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At least 19 recordsLinked to original sources

PubMed1992

A two-base-pair substitution in T7 promoter by SP6 promoter-specific base pairs alone abolishes T7 promoter activity but reveals SP6 promoter activity.

The phage T7 and SP6 RNA polymerase-promoter systems are very similar in many characteristics, but maintains stringent specificity for each. In order to identify the base pair element that distinguishes between T7 and SP6 promoters, the base pairs at -12, -10, -9, and -8 of the T7 promoter consensus sequence were changed singly and multiply to the SP6 promoter-specific base pairs, and assayed for T7 and SP6 promoter activities. The results indicate that the primary discrimination element is the base pairs at -8 and -9. The two-base-pair substitution alone in T7 promoter by SP6-specific base pairs is sufficient to make the T7 variant be a SP6 promoter, abolishing T7 promoter activity.

Base Composition↗

PubMed2002

Analysis of cis-sequence of subgenomic transcript promoter from the Figwort mosaic virus and comparison of promoter activity with the cauliflower mosaic virus promoters in monocot and dicot cells.

A sub-genomic transcript (Sgt) promoter was isolated from the Figwort mosaic virus (FMV) genomic clone. The FMV Sgt promoter was linked to heterologous coding sequences to form a chimeric gene construct. The 5'-3'-boundaries required for maximal activity and involvement of cis-sequences for optimal expression in plants were defined by 5'-, 3'-end deletion and internal deletion analysis of FMV Sgt promoter fragments coupled with a beta-glucuronidase reporter gene in both transient protoplast expression experiments and in transgenic plants. A 301 bp FMV Sgt promoter fragment (sequence -270 to +31 from the transcription start site; TSS) provided maximum promoter activity. The TSS of the FMV Sgt promoter was determined by primer extension analysis using total RNA from transgenic plants developed for FMV Sgt promoter: uidA fusion gene. An activator domain located upstream of the TATA box at -70 to -100 from TSS is absolutely required for promoter activity and its function is critically position-dependent with respect to TATA box. Two sequence motifs AGATTTTAAT (coordinates -100 to -91) and GTAAGCGC (coordinates -80 to -73) were found to be essential for promoter activity. The FMV Sgt promoter is less active in monocot cells; FMV Sgt promoter expression level was about 27.5-fold higher in tobacco cells compared to that in maize cells. Comparative expression analysis of FMV Sgt promoter with cauliflower mosaic virus (CaMV) 35S promoter showed that the FMV Sgt promoter is about 2-fold stronger than the CaMV 35S promoter. The FMV Sgt promoter is a constitutive promoter; expression level in seedlings was in the order: root>leaf>stem.

Base Sequence↗

PubMed2006

[Comparison of the relative luciferase activity in secondary CEF by different heterogenous strong promoters, MDV gB promoter and the composed promoters].

To improve the protection efficiency of the recombinant Marek' s disease viruses (MDV) in chickens with or without maternal antibodies,the work of selecting the optimal promoters for the construction of recombinant MDV was carried out. Combined with the efficient genetic manipulation, the composed promoters was constructed by use of the MDV gB core promoter with the regulatory elements from the early immediately promoter and enhancer of hCMV, the promoter and enhancer of SV40 or the partial enhancer of hCMV. And these composed promoters were ligased to the luciferase to construct the eukaryotic expressing vectors and named PhCMV-gB, Psv-gB and Pen-gB, respectively. In vitro, these vectors and internal standard plasmid (pSV-beta-LacZ) were transiently co-transfected into secondary CEF by FuGene 6 Transfection Reagent. Furthermore, cells were harvested 48 hours after transfection. Then the luciferase activity was detected by a luciferase assay kit, at the same time, the beta-galactosidase enzyme activity was detected by a beta-galactosidase enzyme assay kit, and the luciferase activity was corrected by the beta-galactosidase enzyme activity to get the relative luciferase activity. The relative luciferase activity was used as the transcriptional activity. By comparison of the relative luciferase activity of every promoter, it was found that these composed promoters could more effectively drive the reporter gene expression than the full legth of gB promoter did. Among them, PhCMV-gB robustly drove the reporter gene expression. On the other hand, PSV-gB and Pen-gB appeared to have the same strength; But compared with the commercial strong promoters, the transcriptional activity of the composed promoter were less than as or the same as that of the strong promoters. Therefore, at a sense, it can be proposed that these composed promoters have not only the characteristic of MDV gB promoter, but also that of the commercial strong promoters. These provide the choices for further developing the new-type recombinant MDV vaccine.

