Insertion Of Promoter Research Articles

Expression of the pyrG gene determines the pool sizes of CTP and dCTP in Lactococcus lactis.

The pyrG gene from Lactococcus lactis encodes CTP synthase (EC 6.4.3.2), an enzyme converting UTP to CTP. A series of strains were constructed with different levels of pyrG expression by insertion of synthetic constitutive promoters with different strengths in front of pyrG. These strains expressed pyrG levels in a range from 3 to 665% relative to the wild-type expression level. Decreasing the level of CTP synthase to 43% had no effect on the growth rate, showing that the capacity of CTP synthase in the cell is in excess in a wild-type strain. We then studied how pyrG expression affected the intracellular pool sizes of nucleotides and the correlation between pyrG expression and nucleotide pool sizes was quantified using metabolic control analysis in terms of inherent control coefficients. At the wild-type expression level, CTP synthase had full control of the CTP concentration with a concentration control coefficient close to one and a negative concentration control coefficient of -0.28 for the UTP concentration. Additionally, a concentration control coefficient of 0.49 was calculated for the dCTP concentration. Implications for the homeostasis of nucleotide pools are discussed.

Evidence for contribution of an internal ribosome entry site to intercellular transport of a tobamovirus.

Previously, it has been shown that tobacco mosaic virus (TMV) U1 and crucifer-infecting TMV contain a 75 nt internal ribosome entry site (IRES) upstream of movement protein (MP) gene (IRES(U1)(MP,75) and IRES(CR)(MP,75), respectively). A movement-deficient TMV mutant, KK6, has been constructed previously [Lehto, K., Grantham, G. L. & Dawson, W. O. (1990). Virology 174, 145-157] by insertion of the second coat protein subgenomic promoter (CP SGP-2) upstream of the MP gene, in addition to the natural CP SGP-1. Here, the authors compare the efficiency of movement function expression by KK6 and a derivative, K86, obtained by insertion of IRES(CR)(MP,75) between the CP SGP-2 and MP genes resulting in restoration of IRES(CR)(MP,75) function in the 5'-untranslated sequence of the I(2) subgenomic RNA of K86. The data indicate that the efficiency of K86 movement was largely restored by this insertion, which was apparently due to the translation-enhancing ability of IRES(CR)(MP,75).

Disruption of the Mechanism of Long Range Activation within the Human α-Globin Complex

The human alpha-globin complex lies at the tip of the short arm of chromosome 16. It comprises three functional globin genes (5'-zeta2-alpha2-alpha1-3'), the expression of which is strictly dependent on a positive regulatory element located 40-kb upstream, HS-40. This DNase I-hypersensitive site is the only known regulatory element displaying strong erythroid-specific enhancer activity within the human alpha-globin complex. How this enhancer activity is shared among different erythroid genes present in the same cluster without affecting the ubiquitous genes present within and around the complex is poorly understood. To address this issue, we used hybrid murine erythroleukemia cells containing a single copy of human chromosome 16 and targeted the insertion of different sequences downstream of HS-40 by recombinase-mediated cassette exchange. We thus demonstrate that (i). HS-40-mediated erythroid-specific activation of the alpha-globin genes is impaired solely by the insertion of a promoter sequence and not a coding sequence, unless it is methylated, and that (ii). the degree of transcriptional repression observed seems to be related directly to the transcriptional rate of the inserted promoter. Taken together, these results emphasize the importance of promoter sequences as the main targets for the activation mechanism of the human alpha-globin genes by HS-40.

Sp1-dependent regulation of the RTP801 promoter and its application to hypoxia-inducible VEGF plasmid for ischemic disease.

