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At least 937 records · Page 52Linked to original sources
PubMed1998

The TATA-less, GC-rich porcine dipeptidylpeptidase IV (DPPIV) promoter shows bidirectional activity.

Dipeptidylpeptidase IV (DPPIV) is an exopeptidase highly expressed in the brush-border membrane of the small intestine and in the proximal renal tubules. In this paper the 5'-flanking region of the DPPIV gene containing the promoter was sequenced and functionally characterized. The porcine DPPIV promoter lacks a consensus TATA-box, but contains two TATA-like sequences. Evidence for multiple transcription initiation sites was found. Different deletion constructs of the DPPIV 5'-flanking region in front of the CAT gene were analyzed for transient CAT-expression after transfection of the intestinal Caco-2 cell line. These experiments showed that a 89 base pair construct (-91 to -3 relative to the translation initiation site) is sufficient for promoter activity. In the reverse orientation this construct also stimulates transcription with a similar effectivity indicating that the DPPIV promoter has a bidirectional function. The bidirectional function was further demonstrated by the introduction of the -91 to -3 construct into the bidirectional vector system pLUC/CAT-3. In the hepatoma cell line HepG2 two selected deletion constructs (-857 to -3; -282 to -3) were analyzed in the normal orientation using the CAT gene as a reporter gene. The transfection experiments showed that deletion of the region -857 to -282 raised the promoter activity 3-fold. The GC-rich 5'-flanking region was further analyzed and we demonstrate that the DPPIV promoter contains a region with the characteristics of an unmethylated CpG island.

Animals↗
PubMed2004

STAT1 binds to the herpes simplex virus type 1 latency-associated transcript promoter.

The authors hypothesized that environmental stimuli induce cytokines that act through an intracellular cascade, which includes signal transducers and activators of transcription (STATs), to change herpes simplex virus (HSV) gene expression, thereby inducing viral reactivation. The HSV type 1 (HSV-1) latency-associated transcript (LAT) gene regulates viral reactivation within neurons via an unknown mechanism. HSV-1 deletion mutants that are missing key portions of the LAT gene, particularly the 3' region of the LAT promoter, do not reactivate normally in vivo. The authors hypothesized that STAT transcription factors may bind in this region to regulate viral reactivation. Electrophoretic mobility shift assay (EMSA) experiments were performed by incubating mouse trigeminal ganglion (TG) nuclear extracts with each of three overlapping sequences representing the 3' region of the HSV-1 LAT promoter (referred to as oligos L1, L2, and L3). The ganglionic nuclear extracts bound specifically to oligos L1 and L3, but not L2. Oligos L1 and L3 contain predicted STAT binding sequences whereas L2 does not. Specific binding to oligo L3 (including the TATA box sequence) was supershifted by incubating with anti-STAT1 antibodies, but not by incubating with anti-STAT3 or anti-STAT5a antibodies. Specific L3 binding was reduced by competing with excess unlabeled STAT1 consensus sequences. These results indicate that STAT1, probably as part of a complex, is capable of binding to the LAT promoter on or near the TATA box. Further studies are required to determine if STAT1 is required for LAT expression in vivo. This work supports the hypothesis that interferons act through STAT1 to regulate the expression of HSV-1 LAT.

Animals↗
PubMed1998

Interferon-gamma-dependent inducible expression of the human interleukin-12 p35 gene in monocytes initiates from a TATA-containing promoter distinct from the CpG-rich promoter active in Epstein-Barr virus-transformed lymphoblastoid cells.

