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PubMed1988

Mechanism of expression of the overlapping genes of Bacillus subtilis aspartokinase II.

The mechanism of expression of the overlapping genes that encode the alpha and beta subunits of aspartokinase II of Bacillus subtilis was studied by specific mutagenesis of the cloned coding sequence. Escherichia coli or B. subtilis VB31 (aspartokinase II-deficient), transformed with plasmids carrying either a deletion of the translation start site and about one-half of the coding region for the larger alpha subunit or a frameshift mutation early in the alpha subunit coding region, produced the smaller beta subunit in the absence of alpha subunit synthesis, indicating that beta subunit is not derived from alpha subunit and that its synthesis does not depend on the alpha subunit translation initiation site. The beta subunit translation start site was identified by oligonucleotide-directed mutagenesis of the putative translation start codon. Modification of the nucleotide sequence encoding methionine residue 247 of the alpha subunit from ATG to either TTA or AAT (but not GTG) abolished beta subunit synthesis but had no effect on the production of alpha subunit. This observation is consistent with peptide chain initiation by N-formylmethionine, which specifically requires an ATG or GTG sequence, and indicates that translation of the beta subunit starts at a site corresponding to Met247 of the alpha subunit. Initial studies on the function of the aspartokinase II subunits, using E. coli as a heterologous host, showed that beta subunit was not essential for the expression of the catalytic function of aspartokinase, measured in vitro and in vivo, nor for its allosteric regulation by L-lysine. Whether the beta subunit has a function specific to B. subtilis needs to be explored in a homologous expression system.

Aspartate Kinase↗

PubMed2002

An intrinsic control element for translational initiation in class 1 integrons.

Integrons are genetic elements able to capture anti-biotic resistance and other genes and to promote their transcription. Here, we have investigated integron-dependent translation of an aminoglycoside 6'-N-acetyltransferase gene (aac(6')-Ib7) inserted at the attI1 site. N-terminal sequencing revealed that translation of this gene was initiated at a GTG codon, which is not part of a plausible translation initiation region (TIR). A short open reading frame (called ORF-11) overlapping the attI1 site was probed by site-directed mutagenesis for its contribution to aac(6')-Ib7 translation. When ORF-11 and its TIR were deleted en bloc, translational efficiency dropped by over 80%, as determined with an acetyltransferase- luciferase fusion product. Invalidation of the ATG start codon of ORF-11 or its putative Shine-Dalgarno sequence resulted in a decrease of over 60%, whereas the decrease was much less pronounced when the amino acid sequence of the putative ORF-11-encoded peptide was altered or when the distance between ORF-11 and aac(6')-Ib7 was doubled. This demonstrates that aac(6')-Ib7 translation is dependent upon the translation of ORF-11, but almost certainly not upon the corresponding peptide. These results lead us to conclude that an intrinsic short ORF present in the 5'-conserved segment of many class 1 integrons may substantially enhance expression at the translational level of captured TIR-deficient anti-biotic resistance genes.

Amino Acid Sequence↗

PubMed1999

Prevalence of core promotor and precore mutants of hepatitis B virus in thailand by RFLP and sequencing.

Hepatitis B virus has been known to frequently undergo mutations of its genome at various sites, mostly due to it employing a reverse transcriptase devoid of proofreading capacity in the course of its replication. The purpose of the present study has been to screen 257 HBsAg-positive chronic liver disease patients, more specifically 78 cases chosen at random out of those negative for HBeAg and 33 of the HBeAg-positive cases serving as controls for three discreet point mutations in the precore/core region of hepatitis B virus. To that end, HBV DNA extracted from sera was amplified by polymerase chain reaction (PCR) using semi-nested primers and subsequently subjected to restriction fragment length polymorphism (RFLP) analysis, 36 HBeAg-negative versus 30 HBeAg-positive sera, respectively, as well as to direct sequencing in some samples randomly selected to corroborate the RFLP results. Our results showed double mutations at positions 1762 and 1764 of the core promoter in between 25/36 (69.4%) and 19/25 (76%) of the sera tested, a missense mutation of the start codon in between 8/36 (22.2%), and 5/25 (20%) and a mutation turning codon 1896 into a stop codon in between 9/36 (25%) and 6/25 (24%) determined by RFLP and sequencing, respectively. These data indicate the double mutation at positions 1762 and 1764 to be the most prevalent among HBeAg-negative chronic hepatitis patients in Thailand whereas, in contrast to reports from other Asian countries, the mutation at nucleotide 1896 occurred in a mere 25%, while on the other hand the mutation abolishing the start of protein synthesis was observed to occur at a higher frequency than determined in several other geographical areas.

