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PubMed1999

Selective promoter usage of the human estrogen receptor-alpha gene and its regulation by estrogen.

Three promoters have been identified for the human estrogen receptor-alpha gene. The positions of promoters A and B are known whereas that of the recently identified promoter C is not. Cloning and hybridization experiments demonstrated that promoter C is located more than 21 kb upstream of promoter A. The use of the three promoters was examined in estrogen receptor-positive breast cancer cell lines, cell lines derived from other malignancies, and some normal tissues by RT-PCR and transient transfection. All estrogen-responsive breast cancer cell lines used all three promoters, apart from ZR-75 cells, which did not use promoter B in one of two sublines examined. Cell lines derived from other malignancies and other normal tissues that express lower levels of estrogen receptor-alpha showed more selective promoter usage. This suggests that the level of expression of estrogen receptor-alpha is determined by the number of promoters used, rather than the selective use of specific promoters. We also show that promoter C is used more widely than suggested by others. Analysis of a series of estrogen receptor-positive primary breast tumors showed that all three promoters were used in all the tumors. All three promoters were modulated by estrogen in estrogen-responsive breast cancer cell lines: all three promoters were down-regulated by estrogen in MCF-7 cells in which estrogens reduce receptor expression whereas all promoters used were upregulated in T47D, ZR-75, and EFM-19 cells in which estrogens increase receptor expression. This suggests that it is the repertoire of transcription factors present within a cell rather than the selective use of a specific promoter that determines whether estrogen receptor-alpha expression is increased or decreased by estrogen.

Breast Neoplasms↗
PubMed1989

Relative efficiency of utilization of promoter and termination sites by bacteriophage T3 RNA polymerase.

Bacteriophage T3 RNA polymerase promoters have been classified as class II and class III on the basis of their relative location in T3 DNA as well as on the function of the protein products encoded by the messages transcribed from them. In the present work, the efficiency of utilization of several class II and class III promoters by bacteriophage T3 RNA polymerase was compared with regard to (a) rate of initiation of transcription as determined by [32P]PPi exchange with GTP; (b) complex formation between polymerase and promoters in the presence of GTP; and (c) competition between different promoters for T3 RNA polymerase in a standard transcription assay. The results of these experiments indicated that the class II promoters at 1.05 and 22.8 T3 map units, whose promoter sequences are remarkably similar to the consensus class III promoter sequences, are nearly as strong as typical class III promoters. In contrast, the class II promoter at 14.3 T3 map units, whose promoter sequence differs from the consensus class III promoter sequence by having a C:G base pair instead of a usual A:T base pair at the -1 position, was considerably weaker than the class III promoter. When the C:G base pair at this position was changed to A:T using site-directed mutagenesis, the rate of initiation of RNA synthesis from the mutant promoter was similar to that of a typical class III promoter. In agreement with this observation, it was observed that changing the A:T base pair at the -1 position of a strong class II promoter, at 1.05 T3 map units, to C:G decreased the rate of RNA synthesis from this promoter by about 65%. These observations indicate that the nucleotide residues at the -1 position play a critical role in determining the efficiency of promoter utilization by T3 RNA polymerase. The two termination sites recognized in vitro by bacteriophage T3 RNA polymerase on the T3 genome have been cloned, sequenced, and mapped. Analysis of the DNA nucleotide sequence surrounding the termination site at 59.7 map units indicated that the putative RNA transcript arising from this region can be arranged into a GC-rich stem-loop structure followed by a U-rich 3' tail. However, a major fraction of T3 RNA polymerase molecules read through this terminator in vitro to transcribe regions of T3 DNA beyond this terminator. In contrast to termination at 59.7 map units, termination of transcription at 100 T3 map units does not occur in response to any putative terminator structure or sequence.(ABSTRACT TRUNCATED AT 400 WORDS)

Base Sequence↗
PubMed1993

Vitamin E is a complete tumor promoter in mouse skin.