Interleukin-12 (IL-12) production by human monocytes is stringently regulated through the inducibility of both subunits, p35 and p40, and expression of p35 mRNA is the limiting factor for the secretion of the bioactive IL-12 p70 heterodimer. Optimal induction of p35 mRNA requires priming of the monocytes by interferon-gamma (IFN-gamma), followed by brief exposure to lipopolysaccharide or other bacterial products. To investigate control of p35 gene expression, we isolated genomic clones containing the human p35 gene and determined the 5' end of the mRNA expressed in monocytes. We discovered that a unique p35 transcript is induced in monocytes that begins downstream of a consensus TATA box that lies within the 5' end of the cDNA originally cloned from Epstein-Barr virus (EBV)-transformed B cells. Analysis of p35 mRNA by Northern blotting showed that the message from monocytes is approximately 200 bases shorter than message derived from the EBV-transformed B-cell line VDS. The initiation sites downstream from the TATA box were confirmed by RNase protection and 5' RACE. The data indicate that p35 transcription can initiate from different sites depending on the cell type and that the shorter inducible transcript in monocytes is the one that accumulates after stimulation. Protein translation of these two forms may result in proteins of different sizes with potential implications for the regulation of IL-12 secretion and function.

Base Sequence↗
PubMed1997

Identification of a novel repressive element that contributes to neuron-specific gene expression.

Multiple signaling pathways are thought to control the selective expression of genes over the course of neuronal differentiation. One approach to elucidating these pathways is to identify specific cis-acting elements that serve as the final targets for these signaling pathways in neural-specific genes. We now identify a novel repressive element from the growth-associated protein 43 (GAP-43) gene that can contribute to neuron-specific gene expression by inhibiting transcription in a wide range of non-neuronal cell types. This repressive element is located downstream of the GAP-43 TATA box and is highly position-dependent. When transferred to viral promoters this element preferentially inhibits transcription in non-neuronal cells. Electrophoretic mobility shift assays show that the repressive element comprises at least two protein recognition sites. One of these is a novel sequence motif that we designate the SNOG element, because it occurs downstream of the TATA boxes of the synaptosomal-associated protein of 25 kDa and neuronal nitric oxide synthase genes, as well as the GAP-43 gene. The GAP-43 repressive element is distinct in sequence and position dependence from the repressor element 1/neuron-restrictive silencer element previously described in other neural genes and therefore is a likely target for a distinct set of signaling pathways involved in the control of neuronal differentiation.

Animals↗
PubMed1994

Characterization of the internal promoter of simian foamy viruses.

Simian and human foamy viruses (HFV and SFV), genetically related members of the spumavirus genus of retroviruses, have complex genome structures which encode the gag, pol, and env genes for virion proteins as well as additional open reading frames. One of these open reading frames is a viral transactivator, encoded by genes designated taf for SFV and bel-1 for HFV, which augments transcription directed by the long terminal repeat (LTR) through cis-acting targets in the U3 domain of the LTR. Recently, an internal transcriptional promoter has been identified in sequences within the 3' end of the HFV env gene (M. Lochelt, W. Muranyi, and R. M. Flugel, Proc. Natl. Acad. Sci. 90:7317-7321, 1993). We have demonstrated by using transient expression assays in several tissue culture cell lines and by analyzing viral transcripts in infected cells that SFV-1 from a rhesus macaque and SFV-3 from an African green monkey also encode an internal promoter in the env gene. Transcription directed by the internal promoters of SFV-1 and SFV-3 is activated by the taf-1 and taf-3 gene products, respectively, in several cell types. The importance of a TATA box for the SFV-1 internal promoter was established by site-specific mutagenesis, and the 5' ends of transcripts initiating in the internal promoter have been determined. cis-acting sequences in the SFV-1 env gene required for the response to taf-1 are contained within a 121-bp element located 5' to the TATA box in the internal promoter. This taf-1-responsive element in the internal promoter functions in a position- and orientation-independent fashion in a heterologous promoter and thus has the properties of an enhancer which depends on taf-1 activity. Alignments reveal that the SFV-1 internal promoter and the SFV-1 LTR have little sequence relatedness. Cross-transactivation studies show that the transactivators of SFV-1 and HFV function on the internal promoter and LTR of the homologous virus but not on the heterologous virus. In summary, the genomes of simian and human foamy viruses direct viral transcription through both the promoter in the LTR and an internal promoter within the env gene, and each promoter contains unique enhancer-like elements regulated by the viral transactivator.

