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PubMed2006

Identification of human hepatic stimulator substance gene promoter and demonstration of dual regulation of AP1/AP4 cis-acting element in different cell lines.

Human hepatic stimulator substance (hHSS) is a newly identified growth-promoting factor in the liver. HSS is capable of stimulating hepatic regeneration in partial hepatectomized rats, thus, promoting growth of hepatic tumor cells. To understand and elucidate the transcriptional regulation of hHSS gene, the 4890bp of 5'-flanking region of the gene have been isolated and sequenced. The transcriptional start site, located at 248nt upstream from the ATG starting codon, was identified by 5'-rapid amplification cDNA end (5'-RACE). The classical promoter sequences, such as TATA box or GAATT were not identified in the promoter region, instead a GC-rich segment was formed (>70%) by expanding to a longer than 400bp, and immediately upstream from the ATG start codon. The transient transfection assays, using promoter deletion constructs, showed that hHSS promoter was efficiently capable in driving the reporter expression not only in HepG2 cells, but also in Cos7 cells. A region spanning nucleotides in the range of -447 to -358bp revealed a negative regulation on promoter activity in HepG2 cells, but with positive regulation in Cos7 and Hela cells. The promoter activity was obviously influenced by AP1/AP4 (-375/-369nt) mutation in these three cell lines. EMSAs showed that the site was recognized by AP1 in HepG2 cell, and only by an AP4 protein in Cos7 cells. The c-Jun bound to the promoter was further verified by supershift in HepG2 cells and human liver tissue. Chromatin immuno-precipitation (ChIP) demonstrated that there was a direct association of c-Jun with hHSS promoter in HepG2 cells. The c-Jun strongly suppressed hHSS promoter activity in transient expression analyses in HepG2 cells. Mutations in the AP1 binding sites rescued suppression caused by c-Jun, suggesting this was a direct regulation of the hHSS promoter. In contrast, there was no significant effect in c-Jun over-expressed Cos7 and Hela cells. The tissue-specific function of c-Jun in hHSS promoter activity may in part help explain the differences in biology function of hHSS between liver and non-liver cells.

Animals↗
PubMed2002

Differential activation of the IkappaBalpha and mouse mammary tumor virus promoters by progesterone and glucocorticoid receptors.

The glucocorticoid and progesterone receptors (GR and PR) are structurally homologous and bind a common hormone response element (HRE). The mechanisms by which receptors activate specific promoters when multiple steroids are present in a cell is a critical question in endocrinology. To investigate how co-existing steroid receptors regulate gene transcription, we have compared two hormone-responsive promoters in T47D/A1-2 human breast cancer cells expressing both the GR and PR. The promoters chosen were those for the mouse mammary tumor virus (MMTV) and the gene for IkappaBalpha, the inhibitor of the ubiquitous transcription factor, nuclear factor kappa B (NFkappaB). Several differences between glucocorticoid and progestin activation of the IkappaBalpha and MMTV promoters were revealed. Both steroids activated the endogenous IkappaBalpha promoter, while only glucocorticoids activated a stably integrated MMTV promoter. In combination, progestins enhanced glucocorticoid activation of IkappaBalpha, but antagonized glucocorticoid activation of MMTV. These differences in steroid receptor competition were further demonstrated when levels of the PR were reduced by prolonged treatment with progestin. Under these conditions, the PR no longer competes effectively with the GR for activation of the MMTV promoter. However, on the IkappaBalpha promoter, the GR and PR still activate the promoter in a cooperative fashion. Another difference between the two promoters is their chromatin structure. In this cell line, the MMTV promoter chromatin is "closed" and insensitive to restriction enzyme cleavage, while the IkappaBalpha promoter is "open." Using PR antagonists, we demonstrate that at least one cofactor complex, the BRG-1 chromatin remodeling complex, differentially contributes to activation of both promoters.

Animals↗
PubMed1983

Relationship between promoter structure and template specificities exhibited by the bacteriophage T3 and T7 RNA polymerases.