The dorsal skins of 6-8 week old female SENCAR mice were initiated with a single application of 10 nmol of 7,12-dimethylbenz[a]anthracene (DMBA) and subsequently promoted twice/week with topical applications of vitamin E (dl-alpha-tocopherol, 80 mumol/treatment). Vitamin E from two separate commercial suppliers was tested. For comparison, a group of similar mice, also initiated with DMBA, was promoted twice/week with the known tumor promoter 12-O-tetradecanoylphorbol-13- acetate (TPA, 2 micrograms/treatment). Papillomas appeared 39 and 50 days respectively after promotion began with vitamin E from the two different sources, as compared with 32 days in the group receiving TPA promoted. Hundred per cent of TPA-promoted animals and 92-96% of the vitamin E-promoted mice developed tumors. A maximum of 15 papillomas/animal appeared in the TPA-promoted mice. The two vitamin E preparations were somewhat less effective than TPA and showed different relative potencies, producing about seven and 12 papillomas per animal respectively. Unlike TPA, vitamin E showed very little ability to produce an inflammatory response in skin. To test whether initiated cells that did not appear as papillomas after vitamin E promotion were still viable, and had proceeded past stage I of promotion (conversion), the group that developed 12 papillomas/animal from vitamin E promotion was further promoted with mezerein, a stage II promoter. In this group, the papilloma frequency then increased to approximately 17/animal. The animals were followed over the course of their lifespan and monitored for skin carcinomas. In the TPA-promoted group 64% of the mice developed carcinomas, while the two vitamin E-promoted groups showed 48 and 60% incidence respectively. These results indicate that topically applied vitamin E acts as a complete tumor promoter in DMBA-initiated mouse skin, with an efficiency approaching that of TPA. Since vitamin E is a powerful antioxidant, they also suggest that reduction of cellular oxidant levels may trigger the tumor promotional process, and it may therefore be prudent to avoid repetitive or prolonged topical exposure of human skin to antioxidants like vitamin E.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed2004

A preproenkephalin-neurofilament chimeric promoter in a helper virus-free herpes simplex virus vector enhances long-term expression in the rat striatum.

Helper virus-free herpes simplex virus (HSV-1) plasmid vectors are an attractive system for gene transfer into neurons in the brain, but promoters that support long-term, neuronal-specific expression are required. Elucidation of general principles that govern long-term expression would likely assist efforts to develop improved promoters. Although expression from many promoters in HSV-1 vectors is unstable, two neuronal subtype-specific promoters, the preproenkephalin (ENK) promoter and the tyrosine hydroxylase (TH) promoter, support long-term expression. We have previously shown that 5' upstream sequences in the TH promoter are required for long-term expression, and addition of these upstream sequences to a neurofilament heavy gene (NF-H) promoter enhances long-term, neuronal-specific expression. The goal of this study was to determine if the upstream sequences from the TH promoter contain a unique element that enhances expression, or if other neuronal promoters also contain sequences that can enhance expression. To this end, we tested 5' upstream sequences in the ENK promoter. We isolated a vector that fuses upstream sequences from the ENK promoter to the NF-H promoter. This vector supported expression in the striatum for 2 months after gene transfer, the longest time point evaluated. Expression was neuronal specific. As ENK and TH are a peptide neurotransmitter and a classical neurotransmitter biosynthetic enzyme, respectively, these results suggest that a significant number of promoters for neurotransmitter biosynthetic genes may contain elements that can enhance expression from HSV-1 vectors. The strategy of using upstream sequences from neuronal subtype-specific promoters to enhance expression from heterologous promoters is discussed.

Animals↗
PubMed1990

Discrimination between bacteriophage T3 and T7 promoters by the T3 and T7 RNA polymerases depends primarily upon a three base-pair region located 10 to 12 base-pairs upstream from the start site.

The bacteriophage T3 and T7 RNA polymerases are closely related, yet are highly specific for their own promoter sequences. To understand the basis of this specificity, T7 promoter variant that contain substitutions of T3-specific base-pairs at one or more positions within the T7 promoter consensus sequence were synthesized and cloned. Template competition assays between variant and consensus promoters demonstrate that the primary determinants of promoter specificity are located in the region from -10 to -12, and that the base-pair at -11 is of particular importance. Changing this base-pair from G.C, which is normally present in T7 promoters, to C.G, which is found at this position in T3 promoters, prevented utilization by the T7 RNA polymerase and simultaneously enabled transcription from the variant T7 promoter by the T3 enzyme. Substitution of T7 base-pairs with T3 base-pairs at other positions where the two consensus sequences diverge affected the overall efficiency with which the variant promoter was utilized by the T7 RNA polymerase, but these changes were not sufficient to permit recognition by the T3 RNA polymerase. Switching the -11 base-pair in the T3 promoter consensus to the T7 base-pair prevented utilization by the T3 RNA polymerase, but did not allow the T3 variant promoter to be utilized by the T7 RNA polymerase. This probably reflects a greater specificity of the T7 RNA polymerase for base-pairs at other positions where the promoter sequences differ, most notably at -15. The magnitude of the effects of base substitutions in the T7 promoter on promoter strength (-11C much greater than -10C greater than -12A) correlates with the affinity of the T7 polymerase for the promoter variants, suggesting that the discrimination of the phage RNA polymerases for their promoters is mediated primarily at the level of DNA binding, rather than at the level of initiation.

