Major Transcription Start Point Research Articles

Negative and positive regulation of the non-osmoregulated ompS1 porin gene in Salmonella typhi: a novel regulatory mechanism that involves OmpR.

The Salmonella typhi ompS1 gene codes for an outer membrane protein of the OmpC/OmpF porin family. It is expressed at very low levels, relative to the major porins. However, deletion analysis of the 5' regulatory region showed that the gradual removal of nucleotides -310 to -88, upstream from the P1 major transcriptional start-point, resulted in a stepwise increase in expression, reaching levels 10-fold above those for the ompC major porin gene. Hence, this 222 bp segment contains cis-acting regulatory elements involved in negative control. Primer extension analysis revealed the presence of three promoters: P1 activity was OmpR dependent; P2 was expressed at a lower level in the absence of OmpR; and P3 had a minor constitutive activity. OmpR bound preferentially to box II, an 18 bp F1/C1 canonical site, the removal (-88 to -66) of which resulted in a decrease in expression thus supporting its role in positive control. Expression of ompS1 was not induced by a set of stress conditions, including a shift in osmolarity, nor was the IHF regulator involved in negative control. An ompS1 homologue was found in E. coli K-12, which contains a nonsense codon and a shift in the reading frame, whereas Salmonella typhimurium contains an open reading frame in this region. Thus, S. typhi ompS1 provides novel features in OmpR regulation.

Characterisation of the murine gene encoding the intracellular hyaluronan receptor IHABP (RHAMM).

We have recently shown that the published cDNA sequence encoding the murine cell surface receptor for hyaluronan-mediated motility (RHAMM) in fact represents a partial sequence of the cDNA encoding a new intracellular hyaluronic acid binding protein (IHABP). Here we publish the genomic organisation, including 700bp sequences of the promoter region, of the IHABP gene. The IHABP gene consists of 18 exons and spans more than 25kb. Part of the IHABP gene is identical with the published data on RHAMM. The IHABP gene apparently possesses one promoter region with one major transcriptional start point. IHABP is ubiquitously expressed at the mRNA and the protein level in all murine tissues, suggesting that the function of this intracellular hyaluronan binding protein is not restricted to migrating cells.

Structure and function in Escherichia coli of plasmids containing pyrimidine/purine-biased stretch originated from the 5'-flanking region of the basidiomycete ras gene.

The Basidiomycete ras gene possesses a pyrimidine-rich stretch (CT-motif) with a short (7 bases) mirror repeat in which its major transcription start point is contained. To analyze the tertiary structure induced by the CT/AG-biased sequence and its effect on gene expression in supercoiled plasmids in Escherichia coli, the DNA fragment containing the ras CT/AG sequence was inserted into the EcoRI site on pBR322 in both orientations and the resulting pBR322 derivatives, named pBR-CT[ras] and pBR-invCT[ras] were introduced into E. coli strains DM800 (deltatopA gyrB225) and JM109 (topA+ gyrA96). In pBR-CT [ras] the pyrimidine-rich sequence is on the pBR322 tetracycline-resistance gene (tet)coding strand and in pBR-invCT[ras] the complementary purine-rich sequence is on this strand. DNAs of pBR-CT[ras] and pBR-invCT[ras] isolated from DM800 were frequently cleaved with single-strand-specific S1 nuclease within the CT/AG sequence, showing the formation of extended open structure. Compared with those carrying pBR322, DM800 and JM109 carrying pBR-CT [ras] showed much higher levels of tetracycline resistance (Tcr), while both strains carrying pBR-invCT[ras] showed clearly lower levels of Tcr. pBR-CT [ras] and pBR-invCT [ras], however, conferred reduced activity of beta-lactamase on DM800 and JM109. pBR-CT [ras] derivatives lacking the counterpart of the mirror repeat did not form the S1-cleavable open structure within the CT/AG sequence and conferred pBR322-like Tcr and beta-lactamase activity. The tertiary structure formed in the CT/AG sequence via the mirror repeat was suggested to affect the expressions of pBR322-tet and -bla genes.

A functional YY1 binding site is necessary and sufficient to activate Surf-1 promoter activity in response to serum growth factors.