Gene therapy using vascular endothelial growth factor (VEGF) is a new potential treatment of ischemic disease. To be safe and effective, VEGF expression should be enhanced locally in ischemic tissue. In this study, we identified the cis-regulatory element for the hypoxia induction of the RTP801 promoter. In addition, pRTP801-VEGF was evaluated as a therapeutic plasmid in vitro. The cis-regulatory element for hypoxia induction was identified by deletion and mutation analyses. Antisense oligonucleotide co-transfection assay was performed to evaluate the role of Sp1. pRTP801-VEGF was constructed by the insertion of the RTP801 promoter into the VEGF plasmid. The hypoxia-inducible expression of VEGF was evaluated by in vitro transfection assay. In luciferase assay, the region between -495 and -446 was responsible for the hypoxia-induced transcription. The mutation of the Sp1 site in this region reduced hypoxia-induced transcription. In addition, co-transfection with antisense Sp1 oligonucleotide suggests that hypoxia induction of the RTP801 promoter is mediated by Sp1. In vitro transfection showed that pRTP801-VEGF had higher VEGF expression than pEpo-SV-VEGF. In addition, VEGF expression by pRTP801-VEGF was induced under hypoxia. With strong basal promoter activity and induction under hypoxia, pRTP801-VEGF may be useful for gene therapy for ischemic disease.

193. Hypoxia-Inducible VEGF Gene Therapy Using the RTP801 Promoter

Background; The angiogenic gene therapy using VEGF is a new potential treatment for ischemic disease. Gene expression in ischemic tissue must be locally enhanced and regulated to be safe and effective. Previously, it was reported that the RTP801 promoter was strongly induced under hypoxic conditions. This strong induction of the RTP801 promoter suggests its applicability to the regulation of VEGF gene in the VEGF gene therapy. In this study, we evaluated the role of the RTP801-VEGF plasmid as a therapeutic plasmid in vitro and in vivo study. Method; To identify the cis-regulatory element, the RTP801 promoter was analyzed by various methods. pRTP801-VEGF was constructed by the insertion of the RTP801 promoter upstream of the VEGF cDNA. pEpo-SV-VEGF and pSV-VEGF were used as positive controls and WSLP only injection without plasmid was used as a negative control the in vivo model. In vitro cell transfection study, polyethylenimine (PEI, 25,000 Da) was used as a gene carrier and 293T cells were used. In an in vivo VEGF expression study, we used water-soluble lipopolymer (WSLP) as a gene carrier. The circumflex artery of a New Zealand white rabbit was ligated with a 6-0 Prolene suture and WSLP-plasmid complex (150 ug/750 ul) was injected into the infarcted myocardium using a 30-guage needle. The hearts were harvested 4 days after the injections and homogenized in lysis buffer. The expression level of the VEGF gene was measured by ELISA. Result; In luciferase assay, the region between -495 and -445 was responsible for the hypoxia-induced transcription. In this region, there was a potential Sp1 binding element. The mutation of the Sp1 site reduced the hypoxia-induced transcription of the RTP801 promoter. In addition, co-transfection with antisense Sp1 oligonucleotide decreased the promoter activity. These results suggest that the hypoxia induction of the RTP801 promoter is mediated by Sp1. The pRTP801-VEGF had stronger VEGF expression than pEpo-SV-VEGF, which contained the Epo (erythropoietin) enhancer and the SV 40 promoter system. In addition, the VEGF expression by pRTP801-VEGF was induced under hypoxic condition. In the animal study, the expression level of VEGF by pRTP801-VEGF in the ischemic myocardium was 5.7 times higher than controls (p < 0.001) and 2.5 times higher than the pSV-VEGF (p < 0.01) and 2 times higher than the pEpo-SV-VEGF (p < 0.05). Conclusion; Therefore pRTP801-VEGF will be useful for the VEGF gene therapy for ischemic heart disease with strong induction under hypoxic condition.

Novel integrase-defective lentiviral episomal vectors for gene transfer.

