phosphatase in neurite outgrowth. Targeted inhibition of CalN promotes neurite outgrowth in PC12 cells in the presence of NGF, as early as 24 h after transfection. Inhibition of CalN-mediated enhancement of neurite outgrowth in PC12 cells reaches a maximum effect within the first 4 to 6 days after transfection, and does not cause adverse effects when highly expressed for up to 12 days. Cyclosporin A, a nontargeted CalN inhibitor, increases the number of neurites in mock transfected cells by 1.5 fold, while in transfected PC12 cells, the expression of the CalN inhibitor peptide increases the neurite number by 1.8 fold. These data demonstrate that CalN is an important regulator of the neurotrophic response in cholinergic cells and may prove valuable in developing treatment strategies to promote recovery from neurological injury.

Animals↗
PubMed1996

Cloning of a marine cyanobacterial promoter for foreign gene expression using a promoter probe vector.

A marine cyanobacterial promoter was cloned to allow efficient foreign gene expression. This was carried out using chloramphenicol acetyl transferase (CAT) as a marker protein. For rapid and simple measurement of CAT activity, a method based on a fluorescently labeled substrate was improved by utilizing HPLC equipped with a flow-through fluorescent spectrophotometer. This method was used in conjunction with a newly constructed promoter probe vector. Cyanobacterial transformants, harboring plasmid containing a cloned 2-kbp marine cyanobacterial genomic fragment, showed a 10-fold higher CAT activity, compared with that achieved using the kanamycin-resistant gene promoter. From the sequence analysis of the cloned fragment, a putative promoter region was found.

Base Sequence↗
PubMed2006

[Evidence and evaluation in health promotion and prevention: the example of the health promotion initiative "healthy living in Bavaria"].

BACKGROUND: The use of evidence and evaluation is rapidly gaining importance in the field of health promotion and prevention. The evidence base, evaluation methods as well as the capacity and required competencies among practitioners are still being developed. AIMS: Beyond its specific health-related targets, the health promotion initiative "Healthy Living in Bavaria" aims to integrate the use of evidence and evaluation as key components into project funding as part of a total quality management concept. Another objective is the creation of new evidence for the field of health promotion and prevention. APPROACH: A core element of the quality management concept is the initiative's multistage evidence and evaluation model which embraces the initiative as a whole as well as the individual projects. The model is operationalized through a set of matching instruments for application, reporting, evaluation, and appraisal. CONCLUSION: Nationally as well as internationally, evidence and evaluation stand out as central aspects of quality improvement instruments in health promotion and prevention. Characteristic of Bavaria's quality improvement approach is the seamless process from the application to the evaluation under consideration of scientific as well as pragmatic aspects.

Germany↗
PubMed2006

Single nucleotide polymorphisms in the interleukin-6 gene promoter, tumor necrosis factor-alpha gene promoter, and transforming growth factor-beta1 gene signal sequence as predictors of time to onset of aseptic loosening after total hip arthroplasty: preliminary study.

BACKGROUND: Aseptic loosening resulting from inflammatory response to the implant wear debris is the major cause of late total hip arthroplasty (THA) failure. We examined single nucleotide polymorphisms in genes encoding for three involved cytokines--interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha), and transforming growth factor-beta1 (TGF-beta1)--as potential predictors of time to onset of aseptic instability. METHODS: A total of 41 patients/45 total hip endoprostheses (same type, same surgeon) were followed up for as long as 18 years. They were genotyped for the IL-6 promoter (-597G-->A) and (-572G-->C), TNF-alpha promoter (-308G-->A), and TGF-beta1 signal sequence (29T-->C) transitions. Cox regression was performed on the prosthesis survival. RESULTS: Overall, 22 of 45 prostheses developed aseptic instability. Cumulative survivals at 10 and 15 years after THA were 95.6% and 66.6%, respectively. The effect of a particular polymorphic site was estimated with adjustment for sex, age at THA, reason for THA, and the effects of other analyzed sites. The hazard ratio (HR) for genotype T/T versus "C-allele carriage" at the TGF-beta1 site was 8.23 [95% confidence interval (CI) 1.45-46.8] (P=0.017) or 5.70 (1.39-23.4) (P=0.016) when the IL-6 promoter sites were considered as a "combination of genotypes (-597)|(-572)." The most prevalent combination of genotypes at IL-6 sites was G/A (-597)|C/C (-572). HR for this combination (versus other combinations) was 5.43 (1.73-17.0) (P=0.004) when "TGF-beta1 (29T-->C)" was considered as a three-level factor (three possible genotypes), and 4.92 (1.71-14.1) (P=0.003) when TGF-beta1 site was considered as a two-level factor (T/T and "C-allele carriage"). The HR for the "A-allele carriage" at TNF-alpha (-308G-->A) could not be determined (only two patients had the G/G genotype). CONCLUSIONS: This preliminary study is the first to suggest that the TGF-beta1 signal sequence (29T-->C) and IL-6 promoter (-597G-->A)|(-572G-->C) transitions are predictive for the time to onset of aseptic instability after THA.

Adult↗
PubMed1988

The late promoter of the human papovavirus BK is contained within the early promoter enhancer region.

