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At least 289 records · Page 16Linked to original sources
PubMed1994

Molecular cloning and functional analysis of the promoter of rat skeletal muscle voltage-sensitive sodium channel subtype 2 (rSkM2): evidence for muscle-specific nuclear protein binding to the core promoter.

rSkM2 is a tetrodotoxin-resistant rat skeletal muscle voltage-sensitive sodium channel that is expressed in immature and denervated skeletal muscle and in adult heart. We have isolated a 3.7-kb gene segment that contains the first exon, multiple transcription initiation sites, the core promoter (nt -102 to +1), GC-rich elements (Sp1 recognition sites), three overlapping C-rich motifs (important for muscle-specific expression of some muscle genes), and multiple CANNTG (E-box) motifs (MyoD binding sites). A deletion analysis of the 5' upstream 2.8-kb segment, driving the rSkM2 core promoter, has localized a muscle-restrictive enhancer element (MRSE) at least 2 kb upstream from the core promoter. The core promoter is silenced by an additional cis element (-645/-506). The positive and negative cis-elements together drive transcription of the chloramphenicol acetyltransferase (CAT) reporter gene from the core promoter at about the same level as does the core promoter alone in a skeletal muscle differentiation stage-specific manner. Gel-shift assays have identified sequence- and cell-type-specific proteins that bind to a 16-bp region (-44/-29) containing C-rich motifs. Muscle-specific complexes formed from muscle cell nuclear extracts and a 16-bp element (-44/-29) are competed by unlabeled -44/-29 oligonucleotide but not by several mutant oligonucleotides that implicate nucleotides -40 to -38 and -34 to -32 in the binding of a nuclear protein (designated SkM2 transcription factor 1, SkM2-TF1). We conclude that rSkM2 gene expression depends on the interactions of positive and negative transcriptional regulators with tissue- and developmental stage-specific core promoter elements.

Animals↗
PubMed1997

The murine L-plastin gene promoter: identification and comparison with the human L-plastin gene promoter.

Plastins (or fimbrins) are a family of actin-binding proteins that are conserved from yeast to humans. In mammals, three tissue-specific plastin isoforms have been identified. The L isoform (L-plastin) is normally expressed only in leukocytes but is also found in >90% of neoplastic nonleukocyte human cells. Because L-plastin expression in tissue-specifically regulated in both humans and rodents, it is likely that similar mechanisms regulate L-plastin gene expression in human and rodent cells and that they could be identified by comparing the function and nucleotide sequences of the human and murine L-plastin gene promoters. Previously, we reported the isolation and characterization of the human L-plastin gene promoter. In this study, we isolated a murine L-plastin 5' end cDNA and used it as a probe to isolate several murine genomic clones. A representative clone contained 7 kb of the flanking region, 0.1 kb of the first exon, and 9.9 kb of the first intron. A continuous 1,354-bp sequence was identified around the first exon. Five transcription initiation sites were found 40 to 73 bp downstream from a perfect TATA box. Alignment of the sequence with its human counterpart revealed approximately 60% homology in a 1-kb region spanning the first exon and the flanking region. The TATA box, one ER binding site, and two ETS binding sites were completely conserved. An Sp1 binding sequence in the human promoter was partially conserved in the murine promoter but could still bind to Sp1. A second ER binding sequence, lying 5' adjacent to the TATA box in the human promoter, was conserved only at the 3' half-site in the murine promoter; the 5' half-site was changed into a potential AP1 binding site. This AP1/ER hybrid sequence was incapable of binding to ER. However, both human and murine promoters were found to function equally well in either human or murine leukocytes.

3T3 Cells↗
PubMed2005

Promoter modeling: the case study of mammalian histone promoters.

MOTIVATION: Histone proteins play important roles in chromosomal functions. They are significantly evolutionarily conserved across species, which suggests similarity in their transcription regulation. The abundance of experimental data on histone promoters provides an excellent background for the evaluation of computational methods. Our study addresses the issue of how well computational analysis can contribute to unveiling the biologically relevant content of promoter regions for a large number of mammalian histone genes taken across several species, and suggests the consensus promoter models of different histone groups. RESULTS: This is the first study to unveil the detailed promoter structures of all five mammalian histone groups and their subgroups. This is also the most comprehensive computational analysis of histone promoters performed to date. The most exciting fact is that the results correlate very well with the biologically known facts and experimental data. Our analysis convincingly demonstrates that computational approach can significantly contribute to elucidation of promoter content (identification of biologically relevant signals) complementing tedious wet-lab experiments. We believe that this type of analysis can be easily applied to other functional gene classes, thus providing a general framework for modelling promoter groups. These results also provide the basis to hunt for genes co-regulated with histone genes across mammalian genomes.