The human Surf-1 and Surf-2 housekeeping genes are divergently transcribed and share a bi-directional, TATA-less promoter. Housekeeping promoters typically contain complex arrays of transcription factor binding sites and several studies have suggested that many of these sites might be functionally redundant. The Surf-1/Surf-2 promoter region contains four factor binding sites; members of the ETS family of transcription factors bind to two of these sites whilst YY1 binds to a third site immediately downstream of the major Surf-1 transcription start point. Here we show that Sp1 binds to the fourth transcription factor binding site. Although YY1 and Sp1 have previously been shown to interact both in vitro and in vivo, these proteins function independently at the Surf-1/Surf-2 promoter. The binding of Sp1 alone is sufficient to bring about full promoter activity in the Surf-2 direction. In contrast, both Sp1 and ETS proteins are required to bring about full promoter activity in the Surf-1 direction. The YY1 binding site is not required for basal transcription in either direction. The YY1 binding site is, however, both necessary and sufficient to confer growth factor inducibility on transcription in the Surf-1 direction. Our data suggest that functionally redundant transcription factor binding sites might not be a general feature of housekeeping promoters.

The Rat Arylalkylamine N-Acetyltransferase Gene Promoter: cAMP ACTIVATION VIA A cAMP-RESPONSIVE ELEMENT-CCAAT COMPLEX

A 10-100-fold rhythm in the activity of arylalkylamine N-acetyltransferase (AA-NAT; EC 2.3.1.87) controls the rhythm in melatonin synthesis in the pineal gland. In some mammals, including the rat, the high nocturnal level of AA-NAT activity is preceded by an approximately 100-fold increase in AA-NAT mRNA. The increase in AA-NAT mRNA is generated by norepinephrine acting through a cAMP mechanism. Indirect evidence has suggested that cAMP enhances AA-NAT gene expression by stimulating phosphorylation of a DNA-binding protein (cAMP-responsive element (CRE)-binding protein) bound to a CRE. The nature of the sites involved in cAMP activation was investigated in this report by analyzing the AA-NAT promoter. An approximately 3700-base pair fragment of the 5'-flanking region of the rat AA-NAT gene was isolated, and the major transcription start points were mapped. The results of deletion analysis and site-directed mutagenesis indicate that cAMP activation requires a CRE.CCAAT complex consisting of a near-perfect CRE and an inverted CCAAT box located within two helical turns.

Genomic characterization of the mouse homolog of the Saccharomyces cerevisiae recombination and double-strand break repair gene RAD52

The yeast Saccharomyces cerevisiae RAD52 gene is involved in recombination and DNA double-strand break repair. Recently, mouse and human homologs of the yeast RAD52 gene have been identified. Here we present the genomic organization of the mouse RAD52 gene. It consists of 12 exons ranging in size from 67 to 374 bp spread over a region of approximately 18 kb. The first ATG is located in exon 2. Analysis of the promoter region revealed no classical promoter elements such as CCAAT or TATA boxes. Transcriptional mapping analysis revealed one major transcription start point. Analogous to the situation in yeast, transcription of the RAD52 gene in human skin fibroblasts and mouse Ltk− cells was not induced by methyl methanesulfonate treatment. Furthermore, no specific alteration in human RAD52 expression levels throughout the cell cycle was observed.

Identification of a second transcriptional start site for the insecticidal protein gene cryIVA of Bacillus thuringiensis subsp. israelensis

Expression of cryIVA, one of the insecticidal protein genes of B. thuringiensis subsp. israelensis, is regulated at the transcriptional level. The cryIVA gene is specifically transcribed during the stationary phase of this bacterium. As shown in our previous report [Yoshisue et al. (1993a)], the transcription from the −364 position of the cryIVA gene is conducted by the major promoter P1 that is functional during middle stages of the stationary phase of B. thuringiensis. In the present study, we have identified a second transcriptional start point P2 for the cryIVA gene in addition to P1, the major transcriptional start point. The transcription from P2 of the cryIVA gene occurred later than that from P1, during later stages of stationary phase of B. thuringiensis subsp. israelensis. The −10 and −35 nt sequences upstream from P2 of cryIVA are similar to those of the σ 28-specific promoters of B. thuringiensis genes and of the σ K-specific promoters of B. subtilis genes. It is most likely that the region upstream from P2 of cryIVA contains the nt sequences that determine the σ 28-specific promoter, the second one, for the cryIVA gene.

Structural organization and tissue-specific expression of the gene encoding rat cysteine dioxygenase

Cysteine dioxygenase (CDO) is a key enzyme involved in the metabolism of l-cysteine. Genomic clones containing the 5′-flanking sequence of the rat CDO gene were isolated and characterized. The CDO gene spanned about 15 kb, and comprised 5 exons. All boundaries between the exons and introns matched the GT/AG rule. The major transcription start point ( tsp) was A at 213 bp upstream from the ATG codon. The 5′-flanking region contained a TATA-box-like sequence and putative cis-acting regulatory elements. The 3' end of CDO was polyadenylated at several sites. Northern blots of RNA from rat tissues revealed the highest CDO mRNA level in the liver. Significant levels were observed in the kidney, lung and brain, implying tissue-specific differences in CDO promoter function.