High levels of circular viral extrachromosomal DNA (E-DNA) are normally produced after infection with integration-competent and -incompetent lentiviruses. Although E-DNA has been shown to be transcriptionally active, it lasts for only a short time in replicating cells. Here, we report an integrase (IN)-defective lentiviral episomal vector in which insertion of the simian virus 40 (SV40) promoter, containing the origin of replication (ori), is associated with long-term expression and persistence of E-DNA in the presence of SV40 large T antigen (TAg) from 293T cells. 293 and 293T cell lines transduced with IN-competent lentiviral vectors expressing green fluorescent protein (GFP) or luciferase from the cytomegalovirus (CMV) or SV40 promoter gave similar levels of transduction and expression. In contrast, only transient reporter expression occurred when using the CMV IN-defective control vector in both 293 and 293T cells. However, reporter gene expression was maintained for more than 8 weeks in 293T, but not 293, cells transduced with the IN-defective lentiviral vector containing the SV40-ori promoter. Polymerase chain reaction for two-long terminal repeat (2LTR) extrachromosomal circular forms, a marker of lentiviral E-DNA, and fluorescence in situ hybridization analysis confirmed the persistence and episomal nature of circular E-DNA up to 60 days after transduction. Taken together, these results indicate that insertion of the SV40-ori promoter in a lentiviral vector contributes to long-term expression by promoting episomal replication when TAg is provided in trans. Lentiviral episomal vectors may serve as specific tools for therapeutic approaches to diseases, particularly those associated with episomal replication of DNA viruses including papillomaviruses, polyomaviruses, and herpesviruses.

Hepatitis B Virus-Based Vectors Allow the Elimination of Viral Gene Expression and the Insertion of Foreign Promoters

It has recently been shown that hepatitis B virus (HBV)-based vectors are suitable for a hepatocyte specific gene transfer. As candidate vectors for gene therapy, however, they should no longer express any viral gene products. In addition, the insertion of promoters that do not originate from HBV is needed to allow a variation of the level of gene expression. Furthermore, production of high-titer stocks is required. To eliminate viral gene expression, we knocked out all HBV open reading frames (ORFs) in the transfer construct used to express recombinant HBV RNA. To minimize the chance of homologous recombination, we generated an improved helper construct. With these constructs, recombinant viruses containing single or combined knockouts of the viral ORFs were produced at titers equal to wild-type HBV produced in parallel. We identified a site in the HBV genome that allows insertion of foreign promoter elements without interfering with maturation of infectious HBV particles. Successful gene transfer was demonstrated on infection of primary human hepatocytes using recombinant HBV in which all viral ORFs were knocked out and a foreign promoter controlled transgene expression. These improvements represent a major step toward the development of HBV vectors as candidates for human gene therapy.

Insertion of an exogenous promoter in the E1A regulatory region of adenovirus does not disturb viral replication despite reduced E1A transcription

Insertion of an exogenous promoter into adenoviral regulatory regions may result in altered specificity and activity of the integrated promoter-mediated transcription in the context of recombinant adenoviruses (Ad). The alteration is due to the influence of the viral regulatory elements. Specificity of oncolytic Ad, in which the E1A expression is designed to be controlled by a tumor-specific promoter, could thus be modulated by the Ad E1A enhancer/promoters. We prepared recombinant Ad bearing a midkine (MK) promoter region in the 3′-side of the E1A promoter and investigated the relationship between transcriptional activity by the E1A promoter-fused MK fragment and the viral replication. Reporter assays revealed that the transcriptional activity of the fused E1A-MK fragment was significantly lower than that of respective E1A and the MK promoters. However, the replication of the Ad bearing the MK promoter was greater than or comparable to that of wild-type Ad. The present study suggests that the replication of oncolytic Ad in tumors is not directly correlated with the promoter activity to transcribe the E1A gene and that insertion of an exogenous promoter downstream to the E1A regulatory region may not always disturb Ad replication.