We have analyzed the sequences necessary for late promoter function and mapped the late mRNA start sites of the human papovavirus BK (prototype). Our results show that, under both replicating and nonreplicating conditions, the BKV late promoter is contained within the same region defined as the enhancer for the early promoter. This region consists of three 68-bp repeats (the middle one of which has an 18-bp deletion) and a 66-bp region containing an enhancer element denoted as c, located to the late side of the 68-bp repeats. Deletions within the early enhancer domain indicate that elements of the late promoter are found throughout the entire region.

Antigens, Viral, Tumor↗
PubMed1991

Regulation of JCVL promoter function: evidence that a pentanucleotide "silencer" repeat sequence AGGGAAGGGA down-regulates transcription of the JC virus late promoter.

The human neurotropic papovavirus JCV contains sequences within the two 98-bp tandem repeats which play a key role in glial-specific transcription of the viral early and late promoters. Previous analysis of the 98-bp sequence has delineated several protein-binding domains that are recognized by nuclear factors present in human brain cells. In the present study, by deletion mutation analysis, we have identified a region within each 98-bp repeat that reduces transcriptional activity of the JCV late promoter (JCVL). Using synthetic oligonucleotides spanning this region, designated "OP," we demonstrate that down-regulation of the JCVL promoter is associated with a pentanucleotide repeat sequence (AGGGAAGGGA) juxtaposed to the poly(dA) tract within the 98-bp tandem repeats. The OP sequence interacts specifically with a protein derived from glial nuclear extract and forms a major 56- to 60-kDa complex. Methylation interference experiment indicates that the three G residues proximal to the poly(dA) tract make major groove contacts with the protein. Single-base-pair substitution of these residues suggests that the complex can form in the presence of two of the three guanosyl residues. The possible role of this protein in regulating the JCV lytic cycle in concert with nearby regulatory elements within JCV promoter region is discussed.

Animals↗
PubMed1987

A transposable promoter and transposable promoter probes derived from Tn1721.

We describe two types of new Tn1721-derivatives capable of random insertion and of generating transcriptional fusions at the site of insertion: transposable promoters (Tn1735) carrying a strong, inducible ptac promoter that turns on adjacent (cryptic) genes; and transposable promoter probes (Tn1736, Tn1737) carrying promoterless genes coding for chloramphenicol acetyl transferase or beta-galactosidase, and used to accurately determine the expression of external promoters. These elements are available with four different selectable resistance markers and on conjugative, temperature-sensitive and multicopy plasmid vehicles. Experiments are described that demonstrate the advantage of random insertions for expressing various genes and for studying gene regulation.

Acetyltransferases↗
PubMed1990

Construction of lacZ promoter probe vectors for use in Synechococcus: application to the identification of CO2-regulated promoters.

It was shown that the Escherichia coli lacZ gene could be expressed in the cyanobacterium Synechococcus R2 PCC7942 both as a plasmid-borne form and also integrated into the chromosome. A promoterless form of the lacZ gene was constructed and used as a reporter gene to make transcriptional fusions with cyanobacterial promoters using a shuttle vector system and also via a process of integration by homologous recombination. Synechococcus R2 promoter-lacZ gene fusions were then used to identify CO2-regulated promoters, by quantitatively assessing beta-galactosidase activity under high and low CO2 conditions using a fluorescence assay. Several promoters induced under low CO2 conditions were detected.

Amino Acid Sequence↗
PubMed2004

Chicken ovalbumin upstream promoter-transcription factor I represses the transcriptional activity of the human muscle glycogen phosphorylase promoter in C2C12 cells.

The responsiveness of the 1.13 kb proximal human muscle glycogen phosphorylase (MGP) gene promoter to the chicken ovalbumin upstream promoter-transcription factor (COUP-TF) repressor, known to be ablated during muscle cell differentiation, was examined. Constitutive expression of COUP-TFI repressed the activity of the promoter in C2C12 muscle cells and sequential deletion analysis mapped the sensitive region between nucleotides -362 and -185, which included a putative consensus COUP-TF binding half-site at -198/-193. Mutation of this site abolished transcriptional response to COUP-TFI of the -362 construct. A -209/-180 probe bound in vitro to COUP-TFI and to protein extracts from proliferating but not fusing myoblasts. Thus, COUP-TF may be involved in repression of the human MGP gene promoter at the myoblast stage.

Animals↗
PubMed2005

Conversion of active promoter-RNA polymerase complexes into inactive promoter bound complexes in E. coli by the transcription effector, ppGpp.

Guanosine tetraphosphate (ppGpp) is a signal of nutritional stress that regulates transcription. An RNA polymerase rudder mutant rpoC (Delta 312-315) is found to suppress ppGpp deficiency phenotypes by restoring both negative and positive activities of promoter fusions in vivo, as if ppGpp were present. Measurements of defects in transcription of the PargT tRNA promoter with mutant RNA polymerase reveal that the mutant enzyme quantitatively mimics the presence of added ppGpp. DNaseI footprints and mobility shifts under RNA polymerization conditions reveal that the promoter-specific transcription defect of the mutant enzyme can be ascribed to the presence of inactive dead-end promoter complexes with features similar to those of a stable closed complex. We propose that formation of such inactive complexes represents an alternative explanation of "stringent RNA polymerase" mutant behavior to those currently published, and it represents a newly discovered mode of action of ppGpp.

DNA Footprinting↗