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PubMed2005

Libraries of synthetic stationary-phase and stress promoters as a tool for fine-tuning of expression of recombinant proteins in Escherichia coli.

Due to their induction characteristics stationary-phase promoters have a great potential in biotechnological processes for the production of heterologous proteins on a large-scale. In order to broaden the utility of stationary-phase promoters in bacterial expression systems and to create novel promoters induced by metabolic conditions, a library of synthetic stationary-phase/stress promoters for Escherichia coli was constructed. 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, while sequences from -37 to -14 were partially or completely randomized. For detection and selection of stationary-phase promoters GFP with enhanced fluorescence was used. The expression pattern of the GFP reporter system was compared with that of the LacZ reporter system. To screen and characterize colonies containing stationary-phase/stress promoters a bioinformatic approach was developed. In total, 33 promoters were selected which cover a broad range of promoter activities and induction times indicating that the strength of promoters can be modulated by partially randomizing the sequence upstream of the -10 region. The induction ratio of synthetic promoters at the transition from exponential to stationary-phase was from 4 to over 6000 and the induction time relative to the entrance into stationary-phase from -1.4 to 2.7 h. Ninety-one percentage of the promoters had no or only low background activity during exponential growth. The broad variability of the promoters offers good possibilities for fine-tuning of gene expression and for applications in industrial bioprocesses.

Escherichia coli Proteins↗
PubMed2005

Identification of two functional estrogen response elements complexed with AP-1-like sites in the human insulin receptor gene promoter.

This study was designed to explore the possible existence and location of estrogen response elements (EREs) in the human insulin receptor (hIR) gene promoter. Transfections of U-937 cells with the reported plasmids phIR(-1819)-GL2, phIR(-1473)-GL2, and phIR(-876)-GL2, that contain the -1819 to -271 bp fragment of the hIR promoter (wild-type promoter) and progressive 5' deletions of this promoter, revealed that while the activity of the wild-type promoter, was repressed 36% by treatment with 17beta-estradiol (E(2)), the activities of progressive 5' deletions of this promoter were reduced by 26% and by 0%, by this hormone. This suggests that E(2) needs the wild-type promoter for full transcriptional repression of this gene and it also suggests the presence of putative EREs in the region between -1819/-877 bp of this promoter. To identify these EREs we performed a computer search, using the SEQFIND programme developed in our laboratory, by homology with the consensus vit-ERE (5'GGTCAnnnTGACC3') of the Xenopus vitellogenin A(2) gene promoter. The results of our search indicated no sequence identical to this consensus ERE, and neither was any sequence found to show 9 or 8 of the 10 bases of this consensus in this promoter. Nevertheless, a putative hIR ERE1 (5'AGTGAaacTGGCC3') showing 7 bases of the consensus vit-ERE, and 10 bases of the optimal binding sequence ERE (5'CA/GGGTCAnnnTGACCT/CG3'), was identified between -1430/-1418bp of the hIR promoter. An AP-1-like site was covering the 3' half-element of this ERE; another AP-1-like site was overlapping the first AP-1-like site, and finally a third AP-1-like site was located beside to the 5' half-element. In addition, another putative hIR ERE2 (5'GCTCCtagCAAAC3') showing 5 bases of the consensus vit-ERE, and 9 bases of the optimal binding sequence ERE, was located upstream of the hIR promoter, between -1567/-1555 bp. An AP-1-like site was located downstream of the 3' half-element of this ERE, and another AP-1-like site was beside the 5' half-element. EMSA analysis using nuclear extracts of E(2)-treated cells and natural sequences, including these putative EREs, indicated that ERbeta - the only isoform expressed in U-937 cells - specifically recognized both EREs because ERbeta-DNA complexes were efficiently competed by the corresponding unlabelled probe and supershifted by the anti-human ERbeta (L-20) antibody. These data provide the first identification of EREs complexed with AP-1-like sites in the hIR promoter, which account for the transcriptional repression of the hIR gene mediated by ERbeta in U-937 cells.