Animals↗
PubMed1990

In vitro transcription and DNA binding characteristics of chloroplast and etioplast extracts from mustard (Sinapis alba) indicate differential usage of the psbA promoter.

The psbA gene which is differentially expressed in vivo in chloroplasts and etioplasts has an unusual promoter, containing both prokaryotic-type '-35' and '-10' elements and a sequence motif that resembles the nuclear TATA box. Single base pair substitutions were introduced into the mustard psbA promoter and the mutants were tested in transcription and DNA binding experiments, using extracts from either chloroplasts or etioplasts. Positions within the '-35' region appear to play an essential role in the chloroplast but not in the etioplast system. Altering the first or second position of the 'TATA box'-like region led to decreased psbA in vitro transcription in either plastid extract. These two mutations, however, did not affect binding of extracts to the (linear) psbA promoter fragment in gel retardation assays. Fragments carrying two other plastid promoters effectively competed psbA promoter binding of the etioplast extract, but more weakly that of the chloroplast extract. Lambda exonuclease mapping shows that the 5' border of the binding region is more upstream with the etioplast than with the chloroplast system, whereas the 3' border appears to be the same. Hence, protein(s) of the two plastid types seem to interact differently with the mustard psbA promoter in vitro and perhaps also in vivo.

Base Sequence↗
PubMed2002

Expression and promoter activation of the Rpe65 gene in retinal pigment epithelium cell lines.

PURPOSE: To examine the expression and promoter activation of the retinal pigment epithelium (RPE)-preferentially expressed Rpe65 gene in the commonly available RPE cell lines. METHODS: Reverse transcription coupled to polymerase chain reaction (RT-PCR) was performed after total RNA extraction from different RPE (ARPE-19, monkey, hTERT-RP1 and D407) and non-RPE (COS-7, HeLa, HepG2 and HS27) cell lines. Promoter activity was assayed by transient transfection of luciferase reporter constructs containing nested deletions of the 5' flanking region of the mouse Rpe65 gene. The involvement of a putative TATA box in the basal promoter expression was studied by site-directed mutagenesis in D407 cells and binding of TATA box-related transcription factors to that region was demonstrated by Electrophoretic Mobility Shift Assays (EMSA). RESULTS: Expression of the human RPE65 cDNA was observed in all the RPE cell lines tested, and in COS-7 cells (monkey RPE65 cDNA). Transient transfections of the mouse Rpe65 promoter/luciferase transgene containing nested deletions of the Rpe65 5' flanking region showed that fragments containing bases -655 to +48 and -1240 to +48 generated specific promoter activity only in the D407 cell line. A promoter fragment from -49 to +48 directed basal promoter activity in all the cell lines tested. Part of this basal activity was due to a putative TATA box that specifically binds transcription factors contained in a D407 nuclear extract. CONCLUSIONS: Although transcription of the Rpe65 gene occurs in all the tested cell lines, we find that the D407 cell line is the only one capable of directing specific mouse Rpe65 promoter activity. This limits the study of the transcriptional regulation of the mouse Rpe65 gene in vitro to this particular cell line.

Animals↗
PubMed2004

Heat capacity effects of water molecules and ions at a protein-DNA interface.