To explore the basis for the template specificities of the bacteriophage T3 and T7 RNA polymerases (EC 2.7.7.6), we determined the nucleotide sequences of six promoters recognized by the T3 RNA polymerase and compared them with the previously determined promoter sequences recognized by the bacteriophage T7 RNA polymerase. Recombinant plasmids containing random Hpa II and Taq I fragments of T3 DNA were screened for T3 promoter activity in vitro in a transcription assay using purified T3 RNA polymerase. Five promoters for the T3 RNA polymerase were identified in this manner and their sequences were determined; the sequence of an additional promoter was determined directly from a genomic DNA fragment. In five of the T3 promoters an identical 16-base-pair sequence (A-C-C-C-T-C-A-C-T-A-A-A-G-G-G-A) extends from -12 to +4 (initiation occurring with GTP at +1); this sequence is preceded by a 6-base-pair A + T region. The remaining promoter contains an inserted C at position -1 and an A at the +1 position. The sequence of the 5' end of the RNA transcript from the latter promoter confirms that transcription is initiated with ATP at the +1 position. Previously, late T3 or T7 transcripts had not been found to initiate with ATP. The highly conserved T3 promoter sequence was compared to the T7 promoter consensus sequence. The fundamental difference between the two kinds of phage promoters is the occurrence of G-A at positions -11 and -10 in the T7 promoter, whereas there is a single C at position -10 in the T3 promoter.

Base Sequence↗
PubMed1983

The role of growth of normal and preneoplastic cell populations for tumor promotion in rat liver.

A number of different compounds, including phenobarbital, hypolipidemic drugs such as clofibrate and nafenopin, the sex steroids progesterone, cyproterone acetate, estradiol and mestranol, chlorinated hydrocarbons such as DDT, hexachlorocyclohexane, and TCDD and the antioxidant butylhydroxytoluene, appears to promote the development of liver tumors from previously induced initiated cells. The mechanisms of tumor promotion by several representative prototypes of these compounds were studied in rat liver in vivo. All liver tumor promoters mentioned above stimulate growth of normal liver. The growth response is due to cellular hypertrophy and/or increased rate of DNA (and cell) replication and/or decreased rate of cell death. Hepatocytes in foci or islands of altered cells (putatively preneoplastic) show higher rates of replication than normal liver cells; various different liver tumor promoters cause a further increase of proliferation of focal cells. The increased proliferative activity is found in different island phenotypes and thus seems to be a useful marker of the putative preneoplastic state. The focal cells respond to several factors limiting proliferation in normal liver, suggesting that they are not autonomous with respect to growth control. Early preneoplastic foci grow slowly without promotion, despite the relatively high rates of cell replication. Thus their cells seem to have a much shorter life-time than normal hepatocytes or to undergo reversion to the normal phenotype. Promoters seem to accelerate island enlargement by increasing cell replication and delaying cell death or remodeling. Thus, tumor promoters enhance the manifestation of the proliferation advantage of the putative initiated cell population. In addition, promoters cause increases in the number of detectable islands. This can partially be explained by enlargement of existing islands, but phenotypic changes that would enhance the probability of detection of remodelling islands and growth of dormant initiated cells, probably contribute to the apparent increase of island number. Putative preneoplastic foci of unknown origin are frequent in the liver of aged Wistar rats. They are morphologically and functionally very similar to those induced by carcinogens and are responsive to the mitogenic effect of tumor promoters. Promotion of these "spontaneous" foci may explain tumor appearance after long-term application of promoters.The findings may provide a basis for improved identification of initiated hepatocytes (and of initiating hepatocarcinogens) and for detection of tumor promoters. All suspected liver tumor promoters tested so far induced enhanced preneoplastic cell proliferation after single doses. The long-term carcinogenicity bioassay as currently performed does not discriminate between initiating and promoting properties of a test compound if the animals used develop spontaneous preneoplastic lesions in the organ affected.

Animals↗
PubMed1987

Characterization of skin tumor promotion and progression by chrysarobin in SENCAR mice.