Base Composition↗
PubMed1997

PromFD 1.0: a computer program that predicts eukaryotic pol II promoters using strings and IMD matrices.

MOTIVATION: A large number of new DNA sequences with virtually unknown functions are generated as the Human Genome Project progresses. Therefore, it is essential to develop computer algorithms that can predict the functionality of DNA segments according to their primary sequences, including algorithms that can predict promoters. Although several promoter-predicting algorithms are available, they have high false-positive detections and the rate of promoter detection needs to be improved further. RESULTS: In this research, PromFD, a computer program to recognize vertebrate RNA polymerase II promoters, has been developed. Both vertebrate promoters and non-promoter sequences are used in the analysis. The promoters are obtained from the Eukaryotic Promoter Database. Promoters are divided into a training set and a test set. Non-promoter sequences are obtained from the GenBank sequence databank, and are also divided into a training set and a test set. The first step is to search out, among all possible permutations, patterns of strings 5-10 bp long, that are significantly over-represented in the promoter set. The program also searches IMD (Information Matrix Database) matrices that have a significantly higher presence in the promoter set. The results of the searches are stored in the PromFD database, and the program PromFD scores input DNA sequences according to their content of the database entries. PromFD predicts promoters-their locations and the location of potential TATA boxes, if found. The program can detect 71% of promoters in the training set with a false-positive rate of under 1 in every 13,000 bp, and 47% of promoters in the test set with a false-positive rate of under 1 in every 9800 bp. PromFD uses a new approach and its false-positive identification rate is better compared with other available promoter recognition algorithms. The source code for PromFD is in the 'c+2' language.

Algorithms↗
PubMed1992

Autonomous activity of the alternate aldolase A muscle promoter is maintained by a sequestering mechanism.

The mouse aldolase A gene contains two closely-spaced alternate promoter/first exons. The more distal of the two, the M promoter, is muscle-specific while the 3' promoter, the H promoter, is expressed constitutively. Various segments from these promoter regions were linked to a reporter gene and used to transfect the myogenic cell line C2C12 and the hepatoma cell line BWTG3. A muscle-specific enhancer, MEN1, responsible for 80% of promoter M activity and containing 4 consensus MyoD binding sites was localized between -2578 to -2723 of the M promoter. Another muscle-specific enhancer and a restrictive element, MEN2/MSE, were found in the interval -1100 to -350. The MSE restrictive element was found to prohibit inappropriate up-regulation of the M promoter by selectively sequestering it from H promoter elements in both myoblasts and myotubes. Among the H promoter elements was found an enhancer, HEN, situated between -533 and -200 which did not function in myotubes. These studies also show that H promoter elements can act synergistically with a non-specific element, MAE, located between -350 and -130 of the M cap site greatly stimulating M promoter transcription in all cell types when the MSE restrictive element was absent. Through the analysis of interactions between these elements and the aldolase A and HSV-TK promoters we showed that neither the enhancers nor the promoter proximal sequences by themselves contain adequate information to reproduce the native pattern of aldolase A promoter modulation. Rather, the sequestering of the M promoter by the MSE restrictive element and the relative positioning and context of promoters M and H appear critical to the regulated expression of aldolase A.

Animals↗
PubMed2001

Mechanisms controlling differential promoter-occupancy by the yeast forkhead proteins Fkh1p and Fkh2p: implications for regulating the cell cycle and differentiation.