The interaction of the TATA-box binding protein from the thermophilic and halophilic archaea Pyrococcus woesei (PwTBP) with an oligonucleotide containing a specific binding site is stable over a very broad range of temperatures and ionic strengths, and is consequently an outstanding system for characterising general features of protein-DNA thermodynamics. In common with other specific protein-DNA recognition events, the PwTBP-TATA box interaction is accompanied by a large negative change in heat capacity (deltaCp) arising from the total change in solvation that occurs upon binding, which in this case involves a net uptake of cations. Contrary to previous hypotheses, we find no overall effect of ionic strength on this heat capacity change. We investigate the local contributions of site-specific ion and water binding to the overall change in heat capacity by means of a series of site-directed mutations of PwTBP. We find that although changes in the local ion binding capacity affect the enthalpic and entropic contributions to the free energy of the interaction, they do not affect the change in heat capacity. In contrast, we find remarkably large heat capacity effects arising from two particular symmetry-related mutations. The great magnitude of these effects is not explicable in terms of current semi-empirical models of heat capacity change. Previously reported X-ray crystal structures show that these mutated residues are at the centre of an evolutionarily conserved network of water-mediated hydrogen bonds between the protein and the DNA backbone. Consequently, we conclude that, in addition to water molecules buried in the protein-DNA interface that have been previously shown to influence heat capacity, bridging water molecules in a highly polar surface environment can also contribute substantially to negative heat capacity change on formation of a protein-DNA complex.

Archaeal Proteins↗
PubMed1998

Transcriptional regulation of human adrenomedullin gene in vascular endothelial cells.

Adrenomedullin (AM) is a recently identified vasodilator peptide produced in cardiovascular organs such as the heart, vascular wall, kidney and lung. Because plasma levels of AM are not different between various portions of the cardiovascular system, vascular cells are supposed to be the main source of circulating AM. To elucidate the regulatory mechanism of human AM gene expression, functional elements of 5'-flanking region of AM gene were studied in human aortic endothelial cells (HAEC). Northern blot analysis revealed considerable AM mRNA expression in cultured HAEC, and 2 transcription start sites were recognized at 21 and 25 bp downstream from the TATA box. The 1534 bp 5'-flanking region DNA inserted in the luciferase vector showed significant promoter activity when transfected into HAEC using liposome. Serial deletion of the inserted 5'-flanking region DNA length resulted in reduction in promoter activity by 41% between 110 and 90 bp and further reduction by 42% between 66 and 29 bp. The 19 bp DNA fragment without TATA box had almost no promoter activity. Gel shift assay revealed presence of HAEC nuclear proteins specifically bound to nuclear factor for interleukin-6 expression (NF-IL6) consensus sequence existing from -85 to -93 bp of the AM gene 5'-flanking region and activator protein 2 (AP-2) consensus sequence clustered from -33 to -68 bp. Furthermore, mutation of NF-IL6 consensus sequence in the 5'-flanking region resulted in 42% reduction in the expression of luciferase activity. These findings suggest that NF-IL6 and AP-2 sites in the promoter region are the functional elements in the transcriptional regulation of human AM gene in vascular endothelial cells.

Adrenomedullin↗
PubMedDate not supplied

Involvement of protein-DNA interaction in adeno-associated virus Rep78-mediated inhibition of HIV-1.

OBJECTIVE: It has been well documented by several laboratories that adeno-associated virus (AAV) is able to inhibit HIV-1 replication and gene expression. This effect has been mapped to the AAV-encoded Rep78 protein. However, the mechanism by which Rep78 is able to inhibit HIV-1 is unclear. As Rep78 is a DNA binding transcription factor, the objective of this study was to investigate where Rep78 might bind within the HIV-1 long terminal repeat (LTR) sequences and to judge the importance of this protein-DNA interaction. STUDY DESIGN/METHODS: Rep78's binding to HIV-LTR DNA was analyzed by electrophoretic mobility shift assay (EMSA). The importance of this protein-DNA interaction was analyzed using a Rep78 mutant defective for binding HIV-LTR DNA in an assay for monitoring gene expression (chloramphenicol acetyltransferase [CAT] assay). RESULTS: The preferred site for Rep78 binding was found to be adjacent to the HIV-LTR TATA box, within nt -54 to -34 relative to the site of transcription initiation. Furthermore, a Rep78 mutant with substitutions at amino acid residues 64 and 65 which was found defective for binding HIV-LTR DNA, was also found to be defective for inhibition of tat transactivated HIV-LTR gene expression. CONCLUSION: These data strongly suggest that Rep78's DNA binding ability is important for its mechanism of inhibition. Furthermore, the TATA box region of the HIV-LTR, to which Rep78 preferentially binds, is a likely target through which the inhibition takes place.

