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At least 145 records · Page 8Linked to original sources
PubMed1987

The conserved decanucleotide from the immunoglobulin heavy chain promoter induces a very high transcriptional activity in B-cells when introduced into an heterologous promoter.

A conserved decanucleotide (ATGCAAATNA) is present 45-60 nucleotides upstream from the transcription startpoint in all immunoglobulin heavy chain promoters (VH promoters). We have introduced this decanucleotide (cd sequence) at a similar position into the upstream flanking sequence of the mouse Renin-1 gene. This gene is only transcribed in highly specialized tissues, and the fragment used here (-449 to +30 with respect to the main transcription startpoint) has little promoter activity in fibroblastic or myeloma cell lines, even if coupled to a functional enhancer. In contrast, after insertion of the decanucleotide, this fragment, while still inactive in non-lymphoid cells, becomes a potent promoter in B-cells when associated with SV40 or immunoglobulin heavy chain enhancer. In all respects, the engineered fragment behaves like an authentic VH promoter isolated in this laboratory, except that it is even more active in B-cells. Deletion experiments show that all renin sequences are dispensable for the activity of the chimaeric promoter, except probably for the renin TATA box which defines the precise transcription startpoint. We conclude that the decanucleotide is sufficient to activate a promoter in B-cells but not in non-B-cells, and therefore that no other element is needed to account for the B-cell specificity of the VH promoter. In addition, our results suggest that the lack of activity of the renin promoter in non cognate cells is not due to the binding of a repressor.

Animals↗
PubMed1987

Promoter recognition and promoter strength in the Escherichia coli system.

The strength of Escherichia coli promoters in vivo as well as the rates of association between RNA polymerase and promoter sequences differ by more than an order of magnitude. Since efficient promoter recognition and rapid binding of the enzyme might be a prerequisite for exceptional promoter strength we have determined the forward rate constants kon (as well as koff) for nine promoters including PL, PA1, and PN25 from phages lambda, T7, and T5, respectively as well as Pbla and PlacUV5 from E. coli. The second order forward rate constants span a 30-fold range from 1 X 10(7) M-1 s-1 for Pbla and PL up to 2.9 X 10(8) M-1 S-1 for PN25. Little correlation between 'promoter recognition' as defined by the rate of complex formation of a promoter sequence with RNA polymerase and its strength in vivo as defined by the rate of RNA synthesis has been found. This adds to the evidence that the complex functional pathway encoded in a promoter sequence can be limited at various levels and that promoter strength in vivo is the result of an optimization process involving more than just one functional parameter.

Bacteriophage lambda↗
PubMed1996

Construction of a broad-host-range promoter-probing vector and cloning of promoter fragments of Xanthomonas campestris.

A broad-host-range promoter-probing vector, pMY3 (8.0 kb), was constructed for cloning of DNA fragments containing promoter sequences of Xanthomonas campestris pv. campestris. This vector (pMY3) consists of the RK2 replicon, promoterless luxAB genes, the thr attenuator to block the transcription of RNA into the luxAB region, and multiple cloning sites for cloning of the fragment carrying promoter sequences. The feasibility of using pMY3 as a promoter-probing vector in both E. coli and Xc17 was demonstrated by using the lac promoter of E. coli, and the amy promoter of X. campestris in Xc17. Among the 63 promoter-containing fragments cloned from Xc17, only 9 were able to express in E. coli. It appears that X. campestris can recognize most E. coli type promoters, but, E. coli can recognize only a small portion of the X. campestris type promoters.

Cloning, Molecular↗
PubMed1999

Identification of the physiological promoter for spinocerebellar ataxia 2 gene reveals a CpG island for promoter activity situated into the exon 1 of this gene and provides data about the origin of the nonmethylated state of these types of islands.

In order to further use the spinocerebellar ataxia 2 (SCA2) promoter for transgenic mice models of "CAG repeat" neurodegeneration, different fragments of this 5' end were ligated into pGL3-Luc plasmid to obtain the better promoter-activity of the physiological promoter for SCA2. Base-par composition of the SCA2-5' region, and promoter prediction algorithms such as TSSW and TSSG, together with the high firefly luciferase expression after 48 hours of transient transfection in mammalian cells lines, showed a typical CpG island for promoter-activity. The promoter activity was specifically localized into the exon 1 of the SCA2 gene. The higher expression of firefly luciferase in the embryonal F9 cells by the use of SCA2 promoter, rather than by the use of CMV promoter may be related with the origin of the nonmethylated CpG island during the early embryogenesis. Analysis of the 5' region from HD gene revealed to a CpG island, which could be containing the physiological promoter for this gene.