Animals↗

PubMed1990

Characteristics of a strong promoter from figwort mosaic virus: comparison with the analogous 35S promoter from cauliflower mosaic virus and the regulated mannopine synthase promoter.

A segment of DNA from the genome of figwort mosaic virus (FMV) strain M3 possesses promoter activity when tested in electroporated protoplasts from, and transgenic plants of, Nicotiana tabacum cv. Xanthi nc. The 1.1 kb DNA segment, designated the '34S' promoter, is derived from a position on the FMV genome comparable to the position on the cauliflower mosaic virus (CaMV) genome containing the 35S promoter. The 34S and 35S promoters show approximately 63% nucleotide homology in the TATA, CCACT, and -18 to +1 domains, but in sequences further upstream the homology drops below 50%. Promoter activities were estimated using beta-glucuronidase and neomycin phosphotransferase II reporter gene systems. The activity of the 34S promoter segment approximates that of the 35S promoter in both protoplast transient expression assays and in stably transformed tobacco plants. Truncation of 5' sequences from the 34S promoter indicates that promoter strength depends upon DNA sequences located several hundred nucleotides upstream from the TATA box. In leaf tissue the 34S promoter is 20-fold more active than the mannopine synthase (MAS) promoter from Agrobacterium tumefaciens T-DNA. The 34S promoter lacks the root-specific and wound-stimulated expression of the MAS promoter, showing relatively uniform root, stem, leaf, and floral activities.

Base Sequence↗

PubMed2000

Erythroid Kruppel-like factor is recruited to the CACCC box in the beta-globin promoter but not to the CACCC box in the gamma-globin promoter: the role of the neighboring promoter elements.

The programmed expression of the five beta-like globin genes (epsilon, (A)gamma, (G)gamma, delta, and beta) is characterized by a series of switches that are developmentally regulated. The (A)gamma- and (G)gamma- (fetus) to beta-globin (adult) switch depends on transcription factor erythroid Krüppel-like factor (EKLF), which, like Sp1, binds to CACCC boxes. EKLF is essential for the expression of the beta-globin but not the gamma-globin gene. Because both gamma-globin and beta-globin promoters contain the CACCC box, and their promoter elements are similar, it is not known why the two promoters behave so differently. In this report, we searched for the functional differences between the two promoters by studying their ability to recruit EKLF. We used the in vivo PIN*POINT assay to show that EKLF is recruited to the beta-globin promoter but not to the gamma-globin promoter. We show that this selectivity is a result of differences in surrounding promoter elements and not CACCC box alone. One of the differences between the two promoters with a functional consequence is the CCTTG repeat that is present in the gamma-globin promoter but not in the beta-globin promoter. The repeat, when inserted in the beta-globin promoter, decreases EKLF recruitment to and activity of the beta-globin promoter, suggesting that the repeat functions as a suppressor element. The CCTTG repeat can also suppress the SV40 promoter in cis, and the suppressor factor binding to the repeat can be squelched with a plasmid containing a high copy number of the repeat. These findings may have implications in designing drug targets for treatment of beta-globin disorders.

Animals↗

PubMed2005

A time to be promoted. The Prospective Study of Promotion in Academia (Prospective Study of Promotion in Academia).