DNA, Viral↗

PubMed2002

Dax1 expression is dependent on steroidogenic factor 1 in the developing gonad.

The nuclear hormone receptor DAX1 has been implicated in mammalian gonad development and sex determination. The expression of the gene in the gonad follows a dynamic pattern in time and place in the embryo and the adult. We have undertaken the first in vivo study of the regulation of Dax1 expression. Using a transgenic mouse approach we have identified a novel 500-bp region 4 kb upstream of the mouse Dax1 start codon that is essential for LacZ reporter gene expression in the embryonic gonad. Within this region, a highly conserved steroidogenic factor 1 (SF1) consensus-binding site is necessary to direct LacZ expression to the embryonic gonad implicating SF1 in the regulation of Dax1 in the developing gonad. Consistent with this, Dax1 is expressed at much reduced levels in gonads of embryos that are deficient in SF1. In addition, our results show that SF1 consensus-binding sites close to the start of Dax1 transcription are important in regulating levels of expression in the developing gonad. These studies have identified the critical in vivo regulatory region for expression of Dax1 in the early gonad and provide novel information on how a specific enhancer element acts in different cell types at different stages of development.

Animals↗

PubMed1997

Site-directed mutations reveal long-range compensatory interactions in the Adh gene of Drosophila melanogaster.

Long-range interactions between the 5' and 3' ends of mRNA molecules have been suggested to play a role in the initiation of translation and the regulation of gene expression. To identify such interactions and to study their molecular evolution, we used phylogenetic analysis to generate a model of mRNA higher-order structure in the Adh transcript of Drosophila melanogaster. This model predicts long-range, tertiary contacts between a region of the protein-encoding sequence just downstream of the start codon and a conserved sequence in the 3' untranslated region (UTR). To further examine the proposed structure, site-directed mutations were generated in vitro in a cloned D. melanogaster Adh gene, and the mutant constructs were introduced into the Drosophila germ line through P-element mediated transformation. Transformants were spectrophotometrically assayed for alcohol dehydrogenase activity. Our results indicate that transformants containing a silent mutation near the start of the protein-encoding sequence show an approximately 15% reduction in alcohol dehydrogenase activity relative to wild-type transformants. This activity can be restored to wild-type levels by a second, compensatory mutation in the 3' UTR. These observations are consistent with a higher-order structure model that includes long-range interactions between the 5' and 3' ends of the Adh mRNA. However, our results do not fit the classical compensatory substitution model because the second mutation by itself (in the 3' UTR) did not show a measurable reduction in gene expression.

Alcohol Dehydrogenase↗

PubMed2005

Binding sites of VanRB and sigma70 RNA polymerase in the vanB vancomycin resistance operon of Enterococcus faecium BM4524.

The vanB operon of Enterococcus faecium BM4524 which confers inducible resistance to vancomycin is composed of the vanR(B)S(B) gene encoding a two-component regulatory system and the vanY(B)WH(B)BX(B) resistance genes that are transcribed from promoters P(RB) and P(YB) respectively. In this study, primer extension revealed transcription start sites at 13 and 48 bp upstream from the start codon of vanR(B) and vanY(B), respectively, that allowed identification of -10 and -35 promoter motifs. The VanR(B) protein was overproduced in Escherichia coli, purified and phosphorylated (VanR(B)-P) non-enzymically with acetylphosphate. VanR(B)-P and VanR(B) specifically bound to P(RB) and P(YB) promoters. VanR(B) bound at a single site at position -32.5 upstream from the P(RB) transcriptional start site and at two sites at positions -33.5 and -55.5 upstream from that of P(YB). The proximal VanR(B) binding site overlapped the -35 region of both promoters. VanR(B) was converted from a monomer to a dimer upon acetylphosphate treatment. VanR(B)-P had higher affinity than VanR(B) for its targets and appeared more efficient than VanR(B) in promoting open complex formation with P(RB) and P(YB). In the absence of regulator, E. coli RNA polymerase was able to interact with P(RB) but not with P(YB). Phosphorylation of VanR(B) significantly increased promoter interaction with RNA polymerase and led to an extended and modified footprint. In vitro transcription assays showed that VanR(B)-P activates P(YB) more strongly than P(RB). Analysis of the protected regions revealed one copy of a 21 bp sequence in the P(RB) promoter and two copies in the P(YB) promoter which may serve as recognition sites for VanR(B) and VanR(B)-P binding that are required for transcriptional activation and expression of vancomycin resistance.