The roles of DNA and Mcm1p interactions in determining the overlapping and distinct functions of the yeast cell cycle regulatory transcription factors Fkh1p and Fkh2p were examined. Full-length recombinant Fkh1p and Fkh2p were purified and their binding to bona fide promoters examined in vitro. Each protein bound a variety of target promoters with similar specificity in vitro, consistent with the observation that these proteins bind common promoters in vivo. However, in vivo, the Fkh1p and Fkh2p occupied different target promoters to different extents, suggesting that each was primarily responsible for controlling a different set of genes. Additional in vitro studies provided a mechanistic explanation for this differential promoter-occupancy. Specifically, the Fkh2p, but not the Fkh1p, was capable of binding cooperatively with Mcm1p. The Mcm1p-Fkh2p cooperative binding was enhanced by, but did not require, the presence of a Mcm1p-binding site within a target promoter. Consistent with these data, Mcm1p was present at Fkh-controlled promoters in vivo regardless of whether they contained Mcm1p-binding sites, suggesting a role for Mcm1p at promoters not thought previously to be under Mcm1p control. Analysis of Fkh1p and Fkh2p binding to promoter targets in vivo by use of mutant strains indicated that the two proteins compete for promoter-occupancy at a number of target promoters. We postulate that Fkh1p and a stable Fkh2p/Mcm1p complex compete for binding to target promoters and that the levels and/or binding activity of Fkh1p, but not Fkh2p, are most limiting for promoter-occupancy in vivo. Interestingly, the in vitro DNA-binding assays, using a variety of promoter targets, revealed that bona fide Fkh target promoters contained two or more Fkh-binding sites that allowed the Fkh1p and Fkh2p proteins to form multiple protein-DNA complexes in vitro. Multiple Fkh-binding sites may be a distinguishing feature of bona fide Fkh promoters in yeast and other organisms.

Base Sequence↗
PubMed1996

Genetic analysis of the Mycobacterium smegmatis rpsL promoter.

The DNA sequence of the promoter region of the Mycobacterium smegmatis rpsL gene, which encodes the S12 ribosomal protein, was determined. Primer extension analysis and S1 nuclease protection experiments identified the 5' end of the rpsL mRNA to be 199 bp upstream of the translation initiation codon. The rpsL promoter contained sequences upstream of this start point for transcription that were similar to the canonical hexamers found at the -10 and -35 regions of promoters recognized by Esigma70, the major form of RNA polymerase in Escherichia coli. To define the promoter of the rpsL gene, DNA fragments containing progressive deletions of the upstream region of the rpsL gene were inserted into a plasmid vector containing a promoterless xylE gene. These insertions revealed that the 200 bp of DNA sequence immediately upstream from the translation initiation codon was not essential for promoter function. In addition, 5' deletions removing all but 34 bp upstream of the transcription start point retained greater than 90% promoter activity, suggesting that the -35 hexamer was not essential for promoter activity. To determine which nucleotides were critical for promoter function, oligonucleotide-directed mutagenesis and mutagenic PCR amplification were used to produce point mutations in the region upstream of the start point of transcription. Single base substitutions in the -10 hexamer, but not in the -35 hexamer, severely reduced rpsL promoter activity in vivo. Within the -10 hexamer, nucleotide substitutions causing divergence from the E. Coli sigma70 consensus reduced promoter activity. The DNA sequence immediately upstream from the - 10 hexamer contained the TGn motif described as an extended -10 region in prokaryotic promoters. Mutations in this motif, in combination with a transition at either the -38 or -37 position within the -35 hexamer, severely reduced promoter activity, indicating that in the absence of a functional -35 region, the rpsL promoter is dependent on the TGn sequence upstream from the -10 hexamer. Comparison of the nucleotide sequence of the rpsL promoter region of M. smegmatis with the homologous sequences from Mycobacterium leprae, Mycobacterium bovis, and Mycobacterium tuberculosis showed the presence in these slowly growing mycobacterial species of conserved promoter elements a similar distance upstream of the translation initiation codon of the rpsL gene, but these other mycobacterial promoters did not contain the extended -10 motif.

Base Sequence↗
PubMed1988

Inactivation by sequence-specific methylations of adenovirus promoters in a cell-free transcription system.

Studies on the biochemical mechanism of promoter inhibition or inactivation by sequence-specific promoter methylations necessitated the development of a cell-free transcription system that responded to in vitro promoter methylations. Such systems were hitherto not available. In nuclear extracts from HeLa cells, the activities of two adenovirus type 2 promoters in the nonmethylated and methylated forms were compared. The late E2A promoter in vitro methylated at three 5'-CCGG-3' (HpaII) sequences at nucleotides -215, +6, and +24, or the major late promoter in vitro methylated at nucleotide -52 in the 5'-CCGG-3' sequence or at nucleotide -13 in the 5'-GCGC-3' (HhaI) sequence exhibited strikingly lower activities than did the nonmethylated constructs or exhibited no activity at all. The designated nucleotide positions were calculated relative to the cap sites of the two promoters. Upon in vitro transcription, the methylation pattern of the E2A late promoter was shown to be stable. For the inhibitory effects by sequence-specific methylations to be elicited, circular templates had to be used, the DNA titers had to be at critical levels for each extract, and high protein concentrations had to be maintained. When a template mixture of the nonmethylated major late promoter and the 5'-CCGG-3' methylated late E2A promoter of adenovirus type 2 DNA was used, the major late promoter was active and the methylated late E2A promoter was inhibited or inactivated. Activity levels of the two different promoters could be assessed simultaneously in the same assay due to differences in lengths between the products of transcription from the late E2A and major late promoters. Thus inhibition in the cell-free system could be proven to be specific for the methylated promoter. We are currently pursuing the hypothesis that cellular factors are crucial in recognizing methylated promoters and somehow participate in their inactivation.