OBJECTIVE: The study's objectives were to determine (1) the rate at which department of medicine faculty in the United States are promoted, (2) if clinician-educators (CEs) are promoted to Associate Professor at the same rate as clinician-investigators (CIs), and (3) the variables that predict promotion. METHODS: The Prospective Study of Promotion in Academia was a part-retrospective, part-prospective (from 2000 to 2003) cohort study. Six-hundred and four Internal Medicine junior faculty across the United States who had been registered as new appointees with the Association of American Medical Colleges in 1995 were invited to participate. Twenty-one percent of these had already left their institution when the study began. One hundred and eighty-three Internal Medicine faculty from 87 institutions in 35 states enrolled. The main outcome measure was the time from appointment as Assistant Professor to promotion to Associate Professor. RESULTS: Follow-up was complete for all 183 faculty. Among the faculty that achieved promotion, the estimated median time to promotion was 6.0 years (95% Conf. Int.=5.8 to 6.2). The unadjusted sixth-year promotion rate for CEs was 16%, while for CIs it was 26% (P=.002). Independent negative predictors of promotion included low amount of research time (Hazard Ratio [HR] =0.3, 95% Conf. Int.=0.2 to 0.5), having a manuscript review service (HR=0.4, 95% Conf. Int.=0.2 to 0.7), never meeting with Chairman/Chief about promotion (HR=0.4, 95% Conf. Int.=0.2 to 0.7), low job satisfaction (HR=0.5, 95% Conf. Int.=0.3 to 0.9), and working in the Northeast (HR=0.6, 95% Conf. Int.=0.4 to 1.1). Positive predictors included making between 130 dollars and 149,000 dollars per year (HR=1.9, 95% Conf. Int.=1.1 to 3.4), working more than 60 h/wk (HR=1.9, 95% Conf. Int.=1.1 to 3.0), having a career mentor available (HR=1.8, 95% Conf. Int.=1.1 to 2.9), and having access to a grant office (HR=1.6, 95% Conf. Int.=1.0 to 2.6). CONCLUSION: CEs and CIs appear to be promoted at different rates. The characteristics that are independently associated with earlier promotion may be helpful for institutions and individual faculty that are committed to achieving promotion efficiently.

Adult↗

PubMed1985

Release of fibronectin is linked to tumor promotion: response of promotable and non-promotable clones of a mouse epidermal cell line.

By use of indirect immunofluorescence technique and enzyme-linked immunosorbent assay we show that JB 6 mouse epidermal cells have cell surface fibronectin (FN) and release FN into the culture medium. The addition of 10(-8) M 12-O-tetradecanoylphorbol-13-acetate (TPA) to promotable clones caused a 2-fold enhancement of the FN release over solvent control. On the other hand, in non-promotable clones, TPA in concentrations of 10(-8) M or 10(-7) M did not cause increased FN release. Mezerein, a non-phorbol diterpene and second-stage tumor promoter was also found to be active in causing enhanced FN release in promotable but not in non-promotable clones. The vitamin A derivative retinoic acid (RA) antagonized the TPA-caused FN-release in promotable clones. RA had, however, no effect on the basic release patterns, when given alone or given to non-promotable clones together with TPA. These results suggest that the increased release of FN may be a required event for promotion to transformation. Our view is derived from the observation that promotable clones of the JB 6 cell line release increased amounts of FN into their medium upon promoter exposure while non-promotable clones are unaffected.

Animals↗

PubMed1995

Long-range interaction between two promoters: activation of the leu-500 promoter by a distant upstream promoter.

The leu-500 mutation can be suppressed in S. typhimurium topA. Previous studies have demonstrated that the plasmid-borne leu-500 minimal promoter cannot be activated in topA mutants unless adjacent (< 250 bp) transcription occurs away from the leu-500 promoter (short-range promoter interaction). To search for a potential upstream promoter responsible for activation of leu-500 in the chromosomal context, we have identified the ilvlH promoter, located 1.9 kb upstream of leu-500 (long-range promoter interaction). Different from short-range promoter interaction, which is abolished by DNA sequence insertions, the long-range promoter interaction is mediated by the intervening DNA sequence. These studies suggest that the long-range interaction between a pair of divergently arrayed promoters is probably mediated by a complex process involving relay of DNA supercoiling by the DNA sequence located between the two promoters.