Algorithms↗
PubMed1997

Design of CytR regulated, cAMP-CRP dependent class II promoters in Escherichia coli: RNA polymerase-promoter interactions modulate the efficiency of CytR repression.

In CytR regulated promoters in Escherichia coli, the cAMP-CRP complex acts as a transcriptional activator as well as a co-repressor for the CytR protein. Repression by CytR depends on the formation of nucleoprotein complexes in which CytR binds cooperatively to the DNA with one or two cAMP-CRP complexes. Here, we demonstrate that in order to establish CytR regulation in a cAMP-CRP dependent class II promoter with a single CRP site (CRP site centred around position -40.5) in which the CytR operator is located upstream of the CRP site, high affinity binding sites for both regulators are required. The efficiency of CytR regulation was observed to be modulated by RNA polymerase (RNAP)-promoter interactions. Specifically, in class II promoters with a single CRP site, the efficiency of CytR regulation was found to correlate inversely with cAMP-CRP independent promoter activity. These observations can be reconciled in a competition model for CytR regulation in which CytR and RNAP compete for cooperative binding with cAMP-CRP to the promoters in vivo. In this model, two mutually exclusive ternary complexes can be formed: a CytR/cAMP-CRP/promoter repression complex and an RNAP/cAMP-CRP/promoter activation complex. Thus, CytR regulation critically depends on formation of a repression complex that binds the promoter with sufficiently high affinity to exclude formation of the competing activation complex. We suggest that the transition from repression to activation involves a switch in the protein-protein interactions made by cAMP-CRP from CytR to RNAP. On the basis of the regulatory features of the promoters analysed here, we speculate about the advantages offered by the structural complexity of natural CytR/cAMP-CRP regulated promoters.

Base Sequence↗
PubMed1996

Characterization of bacteriophage T4 early promoters in vivo with a new promoter probe vector.

We report on the construction of promoter probe vector pKWIII, useful in cloning and analyzing strong promoters for Escherichia coli RNA polymerase. Also T4 early promoters that proved to be difficult to clone with other vectors could be tested. The promoter activities obtained with this convenient and nonradioactive system largely correspond to those determined by pulse-labeling of transcripts in the same system. Results with well-characterized control promoters are in good agreement with values given by other authors. We present relative activities of several early promoters of phage T4 and compare these to promoter activities of other phages. Sorting the T4 promoters according to strength suggests the importance of distinct sequence elements to promoter functioning. They are centered around positions -52, -42, and -15.

Bacteriophage T4↗
PubMed1999

Isolation and characterization of Synechococcus PCC7942 promoters: tRNApro gene functions as a promoter.

Promoter-active fragments of Synechococcus PCC7942 were isolated by transcriptional gene fusion to the promoterless beta-glucuronidase (GUS) gene of E. coli, which was used as a reporter gene. Several of the isolated promoter-active fragments expressed GUS activity in Synechococcus comparable to that of the lambdaPR promoter. Only 10% of the isolated promoter-active fragments also functioned in E. coli. The transcription initiation sites of the two promoter-active fragments, D13 and E3, were identified. The major transcription initiation sites of D13 and E3 in Synechococcus were located within the nucleotides TTTG and TTG respectively, which were identical to those corresponding to E. coli. The inferred -10 and -35 regions of D13 were TAAACT and TTGTAG respectively, which conformed to the E. coli sigma70 promoter. Immediately upstream of the E3 transcription initiation sites was the tRNApro (GGG) gene, which contained two regions exhibiting strong homology to the major promoter elements in eukaryotic tRNA genes, but did not contain the E. coli promoter element. Thus, the tRNApro gene can act as a promoter.

Amino Acid Sequence↗
PubMed2005

Isolation and characterization of the bovine Stearoyl-CoAdesaturase promoter and analysis of polymorphisms in the promoter region in dairy cows.