Animals↗
PubMed2000

A tyrosine hydroxylase-neurofilament chimeric promoter enhances long-term expression in rat forebrain neurons from helper virus-free HSV-1 vectors.

Helper virus-free herpes simplex virus (HSV-1) plasmid vectors are attractive for neural gene transfer, but a promoter that supports neuronal-specific, long-term expression is required. Although expression from many promoters is unstable, a 6.8-kb, but not a 766-bp, fragment of the tyrosine hydroxylase (TH) promoter supports long-term expression. Thus, 5' upstream sequences in this promoter may enhance expression. In this study, we evaluated expression from vectors that contain 5' upstream sequences from this promoter (-0.5 to -6.8 kb) inserted at the 5' end of either a neurofilament heavy subunit (NF-H) promoter or the cytomegalovirus (CMV) immediate early promoter. The TH-NFH promoter supported expression for 6 months in the striatum, 2 months in the hippocampus, and for 1 month in both perirhinal and postrhinal cortex (the longest time points examined). Expression was targeted to neurons. The enhanced expression may require specific sequences in the TH promoter fragment because replacing this fragment with a similar sized fragment of bacteriophage lambda DNA did not enhance expression. The reverse orientation of the TH promoter fragment also enhanced expression. Insertion of insulators from the chicken beta-globin locus between the TH-NFHlac transcription unit and the vector backbone may support a modest additional enhancement in expression. Other eucaryotic sequences may also enhance expression; a S. cerevisiae (40-kb fragment)-NFH promoter enhanced expression. In contrast, the TH-CMV promoter did not enhance expression. Thus, the TH-NFH promoter may support some physiological studies that require long-term expression in forebrain neurons.

Animals↗
PubMed1998

Regulation of PAX-6 gene transcription: alternate promoter usage in human brain.

We have isolated and characterized the 5'-flanking regulatory region of the human PAX-6 gene. Mapping of transcription initiation sites revealed the existence of an additional non-coding 5' exon, exon 1A. Functional analyses indicated that PAX-6 transcription is regulated by two distinct promoters, A and B, resulting in alternative transcription of exon 1A or 1B and joint transcription of exons 2 to 13. While a single initiation site was identified for exon 1A, transcription of exon 1B appears to be initiated from more than one site downstream of the promoter B-associated TATA motif. Multiple potential binding sites for transcription factors were found in the regions of promoter A and B. Although a 1.1-kb fragment of promoter A and a 1.5 kb fragment of promoter B, which had been fused to a reporter gene and transiently expressed in cell lines, displayed constitutive promoter activity, transcription of PAX-6 driven by promoter B was considerably higher than by promoter A in various regions of human postmortem brain. Transcript PAX-6B was primarily expressed in cerebellar cortex, whereas relatively low concentrations were detected in other brain areas. Functional dissection by serial deletions revealed several clusters of both activating elements and cell-selective silencers within the regulatory regions upstream of exon 1A and 1B. Coexpression of the promoter B constructs with a vector expressing PAX-6 modulated promoter B activity, thus indicating autoregulation by PAX-6 transcription. In conclusion, our findings suggest that PAX-6 transcription is regulated by alternate usage of promoter A and B, and that in adult human brain expression of PAX-6 is primarily controlled by promoter B. Alternate promoter usage and differential PAX-6 transcription are likely to play a critical role in brain development and neuroplasticity.

3T3 Cells↗
PubMed2006

T7 RNA polymerase-induced bending of promoter DNA is coupled to DNA opening.