Dynamic Alterations of Replication Timing in Mammalian Cells

Background: The eukaryotic genome is divided into distinct replication timing domains, which are activated during S phase in a strictly conserved order. Cellular differentiation can alter replication timing in some loci, but recent experiments yielded conflicting data regarding the relationship between gene expression and replication timing. The genetic and epigenetic determinants of replication timing in mammalian cells have yet to be elucidated.Results: We developed a mammalian experimental system in which the timing of DNA replication can be altered in a controlled manner. This system utilizes sequences from the human β-globin locus that exhibit orientation-dependent transcriptional silencing when inserted into the murine genome. We found that before insertion, the murine target site replicated late during S phase. After insertion, replication timing depended on the orientation of the transgene. In a transcription-permissive orientation, the transgene and flanking sequences replicated early. In the reverse (silencing-prone) orientation, these sequences replicated late. Early replication correlated with histone modifications of the transgene chromatin but could be observed in the absence of the β-globin promoter. Importantly, the replication timing switch did not require a replication origin within the transgene.Conclusions: Transgene insertions into mammalian heterochromatin can alter the timing of DNA replication at the insertion site. This differentiation-independent replication timing switch did not necessitate insertion of an active promoter or a replication origin. These observations suggest that the timing of DNA replication can be manipulated by changes in DNA sequence, but that the determinants of replication timing are distinct from the sequences that specify replication initiation sites.

Transcriptional tools for aging research

The process of aging is controlled and influenced by genes and gene expression. Dissecting the roles individual genes play in aging requires molecular tools for manipulation of gene expression in a wide variety of experimental model systems. These tools include homologous recombination-based methods for the activation and repression of gene expression, gene activation by random insertion of strong promoters, the use of engineered dominant negative mutants, inhibition with antisense oligonucleotides, minor groove binding small molecules and via RNAi and the use of engineered transcription factors to either activate or repress endogenous gene expression.

The effect of turbulence promoter on cross-flow microfiltration of skim milk

The effect of turbulence promotion on cross-flow microfiltration of skim milk was studied experimentally. The use of Kenics static mixer as turbulence promoter in inorganic (Membralox™) membranes with pore sizes 50, 100 and 200 nm provided a significant permeate flux improvement. In experiments at the same Reynolds number, the flux improvements of more than 500% were achieved for Re numbers in the range 3000–10 000. The increase of power consumption for fluid flow due to the insertion of turbulence promoter was also discussed. The effect of turbulence promoter on microfiltration performance was checked at the same hydraulic dissipated power. The flux improvements of more than 300% were achieved for hydraulic dissipated power in the range from 0.13 to 0.95 W indicating that using the turbulence promoter is more energy efficient than operation without the turbulence promoter. The fouling resistance analysis approved that resistance of the surface micellar layer dominates the overall fouling resistance. The use of turbulence promoter caused a large scouring of the membrane surface provided significant reduction of reversible fouling. Also, when the turbulence promoter was used, the membrane cleaning efficiency was improved compared with experiment without the promoter.

Fibrinogen and Factor VII Promoter Polymorphisms in Women With Preeclampsia

In Brief OBJECTIVE To determine whether genetic variability in the promoter regions of the genes encoding fibrinogen and factor VII contribute to individual differences in susceptibility to the development of preeclampsia. METHODS The study involved 133 preeclamptic and 115 healthy control pregnant women who were genotyped for the G-455A polymorphism in the β-fibrinogen gene promoter and for a decamer insertion or deletion polymorphism at position −323 in the factor VII gene promoter. We used χ2 analysis to assess genotype frequency differences between preeclamptic women and controls. RESULTS The allelic distribution of the fibrinogen A-455G polymorphism was similar in the two groups, with the frequency of the variant A allele being 18.8% in the preeclampsia group and 20.9% in the control group. We did not find any association between the presence of the factor VII insertion allele and preeclampsia (5.6% versus 6.1%). Accordingly, the genotype distribution of the fibrinogen G-455A and factor VII polymorphisms in the preeclamptic and control groups was similar (P = .852 and P = .308). CONCLUSION The G-455A polymorphism of the fibrinogen gene promoter and the decamer insertion or deletion polymorphism of the factor VII gene promoter are unlikely to be major genetic predisposing factors for preeclampsia in subjects from eastern Finland. There was no association between the G-455A polymorphism of the fibrinogen gene promoter or the decamer insertion or deletion polymorphism of the factor VII gene promoter and the occurrence of preeclampsia.