Conjugated linoleic acid (CLA) in milk arises through microbial biohydrogenation of dietary polyunsaturated fatty acids (PUFA) in the rumen, and by the action of mammary Stearoyl-CoA desaturase (Scd). A large variation (up to tenfold) in the concentration of this fatty acid in milk has been observed, even in cows receiving the same diet. The reasons for this variation are not well understood. In this study the bovine core promoter region was isolated by a genome walking strategy from genomic DNA Genome Walker libraries and then cloned and characterized. This core promoter sequence extended approximately 600 bp upstream of the translation start site. The presence of putative transcription factor binding sites conserved in bovine, human, and mouse promoters was observed. Evidence that this promoter fragment was functional in vivo was obtained from expression studies in a mammary cell line. The promoter sequence of the scd gene was compared between cows selected for the ability to produce high fatty acid methyl esters (FAME) (2.22-2.72) in their milk, with the same promoter region of low-FAME-producing cows (0.81-1.12). However, such comparisons of the sequences of the scd promoter region of cows producing high milk CLA compared with low CLA revealed no polymorphisms in this promoter segment. Furthermore, no sequence polymorphisms were observed among the scd promoter region of Holstein Friesian, Montbeliarde, Normande, Norwegian Red, Charlois, Limousin and Kerry breeds.

Animals↗
PubMed1996

Isolation of strong promoters from Clavibacter xyli subsp. cynodontis using a promoter probe plasmid.

To isolate promoters from Clavibacter xyli subsp. cynodontis (C. xyli subsp. cynodontis), we constructed a new promoter probe plasmid and made a C. xyli subsp. cynodontis promoter probe library. Two promoters gave over 2500-times stronger expression than the parental plasmid. The promoters were sequenced and compared to other bacterial promoters. These C. xyli subsp. cynodontis promoter regions are GC-rich and do not resemble E. coli promoters, but do resemble a few individual promoters found in streptomycetes.

Actinomycetales↗
PubMed1987

Control of cloned gene expression by promoter inversion in vivo: construction of improved vectors with a multiple cloning site and the Ptac promoter.

We have constructed three gene-expression plasmids which contain (an) invertible promoter(s) and a multiple cloning site. We used either the plac promoter or the ptac-plac tandem promoters, the latter directing a more than fourfold increase in expression of the galK reporter gene in Escherichia coli host. All these plasmids were derived from the pNH7a expression plasmid of Podhajska et al. [Gene 40 (1985) 163-168]. Like pNH7a, these vectors have three novel properties: (i) in the 'OFF phase', the promoter is facing away from the gene to be expressed, (ii) the 'ON phase' is attained by the rapid and efficient inversion of the promoter mediated by the phage lambda Int product and the flanking attP and attB sites, which have a divergent orientation, and (iii) only a short heat pulse is required for the efficient inversion of the promoter and switching from the OFF to the ON phase. As for the pNH7 a vector, the present plasmids contain the nut-N transcriptional antitermination system, which permits efficient gene expression even if terminator(s) happen to be present between the promoter(s) and the expressed gene. The promoter inversion is rapid and over 95% efficient, as assayed by restriction analysis and galactokinase assay. Many genes could be conveniently cloned in the multiple cloning site, and then either kept totally silent or expressed in a rigidly controlled manner. Moreover, the pNH8, pNH16 and pNH18 plasmids, with already inverted promoters, could be used for expression of cloned genes, either in an unregulated manner or regulated by the lac repressor. They would be particularly useful for genes associated with terminators affecting their expression.

Attachment Sites, Microbiological↗
PubMed1993

Use of a promoter-probe vector system in the cloning of a new NifA-dependent promoter (ndp) from Bradyrhizobium japonicum.

Many of the symbiotic nitrogen-fixation genes in the soybean root nodule bacterium, Bradyrhizobium japonicum, are transcribed from -24/-12 promoters that are recognized by the sigma 54-RNA polymerase and activated by the transcriptional regulator protein, NifA. Several lines of evidence suggest that the B. japonicum genome has more than those seven NifA-regulated promoters which were characterized previously. Here, we present a strategy aimed at the cloning of new NifA-activated promoters. It makes use of (i) a promoter-probe vector into which random B. japonicum genomic fragments were cloned in front of a promoterless reporter gene and (ii) a screening procedure that allowed us to distinguish constitutive promoters from promoters that were specifically activated by NifA under microaerobic or anaerobic conditions. With certain modifications, the system may be generally applicable to clone positively regulated, anaerobically induced genes. A novel NifA-dependent promoter region (ndp) of B. japonicum was found by these means. The transcription start point was mapped, and its 5'-flanking DNA carried a -24/-12-type promoter sequence plus potential binding sites for NifA and integration host factor. Further transcript analyses confirmed that maximal transcription from this promoter occurred only in the presence of NifA and sigma 54 during anaerobic growth of B. japonicum. In Escherichia coli, expression of beta-galactosidase derived from a transcriptional ndp::lacZ fusion was activated 11-fold by B. japonicum NifA, and this activation also required sigma 54 but was independent of NtrC. The DNA around ndp shared no similarity with known sequences in databases.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Proteins↗
PubMed1998

Cloning, sequencing and characterization of the rat hereditary hemochromatosis promoter: comparison of the human, mouse and rat HFE promoter regions.