To initiate transcription, T7 RNA polymerase (RNAP) forms a specific complex with its promoter DNA and melts several base pairs near the initiation site to form an open complex. Previous gel electrophoresis studies have indicated that the promoter DNA in the initiation complex is bent [Ujvari, A., and Martin, C. T. (2000) J. Mol. Biol. 295, 1173-1184]. Here we use fluorescence resonance energy transfer (FRET) to investigate the conformation of promoter DNA in the closed and open complexes of T7 RNAP. We have used steady state and time-resolved fluorescence approaches to measure the FRET efficiency in a doubly dye-labeled duplex promoter and in a premelted bubble promoter. Changes in the FRET efficiency and hence the DNA end-to-end distance changes are small when the duplex promoter forms a complex with T7 RNAP. On the other hand, FRET changes are relatively larger when the bubble promoter binds T7 RNAP or when initiating nucleotides are added to the duplex promoter-T7 RNAP complex. The shortening of DNA end-to-end distances is indicative of DNA bending in the bubble DNA complex and in the duplex promoter complex with the initiating nucleotides. Our results are consistent with the model in which in the absence of initiating nucleotides there is a distribution of closed and open complexes, and the promoter DNA is bent slightly by <40 degrees in the closed complex but bent more sharply by 86 degrees in the open complex. The energetics of DNA bending suggests that a significant part of the available free energy from promoter and polymerase interactions is utilized in DNA bending and/or untwisting. We propose that promoter opening occurs spontaneously upon DNA bending and/or untwisting as free energy is gained through interactions of the melted promoter with the T7 RNAP active site.

2-Aminopurine↗
PubMed1999

Structure and promoter/leader deletion analysis of mirabilis mosaic virus (MMV) full-length transcript promoter in transgenic plants.

A full-length transcript (FLt) promoter fragment was isolated from a genomic clone of mirabilis mosaic virus (MMV), a double-stranded DNA plant pararetrovirus belonging to the caulimovirus family. The boundaries required for maximal promoter expression were defined by 5' and 3' deletion analysis of the MMV promoter fragments coupled to a GUS reporter gene. The expression patterns of these chimeric gene constructs were evaluated both in transgenic Nicotiana tabacum cv. Samsun NN plants and in protoplast transient expression experiments. A 360 bp FLt promoter fragment (sequence -297 to +63 from the transcription start site) was found sufficient for strong promoter activity. The transcription start site (TSS) of the MMV FLt promoter was determined by primer extension analysis using total RNA isolated from transgenic plants containing a MMV promoter:uidA fusion gene. Analysis of the 5' and 3' deletion constructs showed that an upstream region (sequence -248 to -193 from the transcription start site) is required for the MMV FLt promoter activity along with the as-1, TATA box regions. In addition, a 31 bp sequence (+33 to +63 from the transcription start site) located downstream of a TATA box is also essential for the maximum expression of the MMV FLt promoter. Analysis of transcripts (mRNA) from these chimeric constructs also indicated that the MMV FLt promoter fragment (-297 to +63 from the transcription start site) has the highest promoter activity. In a comparative analysis the MMV FLt promoter showed much greater activity than the CaMV 35S promoter.

3' Untranslated Regions↗
PubMed2004

The strong efficiency of the Escherichia coli gapA P1 promoter depends on a complex combination of functional determinants.

The Escherichia coli multi-promoter region of the gapA gene ensures a high level of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) production under various growth conditions. In the exponential phase of growth, gapA mRNAs are mainly initiated at the highly efficient gapA P1 promoter. In the present study, by using site-directed mutagenesis and chemical probing of the RPo (open complex) formed by Esigma70 (holoenzyme associated with sigma70) RNAP (RNA polymerase) at promoter gapA P1, we show that this promoter is an extended -10 promoter that needs a -35 sequence for activity. The -35 sequence compensates for the presence of a suboptimal -10 hexamer. A tract of thymine residues in the spacer region, which is responsible for a DNA distortion, is also required for efficient activity. We present the first chemical probing of an RPo formed at a promoter needing both a -10 extension and a -35 sequence. It reveals a complex array of RNAP-DNA interactions. In agreement with the fact that residue A-11 in the non-template strand is flipped out in a protein pocket in previously studied RPos, the corresponding A residue in gapA P1 promoter is protected in RPo and is essential for activity. However, in contrast with some of the previous findings on RPos formed at other promoters, the -12 A:T pair is opened. Strong contacts with RNAP occur both with the -35 sequence and the TG extension, so that the sigma4 and sigma2 domains may simultaneously contact the promoter DNA. RNAP-DNA interactions were also detected immediately downstream of the -35 hexamer and in a more distal upstream segment, reflecting a wrapping of RNAP by the core and upstream promoter DNA. Altogether, the data reveal that promoter gapA P1 is a very efficient promoter sharing common properties with both extended -10 and non-extended -10 promoters.