The characteristics of the skin tumor promotion response with anthrone derivatives has been further examined in SENCAR mice. Chrysarobin (1,8-dihydroxy-3-methyl-9-anthrone) was an effective skin tumor promoter when applied twice weekly with dose-dependent increases in both papillomas and squamous cell carcinomas between 25 and 100 nmol/mouse. A similar dose-response relationship for papilloma and carcinoma formation was observed when chrysarobin was applied once weekly. Interestingly, chrysarobin was approximately twice as active as a skin tumor promoter when applied once weekly versus twice weekly. Doses of 25,100, and 220 nmol/mouse gave maximal papilloma responses of 2.90, 8.15, and 9.38 versus 0.73, 4.70, and 5.42 papillomas/mouse, respectively, in mice initiated with 25 nmol 7,12-dimethylbenz(a)anthracene. Thus, unlike 12-O-tetradecanoylphorbol-13-acetate (TPA), where a twice weekly application frequency is optimal, application of anthrone promoters such as chrysarobin once weekly is a more optimal frequency for papilloma development. Chrysarobin was also a much more effective skin tumor promoter when the start of promotion was delayed by an additional 10 weeks. Thus, groups of mice initiated with 10 nmol 7,12-dimethylbenz(a)anthracene and having promotion started in either the 3rd or the 13th week after initiation had maximal responses of 5.6 or 11.0 papillomas/mouse, respectively. In addition, the rate of papilloma development was faster in the delayed promotion group. The progression of papillomas to carcinomas was examined in all chrysarobin-treated groups and compared with three groups of mice treated with 3.4 nmol TPA. After 60 weeks of promotion, the anthrone promoter-treated groups had carcinoma:papilloma ratios 2.5 to 5.0 times higher than the TPA-treated groups. This was due primarily to the fact that similar carcinoma responses were observed in both anthrone- and TPA-treated mice at optimal promoting doses whereas the papilloma responses were significantly lower in the former groups. The data suggest that anthrone derivatives are very efficient tumor promoters. The results are further discussed in terms of mechanisms of skin tumor promotion.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1988

The protein kinase C activator L-alpha-dioctanoylglycerol: a potent stage II mouse skin tumor promoter.

Endogenous diacylglycerol, as produced during ligand-stimulated hydrolysis of phosphatidylinositol, is a physiological activator of protein kinase C, a receptor for the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA). Diacylglycerol mimics many effects of phorbol ester TPA, but it is not known whether diacylglycerol is a mouse skin tumor promoter. The present studies determined the mouse skin tumor-promoting activity of L-alpha-dioctanoylglycerol (DG), a membrane-permeable diacylglycerol derivative. In two independent experiments with female SENCAR mice, DG at a 2 mumol dose, when applied twice weekly to the initiated mouse skin, failed to promote mouse skin tumor formation. Similarly, DG lacked Stage I tumor-promoting activity; twice weekly applications of DG for 2 wk to the initiated mouse skin followed by twice weekly applications of mezerein (3.3 nmol) for as long as 27 wk elicited only a few papillomas per mouse. DG was found to be a potent Stage II mouse skin tumor promoter. In a typical two-stage tumor promotion experiment, SENCAR mice were initiated by application of 20 nmol of DMBA to their shaved backs. Two wk after initiation, 3.3 nmol of TPA were applied twice weekly for 2 wk (Stage I), and then 2 mumol of DG or 3.3 nmol of mezerein were applied to the skin twice weekly for the entire duration of the experiment (Stage II). Stage II tumor promotion with mezerein and DG resulted in 13.33 +/- 0.88 and 11.13 +/- 1.25 papillomas per mouse, respectively, at 19 wk and the carcinoma incidence, 43% and 33%, respectively, at 27 wk of promotion. The tumor-promoting activity of DG was compared with the parent alcohol glycerol, and it was found that glycerol, at a dose as high as 11 mumol, was not a complete, Stage I or Stage II mouse skin tumor promoter. Both TPA and DG, when applied to mouse skin, induced epidermal ornithine decarboxylase activity. Both TPA and DG activate protein kinase C, but the results presented indicate that TPA and DG differ in their tumor-promoting properties. DG, like mezerein, is a Stage II mouse skin tumor promoter.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1983

Promotion of hepatocarcinogenesis in vivo and in vitro.