We have cloned and sequenced 1398bp of the rat HFE gene promoter region. The alignment of the rat promoter HFE sequence with the HFE promoter sequence from human and mouse detected several highly conserved sequences present at orthologous or heterologous positions in the three species. Subsequent analysis of the conserved promoter sequences identified the presence of 10 novel transcription elements present in the promoter regions of the human, mouse and rat HFE genes (GATA, NF-IL6, AP1, AP2, CREB, PEA3, gamma-IRE, GFI1, HNF-3beta, HFH2). Different gel retardation analyses performed with rat-liver nuclear extracts have confirmed the presence of factors binding to some of these transcription elements. This represents the first data concerning the identification of potential transcriptional elements of the HFE promoter in these three species. The expression pattern of the transcription factors corresponding to the novel elements identified in the HFE promoter is consistent with the potential role of the HFE promoter in the transcription regulation and function of the HFE gene. Knowledge of the identified conserved elements in the HFE promoter from human, mouse and rat provides the basis for subsequent in-vitro or in-vivo studies leading to identification of the detailed mechanisms involved in the regulation of the iron metabolism and the design of potential future alternative therapies.

Animals↗
PubMed1999

Nucleotide sequences surrounding the nonanucleotide promoter motif influence the activity of yeast mitochondrial promoter.

The highly conserved nonanucleotide (5'-TATAAGTAA[+2]) promoter sequence dictates initiation of gene-specific transcription by the mitochondrial (mt) RNA polymerase in yeast mitochondria. However, transcriptional efficiency of the nonanucleotide promoter in different mt genes varies severalfold. To explore the regulatory role of the promoter-proximal template sequence in mt transcription, different deletion, nucleotide (nt) substitution, and tandem promoter constructs were analyzed under in vitro transcription reaction conditions. It has been found that the conserved nonanucleotide promoter plus more than 9 nt of nonconserved sequence 3' to the promoter were absolutely essential for mt gene-specific transcription. In addition, approximately 300 nt of nonspecific DNA sequence 5' to the promoter was also important for efficient transcription. Interestingly, introduction of consecutive T residues in the early transcribed sequence of the template strongly inhibited mt transcription at low nt concentrations (i.e., 5 microM UTP). In contrast, neither other nt clusters nor a bacterial terminator-like sequences at that location inhibited mt transcription. Under the nonproductive reaction conditions, the full-length transcript from the mt polyT template was drastically reduced with the formation of several short abortive oligoribonucleotides. These results suggest that the transcriptional efficacy of the yeast mt promoter is influenced by sequence 3' to the promoter.

3' Untranslated Regions↗
PubMed2005

Escherichia coli RNA polymerase contacts outside the -10 promoter element are not essential for promoter melting.

We examined the relative affinity of model promoter constructs for binding Escherichia coli RNA polymerase (RNAP) holoenzyme. Model promoter constructs were designed to mimic DNA structures characteristic for different steps of transcription initiation. DNA duplexes in which a chemical cross-link was introduced just downstream from -10 hexamer to prevent DNA melting upon RNAP binding were used to mimic RNAP-promoter contacts in a closed complex. Fork junction DNA molecules with double-stranded/single-stranded junction between -11 and -10 position were used to study interactions of RNA polymerase with DNA in open complex. The -35 and -10 promoter regions were found to be equally important for the initial RNAP binding. The recognition of -35 promoter region was independent of structural context of the model promoter fragment. In contrast, free energy of RNAP binding to -10 hexamer was highly dependent on DNA structure. The relative importance of -10 region for sequence-specific interaction with the polymerase was the lowest for constructs mimicking closed complex and the highest for the constructs mimicking open complex. The relative importance of region -10 was also dependent on the presence of -35 consensus element indicating a communication between different DNA binding determinants of polymerase during open complex formation. Short double-stranded promoter fragments comprising only -35 and -10 or only -10 consensus elements underwent melting in a complex with polymerase indicating that the core of promoter melting activity of the polymerase is localized to a very small subset of all promoter-polymerase contacts.

Binding, Competitive↗
PubMed2002

The promoter-proximal KCS element of the PKR kinase gene enhances transcription irrespective of orientation and position relative to the ISRE element and is functionally distinct from the KCS-like element of the ADAR deaminase Promoter.