Fibrinogen and factor VII promoter polymorphisms in women with preeclampsia

OBJECTIVE:To determine whether genetic variability in the promoter regions of the genes encoding fibrinogen and factor VII contribute to individual differences in susceptibility to the development of preeclampsia.METHODS:The study involved 133 preeclamptic and 115 healthy control pregnant women who were genotyped for the G-455A polymorphism in the β-fibrinogen gene promoter and for a decamer insertion or deletion polymorphism at position −323 in the factor VII gene promoter. We used χ2 analysis to assess genotype frequency differences between preeclamptic women and controls.RESULTS:The allelic distribution of the fibrinogen A-455G polymorphism was similar in the two groups, with the frequency of the variant A allele being 18.8% in the preeclampsia group and 20.9% in the control group. We did not find any association between the presence of the factor VII insertion allele and preeclampsia (5.6% versus 6.1%). Accordingly, the genotype distribution of the fibrinogen G-455A and factor VII polymorphisms in the preeclamptic and control groups was similar (P = .852 and P = .308).CONCLUSION:The G-455A polymorphism of the fibrinogen gene promoter and the decamer insertion or deletion polymorphism of the factor VII gene promoter are unlikely to be major genetic predisposing factors for preeclampsia in subjects from eastern Finland.

Amphotropic murine leukemia virus replication in human mammary epithelial cells and the formation of cytomegalovirus-promoter recombinants.

Amphotropic murine leukemia virus (MLV) can replicate in human cells and is a potential contaminant in vector preparations for human gene transfer studies. We have recently shown that replication of amphotropic MLV in specific human sarcoma and lymphoma lines is possible in the absence of the viral 75-bp transcription enhancer elements. Here, we have tested the replication of an amphotropic MLV, MLV-(MOA), and an enhancer-deficient mutant of this virus in human breast carcinoma-derived cell lines. The proviral expression plasmids use a cytomegalovirus (CMV) promoter for the initial transcription of virus RNA. We found that all cells analyzed are permissive for replication of MLV-(MOA). Enhancer-deficient virus is unable to replicate. However, in two lines the replication defect can be rescued by the spontaneous insertion of a CMV promoter and enhancer into the U3 region. This recombinant virus MLV-(RCMV) replicates with kinetics similar to that of MLV-(MOA) but is restricted to specific cell lines. The potential formation of RCMV recombinants during MLV vector preparation must be considered.

Effect of deletion and the site of insertion in double copy anti-tat retroviral vectors: viral titres and production of anti-tat mRNA

In attempts to further develop murine leukemia virus (MLV) based retroviral vectors for gene therapy, we investigated vector production and antisense expression from retroviral constructs with U3 deletions or insertions. Promoter elements in the U3 region of the 3' LTR of the vector pLXSN were deleted and replaced with DNA encoding the HIV anti-tat gene under control of the tRNAmet promoter to produce a double copy self inactivating vector (DC-SIN). DC-SIN constructs were compared to vectors containing the anti-tat cassette inserted at 5 different sites of the U3 region (DC-insertions). Titres of DC-SIN and DC-insertion vectors were similar but approximately 10 fold lower than parental pLXSN. Cells transduced with DC-SIN and DC-insertion vectors all expressed anti-tat mRNA. Transcripts from the MLV-LTR were detected in cells transduced with DC-insertion but not DC-SIN vectors or a vector with the anti-tat cassette between CAAT and TATA boxes of the promoter, indicating inactivation of the viral promoter in the latter vectors. Cells transduced with constructs of either design showed comparable efficacy of protection against HIV challenge. Thus, no U3 insertion site was preferred for virus production. Insertion of a tRNA promoter between CAAT and TATA boxes and the DC-SIN design which would not introduce an active RNA pol II promoter into the genome are attractive for further development of safe gene therapy agents.

Replication of enhancer-deficient amphotropic murine leukemia virus in human cells.