Bacterial Proteins↗

PubMed1989

Nucleotide sequence of the McrB region of Escherichia coli K-12 and evidence for two independent translational initiation sites at the mcrB locus.

The McrB restriction system of Escherichia coli K-12 is responsible for the biological inactivation of foreign DNA that contains 5-methylcytosine residues (E. A. Raleigh and G. Wilson, Proc. Natl. Acad. Sci. USA 83:9070-9074, 1986). Within the McrB region of the chromosome is the mcrB gene, which encodes a protein of 51 kilodaltons (kDa) (T. K. Ross, E. C. Achberger, and H. D. Braymer, Gene 61:277-289, 1987), and the mcrC gene, the product of which is 39 kDa (T. K. Ross, E. C. Achberger, and H. D. Braymer, Mol. Gen. Genet., in press). The nucleotide sequence of a 2,695-base-pair segment encompassing the McrB region was determined. The deduced amino acid sequence was used to identify two open reading frames specifying peptides of 455 and 348 amino acids, corresponding to the products of the mcrB and mcrC genes, respectively. A single-nucleotide overlap was found to exist between the termination codon of the mcrB gene and the proposed initiation codon of the mcrC gene. The presence of an additional peptide of 33 kDa in strains containing various recombinant plasmids with portions of the McrB region has been reported by Ross et al. (Gene 61:277-289, 1987). The analysis of frameshift and deletion mutants of one such hybrid plasmid, pRAB-13, provided evidence for a second translational initiation site within the McrB open reading frame. The proposed start codon for translation of the 33-kDa peptide lies 481 nucleotides downstream from the initiation codon for the 51-kDa mcrB gene product. The 33-kDa peptide may play a regulatory role in the McrB restriction of DNA containing 5-methylcytosine.

Bacterial Proteins↗

PubMed2006

The constitutive expression of anticoagulant protein S is regulated through multiple binding sites for Sp1 and Sp3 transcription factors in the protein S gene promoter.

Protein S (PS) is a vitamin K-dependent plasma protein that inhibits blood coagulation by serving as a nonenzymatic cofactor for activated protein C in the protein C anticoagulant pathway. Low PS levels are a risk factor for the development of deep venous thrombosis. The regulation of PS levels through transcriptional regulation of the PS gene was investigated in this report. A minimal PS gene promoter 370 bp upstream from the translational initiation codon was sufficient for maximal promoter activity in transient transfections regardless of the cell type. A pivotal role for Sp1 in the constitutive expression of the PS gene was demonstrated through electrophoretic mobility shift assay experiments, transient expression of mutant PS promoter-reporter gene constructs, and chromatin immunoprecipitations in HepG2 cells. At least four Sp-binding sites were identified. The two sites most proximal to the translational start codon were found to be indispensable for PS promoter activity, whereas mutation of the two most distal Sp-binding sites had a negligible influence on basal promoter activity. In addition, all other major promoter-binding proteins that were found by electrophoretic mobility shift assay could be positively identified in supershift assays. We identified binding sites for the hepatocyte-specific forkhead transcription factor FOXA2, nuclear factor Y, and the cAMP-response element-binding protein/activating transcription factor family of transcription factors. Their relevance was investigated using site-directed mutagenesis.

Base Sequence↗

PubMed1990

Construction of Escherichia coli vectors for expression and mutagenesis: synthesis of human c-Myc protein that is initiated at a non-AUG codon in exon 1.

Three types of Escherichia coli vector for both gene expression and mutagenesis were constructed from a plasmid/phage chimera vector pUC118. Each vector contains the lac (pTD-lac), tac (pTD-tac), or T7 promoter (pTD-T7). Downstream from the promoter, these vectors have sequences in common, including a Shine-Dalgarno (SD), multiple cloning sequence, sequence-primer binding site, transcription termination signal, and M13 origin of replication. Using single-stranded circular DNA obtained by infection with helper phage, oligodeoxyribonucleotide (oligo)-directed mutagenesis allows the appropriate fusion between the vector SD sequence and the start codon in the inserted fragment. Since a complementary oligo representing a large deletion is generally used for this construction, the extra nucleotides in the opposing strand form a loop structure. Thus, we have designated this mutagenesis as 'loop-out mutagenesis'. Expression plasmid encoding the larger human c-Myc protein that is initiated at a non-AUG codon in exon 1 and its derivatives were constructed using a pTD-T7 vector. Expression experiments indicated that the wild-type (wt) protein was synthesized poorly after induction with isopropyl-beta-D-thiogalactopyranoside, while one of the derivatives, p62M1T, in which a threonine residue was added at the N terminus of the wt protein, was produced in a large quantity in E. coli.