DNA, Viral↗
PubMed2000

Molecular diagnosis of a familial nonhemolytic hyperbilirubinemia (Gilbert's syndrome) in healthy subjects.

Recent research has shown that congenital nonhemolytic low grade hyperbilirubinemias in patients with Gilbert's syndrome (GS) are linked to mutations in the TATA box upstream of the uridine 5'-diphosphoglucose glucuronosyltransferase (UGT1A1) gene leading to an impaired bilirubin glucuronidation. In routine clinical practice GS patients can, however, only be suspected by exclusion of other causes of hyperbilirubinemia or substantial liver diseases. We developed a new, sensitive, convenient, and economic polymerase chain reaction (PCR) method for the rapid and reliable identification of gene polymorphisms in the TATA box of the UGT1A1 gene using fluorescence resonance energy transfer (FRET). With this procedure the genotype frequency in a cohort of 266 unrelated individuals from Southern Germany was investigated and the allelic distribution for individual genotypes was 43:45:12 for the (TA)(6)TAA:(TA)(6)TAA/(TA)(7)TAA:(TA)(7)TAA alleles, respectively. The homozygous (TA)(7)TAA genotype was strongly associated with suspected Gilbert's patients and its prevalence in our cohort of 266 Southern German individuals was 12.4%. In this cohort total mean serum bilirubin levels ranged from 5 micromol/L (wild-type 6/6 allele) to 57 micromol/L serum total bilirubin (mutant 7/7 homozygous allele). Median (interquartile range) serum total bilirubin levels were 12 (6) and 21 (13) for the homozygous wild-type and mutant allele, respectively. Our assay enables individual guidance for dose adjustment in suspected GS patients undergoing long-term drug therapies, especially if glucuronidation via UGT1A1 is a major metabolic pathway.

Adult↗
PubMed2003

Shikonins, phytocompounds from Lithospermum erythrorhizon, inhibit the transcriptional activation of human tumor necrosis factor alpha promoter in vivo.

Tumor necrosis factor alpha (TNF-alpha) contributes to the pathogenesis of both acute and chronic inflammatory diseases and has been a target for the development of new anti-inflammatory drugs. Shikonins, the naphthoquinone pigments present in the root tissues of Lithospermum erythrorhizon Sieb. et Zucc. (Boraginaceae), have been reported to exert anti-inflammatory effects both in vitro and in vivo. In this study, we evaluated the effects of shikonin and its derivatives on the transcriptional activation of human TNF-alpha promoter in a gene gun-transfected mouse skin system by using a luciferase reporter gene assay. The crude plant extract of L. erythrorhizon as well as derived individual compounds shikonin, isobutyryl shikonin, acetyl shikonin, dimethylacryl shikonin and isovaleryl shikonin showed significant dose-dependent inhibition of TNF-alpha promoter activation. Among the tested compounds, shikonin and isobutyryl shikonin exhibited the highest inhibition of TNF-alpha promoter activation and also showed significant suppression of transgenic human TNF-alpha mRNA expression and protein production. We demonstrated that shikonin-inhibitory response was retained in the core TNF-alpha promoter region containing the TATA box and a 48-bp downstream sequence relative to the transcription start site. Further our results indicated that shikonin suppressed the basal transcription and activator-regulated transcription of TNF-alpha by inhibiting the binding of transcription factor IID protein complex (TATA box-binding protein) to TATA box. These in vivo results suggest that shikonins inhibit the transcriptional activation of the human TNF-alpha promoter through interference with the basal transcription machinery. Thus, shikonins may have clinical potential as anti-inflammatory therapeutics.