Structure and characterization of rat cyclin D3 promoter

To investigate the transcription initiation mechanism of the gene encoding cyclin D3, one of the major G 1 cyclins to promote the G 1/S transition, we cloned the 5′ portion of rat cyclin D3 gene and analyzed the promoter region. The major transcription start point of the gene was identified by the primer extension experiment to be 207 bp upstream from the ATG start codon and its promoter region was found to have no canonical TATA box. The DNA mobility shift assay and DNase I footprinting analysis using nuclear extracts from Nb2 cells stimulated by prolactin (PRL) have clearly revealed that the promoter possesses two binding sites for the PRL-induced transcription factors. One is located between −1 and −59 bp ( Region I) and the other between −328 and -380 bp ( Region II). The induction of transcription factors by PRL was blocked by simultaneous addition of cycloheximide, suggesting that protein synthesis was necessary for the release of these PRL-responsive transcription factors. Region I contained putative binding elements for ATF/CREB, SP1 and AP2, and Region II contained elements for a MCBF/MGF and a novel factor. These results suggest that the cyclin D3 gene is a TATA-less gene whose transcription is regulated by multiple mitogen-inducible transcription factors including an unknown factor.

Structural organization of the rae28 gene, a putative murine homologue of the Drosophila polyhomeotic gene.

A putative murine homologue of the Drosophila polyhomeotic gene, named rae28, has been isolated from a genomic library of 129/SV mouse and its structural organization has been analyzed. rae28 is a single gene of approximately 22 kb long and consists of 15 exons. Its 5'-flanking region lacks typical transcriptional regulatory sequences, such as TATA and CCAAT boxes, but contains GC-rich sequences and seven putative binding sites for a transcription factor, Sp1. One major transcription start point has been determined. The overall exon-intron organization suggested that three different Rae28 mRNAs are generated through alternative splicing. Furthermore, the rae28 gene has been located on the R-positive F3 band of mouse chromosome 6 by the direct R-banding fluorescence in situ hybridization methods.

Endoglucanase II (EGII) of Penicillium janthinellum: cDNA sequence, heterologous expression and promotor analysis.

The cDNA coding for the endoglucanase EGII of P. janthinellum was cloned and sequenced. The open reading frame comprises 1230 nucleotides and the deduced amino-acid sequence shows an overall homology of 63% with the T. reesei egl2. The cellulose-binding domain of EGII represents a typical member of the A family of cellulases. The egl2 gene is only induced by cellulose or cellobiose and not by sophorose. A promotor fragment including 1 kb was cloned and sequenced. Three major transcription startpoints were identified. Five motifs matching the binding site of the carbon-catabolite repressor CREA of A. nidulans were detected. Their potential implication in repression was analyzed by bandshift assays.

Structure and regulation of the porcine skeletal α-actin-encoding gene

The postnatal increase in skeletal α-actin (Sk-α-Act) synthesis in pigs is due, in part, to increased transcription. To characterize the factors responsible for its transcriptional regulation, we have cloned and determined the nucleotide sequence of a 5.2-kb HindIII genomic DNA fragment which contains the complete coding region of Sk-α-Act, distributed over seven exons, plus 1.9 kb of 5′ flanking region and 0.5 kb of 3′ flanking sequence. The major transcription start point ( tsp) of Sk-α-Act was determined to be 840 bp 5′ to the ATG start codon by primer extension and RNase protection analysis. To demonstrate that the Sk-α-Act promoter was functional, L6 myoblasts, C2C12 myoblasts and HeLa cells were transfected with a construct ( pPSKAFL-CAT) linking the 5′ Sk-α-Act promoter to the chloramphenicol acetyltransferase reporter gene ( cat). Cell lysates from L6 myoblasts, L6 myotubes, C2C12 myoblasts, C2C12 myotubes, and HeLa cells were analyzed for CAT activity. CAT activity was detected only in C2C12 myotubes. Thus, the porcine Sk-α-Act promoter is regulated in a developmental and cell-type specific manner

The human S3a ribosomal protein: sequence, location and cell-free transcription of the functional gene