Adenoviridae↗
PubMed1998

Activation of beta-globin promoter by erythroid Krüppel-like factor.

Erythroid Krüppel-like factor (EKLF), an erythroid tissue-specific Krüppel-type zinc finger protein, binds to the beta-globin gene CACCC box and is essential for beta-globin gene expression. EKLF does not activate the gamma gene, the CACCC sequence of which differs from that of the beta gene. To test whether the CACCC box sequence difference is the primary determinant of the selective activation of the beta gene by EKLF, the CACCC boxes of beta and gamma genes were swapped and the resulting promoter activities were assayed by transient transfections in CV-1 cells. EKLF activated the beta promoter carrying a gamma CACCC box at a level comparable to that at which it activated the wild-type beta promoter, whereas EKLF failed to activate a gamma promoter carrying the beta CACCC box, despite the presence of the optimal EKLF binding site. Similar results were obtained in K562 cells. The possibility that overexpressed EKLF superactivated the beta promoter carrying the gamma CACCC box, or that EKLF activated the mutated beta promoter through the intact distal CACCC box, was excluded. To test whether the position of the CACCC box in the beta or gamma promoter determined EKLF specificity, the proximal beta CACCC box sequence was created at the position of the beta promoter (-140) which corresponds to the position of the CACCC box on the gamma promoter. Similarly, the beta CACCC box was created in the position of the gamma promoter (-90) corresponding to the position of the CACCC box in the beta promoter. EKLF retained weak activation potential on the beta(-140CAC) promoter, whereas EKLF failed to activate the gamma(-90betaCAC) promoter even though that promoter contained an optimal EKLF binding site at the optimal position. Taken together, our findings indicate that the specificity of the activation of the beta promoter by EKLF is determined by the overall structure of the beta promoter rather than solely by the sequence of the beta gene CACCC box.

Animals↗
PubMed1999

E-box and cyclic adenosine monophosphate response elements are both required for follicle-stimulating hormone-induced transferrin promoter activation in Sertoli cells.

Sertoli cells are the epithelial cells responsible for the onset of pubertal development and maintenance of spermatogenesis in the adult. Transferrin is one of the major secretory products expressed by differentiated Sertoli cells. Investigation of the transcriptional control of transferrin gene expression provides insight into the regulation of Sertoli cell differentiation. Analysis of the mouse transferrin (mTf) promoter reveals the presence of a number of conserved response elements that have previously been shown to regulate cell specific expression of the human transferrin (hTf) promoter. One of these elements is the human PRII region, which is a cAMP response element (CRE)-like element that is more than 80% conserved in the mTf promoter. The activation of the hTf promoter by FSH and cAMP in rat Sertoli cells has been shown to be mediated in part through the CRE-like PRII region and binding of the CRE binding protein (CREB). The present study investigates the role of PRII in the activation of mTf promoter by FSH and cAMP in rat Sertoli cells. Mutations in the PRII of the mTf promoter reduced FSH activation by only 50% and cAMP activation by more than 90%. In contrast, the mutant PRII mTf promoter construct was fully activated by a partially purified testicular paracrine activity PModS(S300). Gel shift experiments demonstrated that proteins that can bind a consensus CRE oligonucleotide also bind the PRII region of the mTf promoter. An immunoblot confirmed that CREB binds the PRII and promotes the gel shift observed. The hypothesis developed was that another cis-acting element in addition to the CRE-like PRII is also involved in FSH actions. A conserved response element in both the mTf and hTf promoters is the basic helix-loop-helix (bHLH) responsive E-box sequence. Both FSH and PModS (S300) activity were found to promote a mTf E-box gel shift that contained the E2A gene product the bHLH protein E47. Interestingly, mutations in the E-box of the mTf promoter completely abolished the PModS(S300) activation and partially (52%) inhibited the activation by FSH. In contrast, the mutant E-box mTf promoter construct was fully activated by cAMP. Finally a double mutation of both the PRII and the E-box completely abolished FSH activation of the mTf promoter. These results suggest that optimal activation of the mouse transferrin promoter by FSH requires both CREB binding to the CRE-like PRII region and bHLH binding to the E-box. Information is provided that indicates a number of Sertoli cell promoters contain a close association of E-box and CRE-like elements. Observations are discussed in regards to the potential interactions of the CRE and E-box response elements in mediating FSH actions in Sertoli cells.