Animals↗
PubMed1993

Molecular characterization of the 5'-flanking region of human genomic ETA gene.

A clone containing the promoter, the exon 1 and exon 2 of human genomic ETA gene was isolated and sequenced. The transcription initiation site and TATA box were identified at 528 bp upstream of the initiation codon and 24 bp upstream of the transcription initiation site, respectively. The 534 bp fragment containing 5'-flanking sequence and most of the exon 1 of the human ETA gene showed a promoter activity corresponding to 55% of SV40 early promoter activity when placed upstream of the luciferase gene and transfected into CHO cells. Thus, the TATA box which consists of the TAAAAA sequence is functional for transcription of the human ETA gene. The coding region of the human ETA receptor, therefore, starts from exon 2 which contained the first and the second transmembrane regions. The human ETA gene was demonstrated to be a single pair of copy by Southern blot analysis, and it was localized in chromosome 4 by analysis with a panel of human-rodent somatic cell hybrid lines.

Amino Acid Sequence↗
PubMed2006

Potential effect on cellular response to cadmium of a single-nucleotide A --> G polymorphism in the promoter of the human gene for metallothionein IIA.

Most people generally ingest cadmium in their food. Cadmium that has accumulated in tissues induces the synthesis of metallothioneins (MTs) which are metal-binding proteins that bind tightly to cadmium to inhibit its renal toxicity. Individuals whose ability to induce the synthesis of MTs is low seem likely to be particularly susceptible to the toxic effects of cadmium. In this study, we analyzed the polymorphism of the promoter region of the gene for MT-IIA, the major species of MT in humans, in 119 adult Japanese subjects. We found that about 18% of the subjects had an A --> G single-nucleotide polymorphism in the core region of the promoter near the TATA box. A reporter-gene assay using HEK293 cells showed that replacement of A by G at position -5 reduced the efficiency of the cadmium-induced transcription of the gene for MT-IIA. This single-nucleotide polymorphism inhibited the binding of nuclear proteins to the core promoter region of the gene for MT-IIA. When the promoter region upstream of the TATA box was replaced by a sequence that contained three dioxin-responsive elements, the reporter-gene assay demonstrated that the A --> G single-nucleotide polymorphism resulted in a marked reduction in the rate of dioxin-induced transcription. These results suggest that the A --> G single-nucleotide polymorphism reduces the efficiency of those aspects of the transcription of the gene for MT-IIA that are controlled by general transcription factors.

Adult↗
PubMed1998

Functional analysis of the promoters of the human red and green visual pigment genes.

PURPOSE: To delineate cis-acting DNA elements involved in the expression of the human red and green visual pigment genes and to correlate these with transcription factor binding sites. METHODS: Assays of promoter activity were accomplished by transient transfection into WERI cells. Nested deletion and block mutagenesis were undertaken to delineate critical elements. Transcription factor binding sites were determined by DNase I footprinting and electrophoretic mobility shift (EMSA) analyses. RESULTS: The human retinoblastoma cell line WERI, but not Y-79, was found to express the red and green pigment genes. Transfection assays in WERI cells revealed that the proximal region of the red pigment gene promoter had positive (-130 to -113 and -96 to -23) and negative (-190 to -130 and - 113 to -96) regulatory elements. The green pigment gene promoter was found to be 2 to 4 times more active than was that of the red pigment. This difference in activity was attributable mainly to a T to C substitution at position -3. DNase I protection and EMSA studies demonstrated the binding of several ubiquitous and WERI-enriched proteins to DNA sequences between - 130 and the TATA box. The locus control region (LCR) did not have any enhancer activity in transient transfection. CONCLUSIONS: The WERI cell line is a good model system for the analysis of gene expression of the human cone visual pigment genes. The expression of these genes in a cell-specific fashion seems to be controlled mainly by positive-acting elements in the region between - 130 and the TATA box. The higher activity of the green pigment gene promoter could have evolved to compensate for its longer distance from the activating LCR than that of the red pigment gene promoter (approximately 34 versus 3.5 kb). Although the LCR does not enhance transcription in transient transfection, it binds factors that also recognize the proximal promoter region. These interactions may be important for the establishment of a transcriptionally active domain in a chromatin context.