The intron-containing gene encoding human ribosomal protein S3a (hRPS3a) was isolated by utilizing a PCR-based strategy to detect a gene-specific intron which was subsequently used as a probe for cloning of the entire gene. The h RPS3a gene is composed of six exons and five introns spanning 5013 bp. As described for other hRP-encoding genes, the promoter lacks a canonical TATA sequence and a defined CAAT box. Primer extension experiments, as well as cell-free transcription, revealed that a cytosine functions as the major transcription start point in a polypyrimidine region, but a guanosine at position -1 was also able to initiate transcription. Hybridization analysis of chromosomal DNA from a panel of human-rodent somatic cell hybrids revealed that hRPS3a is encoded by a single locus in the human genome, present on chromosome 4

A new gene, cbl, encoding a member of the LysR family of transcriptional regulators belongs to Escherichia coli cys regulon

The cbl ( cysB-like) gene has been identified in Escherichia coli. The analysis of the cloned cbl sequence revealed strict homology to an ORF of unknown function found initially in Klebsiella aerogenes [Schwacha and Bender, J. Bacteriol. 175 (1993) 2107–2115]. The predicted Cbl protein has structural features of the LysR family of transcriptional activators. It is also strongly similar to the CysB protein, the activator of the cys regulon. The position of cbl on the Ec physical map has been established at a 2070-kb (43.5 min) region between asnU and asnV. The gene is expressed in vivo as a 1-kb monocistronic transcript starting from one major transcription start point. Unexpectedly, the in vivo expression of cbl has shown dependence on CysB, belonging to the same family of proteins. The promoter region of cbl binds purified CysB protein in a manner similar to other CysB-responsive promoters. A cbl disruption mutant was constructed by insertion of a Km Rgene cartridge into the ORF on the chromosome. Phenotypes related to cbl expression suggest the involvement of the gene in an accessory regulatory circuit within the cys regulon engaging, in the last step, the function of the cysM gene encoding O-acetylserine (thiol)-lyase B.

Characterization of the murine gene encoding the hyaluronan receptor RHAMM

We describe the isolation and characterization of the murine gene encoding RHAMM, a hyaluronan receptor which regulates focal adhesion turnover, is required for cell locomotion and is a critical downstream regulator of ras transformation. The RHAMM gene spans at least 20 kb and comprises 14 exons ranging in size from 75 to 1099 bp. Primer extension studies indicate that the major transcription start point is in position -31, relative to the start Met. Northern blot analysis of mouse fibroblast RNA identified two hybridizing species of 4.2 and 1.7 kb. Comparison of cDNA clones and RT-PCR products with the genomic clones identified alternately spliced exons in both the coding and 5′ noncoding regions of RHAMM. In the coding region exon 4 is alternately spliced. The major RHAMM transcript ( RHAMM1) in 3T3 fibroblasts does not contain exon 4 and encodes a protein of 70 kDa. A minor transcript containing exon 4, namely RHAMM v4, encodes a 73-kDa protein, as demonstrated by isoform-specific antibodies. Western analysis demonstrated both a major 70-kDa (RHAMM 1) and minor 73-kDa RHAMM protein (v4) in 3T3 murine fibroblast cell lysates. The functional significance of these two isoforms is currently being investigated.

The SPR3 gene encodes a sporulation-specific homologue of the yeast CDC3/10/11/12 family of bud neck microfilaments and is regulated by ABFI

The SPR3 gene is selectively activated only during the sporulation phase of the Saccharomyces cerevisiae (Sc) life cycle. The predicted amino acid (aa) sequence has homology to microfilament proteins that are involved in cytokinesis and other proteins of unknown function. These include the products of Sc cell division cycle (CDC) genes involved in bud formation (Cdc3p, Cdc10p, Cdcl1p and Cdc12p), Candida albicans proteins that accumulate in the hyphal phase (CaCdc3p and CaCdcl0p), mouse brain-specific (H5p) and lymphocyte (Diff6p) proteins, Drosophila melanogaster (Dm) protein Pnutp (which is localized to the cleavage furrow of dividing cells), a Diff6p homologue (DmDiff6p), and the Sc septin protein (Seplhp), a homologue of the 10-nm filament proteins of Sc. One strongly conserved region contains a potential ATP-GTP-binding domain. Primer extension analysis revealed six major transcription start points (tsp) beginning at −142 relative to the ATG start codon. The sequence immediately upstream from the tsp contains consensus binding sites for the HAP2/3/4 and ABFI transcription factors, a T-rich sequence and two putative novel elements for mid to late sporulation, termed SPR3 and PAL. Electrophoretic mobility shift assay (EMSA) and footprint analyses demonstrated that the ABFI protein binds to a region containing the putative ABFI site in vitro, and site-directed mutagenesis showed that the ABFI motif is essential for expression of SPR3 at the appropriate stage in sporulating cells.