Animals↗
PubMed2000

Molecular analysis of the human myeloperoxidase promoter region.

Myeloperoxidase (MPO) is a granule protein, transiently expressed during the promyelocyte stage of myeloid differentiation. It is transcribed in a stage and lineage specific manner. Studies of MPO gene regulation can help to elucidate the mechanism of normal and abnormal myeloid differentiation. Our preliminary data indicated the lack of basal promoter activity in the region immediately 5' to the MPO cDNA. Here, we report the results of the detailed molecular studies of the human MPO promoter region. To locate potential promoter elements active in HL60 cells, we made promoter deletion constructs ranging in size from 200 bp to 4.5 kb of the 5' region of the hMPO gene, cloned into the chloramphenicol acetyl transferase (CAT) reporter vector. Following electroporation of the promoter constructs into HL60 cells, CAT enzyme production was found only in the construct containing approximately the 1 kb region upstream of the reported MPO cDNA. A separate set of constructs was made to look for putative MPO enhancer elements. Several fragments upstream of the MPO promoter showed prominent transactivation of the TK promoter, indicating a possible enhancer. Tissue specificity of MPO promoter fragments was determined in myeloid cells arrested either before induction of MPO expression (KG1), during MPO expression (HL60), or after it had ceased (U937), as well as in non-MPO expressing non-myeloid cells. The construct containing an approximate 1000 bp fragment of the 5' region of MPO was found to direct CAT expression only in HL60 cells. The 3'-truncations of this promoter region resulted in loss of tissue-specificity, while the promoter activity remained largely unchanged. A negative regulatory element was found upstream of the MPO promoter which repressed heterologous promoters in all the tested cell lines. Enhancer elements showed no tissue- or stage-specificity that were characteristic for native MPO gene. Sequence analysis of the putative MPO promoter region showed a number of potential transcription factor binding sites. Of special interest is the region containing the purine-rich site that can bind proteins from the ets-family of transcription factors and a duplicate GATA-like site. When inserted upstream of a reporter containing the minimal Herpes simplex viral thymidine kinase (HSV-TK) promoter (into pBL2CAT plasmid) this site strongly activated the TK promoter in transfected myeloid cells. Further studies showed that oligonucleotides derived from the MPO promoter region bind multiple proteins in a band-shift assay. Taken together, our experiments located the regulatory elements important for human MPO gene expression in HL60 promyelocytes.

Animals↗
PubMed1992

Initiation of transcription by T7 RNA polymerase as its natural promoters.

A kinetic assay has been developed to measure the strength of natural T7 promoters. By determining the rate of appearance of initiation products in the presence of constant concentrations of T7 RNA polymerase, an incomplete mixture of ribonucleoside triphosphates, and increasing promoter concentrations, a maximum rate of product formation (Vmax) and a promoter concentration giving half of the maximal activity ([P]Vmax/2) can be determined for any cloned T7 promoter. On supercoiled plasmids, it was found that the [P]Vmax/2 measured for the six promoters phi 1.1B, phi 1.3, phi 3.8, phi 6.5, phi 10, and phi 13 ranged from 3.4 +/- 1.1 to 12.0 +/- 2.4 nM while the Vmax values showed no significant trends. On plasmids that had been linearized by cleavage at a single site with a restriction endonuclease, the cloned T7 promoters assayed fell into two broad classes that appear to be characterized by the T7 class II and III promoters. Generally, the class II promoters required higher promoter concentrations to produce half of the maximum rates of initiation ([P]Vmax/2 values) than the class III promoters. The [P]Vmax/2 values for the class II promoters ranged from 20 +/- 2.7 to 23 +/- 3.6 nM, while the [P]Vmax/2 values for the class III promoters phi 10 and phi 13 were 13 +/- 1.6 nM and 7.8 +/- 1.4 nM. The one exception is the class III promoter phi 6.5 whose [P] Vmax/2 (17 +/- 5 nM) falls between the [P]Vmax/2 values of the class II promoters and the strong class III promoters. The Vmax values measured on linear templates are variable, but it appears that phi 10 is more active than the other five promoters.