Algorithms↗
PubMed1999

In vitro characterization of the tobacco rpoB promoter reveals a core sequence motif conserved between phage-type plastid and plant mitochondrial promoters.

We report here the in vitro characterization of PrpoB-345, the tobacco rpoB promoter recognized by NEP, the phage-type plastid RNA polymerase. Transcription extracts were prepared from mutant tobacco plants lacking PEP, the Escherichia coli-like plastid-encoded RNA polymerase. Systematic dissection of a approximately 1 kb fragment determined that the rpoB promoter is contained in a 15-nucleotide segment (-14 to +1) upstream of the transcription initiation site (+1). Point mutations at every nucleotide reduced transcription, except at the -5 position which was neutral. Critical for rpoB promoter function was a CRT-motif (CAT or CGT) at -8 to -6 (transcription <30%), defining it as the promoter core. The core CAT sequence is also present in the maize rpoB promoter, which is faithfully recognized by tobacco extracts. Alignment of NEP promoters identified a CATA or TATA (=YATA) sequence at the rpoB core position, also present in plant mitochondrial promoters. Furthermore, NEP and the phage T7 RNA polymerase exhibit similar sensitivity to inhibitors of transcription. These data indicate that the nuclear RpoZ gene, identified by sequence conservation with mitochondrial RNA polymerases, encodes the NEP catalytic subunit.

Bacteriophages↗
PubMed1992

Molecular analysis of the zeste-white interaction reveals a promoter-proximal element essential for distant enhancer-promoter communication.

We have analyzed the eye and testis enhancers located 1 kb upstream of the transcription start site of the white gene. Both enhancers confer the corresponding tissue-specific expression on a heterologous promoter as well as on the white promoter. The eye determinant consists of multiple elements, each able to stimulate eye-specific expression. It also contains five binding sites for the zeste protein while the immediately adjacent testis element contains none. Site-directed mutation of these zeste binding sites abolishes the zeste-white interaction but does not significantly affect the eye enhancer activity, indicating that they are not important for the eye enhancer activity per se. Other zeste binding sites just upstream of the promoter are not necessary for the zeste-white interaction. We conclude that the overlap of the eye enhancer with the zeste binding sites is responsible for the zeste-white interaction and explains why this interaction affects eye but not testis expression. Sequence deletion or substitution experiments suggested that the white promoter is internal to the transcription start site; the zeste protein is not required for distant enhancer action but a 95-bp promoter-proximal sequence is essential for distant enhancer-promoter interaction. This element may serve as an anchor to stabilize formation of a loop that brings the enhancer to the vicinity of the promoter.

Animals↗
PubMed1993

The density of transcriptional elements in promoter and non-promoter sequences.

Using data currently available from transcriptional element (TE) databases, we have analyzed the density of these elements in both promoter and non-promoter sequences obtained from the GenBank sequence database. The density of putative TEs in non-promoter sequences was about 16% higher than that seen in pseudo-random DNA sequences. Promoter TE density from the transcription startsite to 100 basepairs upstream was found to be about 42% higher than in non-promoter sequences. However, the extensive overlap of putative TE densities between promoters and non-promoters confounds an attempt to use TE density as a simple discriminator.

Algorithms↗
PubMed1999

CIITA-induced occupation of MHC class II promoters is independent of the cooperative stabilization of the promoter-bound multi-protein complexes.

Precise regulation of MHC class II expression plays a crucial role in the control of the immune response. The transactivator CIITA behaves as a master controller of constitutive and inducible MHC class II gene activation, but its exact mechanism of action is not known. Activation of MHC class II promoters requires binding of at least three distinct multi-protein complexes (RFX, X2BP and NF-Y). It is known that the stability of this binding results from cooperative interactions between these proteins. We show here that expression of CIITA in MHC class II- cells triggers occupation of the promoters by these complexes. This observation raised the possibility that the effect of CIITA on promoter occupation is mediated by an effect on the cooperative stabilization of the DNA-bound multi-protein complexes. We show, however, that the presence of CIITA does not affect the stability of the higher-order protein complex formed on DNA by RFX, X2BP and NF-Y. This suggests other mechanisms for CIITA-induced promoter occupancy, such as an effect on chromatin structure leading to increased accessibility of MHC class II promoters. This ability of CIITA to facilitate promoter occupation is undissociable from its transactivation potential. Finally, we conclude that this effect of CIITA is cell-type specific, since expression of CIITA is not required for normal occupation of MHC class II promoters in B lymphocytes.