Base Sequence↗
PubMed1994

Members of the CAAT/enhancer-binding protein, hepatocyte nuclear factor-1 and nuclear factor-1 families can differentially modulate the activities of the rat alpha-fetoprotein promoter and enhancer.

The promoter of the rat alpha-fetoprotein (AFP) gene, which makes the expression of the developmentally regulated AFP gene specific to the liver, is a putative target for transcription factors of the CAAT/enhancer-binding protein (C/EBP), hepatocyte nuclear factor-1 (HNF-1) and nuclear factor-1 (NF-1) families. We have evaluated the influence of these factors on the activity of the AFP promoter by transfection of HepG2 hepatoma cells with the appropriate expression vector plus a CAT plasmid under the control of the AFP promoter. A similar plasmid bearing the rat albumin promoter was used as a control. C/EBP alpha, C/EBP beta and D-binding protein (DBP) acted as trans-activators on the AFP promoter, whereas liver inhibitory protein (LIP), a truncated form of C/EBP beta, was a potent negative regulator of the promoter. C/EBP alpha also bound to and stimulated the activity of the AFP enhancer at -2.5 kb. Interestingly, HNF-1 beta was found to be more potent than HNF-1 alpha in activating the AFP promoter. This effect was specific, as it did not occur with the rat albumin promoter. HNF-1 beta, which is produced earlier than HNF-1 alpha during liver development, would thus have the greater influence on the AFP promoter in early development. Both HNF-1s allowed expression of the AFP promoter in cells of nonhepatic origin. Overexpression of NF-1 induced a specific decrease in the activity of the AFP promoter. This strongly suggests that competition between NF-1 and HNF-1 for binding to their overlapping binding sites on the AFP promoter is critical for modulating its activity. Thus changing combinations of these trans-acting factors may tightly modulate the AFP promoter activity in the course of liver development and carcinogenesis.

Animals↗
PubMed1999

Isolation and characterization of a novel promoter for the bovine growth hormone receptor gene.

The use of alternative promoters represents an important mechanism for the regulation of growth hormone receptor (GHR) gene expression. Two promoters have been isolated previously for the GHR gene: the P1 promoter that drives liver-specific expression, and the P2 promoter that drives ubiquitous expression. In the present study, we isolated a third GHR promoter termed P3. The P3 promoter was GC-rich and TATA-less. The P3 promoter was able to drive the expression of a luciferase reporter gene in cell lines Hep G2, PLC/PRF/5, and BHK-21. In vivo, the P3 promoter initiated transcription from two major sites in exon 1C of the GHR gene in many tissues. In the adult bovine liver, the P3-transcribed GHR mRNA represented only 10% of the total GHR mRNA pool. In non-hepatic tissues such as kidney, skeletal muscle, mammary gland, and uterus, P3-transcribed GHR mRNA represented 30-40% of the total GHR mRNA pool. Within the bovine GHR gene, the P3 promoter was located immediately downstream from the P2 promoter. In transfected cells, the P2 promoter served as an enhancer for the P3 promoter. Existence and co-regulation of two ubiquitous promoters may be a mechanism for achieving a high level of expression of the GHR gene in multiple tissues.

Animals↗
PubMed1999

PU.1 and USF are required for macrophage-specific mannose receptor promoter activity.