Various chemicals including phenobarbital (PB) have been shown to act as promoters in hepatocarcinogenesis in rodents. The effect of initiation and/or promotion of chemicals can be quantitatively measured by scoring the number and size of enzyme-altered islands (EAI). The effect of promotion is proportional to the period of treatment and is much greater when the promoter is given earlier after initiation. A unique aspect of hepatic promoters is their inhibitory action against carcinogenesis when given simultaneously with a carcinogen, reducing both the initiating and promoting activities of the carcinogen. With the aid of the promoting action of PB, carcinogenic activity of pure initiators is demonstrated. Promoting effect of PB was found at as low a dose as 5 ppm, which corresponds to 20 mg per 50 kg person per day, a dose several times less than the common therapeutic dose. In an attempt to analyze the mechanism of promotion and also to develop a model system for detecting environmental promoters in vitro, hepatic cells obtained from animals during carcinogenic treatment were transferred to a culture system. Cells comprising EAI after a certain critical stage gave rise to proliferative epithelial cell foci. Some hepatic promoters showed a growth enhancing effect on these foci while others did not, suggesting both the existence of direct action by some promoters on initiated cells and the presence of different promoter mechanisms. Chronic treatment with PB selectively induced gamma-glutamyl transpeptidase activity in "spontaneous" hepatomas of C3H mice, suggesting that PB may enhance the expression of altered gene functions of initiated cells.

Animals↗
PubMed1983

Cellular and biochemical changes during multistage skin tumor promotion.

Skin tumor promotion in mice which may involve a free radical mechanism can be operationally and mechanistically further divided into at least two stages. The first stage, which is partially irreversible for 4 to 6 weeks, can be accomplished by a single application of 12-O-tetradecanoylphorbol-13-acetate (TPA) or by nonpromting agents such as 4-O-methyl-TPA, calcium ionophore A23187, and hydrogen peroxide, as well as by wounding. These agents plus wounding increase the number of dark basal keratinocytes, which suggest that these cells are important in the first stage of promotion. Recent data also suggest that the dark cells may be the critical target of skin tumor initiators. Prostaglandin E2 was found to specifically enhance stage I and increase the number of dark cells induced by TPA, whereas the protease inhibitor, tosyl phenylalanine chloromethylketone, specifically inhibited stage I of promotion and counteracted the TPA-induced dark cells. We have recently found that TPA and other first stage promoters can decrease the number of epidermal glucocorticoid receptors. Fluocinolone acetamide (FA) was found to inhibit both stages but was more effective in counteracting stage I of promotion. FA also prevents the TPA-induced dark cells and the decrease in glucocorticoid receptors caused by TPA. The second stage of promotion is initially reversible but later becomes irreversible. The weak promoting agent mezerein and the nonpromoting agent 12-deoxyphorbol-13-2,4,6-decatrienoate are effective stage II promoters. Polyamines, gene amplification and epidermal cell proliferation appear to be important events in stage II of promotion. Putrescine was found to specifically enhance stage II, whereas retinoic acid, difluoromethylornithine, and butylated hydroxyanisole specifically inhibited stage II of promotion and the mezerein-induced polyamine levels but not the mezerein-induced hyperplasia. The inhibition of stage II of promotion by antioxidants gives further support for the role of free radicals in tumor promotion. Mezerein was found to be much more effective in amplifying methotrexate resistance than TPA. Although, mezerein can not significantly decrease the number of glucocorticoid receptors or increase the number of dark cells after repetitive treatment, mezerein can maintain the TPA effect in a two-stage promotion protocol.

Animals↗
PubMed2005

Promoter methylation of p16INK4a, RASSF1A, and DAPK is frequent in salivary adenoid cystic carcinoma.