B-Lymphocytes↗
PubMed1989

Up-promoter mutations in the positively-regulated mer promoter of Tn501.

Transcription from the mer promoter of transposon Tn501 is repressed by MerR (the product of the merR gene) in the absence of Hg2+, and activated by MerR in the presence of Hg2+. In the absence of MerR, the mer promoter has weak constitutive activity. The DNA sequence of the mer promoter shows candidate -35 and -10 sequences at the unusually high spacing of 19 base-pairs. We have selected for spontaneous mutations in the mer promoter that confer an up-promoter phenotype. Four different mutants have been isolated. Three of these are single base-pair deletions between the -10 and -35 sequences. A fourth removes the -10 sequence entirely, and places a second potential -10 sequence 17 base-pairs from the -35 sequence. None of these mutant promoters are induced by MerR in the presence of Hg2+. Two of them are repressed by MerR irrespective of the presence or absence of Hg2+. Models for the mode of action of the MerR protein are discussed in the light of these results. Our data support a mechanism in which the MerR protein in the presence of Hg2+ acts to change the conformation of DNA in the mer promoter.

Bacterial Proteins↗
PubMed1997

Non-canonical sequence elements in the promoter structure. Cluster analysis of promoters recognized by Escherichia coli RNA polymerase.

Nucleotide sequences of 441 promoters recognized by Escherichia coli RNA polymerase were subjected to a site-specific cluster analysis based on the hierarchical method of classification. Five regions permitting promoter subgrouping were identified. They are located at -54 +/- 4, -44 +/- 3, -35 +/- 3 (-35 element), -29 +/- 2 and -11 +/-4 (-10 element). Promoters were independently subgrouped on the basis of their sequence homology in each of these regions and typical sequence elements were determined. The putative functional significance of the revealed elements is discussed on the basis of available biochemical data. Those promoters that have a high degree of homology with the revealed sequence elements were selected as representatives of corresponding promoter groups and the presence of other sequence motifs in their structure was examined. Both positive and negative correlations in the presence of particular sequence motifs were observed; however, the degree of these interdependencies was not high in all cases, probably indicating that different combinations of the signal elements may create a promoter. The list of promoter sequences with the presence of different sequence elements is available on request by Email: ozoline@venus.iteb. serpukhov.su.

Base Sequence↗
PubMed1999

Open complex formation during transcription initiation at the Escherichia coli galP1 promoter: the role of the RNA polymerase alpha subunit at promoters lacking an UP-element.

We have studied the role of the C-terminal domain of the alpha subunit (alphaCTD) of Escherichia coli RNA polymerase during transcription initiation at promoters lacking an UP-element. The temperature requirement for open complex formation was used as an indication of the kinetics of this process. We have previously shown that alphaCTD is required for transcription initiation at low temperature at the galP1 promoter, a promoter containing an UP-element. DNase I footprinting has been used to reveal the structure of open promoter complexes and the temperature requirement for open complex formation has been determined using potassium permanganate as a probe. In this work we show that, although alphaCTD is not absolutely required for transcription initiation at promoters lacking an UP-element, it does play a role during transcription initiation. This role is independent of the sequence of the promoter upstream from the -35 region and does not require stable alphaCTD-DNA interactions as determined by DNase I footprinting. The role of alphaCTD at promoters lacking an UP-element is discussed.

Bacterial Proteins↗
PubMed1999

A health promotion programme for oil refinery employees: changes of health promotion needs observed at three years.

The main aim of this three-year follow-up study was to evaluate the long-term effects of a workplace health promotion intervention programme offered by the Neste Oyj corporation's occupational health service. Another aim was to study factors associated with changes in health promotion needs. These were assessed using information obtained by means of questionnaires and laboratory measurements. The target areas assessed were physical activity, musculoskeletal problems, dietary habits, obesity, blood pressure, serum lipids, smoking, quality of sleep and mental well-being. Participants from one oil refinery were offered special health promotion counselling, while those from the other oil refinery studied received only their personal results, written information and instructions. Evaluation of the changes in needs was mainly based on comparison of the results of two examinations performed with an interval of three years. Effects of special health promotion counselling were observed in the target area of physical activity. Elimination of certain health promotion needs was seen in both groups in all of the target areas. The most extensive changes were seen in the target areas of musculoskeletal symptoms, dietary habits, blood pressure and mental well-being. Basic education, occupational status and age-group, as well as the value of tending health were frequent variables explaining the reduction in the need for health promotion activities. Worker participation in health promotion counselling activities provided by occupational health services can be high, as in this study in which the participation rate was 90% and the drop-out rate during the three years only 10%.