Base Sequence↗

PubMed2002

Distinct promoter elements mediate the co-operative effect of Brn-3a and p53 on the p21 promoter and their antagonism on the Bax promoter.

Although the promoters of both the Bax and p21 genes are activated by p53, they differ in the effect on this activation of the POU family transcription factor Brn-3a. Thus, Brn-3a inhibits activation of the Bax promoter by p53 but enhances the ability of p53 to activate the p21 promoter. We demonstrate that repression of p53-mediated activation of the Bax promoter involves a complex upstream sequence in which two Brn-3a response elements flank the p53 response element. In contrast, a minimal p21 promoter is activated by Brn-3a and such activation cannot be abolished without abolishing basal promoter activity. Moreover, synergistic activation by Brn-3a and p53 continues to be observed when the p53-binding sites in the p21 promoter are substituted by the Bax p53 site or by the region of the Bax promoter essential for Brn-3a-mediated repression, indicating that the p21 core promoter plays a central role in this response. The significance of these effects is discussed in terms of the different responses of the Bax and p21 promoters and the overlapping but distinct roles of Brn-3a and p53 in neuronal growth arrest and apoptosis.

Animals↗

PubMed1993

The Klebsiella pneumoniae nifJ promoter: analysis of promoter elements regulating activation by the NifA promoter.

The nifJ and nifH promoters of Klebsiella pneumoniae are divergently transcribed sigma 54-dependent promoters that are positively activated by the NifA protein. NifA binds to upstream activator sequences (UASs), usually located 60-200 bp upstream of the start of transcription. Bound NifA is presented to the RNA polymerase-sigma 54 complex (E sigma 54) via DNA loop formation, mediated by the binding of integration host factor protein (IHF) between E sigma 54 and NifA. The nifJ promoter sequence contains three potential NifA binding sites (UAS1, 2 and 3) and two potential RNA polymerase-sigma 54-binding sites (downstream promoter elements, DPEs 1 and 2). DPE2 is located 420 bp into the coding region and DPE1 overlaps UAS1 by 5 bp. Mutational and footprinting analyses have shown efficient activation of the nifJ promoter requires that NifA is bound at UAS 2 and 3. Transcription is initiated at DPE1. Only a weak interaction of NifA with the UAS overlapping DPE1 was detected. Footprints demonstrated that E sigma 54 forms a closed complex at DPE1 but not DPE2 and that bound E sigma 54 closely approaches the -15 region of DPE1. Stimulation of nifJ promoter activity by IHF was not as great as that observed for other nif promoters. In the absence of IHF nifH promoter sequences stimulated activation of the nifJ promoter. This appeared to require NifA bound at the nifH UAS. Thus, one additional role of IHF may be to partition NifA between the two promoters by constraining the topology of the DNA.

Amino Acid Sequence↗

PubMed2007

The new core promoter element XCPE1 (X Core Promoter Element 1) directs activator-, mediator-, and TATA-binding protein-dependent but TFIID-independent RNA polymerase II transcription from TATA-less promoters.

The core promoter is a critical DNA element required for accurate transcription and regulation of transcription. Several core promoter elements have been previously identified in eukaryotes, but those cannot account for transcription from most RNA polymerase II-transcribed genes. Additional, as-yet-unidentified core promoter elements must be present in eukaryotic genomes. From extensive analyses of the hepatitis B virus X gene promoter, here we identify a new core promoter element, XCPE1 (the X gene core promoter element 1), that drives RNA polymerase II transcription. XCPE1 is located between nucleotides -8 and +2 relative to the transcriptional start site (+1) and has a consensus sequence of G/A/T-G/C-G-T/C-G-G-G/A-A-G/C(+1)-A/C. XCPE1 shows fairly weak transcriptional activity alone but exerts significant, specific promoter activity when accompanied by activator-binding sites. XCPE1 is also found in the core promoter regions of about 1% of human genes, particularly in poorly characterized TATA-less genes. Our in vitro transcription studies suggest that the XCPE1-driven transcription can be highly active in the absence of TFIID because it can utilize either free TBP or the complete TFIID complex. Our findings suggest the possibility of the existence of a TAF1 (TFIID)-independent transcriptional initiation mechanism that may be used by a category of TATA-less promoters in higher eukaryotes.