Base Sequence↗
PubMed1994

The E2 transcriptional repressor can compensate for Sp1 activation of the human papillomavirus type 18 early promoter.

The E6/E7 early promoter (P105) of genital human papillomavirus type 18 contains binding sites for the viral regulator E2, tandemly repeated and closely flanked by two crucial promoter elements; the TATA box downstream and an Sp1 binding site upstream. We showed that binding of purified E2 and Sp1 proteins in vitro to their neighboring sites is mutually exclusive and that Sp1 is displaced by E2. However, this displacement did not result in repression of P105 transcription. In contrast, binding of E2 to its site overlapping the Sp1 binding site activated transcription of P105 derivatives lacking the E2 site most proximal to the TATA box. Surprisingly, a truncated form of E2, deleted of part of the transactivation domain and known as the E2 transcriptional repressor, as well as the E2 DNA-binding domain alone also supported transcription of these P105 derivatives. In the context of P105, the viral E2 protein can thus activate P105 transcription in place of Sp1, even in the absence of its transactivation domain.

Base Sequence↗
PubMed1995

Isolation of chicken hsp90 beta gene promoter.

In order to define the mechanisms responsible for the differential expression of chicken hsp90 alpha and beta genes, a portion of the chicken hsp90 beta genomic sequence, including 3081 bp upstream from the transcription initiation site and 2718 bp of structural gene sequence which covers 7 exons and 6 introns was investigated. The transcriptional initiation site was determined by primer extension, RNAase and S1 nuclease mapping, Northern blot and cloning of 5' end of cDNA. The first intron, as in other hsp90 genes, is located just before the ATG initiation codon. Three Sp1 sites are located near the TATA box. The apparent major divergence with the hsp90 alpha promoter is that, in the hsp90 beta promoter, the only CAAT box and HSE element are located at about 3 and 2 kb upstream the TATA box, respectively. These features may explain why chicken hsp90 beta mRNA is generally less abundant than alpha and is not inducible by heat shock or serum/growth factor stimulation.

Animals↗
PubMed1994

Transcriptional regulation of the dihydrofolate reductase/rep-3 locus.

This review summarizes many studies that have used the mouse, hamster, or human dihydrofolate reductase locus as a model system for the study of basal or regulated transcription. This locus encodes two genes, dhfr and rep-3, that are oriented in opposite directions. The dhfr promoter and the two rep-3 promoters are highly GC-rich and do not contain consensus TATA boxes. The cis-acting elements important in basal transcription of these non-TATA box promoters have been mapped, revealing that Sp1 plays a dominant role, and that sequence elements both upstream and downstream of the initiation site contribute to transcriptional activity. Future studies will be directed toward understanding the assembly of transcription complexes on these promoters. The expression of the dhfr gene following treatment of cells with many different stimuli has been analyzed, and in many, but not all, cases the response has been at the transcriptional level. In those systems where the regulatory cis-acting element has been mapped, the E2F sites flanking the transcription initiation site play a central role in mediating the response. Future studies will be directed at characterizing the regulatory protein E2F and understanding how it interacts with the basal transcription complex at the dhfr promoter. The rep-3 gene contains two promoters that are both growth responsive: one promoter is regulated by E2F, the regulator of the other promoter has not been identified. In summary, the in-depth characterization of the 1 kb containing the dhfr and rep-3 promoters has begun to provide a detailed understanding of growth-responsive transcription.

3T3 Cells↗