Adult↗
PubMed1992

The 72K IE1 and 80K IE2 proteins of human cytomegalovirus independently trans-activate the c-fos, c-myc and hsp70 promoters via basal promoter elements.

Growth-regulating cellular genes or genes encoding proteins involved in cell cycle control are likely to be major targets of viral gene products in the establishment of a cellular state favourable for a permissive infection. We have examined whether infection of permissive fibroblasts with human cytomegalovirus (HCMV) results in trans-regulation of such cellular genes. Here we have shown that the proto-oncogenes c-fos and c-myc are specifically induced during immediate early (IE) and early times of HCMV infection, as has recently been shown for the heat shock protein 70 gene (hsp70). Deletion analyses and transfection assays of all three promoters showed that previously defined control sequences upstream of the constitutive promoters and downstream of the mRNA cap site are not required for this up-regulation by HCMV, such that the minimal inducible promoters of c-fos, c-myc and the hsp70 gene contained only 50 to 60 bp upstream of the transcription start site. Cotransfection assays with vectors expressing HCMV major IE cDNAs showed that the 72K IE1 and 80K IE2 proteins are involved in the up-regulation of these promoters. IE1 and IE2 products independently were able to up-regulate the minimal constitutive promoters of the constructs tested here, but trans-activation by IE1 and IE2 together was synergistic. In the case of the hsp70 promoter, promoter constructs containing a variety of different TATA elements could be activated by the 72K IE1 and 80K IE2 proteins.

Base Sequence↗
PubMed1994

Biochemical and mutational analysis of the polyomavirus core promoter: involvement of nuclear factor-1 in early promoter function.

The polyomavirus enhancer is separated from the early RNA initiation sites by a 120 bp promoter region. To identify the core promoter elements, we introduced base-substitution mutations within the potential elements in the vicinity of the RNA initiation site. Three of these mutants, two with mutations within a putative nuclear factor-1 (NF-1) binding site and the other within the TATA box, exhibited reduced promoter activity by about threefold in the mouse NIH 3T3 cell line. The activity of the other three mutants was either little affected or remained unchanged. Mobility shift assays using specific competitors and antibodies against NF-1 demonstrated the binding of a protein of the NF-1 family at a site adjacent to the TATA box, suggesting a role for NF-1 binding in early promoter function. The effect of these mutations was also evaluated in undifferentiated mouse embryonal carcinoma (F9) cells in the presence of an additional mutation (F441) at nucleotide position 5233. This additional mutation creates a strong binding site for a transcription factor, TEF-1, and helps the virus to grow in this cell line. While the TATA box and the GC box mutants behaved qualitatively in a similar fashion, the NF-1 motif now played a minor role in F9 cells. Western blot experiments demonstrated low levels of NF-1 protein in this cell line. The NF-1 motif partially overlaps a T-antigen binding motif and this motif is not involved in T-antigen-mediated regulation of the early promoter. Our results suggest that a protein of the NF-1 family binds to the core promoter and is important for early transcription in vivo. We further demonstrate that undifferentiated F9 cells contain a very low level of NF-1 and the F441 mutant possibly follows a different mechanism for promoter function in these cells.

3T3 Cells↗
PubMed1989

A novel, non-invasive promoter probe vector: cloning of the osmoregulated proU promoter of Escherichia coli K12.

We have constructed a novel promoter probe plasmid pSB40, containing a unique lac-alpha-tetracycline marker gene tandem, which allows for both positive and negative selection of active promoters. Promoters cloned in pSB40 can be readily mobilized as EcoRI cassettes. Using this vector we have performed a non-invasive analysis of the E. coli chromosome for promoters regulated by osmotic upshift. Only one such promoter, subsequently identified as part of the proU operon, was isolated. A sequence of 253 bp, sufficient to mediate osmotic regulation of the proU promoter, was defined. This E. coli promoter was normally regulated in Salmonella typhimurium, Klebsiella and Citrobacter but not in Shigella. A proU-luxAB fusion plasmid was constructed and used to monitor in vivo real-time kinetics of proU induction following osmotic upshock.

Base Sequence↗
PubMed2000

Health promotion and older people: the role of the general practitioner in Australia in promoting healthy ageing.