Base Sequence↗

PubMed1994

A course of very small doses of DMBA, each of them allegedly with no promoting potency, acts with clear synergistic effect as a strong promoter of DMBA-initiated mouse skin carcinogenesis. A comparison of the tumorigenic and carcinogenic effects of DMBA (7,12-dimethylbenz-alpha-anthracene) and TPA (12-O-tetradecanoyl-phorbol-13-acetate) used as initiators and promoters in classical two-stage experimental protocols.

A carcinogen has recently been defined as an agent that causes a neoplasm by a two-step process involving initiation and promotion. The difference between the two steps is said to be qualitative rather than quantitative. According to this theory, the hydrocarbon carcinogen, 7,12-dimethylbenz-alpha-anthracene (DMBA) has both initiating and promoting properties, and it has been claimed that when the dose level is lowered, the promoting activity is first reduced while its initiating activity is retained, until at a small dose even the promoting property has completely disappeared, and only the initiating property remains. The theory also says, however, that for carcinogens individual doses are additive, and for pure initiators individual doses are additive even at subthreshold doses. Promoters are said to act in a synergistic way on initiated skin, and a threshold exists below which there is no effect. Experiments were carried out to test the validity of these claims. DMBA was applied twice a week in very small (10 or 4 nmol) doses to the skin of hairless mice, with or without previous initiation. When, after DMBA initiation, DMBA or 12-O-tetradecanoyl-phorbol-13-acetate (TPA) were applied twice a week at small equimolar doses, a real synergistic effect, said to characterize promotion, was obtained with DMBA also, and with an even higher tumor crop and many more carcinomas than those elicited by TPA. Promotion with repeated doses of 10 nmol DMBA resulted in very significantly more carcinomas than with 4 nmol DMBA, which again gave more carcinomas than promotion with 10 nmol TPA. Repeated doses of TPA alone gave a 9% tumor rate, and TPA is thus a complete carcinogen. Reverse experiments were not innocuous. These results contradict the hypothesis that very small doses of DMBA have only initiating properties with only additive and not synergistic effects, and that TPA is only a promoter. It is most probable that the difference between the tumorigenic and carcinogenic effects of DMBA and TPA is not qualitative, but quantitative. TPA is highly toxic; it induces cell death followed by pronounced regeneration, and even DNA alterations. It is a complete, but weak carcinogen. DMBA is a strong complete carcinogen, binding covalently to DNA and altering it in various ways, but in equimolar doses it is less acutely toxic than TPA.(ABSTRACT TRUNCATED AT 400 WORDS)

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1997

The finM promoter and the traM promoter are the principal promoters of the traM gene of the antibiotic resistance plasmid R100.

finP multicopy repression and traJ multicopy derepression indicate that the ratio of sense to antisense transcripts is important in the regulation of R100 conjugation. The extension of R100 traM transcripts into traJ shows that promoters in traM can affect this ratio, making the regulation of traM transcription important in the regulation of R100 conjugation. Since R100 traM, traY and tral proteins bind to the traM promoter region, we examined traM transcription in R100-1 traM, traY and tral mutants and compared it with traM transcription in both R100-1 and R100. We verified that the traM and finM promoters provide virtually all the transcripts originating in the R100-1 traM gene. When either is deleted, as in VAR22 or VAR30, the remaining promoter is highly active. We show here that traY positively regulates R100-1 traM transcription, as has been found for F. We found that tral did not regulate R100-1 traM transcription. The measured activity of the native R100 traM promoter was 12% of that in R100-1, whereas the native R100 finM promoter was 45% of that in R100-1. These data and data from the R100-1 traY and tral mutants show that the activities of the two promoters varied independently.

Bacterial Proteins↗

PubMed2000

Downstream promoter sequences facilitate the formation of a specific transcription factor IID-promoter complex topology required for efficient transcription from the megalin/low density lipoprotein receptor-related protein 2 promoter.