The bovine DNA polymerase β promoter: cloning, characterization and comparison with the human core promoter

The core promoter of the human DNA polymerase β (βPol)-encoding gene (POLβ) is regulated through cis-elements for the ATF/CREB protein(s), and GC box-binding and initiation-site-binding proteins. The mechanism of promoter regulation has been studied using a nuclear extract transcription system from HeLa cells [Narayan et al., J. Biol. Chem. 269 (1994) 12755–12763]. To study the homologous promoter (ppolβ) in a bovine system, we cloned and characterized the 5′-flanking region of the bovine gene (polβ). A 15.3-kb fragment of bovine genomic DNA containing the first two exons and 11 kb of 5′-flanking region was isolated from a testis library in bacteriophage λEMBL3. S1 nuclease mapping and primer extension analysis of the 5′-end of the polβ mRNA identified the major transcription start point (tsp), which is located 142-bp 5′ of the translational start codon. In transient expression assays using a bovine cell line, analysis of various 5′-deletion mutants demonstrated that a fragment of only 91-bp 5′ of the tsp had promoter activity similar to that of a 1.37-kb fragment, so that cis-elements for basal transcription are located within this approx. 100-bp core promoter, as in the human promoter (pPOLβ). Comparison of the core promoters from the bovine and human genes revealed striking similarity, including an almost precise match of the tsp, the ATF/CREB-binding and Spl-binding sites, and the spacing separating them.

Characterization of the ?promoter region? of the enolase-encoding gene enol from the anaerobic fungus Neocallimastix frontalis: Sequence and promoter analysis

The sequence of the Neocallimastix frontalis enolase gene promoter was determined up to 1800 nucleotides 5' to the major transcriptional start point. The base composition of the enolase upstream sequence revealed a very A + T-rich profile (13.5% G + C) leading to many putative hairpin structures. The functional organization of the N. frontalis enolase promoter was investigated by heterologous transient-expression assays. DNA fragments obtained by the sequential removal of sequences upstream of the translation start codon were fused to the Escherichia coli lacZ gene and the resulting plasmids were used to transform the ascomycetes Aspergillus nidulans and Penicillium roqueforti and the oomycete Saprolegnia monoica. Transient expression of the lacZ reporter gene was observed in regenerating proteoplasts of S. monoica when using the 0.3 kb or 1 kb upstream of the enolase coding region. In contrast no beta-galactosidase activity was detected in ascomycete protoplasts. DNA hybridization analysis revealed the integration of vector DNA in the genomic DNA of S. monoica and the presence of free copies of the transformation plasmid which could be rescued in E. coli. Our results indicate that the transcriptional machinery of the anaerobic chytrid N. frontalis may differ significantly from that of ascomycetes but that enough conservation exists within the lower fungi to allow a transient-driven expression of a reporter gene in an oomycete fungus.

The turkey prolactin-encoding gene and its regulatory region

Overlapping prolactin (Prl) λ clones were isolated from a turkey genomic library. The 6.7-kb turkey Prl gene consists of five exons. Major transcription start points were located by primer extension 51–53 nucleotides upstream from the Met start codon. No estrogen response element ( ERE) was found, but two regions similar to mammalian Pit-1/GHF-1-binding sites were identified by computer analysis. This suggests that transcription of the turkey Prl gene may be regulated by Pit-1/GHF-1, and not by the estrogen receptor.

The human genes encoding renin-binding protein and host cell factor are closely linked in Xq28 and transcribed in the same direction

The Xq28 chromosomal band represents a C + G-rich region onto which several genes have been mapped. In most cases, the exact relationship between the mapped genes has not yet been established, and neither the regulatory nor the spacer regions between the various transcription units have been defined. In the region around the L1CAM gene (encoding L1 cell adhesion molecule), the transcription units appear, from preliminary analyses, to be quite compact. By sequencing the region at the 3' end of the recently found host cell factor 1-encoding gene ( HCFC1), we report that the renin-binding protein-encoding gene (RBP) major transcription start point lies 2763 bp downstream from the 3 ′ end of HCFC1 and that both are transcribed in the same direction from the telomere to the centromere.