In the current study we report the isolation of 854 base pairs of the rat mannose receptor promoter. Analysis of the sequence revealed one Sp1 site, three PU.1 sites, and a potential TATA box (TTTAAA) 33 base pairs 5' of the transcriptional start site. The tissue specificity of the promoter was determined using transient transfections. The promoter was most active in the mature macrophage cell line NR8383 although the promoter also showed activity in the monocytic cell line RAW. No activity was observed in pre-monocytic cell lines or epithelial cell lines. Mutation of the TTTAAA sequence to TTGGAA resulted in a 50% decrease in activity in transient transfection assays suggesting that the promoter contains a functional TATA box. Using electrophoretic mobility shift assays and mutagenesis we established that the transcription factors Sp1, PU.1, and USF bound to the mannose receptor promoter, but only PU.1 and USF contributed to activation. Transient transfections using a dominant negative construct of USF resulted in a 50% decrease in mannose receptor promoter activity, further establishing the role of USF in activating the rat mannose receptor promoter. Comparison of the rat, mouse, and human sequence demonstrated that some binding sites are not conserved. Gel shifts were performed to investigate differences in protein binding between species. USF bound to the rat and human promoter but not to the mouse promoter, suggesting that different mechanisms are involved in regulation of mannose receptor expression in these species. From these results we conclude that, similar to other myeloid promoters, transcription of the rat mannose receptor is regulated by binding of PU.1 and a ubiquitous factor at an adjacent site. However, unlike other myeloid promoters, we have identified USF as the ubiquitous factor, and demonstrated that the promoter contains a functional TATA box.

Animals↗
PubMed2001

The chromatin structure of the dual c-myc promoter P1/P2 is regulated by separate elements.

The proto-oncogene c-myc is transcribed from a dual promoter P1/P2, with transcription initiation sites 160 base pairs apart. Here we have studied the transcriptional activation of both promoters on chromatin templates. c-myc chromatin was reconstituted on stably transfected, episomal, Epstein-Barr virus-derived vectors in a B cell line. Episomal P1 and P2 promoters showed only basal activity but were strongly inducible by histone deacetylase inhibitors. The effect of promoter mutations on c-myc activity, chromatin structure, and E2F binding was studied. The ME1a1 binding site between P1 and P2 was required for the maintenance of an open chromatin configuration of the dual c-myc promoters. Mutation of this site strongly reduced the sensitivity of the core promoter region of P1/P2 to micrococcal nuclease and prevented binding of polymerase II (pol II) at the P2 promoter. In contrast, mutation of the P2 TATA box also abolished binding of pol II at the P2 promoter but did not affect the chromatin structure of the P1/P2 core promoter region. The E2F binding site adjacent to ME1a1 is required for repression of the P2 promoter but not the P1 promoter, likely by recruitment of histone deacetylase activity. Chromatin precipitation experiments with E2F-specific antibodies revealed binding of E2F-1, E2F-2, and E2F-4 to the E2F site of the c-myc promoter in vivo if the E2F site was intact. Taken together, the analyses support a model with a functional hierarchy for regulatory elements in the c-myc promoter region; binding of proteins to the ME1a1 site provides a nucleosome-free region of chromatin near the P2 start site, binding of E2F results in transcriptional repression without affecting polymerase recruitment, and the TATA box is required for polymerase recruitment.

Base Sequence↗
PubMedDate not supplied

Species-specific regulation of the alpha-2(I) procollagen gene by proximal promoter elements.

Transcriptional regulation of the human alpha 2(I) procollagen proximal promoter involves the interaction of trans-acting factors at the inverted CCAAT box (G/CBE) located at position -80 and an adjacent GGAGGCCC-box at -70. Both these elements have previously been shown to be essential for activity of the human promoter. This study investigated nucleotide differences at three sites (-74, -72 and -71) between the human and mouse promoters that were sufficient to abolish trans-acting factor binding with the mouse sequence (GGAGACGT). Two distinct DNA-protein interactions were detected on the human -107/+54 promoter fragment while a single interaction was observed at the equivalent mouse promoter. One of these factors is the CCAAT-binding factor (CBF) and it's binding was observed on both the human and mouse promoters. Although the GGAGGCCC DNA-binding element was not detected on the mouse promoter, GGAGGCC-binding proteins were present in mouse nuclear extracts as observed by their interaction with the human promoter. Functional analysis of the human and mouse -343/+54 and -107/+54 promoter regions revealed significant differences between species; the human constructs having higher activity than the mouse. The differences in promoter activity between species may in part be a result of the nucleotide differences in the GGAGGCCC-box. Mutations in this region of the human -107/+54 promoter prevented DNA-protein interaction and lowered promoter activity. These results support the hypothesis that the GGAGGCCC-box in the human alpha 2(1) procollagen promoter has a regulatory function and that there exists a species-specific difference in transcription factor binding and regulation of the gene.