The RNA-dependent protein kinase PKR promoter is interferon (IFN) inducible and possesses a novel 15-base pair (bp) constitutive activator element, designated kinase conserved sequence (KCS), in addition to an IFN-stimulated response element (ISRE). Deletion of the KCS element or point mutations within the KCS element greatly reduce both basal and IFN-inducible PKR promoter activity. The IFN-inducible RNA-specific adenosine deaminase ADAR1 promoter possesses a KCS-like (KCS-l) element. The sequences of the KCS and KCS-l elements and their positions relative to the cognate ISRE element are similar between the PKR and ADAR1 promoters. However, substitution of the ADAR1 KCS-l element for the KCS element of the PKR promoter resulted in significantly reduced basal and IFN-inducible promoter activities comparable to either point mutation or entire deletion of the PKR KCS element. The PKR KCS element selectively bound nuclear proteins more efficiently than did the ADAR1 KCS-l element. Reversing the positions of the KCS and ISRE elements of the PKR promoter relative to one another or reversing the orientation of either element while conserving the naturally occurring 4-bp spacing between the two elements did not significantly reduce basal or IFN-inducible promoter activity. Taken together, these results are consistent with the notion that the KCS and ISRE elements of the PKR promoter function as a unit.

Adenosine Deaminase↗
PubMed1989

Pseudorevertants of a lac promoter mutation reveal overlapping nascent promoters.

Four pseudorevertants of a -10 region lacP mutation were isolated. Three of these mutations were found to activate nascent promoters. These mutations were: a -2 G/C----A/T change (-2A) promoting transcription at position +11, a +1 A/T----T/A change (+1T) promoting transcription initiation at position +13, and a +10 C/G----A/T change (+10A) promoting transcription initiation at a complex series of positions. The fourth mutation [a -12 T/A----A/T change (-12A)] promotes transcription initiation at -1. The promoters activated by mutations -12A, -2A and +1T resembled the canonical sigma 70 promoter sequences. The +10A promoter activity is also dependent upon the sigma 70 holoenzyme but can not be readily assigned to a specific promoter sequence.

Escherichia coli↗
PubMed1992

T7 promoter contacts essential for promoter activity in vivo.

T7 RNA polymerase promoters consist of a highly conserved 23 base-pair sequence that spans the site of the initiation of transcription (+1) and extends from -17 to +6. To determine the bases within the T7 consensus promoter that are essential for promoter function a library of mutant T7 promoters was constructed, and the in vivo activity of the mutant promoters was correlated to their sequence. The library of mutant promoters was created by randomly mutagenizing the T7 phi 10 promoter between positions -22 and +6 during the synthesis of oligonucleotides containing the phi 10 promoter. The mutagenized oligonucleotides were then ligated to a promoterless chloramphenicol acetyl transferase gene creating a plasmid (pCM-X#) that can potentially express chloramphenicol acetyl transferase in the presence of T7 RNA polymerase. E. coli containing pCM-X# and a second compatible plasmid carrying T7 gene 1 (T7 RNA polymerase) were screened for chloramphenicol resistance or chloramphenicol sensitivity. The point mutations that were found to inactivate a T7 promoter are a C to A or G substitution at -7, a T to A substitution at -8, a C to A, T, or G substitution at -9, and a G to T substitution at -11.

Base Sequence↗
PubMed1994

Superior expression of juvenile hormone esterase and beta-galactosidase from the basic protein promoter of Autographa californica nuclear polyhedrosis virus compared to the p10 protein and polyhedrin promoters.

The expression characteristics of the p10, polyhedrin and basic protein promoters of Autographa californica nuclear polyhedrosis virus were compared using two reporter enzymes, juvenile hormone esterase (JHE) and beta-galactosidase. In these systems, JHE is exported from the cell and beta-galactosidase is localized to the cytosol. Expression of JHE from the basic, p10 and polyhedrin promoters was first detected in the medium at 13, 19 and 27 h post-infection respectively. The basic protein promoter yielded the highest expression of the three promoters tested for both enzymes, as determined by protein and enzyme activity assays. In addition, yields of beta-galactosidase and JHE under control of the p10 promoter are higher relative to expression under control of the polyhedrin promoter. These data highlight the importance of investigation of viral promoters other than the polyhedrin promoter for high yield protein expression in vitro, and for insecticidal use of recombinant baculoviruses requiring high levels of expression. The results support revision of the current concept that very late viral promoters are always optimal for high yield recombinant protein expression.

Carboxylic Ester Hydrolases↗