BACKGROUND: Promoter methylation is a common mechanism of inactivation of tumor suppressor genes in multiple tumor types. However, little is known about its role in the development of adenoid cystic carcinoma of the salivary gland (ACC). In the current study, the authors investigated whether promoter methylation is common in ACC and whether it may influence ACC development. METHODS: The promoter methylation status of the genes p16(INK4a), RASSF1A, DAPK, and MGMT, which are important in cell growth regulation, apoptosis, and DNA repair, was determined in tissue sections of tumor samples from 60 patients with ACC using methylation-specific polymerase chain reaction. The association between methylation status and patients' clinical and pathologic characteristics were assessed. RESULTS: Of the 60 tumors, DNA methylation of the p16(INK4a) promoter was detected in 28 tumors (47%); the respective values DNA methylation of the RASSF1A, DAPK, and MGMT promoters were 25 tumors (42%), 16 tumors (27%), and 4 tumors (7%), respectively. Forty-six tumors (77%) had DNA methylation in > or = 1 of the 4 promoters, 20 tumors (33%) had DNA methylation in > or = 2 promoters, 6 tumors (10%) had DNA methylation in > or = 3 promoters, and 1 tumor (2%) had DNA methylation in all 4 promoters. RASSF1A promoter methylation was more frequent in high-grade tumors than in low-grade tumors (P = 0.009), in advanced-stage tumors (P = 0.008), and in tumors with metastasis (P = 0.005). CONCLUSIONS: Promoter methylation of p16(INK4a), RASSF1A, and DAPK was common in ACC, and it is possible that such methylation may influence the development of ACC. The high frequency of RASSF1A promoter methylation in high-grade tumors and in tumors with metastasis suggested a role for this gene in the progression of ACC.

Adult↗
PubMed2005

Regulation of human prostate-specific G-protein coupled receptor, PSGR, by two distinct promoters and growth factors.

PSGR is a newly identified human prostate tissue-specific gene belonging to the G-protein coupled receptor (GPCR) family. Overexpression of PSGR is associated with human prostate intraepithelial neoplasia (PIN) and prostate tumors, suggesting PSGR may play an important role in early prostate cancer development and progression. To understand the regulation of tissue-specific expression of human PSGR and its upregulation mechanism in prostate cancers, we characterized the promoter region of PSGR and analyzed the control mechanism for PSGR expression in human prostate tissues/cells. In this report, we demonstrate that two distinct promoters control the transcriptional regulation of PSGR in human prostate cells. The first promoter region includes exon 1 and a TATA box at -31 site. The minimal DNA sequence with promoter activity is about 123 bp upstream of exon 1. Exon 1 contains tissue specific regulatory activity for the first promoter of PSGR gene. The second promoter is located in the upstream region of exon 2, which is a TATA-less and non-GC-rich promoter. Primer extension and RNA protection assays (RPA) revealed that the transcription driven by the second promoter is initiated at the junction of intron and exon 2 within a cluster of nucleotides located about 250 bp upstream from the junction. Both promoters show prostate cell-specific characteristics in our luciferase assays in transfected cells. Furthermore, we investigated the regulation of the promoter activities of the PSGR gene by different growth factors and cytokines, and demonstrated that interleukin-6 (IL-6) activates the promoter activities of PSGR in human prostate cancer cells. These data suggest that two functional promoters regulate the transcriptional expression of PSGR in human prostate tissues and PSGR is a new target for IL-6 transcriptional regulation.

Animals↗
PubMed2005

Effects of the Ad5 upstream E1 region and gene products on heterologous promoters.

BACKGROUND: All recombinant adenovirus vectors contain the upstream region of the E1A gene comprising the viral origin of replication, encapsidation signal, and cis-acting regulatory elements for transcription of the E1A and other early genes. Using different reporter genes, some previous studies demonstrated the maintenance of heterologous promoter specificity in the adenoviral context, while others reported that adenoviral sequences interfere with promoter activity. METHODS: Plasmid DNA-based luciferase reporter gene assays and adenovirus type 5 (Ad5) infection were combined to examine the effect of the Ad5 (nt 1-353) element and/or adenoviral gene products on tissue-specific (Midkine (MK) and COX-2), cell cycle associated (Ki-67 and E2F1) and viral promoters (Ad5 E1, Ad5 E4 and SV40). As a proof of concept, data were verified in the setting of recombinant replication-defective and replication-competent adenoviral vectors. RESULTS: Viral and E2F1 promoter activities were enhanced by the Ad5 (nt 1-353) segment by approximately 100% and 145%, respectively, regardless of its position. A polyadenylation sequence (polyA) upstream of the promoter had no effect, confirming an enhancer element within the Ad5 (nt 1-353) segment. Ad5 (nt 1-353) increased COX-2 promoter activity by 146% but was blocked by an upstream polyA, indicating a cryptic transcription start site. When placing the reporter gene cassette in a replication-defective adenovirus, similar data were obtained. In the plasmid vector-based system, adenoviral gene products transactivated the E2F1 and viral promoters by 194%, 19%, 67%, and 16%, respectively. Tissue-specific promoter activities were not significantly affected by the Ad5 (nt 1-353) segment, nor adenoviral gene products. In concert with these data, we were able to target replication-competent adenoviral vectors with the COX-2 promoter, but not with the cell cycle associated promotor. CONCLUSIONS: The adenovirus E1A upstream regulatory region and gene products interact with some but not all heterologous promoters. Often, the basal promoter activity can be reduced with an upstream polyA. Since the data obtained in our plasmid vector-based assay with internal control and infection with adenovirus could be confirmed in the adenoviral setting, our system might be suitable to speed up the identification of promoters which maintain their specificity in the adenoviral context and circumvent the problems associated with determining infectious adenovirus titers.