Amino Acid Motifs↗
PubMed2000

Sustained expression of human apolipoprotein A-I after adenoviral gene transfer in C57BL/6 mice: role of apolipoprotein A-I promoter, apolipoprotein A-I introns, and human apolipoprotein E enhancer.

Elevation of HDL cholesterol, after adenoviral apolipoprotein A-I (apo A-I) gene transfer, may delay or revert ischemic cardiovascular disease, provided transgene expression is persistent. Previously, we observed transient human apo A-I expression after adenoviral gene transfer with a cytomegalovirus (CMV)-driven construct containing the human apo A-I cDNA. Therefore, the effects of promoters (CMV or 256 base pairs of the human apo A-I promoter), introns of the human apo A-I gene, and the liver-specific human apolipoprotein E (apo E) enhancer on adenovirus-mediated human apo A-I expression were evaluated in C57BL/6 mice. In the presence of the CMV promoter, human apo A-I introns prolonged expression above 20 mg/dl from 14 to 35 days. Addition of one, two, or four copies of the human apo E enhancer in these constructs resulted in a copy-dependent but transient increase in expression for 14 days. The apo A-I promoter induced 3.2-fold lower peak levels of human apo A-I than did the CMV promoter, but insertion of four apo E enhancers in the apo A-I promoter-driven construct resulted in human apo A-I levels above 20 mg/dl for 6 months. The decline between day 6 and day 35 of human apo A-I expression driven by the CMV promoter was due to (1) a 2.5-fold decline in transgene DNA levels that is not observed with apo A-I promoter-driven constructs, and (2) CMV promoter attenuation as evidenced by a 7.6-fold decline in the human apo A-I mRNA/human apo A-I DNA copy number ratio between day 6 and day 35. Hepatotoxicity, as evidenced by up to 10-fold higher serum levels of transaminases on day 6 after gene transfer with CMV promoter-driven constructs than with apo A-I promoter-driven constructs, probably caused the accelerated decline of transgene DNA. In conclusion, gene transfer with an adenovirus comprising the 256-bp apo A-I promoter, the genomic apo A-I DNA, and four apo E enhancers, all of human origin, is associated with low hepatotoxicity and with the absence of promoter shutoff resulting in human apo A-I expression above 20 mg/dl for up to 6 months.

Adenoviridae↗
PubMed1999

Adeno-associated virus 2-mediated transduction and erythroid lineage-restricted expression from parvovirus B19p6 promoter in primary human hematopoietic progenitor cells.

Human parvovirus B19 gene expression from the viral p6 promoter (B19p6) is restricted to primary human hematopoietic cells undergoing erythroid differentiation. We have demonstrated that expression from this promoter does not occur in established human erythroid cell lines in the context of a recombinant parvovirus genome (Ponnazhagan et al. J Virol 69:8096-8101, 1995). However, abundant expression from this promoter can be readily detected in primary human bone marrow cells (Wang et al. Proc Natl Acad Sci USA 92:12416-12420, 1995; Ponnazhagan et al. J Gen Virol 77:1111-1122, 1996). In the present studies, we investigated the pattern of expression from the B19p6 promoter in primary human bone marrow-derived CD34+ HPC undergoing differentiation into myeloid and erythroid lineages. CD34+ cells were transduced with recombinant adeno-associated virus 2 (AAV) vectors containing the beta-galactosidase (lacZ) gene under the control of the following promoters/enhancers: the cytomegalovirus promoter (vCMVp-lacZ), B19p6 promoter (vB19p6-lacZ), B19p6 promoter with an upstream erythroid cell-specific enhancer element (HS-2) from the locus control region (LCR) from the human beta-globin gene cluster (vHS2-B19p6-lacZ), and the human beta-globin gene promoter with the HS-2 enhancer (vHS2-beta p-lacZ). Transgene expression was evaluated either 48 h after infection or following erythroid differentiation in vitro for 3 weeks. Whereas high-level expression from the CMV promoter 48 h after infection diminished with time, low-level expression from the B19p6 and the beta-globin promoters increased significantly following erythroid differentiation. Furthermore, in HPC assays, there was no significant difference in the level of expression from the CMV promoter in myeloid or erythroid cell-derived colonies. Expression from the B19p6 and the beta-globin promoters, on the other hand, was restricted to erythroid cell colonies. These data further corroborate that the B19p6 promoter is erythroid cell-specific and suggest that the recombinant AAV-B19 hybrid vectors may prove useful in gene therapy of human hemoglobinopathies in general and sickle cell anemia and beta-thalassemia in particular.