Amphotropic murine leukemia virus (MLV) replicates in cells from various mammalian species, including humans, and is a potential contaminant in MLV vector preparations for human gene transfer studies. The generation of replication-competent virus is considered less likely with vectors that delete the viral transcription elements. This conclusion is based on data obtained in rodents, where MLV replication depends on the expression of viral genes under the control of 75-bp enhancer elements in the long terminal repeat. We demonstrate here that in some human cells replication of amphotropic MLV is possible in the absence of these enhancer elements. Replication of the enhancer-deficient virus MLV-(MOA)Delta E is observed in selected human sarcoma and B lymphoma lines and proceeds at a lower rate than that of the intact virus. No insertion of a foreign promoter or enhancer into the long terminal repeat was detected. Our data suggest the presence of a secondary enhancer element within the MLV provirus that can in selected human cells mediate virus transcription and replication in the absence of the 75-bp U3 enhancers.

New broad-host-range promoter probe vectors based on the plasmid RK2 replicon

Broad-host-range plasmid RK2-based promoter probe vectors with a known nucleotide sequence were constructed. In the absence of an upstream promoter, the expression of two tested reporter genes ( luc and lacZ) in Escherichia coli was virtually zero, while insertion of the Ptrc promoter resulted in strong inducer-dependent expression. The lacZ-based vectors were mobilized into Pseudomonas fluorescens ST, Pseudomonas putida KT2442, Sphingomonas spp. and Burkholderia spp. LB400, and expression analyses indicated that the properties observed in E. coli are maintained across the species barriers. In addition, the previously established knowledge of RK2 molecular biology allows easy manipulations of features such as plasmid copy number, further extending the application potential of the vectors.

New broad-host-range promoter probe vectors based on the plasmid RK2 replicon.

Broad-host-range plasmid RK2-based promoter probe vectors with a known nucleotide sequence were constructed. In the absence of an upstream promoter, the expression of two tested reporter genes (luc and lacZ) in Escherichia coli was virtually zero, while insertion of the Ptrc promoter resulted in strong inducer-dependent expression. The lacZ-based vectors were mobilized into Pseudomonas fluorescens ST, Pseudomonas putida KT2442, Sphingomonas spp. and Burkholderia spp. LB400, and expression analyses indicated that the properties observed in E. coli are maintained across the species barriers. In addition, the previously established knowledge of RK2 molecular biology allows easy manipulations of features such as plasmid copy number, further extending the application potential of the vectors.

Improved gfp and inaZ broad-host-range promoter-probe vectors.

A new set of broad-host-range promoter-probe vectors has been constructed. One subset contains the pVS1 and p15a replicons and confers resistance to either gentamicin or kanamycin. The other set contains the broad-host-range replicon from pBBR1 and confers resistance to kanamycin, tetracycline, ampicillin, or spectinomycin/streptomycin. Both plasmid sets are highly stable and are maintained without selection for more than 30 generations in several bacterial taxa. Each plasmid contains a promoter-probe cassette that consists of a multicloning site, containing several unique restriction sites, and gfp or inaZ as a reporter gene. The cassette is bound by transcriptional terminators to permit the insertion of strong promoters and to insulate the cassette from external transcription enabling the detection of weak or moderate promoters. The vector suite was augmented with derivatives of the kanamycin-resistant gfp promoter-probe plasmids that encode Gfp variants with different half-life times.

Construction of a promoter-rescue plasmid for Butyrivibrio fibrisolvens and its use in characterization of a flagellin promoter.

The Butyrivibrio fibrisolvens/Escherichia coli shuttle vector pBHerm has been modified to produce a plasmid (pBHE) that can be used for the identification and characterization of promoters in B. fibrisolvens. pBHE allows the insertion of a test promoter immediately upstream of a promoterless erythromycin resistance gene (ermAM). The efficacy of the pBHE plasmid in isolating and characterizing promoters was tested by inserting the flagellin gene (flaA) promoter from B. fibrisolvens OR77. Transcription of the ermAM gene from the flaA promoter was significantly higher than that observed when the ermAM gene was under the control of its own promoter. The flagelling gene of OR77 appears to be transcribed from two different promoters that produce transcripts initiating approximately 130 bp apart. Two mutant flaA promoter constructs, containing mutations in the -10 and -35 regions of either of the two putative promoter regions, showed drastic alterations in both the origin and amounts of the two transcripts produced. Mutations in either promoter affected transcription from both promoters, indicating that both regions contribute to gene expression.