Megalin/low density lipoprotein receptor-related protein 2 (LRP-2) is an endocytic receptor expressed in highly specialized cell types such as parathyroid cells and epithelia of the kidney. Previous experiments identified a nonconsensus TATA element, with the sequence TAGAAAA, as crucial for accurate and efficient transcription from the LRP-2 promoter. Here we show that, in addition to the TAGA element, promoter sequences downstream of the transcription start site contribute significantly to transcription both in vitro and in transfected cells. Deletion and point mutational analyses reveal that the promoter region located between positions +5 and +11 (sequence TTTTGGC) is of particular importance. Complementation experiments in nuclear extracts lacking transcription factor IID (TFIID) activity show that TATA-binding protein-associated factors of TFIID are essential for the function of LRP-2 downstream promoter sequences. Interestingly, DNase I footprinting studies show that the downstream region between positions +5 and +11 does not significantly affect overall TFIID affinity to the promoter but that it profoundly affects the topology of the TFIID x promoter complex not only downstream of the transcription start site, but in particular in the TATA box region. Our observations suggest a model for a novel downstream sequence function, in which TATA-binding protein-associated factor-promoter interactions downstream of the transcription start site modulate TFIID-DNA interactions in the TATA box region.

Base Sequence↗

PubMed1999

Mutational analysis of the phoD promoter in Bacillus subtilis: implications for PhoP binding and promoter activation of Pho regulon promoters.

The PhoP-PhoR two-component regulatory system controls the phosphate deficiency response in B. subtilis. A number of Pho regulon genes which require PhoP approximately P for activation or repression have been identified. The studies reported here were initiated to understand the PhoP-DNA interaction necessary for Pho promoter regulation. The regulatory region of phoD was characterized in detail using oligo-directed mutagenesis, DNase I footprinting, and in vivo transcription assays. These data reveal basic principles of PhoP binding relevant to PhoP's interaction with other Pho regulon promoters. Our results show that: (i) a dimer of PhoP approximately P is able to bind two consensus repeats in a stable fashion; (ii) PhoP binding is highly cooperative within the core promoter region, which is located from -66 to -17 on the coding strand and contains four TT(A/T/C)ACA-like repeats; (iii) specific bases comprising the TT(A/T/C)ACA consensus are essential for transcriptional activation, but the specific base pairs of the intervening sequences separating the consensus repeats are not important for either PhoP binding or promoter activation; (iv) the spacing between two consensus repeats within a putative dimer binding site in the core region is important for both PhoP binding and promoter activation; (v) the exact spacing between two dimer binding sites within the core region is important for promoter activation but less so for PhoP binding affinity, as long as the repeats are on the same face of the helix; and (vi) the 5' secondary binding region is important for coordinated PhoP binding to the core binding region, making it nearly essential for promoter activation.

Alkaline Phosphatase↗

PubMed1999

Dual regulation of promoter II- and promoter 1f-derived cytochrome P450 aromatase transcripts in equine granulosa cells during human chorionic gonadotropin-induced ovulation: a novel model for the study of aromatase promoter switching.