Anemia, Sickle Cell↗
PubMed1998

Heart-specific targeting of beta-galactosidase by the ventricle-specific cardiac myosin light chain 2 promoter using adenovirus vectors.

Adenoviruses are attractive vectors for gene transfer into cardiac muscle. However, their promiscuous tissue tropism, which leads to an ectopic expression of the transgene, is a considerable limitation. To restrict expression to cardiomyocytes, we have constructed two recombinant adenoviruses (Ad-MLC2-250betagal and Ad-MLC2-2100betagal) containing the beta-galactosidase reporter gene under the control of the 250- or 2100-bp rat ventricle-specific cardiac myosin light chain-2v promoter (MLC-2v). Our in vitro and in vivo data have evidenced that the 2100-bp promoter allows stronger beta-galactosidase activity than the 250-bp promoter and that the deleted promoter allows a weak beta-galactosidase expression in skeletal muscle-derived cells in vitro. In contrast to the in vitro results, the highly deleted MLC-2v promoter of 250 pb conserved its heart specificity in in ovo and in vivo when introduced into the adenovirus genome, indicating that the specificity of this promoter is neither altered by the inverted terminal repeat nor by the enhancer of the Ela promoter, both of which located in the 5' flanking region of the promoter. Systemic injections of both recombinant adenoviruses into chicken embryos showed beta-galactosidase expression mainly in the right ventricle of the heart. We have confirmed the cardiac specificity of both promoters in mammalian species after injection of both recombinant adenoviruses into the heart of adult rats in vivo. The comparison of both promoters in vitro and in vivo has shown that the 250-bp MLC-2v promoter is 80% less active than the 2100-bp MLC-2v promoter and has enabled us to conclude that the MLC-2v promoter of 2100 bp is the most appropriate for efficient expression of a reporter gene or a therapeutic cardiac gene (e.g., SERCA2a or minidystrophin gene).

Adenoviridae↗
PubMed1991

Promoters with the octamer DNA motif (ATGCAAAT) can be ubiquitous or cell type-specific depending on binding affinity of the octamer site and Oct-factor concentration.

Immunoglobulin (Ig) gene promoters contain the octamer sequence motif ATGCAAAT which is recognized by cellular transcription factors (Oct factors). Besides the ubiquitous Oct-1 factor, there is also a group of related factors (Oct-2 factors) encoded by a separate gene. The Oct-2 gene is regulated in a cell-type specific manner, and the protein is present in large amounts in B lymphocytes. We have previously shown that simple composite promoters of an octamer/TATA box type are poorly active in non-B cells but are strongly responsive to ectopic expression of Oct-2A factor, a major representative of the lymphocyte Oct-2 factors. In the present study we have tested the activity of a number of composite promoters and natural Ig promoters, and their response to Oct-1 and Oct-2 factors. Unexpectedly, we find that octamer/TATA promoters with a high affinity octamer site direct ubiquitous expression. By contrast, promoter constructions that behave in a B cell-specific manner tend to have a weak octamer binding site. These promoters are responsive to ectopic expression of additional Oct-factor, irrespective of whether it is Oct-1 or Oct-2. Using natural Ig promoters rather than composite promoters, we find that an IgH promoter is well transcribed in non-B cells via the ubiquitous Oct-1 factor, while Ig kappa and Ig lambda light chain promoters require additional Oct factor for maximal expression. It seems therefore likely that during B cell differentiation, Ig heavy chain promoters can be activated by Oct-1, before the appearance of Oct-2 factors. Oct-2 factors then would serve to boost the expression from Ig light chain promoters, which are known to be activated only after successful heavy chain gene rearrangement.