Adenoviridae↗
PubMed1997

Regulation of osteoblast-specific factor-1 (OSF-1) mRNA expression by dual promoters as revealed by RT-PCR.

OSF-1 (osteoblast-specific factor-1), which is also referred to as p18, HBBM, HB-GAM, HBGF-8, HARP, HBNF, and pleiotrophin, is a 121-amino acid polypeptide that can induce neurite outgrowth in vitro and is highly expressed in several tissues during fetal development but exhibits expression restricted to brain and bone tissues in adults. We have reported the genomic structure of mouse OSF-1 gene, in which the open reading frame spans four exons and at least two additional 5'-UTR exons (upstream exon U2 and downstream exon U1) exist. From analysis of isolated cDNAs, two types of cDNAs were identified: one has a sequence for U1 and U2 and the other has a sequence for an intron (present between U1 and U2) and U1. This suggests that the OSF-1 gene utilizes two alternative promoters, a distal and a proximal promoter, designated promoters II and I, respectively, for the translation initiation site (ATG). Promoter II is thought to exist upstream of the intron, while promoter I is present in the intron. RT-PCR was employed to examine which OSF-1 promoters are used during development and in various cell lines. In adult mice (aged 2 months), usage of promoter I was predominant, and OSF-1 mRNAs were expressed in many organs including brain and bone. At one fetal stage (E-19), promoter I was active in the major organs including brain, liver, kidney, and intestine, while promoter II was active only in the brain. In the cell lines examined, usage of promoter I was frequent, while promoter II was active only in a few cell lines such as MC3T3-E1 (cultured for 7 days) and C3H10T1/2. These findings suggest that OSF-1 may play fundamental roles in differentiation, growth and maintenance of adult organs as well as in embryogenesis, and indicate that the expression of OSF-1 is regulated, at least in part, by the usage of different promoters in the mouse.

Aging↗
PubMed2002

Identification of tissue- and cancer-selective promoters for the introduction of genes into human ovarian cancer cells.

OBJECTIVE: One potential limitation of gene therapy for epithelial tumors is the lack of tissue or tumor specificity of treatment. Tumor-selective expression of gene therapies may avoid deleterious side effects and improve the efficacy of the treatment. The aim of this study was to evaluate the tissue and tumor specificity of four different potential gene therapy promoters, to determine their usefulness in tissue-specific gene therapy of epithelial ovarian carcinomas. METHODS: Three potential epithelial cell-selective (hESE1, SLP1, OSP1) and one potential tumor-selective (hTERT) promoter were placed upstream of a luciferase construct to determine relative activity in a wide variety of normal and malignant cell lines. Transient transfection and luciferase assays were carried out in 12 epithelial ovarian (3 SV40 T antigen-transfected normal and 9 malignant) and 8 control cell lines. RESULTS: Luciferase assays revealed that the hTERT promoter presented the highest tumor selectivity. hESE1 and SLP1 promoters showed strong epithelial cell selectivity (hESE1, 16/17; SLP1, 15/17), with the OSP1 (11/17) promoter exhibiting lower epithelial selectivity. Of the potential promoters for gene therapy, hTERT promoter exhibited the strongest transcriptional activity in most of the tumor cell lines. None of the promoters exhibited strict ovarian epithelium selectivity. CONCLUSION: The hTERT promoter may be an optimal promoter for a univector gene therapy approach based on its high tumor selectivity. Utilization of multiple epithelial cell-specific promoters may result in a more tissue-selective gene therapy approach. Using a combination of promoters may prevent potential problems due to expression in nonepithelial stem cells that may constitutively express hTERT.