Adenosine Triphosphate↗
PubMed1979

Utilization of promoter and terminator sites on bacteriophage T7 DNA by RNA polymerases from a variety of bacterial orders.

The transcriptional properties of bacterial RNA polymerases purified from seven different species and representing a variety of bacterial orders have been studied using the well characterized DNA from phage T7 as template. The subunit composition of the different preparations suggests that each RNA polymerase holoenzyme has a promoter structure (betabeta'alpha2sigma) similar to that of the well studied E. coli and B. subtilis enzymes. Each enzyme utilizes DNA from bacteriophage T7 as an effective template for RNA synthesis, although all preparations contain a substantial fraction of inactive enzyme. Electrophoretic analysis of the RNA products made with the different RNA polymerases in vitro using T7 DNA (deletion mutant deltaD111) as template reveals that with minor exceptions, all of the heterologous RNA polymerases utilize the same collection of promoter sites on T7 used by the E. coli host enzyme, and only those promoter sites. The T7 early terminator is also efficiently utilized by each enzyme. Since the different bacterial species from which the RNA polymerases were derived are genetically quite distant, it appears that there is a structural element in the promoter which governs its recognition and which is universally recognized among RNA polymerases of different bacterial species. While the different bacterial RNA polymerases generally utilize the same set of T7 promoter sites, the efficiency of utilization of the different promoters varies considerably for different RNA polymerases and for different reaction conditions with the same RNA polymerase. Hence although the several T7 promoter sites share the ability to be recognized by bacterial RNA polymerases, each shows a unique pattern of utilization and therefore must possess a unique element of promoter structure as well. It has previously been shown (Stahl and Chamberlin, 1977) that T7 promoters A1, C, D and E interact differently with E. coli RNA polymerase as judged by the properties of complexes formed between each promoter and the latter enzyme. Since competition takes place among different promoter sites on a template and since these sites can differ functionally, small changes in reaction conditions or in the structure of the RNA polymerase can lead to significant changes in the rate of utilization of different promoter sites even when these promoter sites share common elements. Because the T7 promoters and terminator are utilized efficiently by such a wide range of RNA polymerases and because each of the several T7 promoters possesses unique properties which govern its utilization by RNA polymerase, analysis of the transcripts formed on a T7 DNA template provides a simple and rapid procedure for detecting and analyzing alterations in bacterial RNA polymerases which affect promoter or terminator recognition or utilization.

Bacteria↗
PubMed1999

Promotion of organophosphate induced delayed polyneuropathy by certain esterase inhibitors.

Certain esterase inhibitors elicit or intensify the clinical expression of various insults to axons. This phenomenon was called promotion of axonopathies because these chemicals are not additive neurotoxicants nor do they interfere with the pharmacokinetics. Characterization of promotion was carried out by using organophosphate induced delayed polyneuropathy (OPIDP) as a model. The search for a physiological explanation of promotion has the following background: (1) Promotion expresses clinically the biochemical lesions which are otherwise well compensated (such as 30/40% neuropathy target esterase (NTE) inhibition by neuropathic organophosphates). (2) Promotion is not specific because axonopathies of different origin are affected. (3) Promoters are effective when given several days before the neuropathic insult. (4) Promotion is less effective in young animals as compared with adults. (5) Promotion occurs when axons, but not necessarily the cell body, are targeted by promoters. (6) Repeated dosing with a promoter failed to produce axonopathy. Based on this evidence it is suggested that promotion might interfere with a mechanism(s) of compensation and/or repair of long axons. The target of promotion of axonopathies is thought to be similar or linked to NTE which is defined as the phenyl valerate esterase activity (PVE) in nervous tissues resistant to paraoxon and sensitive to mipafox (40 and 50 microM, pH 8.0, 20 min, respectively). Mipafox (50 microM) resistant PVEs include some activity sensitive to the promoter phenylmethane sulfonylfluoride (PMSF) but no correlation was found between its inhibition and promotion. A complete titration curve of paraoxon-resistant PVEs by mipafox (0-1 mM) dissected, besides NTE (I50 about 10 microM), another PVE with an I50 of approximately 200 microM. This enzyme was present in hen brain, spinal cord and peripheral nerve, corresponding to about 10, 20 and 30% of NTE activity, respectively, and was sensitive both in vitro and in vivo to promoters and much less so to neuropathic NTE inhibitors. By means of chromatography, other workers have identified in soluble extracts of peripheral nerves two forms of mipafox-sensitive PVEs with different molecular weights and different sensitivity to mipafox. These might correspond to NTE and to the other enzyme. Inhibition in vivo of the latter also correlated with promotion.