Base Sequence↗

PubMedDate not supplied

Mitochondrial gene organization and expression in petunia male fertile and sterile plants.

In cytoplasmic male-sterile Petunia lines, NADH dehydrogenase subunit 3 (nad3) and ribosomal protein S12 (rps12) are cotranscribed with the chimeric gene pcf and located in the region of the mitochondrial genome associated with cytoplasmic male sterility (CMS) in Petunia. In fertile Petunia line 3704, the genes for nad3 and rps12 are cotranscribed with an unidentified open reading frame (orf143). In the homologous region of fertile line 3699, there is an ORF that lacks a genomic DNA-encoded stop codon; instead an RNA editing event creates a stop codon, resulting in an ORF of 161 codons. While expressed sequences homologous to this open reading frame can be detected in sterile lines, a contiguous orf143/orf161 gene does not exist in the CMS-encoding mitochondrial genome. Transcription at the CMS-associated pcf locus and the fertile orf143/nad3/rps12 locus is complex, with multiple 5' and 3' termini. The presence of the nuclear fertility restorer gene affects the abundance of a transcript class with 5' termini--121 nucleotides before the pcf start codon, and greatly reduces the abundance of a pcf gene product with apparent molecular mass of 25 kDa which is present in both vegetative and reproductive tissues of CMS plants. In addition to the 25 kDa protein product, small amounts of precursor and processed pcf products with higher molecular mass have been detected; their possible role in the CMS phenotype is unknown. Current hypotheses for the mechanism of action of CMS-associated and fertility restorer genes are discussed.

Amino Acid Sequence↗

PubMed1986

Nucleotide sequence and expression of a phosphate-regulated gene encoding a secreted hemolysin of Pseudomonas aeruginosa.

A 3.3-kilobase-pair fragment of Pseudomonas aeruginosa DNA containing the phospholipase C (heat-labile hemolysin) gene was sequenced, and the location of the gene was determined. The gene product contains at its NH2 terminus a 38-amino acid sequence which structurally resembles the signal peptides of other secreted proteins but is unusually long and positively charged (6+). The location of the translation start codon was determined by constructing a series of plasmids in which the promoter of a transcription vector was ligated to Pseudomonas DNA containing deletions at the 5' end of the gene. The plasmids were used to transform Escherichia coli, and the resulting clones were assayed for hemolysin activity. In addition, sizes of truncated proteins produced by mutants with translation terminators introduced at specific sites were analyzed in E. coli maxicells. The gene is transcribed, starting just upstream of the hemolysin gene, as an mRNA of approximately 2,800 bases. Analysis of the nucleotide sequence, analysis of mutants in maxicells, and transcriptional studies indicate that the hemolysin is part of an operon composed of two genes. Phosphate regulation of the operon is at the transcriptional level. The location of the 5' end of the transcript was determined by S1 mapping.

Amino Acid Sequence↗

PubMed1992

Replication control of plasmid R1: RepA synthesis is regulated by CopA RNA through inhibition of leader peptide translation.

The replication frequency of plasmid R1 is post-transcriptionally controlled by an antisense RNA, CopA, that binds to the leader region in the RepA mRNA, CopT, and ultimately inhibits the synthesis of the replication initiator protein RepA. We present results demonstrating that CopA controls RepA synthesis indirectly. A reading frame for a 24 amino acid leader peptide (Tap, translational activator peptide) is located in the region between the copA and repA genes. A translational fusion between the tap and lacZ genes was used to demonstrate that tap is translated and controlled by CopA. Stop codons (UAA, UAG and UGA) introduced at three different positions within the tap gene led to a severe decrease in repA expression. Specific suppression of the stop codons reversed the effect. This indicates that tap translation is required for RepA synthesis. Phylogenetic comparisons between IncFII-like plasmids, together with previous in vitro and in vivo results (Ohman and Wagner, 1989, 1991), suggest that a stable RNA stem-loop structure sequesters the repA ribosome binding site irrespective of CopA-CopT duplex formation. The results presented here show that ribosomes translating the tap reading frame have to terminate close to the start codon of repA to permit reinitiation (direct translational coupling), and that transient disruption of the inhibitory RNA stem-loop is insufficient for activation of repA translation. The possibility that direct translational coupling is required because of a suboptimal repA RBS cannot be excluded.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacterial Proteins↗

PubMed1996

Characterization of the chicken fatty acid synthase gene 5' part and promoter region.