Adaptor Proteins, Vesicular Transport↗
PubMed1993

Effects of a single base-pair deletion in the bacteriophage lambda PRM promoter. Repression of PRM by repressor bound at OR2 and by RNA polymerase bound at PR.

We have deleted a single base-pair in the -35 region of the bacteriophage lambda PRM promoter. The deletion (PRM delta 34) creates a better match of PRM to consensus, thereby substantially increasing the activity of the promoter in vitro and in vivo. Since the mutation also increases the overlap between OR2 and the -35 region of PRM, binding of repressor to OR2 no longer activates, but in fact represses PRM. Finally, the mutation decreases the distance between the PRM and PR transcription start sites from 82 to 81 base-pairs. As a consequence, the interaction of RNA polymerase with either promoter in vitro strongly inhibits open complex formation at the other. Kinetic analyses and DNase I protection assays lead to the surprising result that mutual inhibition is not due to steric occlusion. Both promoters can be occupied by RNA polymerase at the same time. Determination of KB and kf revealed that inhibition of PRM delta 34 by PR was manifest in a 100-fold decrease in the value of kf, but at the same time KB was increased tenfold. These data raise the possibility that RNA polymerase molecules bound at the two promoters contact and mutually stabilize each other and that this interaction subsequently inhibits a substep in the isomerization of closed to open complexes. In footprinting assays, each promoter is characterized by sites of enhanced cleavage when that promoter is occupied alone. These enhancements are substantially diminished when both promoters are occupied, suggesting that complexes of each promoter with RNA polymerase alter the structure of complexes formed at the other promoter. Assays of the effects of the delta 34 mutation in vivo indicate that interference between PRM and PR does not limit the rate of open complex formation at PRM in the cell. Apparently, transcription initiation clears the promoter rapidly enough that neither promoter is occupied a significant fraction of the time.

Bacteriophage lambda↗
PubMed1997

Expression of aggregation-prone recombinant proteins at low temperatures: a comparative study of the Escherichia coli cspA and tac promoter systems.

The aggregation-prone fusion protein preS2-S'-beta-galactosidase was used as a model system to compare the efficiencies of the IPTG-inducible tac promoter and the low-temperature-inducible cspA promoter in directing the expression of soluble recombinant polypeptides at reduced growth temperatures in Escherichia coli. At 37 degrees C, the fusion protein was produced at high levels from the tac promoter, but aggregated quantitatively in a biologically inactive form. In contrast, little preS2-S'-beta-galactosidase was synthesized from the cspA promoter at this temperature, presumably due to transcript instability. The highest yields of active enzyme were obtained following temperature downshift from 37 to 30 degrees C for the tac promoter and 25 degrees C for the cspA promoter. At 25 degrees C, the kinetics of accumulation of beta-galactosidase activity, ratios of soluble to insoluble fusion protein, and synthesis rates of preS2-S'-beta-galactosidase were virtually identical for both promoters for a period of 2 h postinduction. Thereafter, the cspA promoter became repressed, whereas synthesis of the fusion protein continued with the tac system. Following transfer to 10 degrees C, the tac promoter was almost completely inhibited while the cspA promoter was able to direct the synthesis of soluble preS2-S'-beta-galactosidase for up to 2 h. However, the levels of active enzyme produced were approximately threefold lower than those measured at 25 degrees C. Overexpression of native CspA had no effect on the accumulation of active preS2-S'-beta-galactosidase from the cspA promoter. It is therefore unlikely that CspA acts as it own positive inducer. Our results indicate that the cspA promoter can efficiently substitute for the tac system at 25 degrees C and may be particularly valuable for the expression of highly aggregation-prone or unstable gene products at 10 degrees C.