Estradiol biosynthesis is a key biochemical trait of developing follicles. To study its regulation in equine follicles, the objectives of this study were to clone and determine the structure of equine cytochrome P450 aromatase (P450AROM), and characterize the regulation of P450AROM and P450 17alpha-hydroxylase/C17-20 lyase (P45017alpha) messenger RNAs (mRNAs) in vivo in equine preovulatory follicles isolated during hCG-induced ovulation. Two distinct P450AROM complementary DNAs (cDNAs) were isolated from an equine preovulatory follicle cDNA library. One clone was 2682 bp in length and included 115 bp of 5'-untranslated region (UTR), 1509 bp of open reading frame encoding a well conserved 503-amino acid protein, and 1058 bp of 3'-UTR. Its 5'-most region represented the equine homolog of exon 1f, previously designated brain specific. The other cDNA clone encoded a truncated protein and contained a distinct 5'-UTR characteristic of transcripts derived from promoter II, previously identified as the predominant ovarian mRNA. Northern blot analyses were performed using preovulatory follicles obtained during estrus between 0-39 h after the administration of hCG and with corpora lutea isolated on day 8 of the estrous cycle (day 0 = day of ovulation). The results showed a biphasic regulation of P450AROM mRNA expression: levels were highest in follicles at 0 h post-hCG, decreased significantly during the ovulatory process at 12 and 24 h (P < 0.05), and increased again between 30-39 h post-hCG and in corpora lutea. When oligonucleotides specific for P450AROM mRNA variants were used as probes, a novel switching phenomenon was observed. Promoter II-derived transcripts accounted for the message present in follicles at 0 h post-hCG and in corpora lutea, whereas promoter 1f-derived mRNA was expressed exclusively during the ovulatory process (30-39 h post-hCG). Levels of P45017alpha mRNA were high in follicles at 0 h, but significantly decreased after hCG treatment (P < 0.05), with lowest levels in follicles at 36 and 39 h post-hCG and in corpora lutea. Northern blots performed on isolated cellular preparations revealed that P450AROM and P45017alpha transcripts were localized exclusively in granulosa cells and theca interna, respectively. Equine aromatase promoters II and 1f were cloned from a genomic library, and putative transcription start sites were characterized by primer extension assays. Sequence analyses identified distinct potential regulatory elements in each promoter. Thus, this study identifies a novel aromatase promoter-switching phenomenon in equine granulosa cells during follicular luteinization and provides a new model in which aromatase promoter switching is induced in vivo.

Amino Acid Sequence↗

PubMed1980

A cell culture assay for tumor-promoter-dependent progression toward neoplastic phenotype: detection of tumor promoters and promotion inhibitors.

Mouse epidermal cell lines have been identified which respond to tumor-promoting (but not nonpromoting) phorbol esters with an irreversible shift in anchorage independence, an in vitro marker of neoplastic phenotype. This response may be analogous to a later stage of tumor promotion in vivo. The shift occurs at TPA concentrations as low as 0.1 ng/ml (1.6 x 10(-10) M). The specificity of the soft agar growth response is not limited to phorbol esters but extends to nonphorbol plant diterpenes such as mezerein, to detergents, to polycyclic hydrocarbons present in cigarette smoke, and to some growth factors. All of the above classes of compounds have been previously shown to have tumor-promoting and/or cocarcinogenic activity in mouse skin in vivo. Clonal heterogeneity for TPA responsiveness has been found. Clones which were highly responsive to phorbol esters were also highly responsive to other classes of promoters, indicating their usefulness both for promoter detection and mechanism studies. The anchorage-independence to response to TPA was inhibited by a series of retinoids whose activity paralleled that for inhibiting tumor promotion in vivo. Both retinoid inhibition and clonal heterogeneity for promoter response are being utilized to study determinants of preneoplastic progression.

Animals↗

PubMed1990

Promoter selectivity of Escherichia coli RNA polymerase: effect of base substitutions in the promoter -35 region on promoter strength.

A set of 18 variant lac UV5 promoters was constructed, each carrying a single base substitution within the -35 region (nucleotide positions from -36 to -31 relative to the transcription start site). Using truncated DNA fragments carrying these variant promoters and purified Escherichia coli RNA polymerase holoenzyme, in vitro mixed transcription assays were performed to determine two parameters governing promoter strength: i.e., the binding affinity to RNA polymerase (parameter I) and the rate of open complex formation (parameter II). The following conclusions were drawn from the data presented: (1) Alteration in the promoter strength of variant promoters is dependent on both the position and base species of substitutions; (2) the consensus sequence (TTGACA) exhibits the highest values for both parameters; (3) base substitutions at nucleotide position -34 cause marked effect on both parameters; (4) cytosine at nucleotide position -32 can not be replaced with other nucleotides without significant reduction of the promoter strength; and (5) base substitution at nucleotide position -31 exerts only a little effect on parameter I. All these findings were confirmed by abortive initiation assays.

Base Sequence↗