Fatty acid synthase activity has been shown to be regulated mainly at the transcriptional level under both dietary and hormonal influences. As a first step towards elucidating the factors involved, we isolated and characterized chicken genomic clones encompassing the 5' part of the chicken fatty acid synthase gene and its flanking region. The entire region of the cloned DNA spans 30 kb, and the first three exons of the gene were mapped to a 6.3-kb genomic fragment. The transcription initiation site was determined after subcloning the cDNA which encodes the 5' end of the mRNA. The first exon, which was 129 bp long, was located approximately 5.3 kb upstream of the second exon, which contained the start codon. In the 5' flanking region, putative TATA and CAAT boxes were located 30 and 92 bp, respectively, upstream of the transcription initiation site. The 5' flanking region contained numerous sequences corresponding to consensus binding sites for transcription factors. Various lengths of flanking sequences extending up to 1028 bp upstream of the transcription initiation site and containing 100 bp of the first exon were linked to the bacterial chloramphenicol acetyltransferase gene; in this study, these constructs were analyzed in transient transfection assays in human hepatoma cells. The proximal 125-bp sequence upstream of the transcription start site was shown to be a basal promoter. The cloning and characterization of the chicken fatty-acid synthase gene provides some further insight into the regulation of fatty acid synthesis in birds as compared to mammals.

Animals↗

PubMed1992

Mutation analysis of the Cys-X2-Cys-X19-Cys-X2-Cys motif in the beta subunit of eukaryotic translation initiation factor 2.

Recessive lethal mutations in the beta subunit of eIF-2 that restore HIS4 expression in the absence of an AUG start codon were isolated from diploid Saccharomyces cerevisiae strains. DNA sequence analysis of these alleles and of eIF-2 beta suppressor alleles isolated from haploid strains, identified point mutations that altered one of six amino acids that map to a Cys-X2-Cys-X19-Cys-X2-Cys "zinc finger" motif and immediately adjacent residues. Five of the affected amino acids are identical in the human and yeast eIF-2 beta protein. Together with earlier studies (Donahue et al., 1988), these point mutations implicate the zinc finger domain of eIF-2 beta in start-site selection during the scanning process. We have supplemented the mutations obtained by genetic selection with an additional set of constructed mutations in this region. Our studies indicate that the cysteine residues and the intervening amino acids of this motif are essential for eIF-2 beta function in translation initiation in vivo. However, the effects observed in cells containing a copy of eIF-2 beta with a deletion of this motif suggest that this mutated form is still able to associate with other components of the initiation complex, imparting defects on translation initiation. Thus, this motif may be required only for later events that lead to initiator codon recognition. Alterations in defined positions, as found in our suppressor alleles, could lead to recognition of non-AUG codons.

Alleles↗

PubMed2002

Downstream codons in the retinoic acid receptor beta -2 and beta -4 mRNAs initiate translation of a protein isoform that disrupts retinoid-activated transcription.

Retinoic acid receptors (RARs) are essential for the differentiation and maintenance of normal epithelium. In studies of RARs in breast cancer, there are striking differences in the expression of certain protein isoforms of the RARbeta gene between cells derived from normal human mammary glands and those derived from breast tumors. While the protein isoforms RARbeta2 and RARbeta4 consist of the longest open reading frames of the RARbeta2 and RARbeta4 mRNAs, respectively, we find that a fraction of scanning ribosomes bypass these upstream RARbeta2 and RARbeta4 protein start codons and initiate translation downstream. This downstream translation initiation site is identical in the RARbeta2 and RARbeta4 transcripts and generates a third RARbeta protein isoform, here termed RARbeta' (formerly human RARbeta4). RARbeta' lacks protein domains found in the N terminus of RARbeta2 and RARbeta4, including one of two zinc fingers required for DNA binding. However, RARbeta' retains the ability to heterodimerize with RXRalpha and interact with transcription cofactors. In reporter gene assays, RARbeta' repressed retinoic acid-activated transcription of co-transfected RARbeta2, RARbeta4, and RARalpha. This repression required the presence of acidic amino acids within the AF2 domain. These findings demonstrate an antagonistic role for RARbeta' in signaling by retinoic acid.