Amino Acid Sequence↗
PubMedDate not supplied

Analysis of promoter sequences from Lactobacillus and Lactococcus and their activity in several Lactobacillus species.

Promoter-active fragments were isolated from the genome of the probiotic organism Lactobacillus rhamnosus strain GG using the promoter-probe vector pNZ272. These promoter elements, together with a promoter fragment isolated from the vaginal strain Lactobacillus fermentum BR11 and two previously defined promoters (Lactococcus lactis and Lactobacillus acidophilus ATCC 4356 slpA), were introduced into three strains of Lactobacillus. Primer-extension analysis was used to map the transcriptional start site for each promoter. All promoter fragments tested were functional in each of the three lactobacilli and a purine residue was used to initiate transcription in most cases. The promoter elements encompassed a 52- to 1,140-fold range in promoter activity depending on the host strain. Lactobacillus promoters were further examined by surveying previously mapped sequences for conserved base positions. The Lactobacillus hexamer regions (-35: TTgaca and -10: TAtAAT) closely resembled those of Escherichia coli and Bacillus subtilis, with the highest degree of agreement at the -10 hexamer. The TG dinucleotide upstream of the -10 hexamer was conserved in 26% of Lactobacillus promoters studied, but conservation rates differed between species. The region upstream of the -35 hexamer of Lactobacillus promoters showed conservation with the bacterial UP element.

Animals↗
PubMed2003

Targeting gene expression of the mouse uroplakin II promoter to human bladder cells.

Differential expression of the desired gene product in the target tissue is central to the concept of gene therapy. One approach is to use a tissue-specific promoter to drive therapeutic genes. To investigate the feasibility of tissue-specific gene therapy for bladder cancer using the mouse uroplakin II (UPII) promoter and its transcriptional control, the efficacy of this promoter as well as fragments in regulating gene expression were qualitatively and quantitatively analyzed in bladder and non-bladder tissue cell lines using DNA transfection. Our results demonstrate that the mouse UPII promoter actively drives gene expression in BIU-87, a bladder cancer cell line. Little promoter activity was detected in the non-bladder tissue cell lines. Furthermore, deleting the 5' end 1.5 kb of the UPII promoter by PCR, the activity was significantly decreased but was bladder-specific. However, deleting the 3' end 143-bp of the UPII promoter, the activity was hardly detected in any tissue cell lines. The activity of the 3' end 143-bp of the UPII promoter was detected in both bladder cancer and stomach cancer cell lines. These data demonstrate that the mouse UPII promoter has a high activity in human bladder cells and a low basal activity in human non-bladder cells. This suggests that targeting the gene expression of the mouse UPII promoter could be used to treat human bladder cancer. The enhancer was contained in the region of the 1.5 kb of the 5' end of the mouse UPII promoter. The core promoter was located in the region of the 143 bp of the 3' end.

Animals↗
PubMed2005

The sequence upstream of the -10 consensus sequence modulates the strength and induction time of stationary-phase promoters in Escherichia coli.

We constructed a library of synthetic stationary-phase promoters for Escherichia coli. For designing the promoters, the known -10 consensus sequence, as well as the extended -10 region, and an A/T-rich region downstream of the -10 region were kept constant, whereas sequences from -37 to -14 were partially or completely randomised. For detection and selection of stationary-phase promoters, green fluorescent protein (GFP) with enhanced fluorescence was used. To establish the library, 33 promoters were selected, which differ in strength from 670 to more than 13,000 specific fluorescence units, indicating that the strength of promoters can be modulated by the sequence upstream of the -10 region. DNA sequencing revealed a preferential insertion of nucleotides depending on the position. By expressing the promoters in an rpoS-deficient strain, a special group of stationary-phase promoters was identified, which were expressed exclusively or preferentially by RNA polymerase holoenzyme Esigma(s). The DNA sequence of these promoters differed significantly in the region from -25 to -16. Furthermore, it was shown that the DNA curvature of the promoter region had no effect on promoter strength. The broad range of promoter activities make these promoters very suitable for fine-tuning of gene expression and for cost-effective large-scale applications in industrial bioprocesses.

Base Sequence↗