Animals↗
PubMed1983

Survey of various chemicals for initiating and promoting activities in a short-term in vivo system based on generation of hyperplastic liver nodules in rats.

The effects of pre- (initiation stage) and post- (promotion stage) administration of 17 chemical carcinogens (including both hepatic and non-hepatic carcinogens), 3 promoters and 2 non-carcinogenic analogs, on the induction of liver hyperplastic nodules were investigated in rats. Test chemicals were administered during the initiation stage after which rats were fed dietary N-2-fluorenylacetamide (2-FAA) for 2 weeks in conjugation with necrogenic CCl4 to enhance production of nodules initiated by test chemicals. Alternatively, effects of test chemicals administered during the promotion phase were evaluated in rats given a combination of dietary 2-FAA for 2 weeks and necrogenic CCl4. This initiation regimen resulted in very few nodules unless a promoter was subsequently used. All chemical carcinogens administered during the initiation stage induced more frequent, larger hyperplastic nodules than did control treatments. However, neither the promoters nor the non-carcinogenic analogs induced hyperplastic nodules if they were administered during the initiation stage. In contrast, hepatocarcinogens and a promoter of hepatocarcinogenesis (phenobarbital) administered in the promotion stage had enhancing (promoting) activity on hyperplastic liver nodules, whereas non-hepatocarcinogens, a promoter for skin carcinogenesis (12-O-tetradecanoylphorbol-13-acetate) and a promoter for urinary bladder carcinogenesis (saccharin) did not. Non-carcinogenic analogs were not active when administered during either the initiation or promotion stages. These findings demonstrated the utility of employing short-term in vivo assays for both initiating and enhancing (promoting) activities of chemicals. By our system, chemicals were classified into 4 main groups, namely, (i) hepatocarcinogens which have both initiating and enhancing activity, (ii) non-hepatocarcinogens with initiating activity only, (iii) promoters of hepatocarcinogenesis which have promoting activity and (iv) non-carcinogens and non liver promoters which did not initiate nor promote nodule development. This is a more discriminating means of assaying carcinogenic activity than is possible with short-term in vitro screening assays for mutagenicity.

Animals↗
PubMed1983

Host factors in the susceptibility of mice to tumour initiating and promoting agents.

It has been known for a long time that skin tumours can be induced by the sequential application of a subthreshold dose of a carcinogen (initiation stage) followed by repetitive treatment with a noncarcinogenic promoter (promotion stage). In mouse skin, the initiation stage requires only a single application of either a direct-acting carcinogen or a procarcinogen and is essentially an irreversible step, which, as data suggest, probably involves a somatic cell mutation. The promotion stage in mouse skin can be accomplished by a wide variety of weak or noncarcinogenic agents and is initially reversible, later becoming irreversible. Current information suggests that skin tumour promoters are not mutagenic but bring about a number of important epigenetic changes, such as epidermal hyperplasia and an increase in levels of polyamines, prostaglandins and dark basal keratinocytes as well as other embryonic conditions. Recently, tumour promotion in mouse skin was shown to consist of at least two stages, each of which can be accomplished by either a known promoter or a weak or nonpromoting agent. Some of the important characteristics of the first stage of promotion are: (1) only one application of a first-stage promoter, such as phorbol ester tumour promoters, calcium ionophore A23187, hydrogen peroxide and wounding, is needed; (2) it is partially irreversible; (3) increases in dark basal keratinocytes and prostaglandins are important; and (4) it can be inhibited by anti-inflammatory steroids and protease inhibitors. The second stage of promotion is initially reversible but later becomes irreversible. Polyamines and epidermal cell proliferation are important events in the second stage of promotion. A number of weak or nonpromoting agents, such as mezerein, are effective second-stage promoters, which can be counteracted by retinoic acid, anti-inflammatory steroids and polyamine synthesis inhibitors. Although skin tumour promotion has been studied extensively in mice, not all strains and stocks of mice are susceptible to phorbol ester tumour promoters. In this regard, C57Bl/6 mice appear to be fairly resistant to these tumour promoters. Not all species are equally susceptible to phorbol ester tumour promotion (mouse greater than rat greater than hamster, and the miniature swine is fairly resistant). It is not presently known if other experimental systems of carcinogenesis or the induction of human cancer go through a series of stages similar to that in the mouse skin.

Animals↗