Autoradiography↗
PubMed1994

Bidirectional transcription from the human immunoglobulin VH6 gene promoter.

The human immunoglobulin (Ig) heavy chain VH6 gene promoter contains an imperfect octamer (AgGCAAAT) and is not dependent on the Ig heavy chain enhancer for activity; reporter constructs containing this promoter are very active in non-B cells. In experiments designed to characterize regions upstream of the transcriptional start site that are important for promoter function, we produced a series of deletion constructs, including one containing sequences between -74 and -146. Surprisingly, this fragment had promoter activity in both orientations. Inspection of the VH6 promoter sequence indicated that there was a possible TATA box in the proper orientation upstream of the imperfect octamer. The -74 to -146 fragment functioned as a promoter in the reverse orientation in three B cell lines and in non-B (HeLa) cells, with a much higher level of activity seen in the HeLa cells. To determine if the promoter could work in both directions simultaneously, reporter genes were positioned up- and downstream of a VH6 promoter fragment. Reporter gene activity was found for both genes in B cells and HeLa cells. Using a reverse transcriptase-polymerase chain reaction procedure (RT-PCR), we found a transcript corresponding to sequences upstream of the VH6 promoter in RNA from both the lymphoblastoid cell line ML-1, which actively transcribes the VH6 promoter, and the REH cell line, which does not. No transcripts were found in the KB epithelial cell line. Two or three mRNA 5' ends were found that mapped between -137 to -143 from the authentic VH6 transcription site, 31-37 nucleotides upstream of the putative TATA box. Inspection of the sequence upstream of the VH6 promoter demonstrated the presence of an open reading frame capable of coding for 96 amino acids. The VH6 promoter represents the second Ig promoter with bidirectional activity.

Amino Acid Sequence↗
PubMed2003

Vertebrate TBP-like protein (TLP/TRF2/TLF) stimulates TATA-less terminal deoxynucleotidyl transferase promoters in a transient reporter assay, and TFIIA-binding capacity of TLP is required for this function.

The TBP-like protein (TLP/TRF2/TLF), which belongs to the TBP family of proteins, is present in all metazoan organisms. Although the human TLP has been reported to interfere with transcription from TATA-containing promoters, the transcription activation potential of TLP in higher animals is obscure. We previously demonstrated that artificially promoter-recruited TLP behaves like an unconventional transcriptional activator. In this study, we investigated the effects of TLP on TATA-less promoters of mouse and human terminal deoxynucleotidyl transferase (TdT) genes by transient reporter assays. As expected, TLP repressed both basal and activator-augmented transcription from the TATA-containing adenovirus major late promoter (MLP) and E1B promoter. On the other hand, however, TLP significantly stimulated both basal and activated transcription from TdT promoters. We investigated the strength of the promoters in chicken DT40 cells that lack the TLP gene. The MLP showed higher activity but the TdT promoter showed lower activity in TLP-null cells than in the wild-type cells. Moreover, ectopic expression of mouse TLP in the TLP-null cells considerably stimulated the TdT promoter. Insertion of a TATA element upstream from the TdT core promoter resulted in a loss of TLP-mediated activation. The mouse TLP was demonstrated to bind specifically to TFIIA with greater strength than TBP. We constructed mutated TLPs having amino acid substitutions that impair TFIIA binding. A representative TLP mutant lacking TFIIA-binding ability could not stimulate transcription from the TdT promoter, whereas that mutation suppressed TLP-mediated transcription repression of TATA promoters. The results of the present study suggest that the vertebrate TLP potentiates exogenous TATA-less promoters and that TFIIA plays an important role in the TLP function.

Animals↗