OBJECTIVE: The Commonwealth Government's Enhanced Primary Care initiative supports measures to enhance the role of general practitioners (GPs) in promoting healthy ageing as part of a population health approach. This paper comments on how the health assessments can be conducted to best effect, to strengthen the role of GPs in primary care and to promote autonomy and independence in older people. METHOD: The relevant literature was collated to produce a review of public health and health promotion approaches and to ascertain the effectiveness of health promotion interventions for older people. A broad definition of health promotion including primary, secondary and tertiary prevention was adopted. RESULTS: The evidence base suggests there is scope for greater targeting of health promotion activities towards older people. The rationale for the Australian GP to assume a major health promotion role with their older patients is provided. Associated barriers and enablers are discussed. CONCLUSIONS: Prevention of disability is a key public health issue. The new MBS items may enable systematic evaluation of function and assist healthy ageing for all older people, including the frail aged. An increase in the preventive advice given to older patients has the potential to increase healthy behaviours and alter health outcomes. IMPLICATIONS: The annual health assessment items on the Medicare Benefits Schedule, by enabling the GP to focus on prevention and coordination of care, have the potential to improve the health, physical, psychological and social function of older Australians.

Aged↗
PubMed1997

Amy as a reporter gene for promoter activity in Nocardia lactamdurans: comparison of promoters of the cephamycin cluster.

Promoter probe vectors containing the pA origin of replication and the Streptomyces griseus promoterless amy gene (encoding alpha-amylase) as reporter have been constructed to study transcription initiation regions in Nocardia lactamdurans. In some of the promoter probe vectors the phage fd terminator has been introduced to avoid readthrough expression from upstream sequences. By using these vectors, four different transcription initiation regions of the cephamycin gene cluster have been studied in N. lactamdurans. The bla gene encoding a beta-lactamase has a relatively strong promoter. Two other separate promoters corresponding to the lat and cefD genes (encoding, respectively, lysine-6-aminotransferase and isopenicillin N-epimerase) showed weak transcription initiation ability. These two promoters are arranged in a bidirectional transcription initiation region located in the center of the cephamycin gene cluster. The cmcH gene (encoding 3-hydroxymethylcephem carbamoyltransferase) upstream region did not contain a functional promoter, suggesting that cmcH is transcribed as a part of a polycistronic mRNA. The native amy promoter is used very efficiently in N. lactamdurans, resulting in secretion of high levels of extracellular alpha-amylase.

Amino Acid Isomerases↗
PubMed1998

Use of the lactococcal nisA promoter to regulate gene expression in gram-positive bacteria: comparison of induction level and promoter strength.

We characterized the regulated activity of the lactococcal nisA promoter in strains of the gram-positive species Streptococcus pyogenes, Streptococcus agalactiae, Streptococcus pneumoniae, Enterococcus faecalis, and Bacillus subtilis. nisA promoter activity was dependent on the proteins NisR and NisK, which constitute a two-component signal transduction system that responds to the extracellular inducer nisin. The nisin sensitivity and inducer concentration required for maximal induction varied among the strains. Significant induction of the nisA promoter (10- to 60-fold induction) was obtained in all of the species studied at a nisin concentration just below the concentration at which growth is inhibited. The efficiency of the nisA promoter was compared to the efficiencies of the Spac, xylA, and lacA promoters in B. subtilis and in S. pyogenes. Because nisA promoter-driven expression is regulated in many gram-positive bacteria, we expect it to be useful for genetic studies, especially studies with pathogenic streptococci in which no other regulated promoters have been described.

Bacillus subtilis↗
PubMed1987

Effects of plasmid propagation of a sporulation promoter on promoter utilization and sporulation in Bacillus subtilis.

Transcription of the sporulation gene spoVG of Bacillus subtilis is induced at the onset of spore formation and depends on the products of the regulatory genes spoOA, spoOB, and spoOH. We describe two effects of propagating the promoter region of spoVG on a multicopy plasmid replicon in B. subtilis cells. One effect is that transcription from the plasmid-borne spoVG promoter is altered with respect to the time of its induction and the dependence on spoO gene products. An example of this effect is that plasmid propagation was observed to relieve substantially the inhibitory effect of a mutation in spoOH, the spoO gene upon which spoVG promoter activity is most strongly dependent. We present results which suggest that propagation on a plasmid replicon causes an alteration in the conformation of spoVG promoter DNA which somehow compensates for the defective spoOH gene product. Plasmid propagation did not, however, entirely eliminate the requirement for the spoOH gene product; little or no spoVG-directed RNA synthesis was observed in cells bearing a putative spoOH deletion mutation, a finding which indicates that SpoOH protein plays an indispensable role in spoVG promoter utilization. Another effect of propagating the promoter region of spoVG on a multicopy plasmid is to inhibit sporulation. S1 nuclease mapping experiments suggest that amplification of spoVG on a multicopy plasmid causes the titration of a transcription factor or minor form of RNA polymerase holoenzyme required for utilization of one of the two overlapping promoters which comprise the spoVG transcription initiation region.

Bacillus subtilis↗