Base Sequence↗

PubMed2007

RamA, the transcriptional regulator of acetate metabolism in Corynebacterium glutamicum, is subject to negative autoregulation.

The RamA protein represents a LuxR-type transcriptional activator of genes involved in acetate metabolism of Corynebacterium glutamicum. Here we analyze the expression of the respective ramA gene and its regulation. Transcription was found to start 71 nucleotides upstream of the translational start codon and to be two- to threefold up-regulated in the presence of acetate in the growth medium. Accordingly, about twofold higher amounts of RamA were observed in C. glutamicum cells grown on acetate instead of glucose. Using cell extracts of C. glutamicum and employing DNA affinity chromatography, we found RamA itself as the main protein which binds to the ramA promoter region. By electrophoretic mobility shift analysis with the ramA promoter region and His-tagged RamA protein, multiple RamA-binding sites were identified in front of the ramA transcriptional start site. Transcriptional cat fusion experiments revealed that ramA promoter activity was about threefold higher in a RamA-deficient mutant of C. glutamicum than in the wild-type, however, acetate-dependent up-regulation of ramA expression was not affected in the RamA-negative mutant. These results indicate that RamA negatively controls the expression of its own gene, but is not involved in acetate-dependent up-regulation of ramA expression.

Acetates↗

PubMed1999

Accumulation of pre-apocytochrome f in a Synechocystis sp. PCC 6803 mutant impaired in cytochrome c maturation.

Cytochrome c maturation involves heme transport and covalent attachment of heme to the apoprotein. The 5' end of the ccsB gene, which is involved in the maturation process and resembles the ccs1 gene from Chlamydomonas reinhardtii, was replaced by a chloramphenicol resistance cartridge in the cyanobacterium Synechocystis sp. PCC 6803. The resulting Delta(M1-A24) mutant lacking the first 24 ccsB codons grew only under anaerobic conditions. The mutant retained about 20% of the wild-type amount of processed cytochrome f with heme attached, apparently assembled in a functional cytochrome b(6)f complex. Moreover, the mutant accumulated unprocessed apocytochrome f in its membrane fraction. A pseudorevertant was isolated that regained the ability to grow under aerobic conditions. The locus of the second-site mutation was mapped to ccsB, and the mutation resulted in the formation of a new potential start codon in the intergenic region, between the chloramphenicol resistance marker and ccsB, in frame with the remaining part of ccsB. In this pseudorevertant the amount of holocyt f increased, whereas that of unprocessed apocytochrome f decreased. We suggest that the original deletion mutant Delta(M1-A24) expresses an N-terminally truncated version of the protein. The stable accumulation of unprocessed apocytochrome f in membranes of the Delta(M1-A24) mutant may be explained by its association with truncated and only partially functional CcsB protein resulting in protection from degradation. Our attempt to delete the first 244 codons of ccsB in Synechocystis sp. PCC 6803 was not successful, suggesting that this would lead to a lack of functional cytochrome b(6)f complex. The results suggest that the CcsB protein is an apocytochrome chaperone, which together with CcsA may constitute part of cytochrome c lyase.

Amino Acid Sequence↗

PubMed2000

Characterization of the c-specific promoter of the gene encoding human endothelin-converting enzyme-1 (ECE-1).

Human ECE-1 is expressed in four isoforms with different tissue distribution and its mRNA and protein levels are altered under certain pathophysiological conditions. To investigate the transcriptional regulation of ECE-1, we studied the regulatory region of ECE-1c, the major ECE-1 isoform. A genomic clone comprising the complete human ECE-1 gene including the putative ECE-1c-specific promoter was obtained. Up to 968 bp upstream of the putative c-specific translation initiation start codon and several serial deletion mutants were subcloned into a reporter vector and transfected into endothelial (BAEC, EA.hy926, ECV304) and epithelial (MDA MB435S, MCF7) cells, showing very strong promoter activity in comparison to the SV40 promoter and to the previously described ECE-1a and 1b promoters. Transfection of serial deletion mutants indicated two positive regulatory regions within the promoter (-142/-240 and -240/490) likely involved in binding GATA and ETS transcription factors. RNase protection assay (RPA) and 5'-RACE revealed multiple transcriptional start sites located at about -110, -140 and -350 bp. Site-directed mutagenesis demonstrated a crucial role for the E2F cis-element for basal ECE-1c promoter activity. Additionally, we found a correlation between isoform-specific ECE-1 mRNA levels and corresponding ECE-1a, 1b, 1c promoter activities.

Aspartic Acid Endopeptidases↗