Putative TATA Box Research Articles

Genomic cloning and characterization of a PPA gene encoding a mannose-binding lectin from Pinellia pedatisecta

A gene encoding a mannose-binding lectin, Pinellia pedatisecta agglutinin (PPA), was isolated from leaves of Pinellia pedatisecta using genomic walker technology. The ppa contained an 1140-bp 5'-upstream region, a 771-bp open reading frame (ORF) and an 829-bp 3'-downstream region. The ORF encoded a precursor polypeptide of 256 amino acid residues with a 24-amino acid signal peptide. There were one putative TATA box and six possible CAAT boxes lying in the 5'-upstream region of ppa. The ppa showed significant similarity at the nucleic acid level with genes encoding mannose-binding lectins from other Araceae species such as Pinellia ternata, Arisaema hererophyllum, Colocasia esculenta and Arum maculatum. At the amino acid level, PPA also shared varying homology (ranging from 40% to 85%) with mannose-binding lectins from other plant species, such as those from Araceae, Alliaceae, Iridaceae, Lillaceae, Amaryllidaceae and Bromeliaceae. The cloning of the ppa gene not only provides a basis for further investigation of PPA's structure, expression and regulation mechanism, but also enables us to test its potential role in controlling pests and fungal diseases by transferring the gene into tobacco and rice in the future.

Genomic organization of nucleolin gene in carp fish: Evidence for several genes

The protein nucleolin, functionally involved in the main steps of ribosome biogenesis, is codified by a single copy gene in mammals. Here we report that at least three different genes codify for this protein in carp fish (Cyprinus carpio). This is the first description of the genomic organization of nucleolin in a teleost. The carp nucleolin gene includes 8.8 kb and contains 16 exons. Promoter cis regulatory elements are similar to constitutive genes, i.e., a putative TATA box, three G/C boxes, and three pyrimidine-rich boxes. As in other species, carp nucleolin gene introns host three snoRNA codifying sequences: U23 from the H/ACA family and two C/D box snoRNAs, U20 and U82. Both U20 and U82 span a complementary sequence with carp 18S rRNA. Additionally, we identified two cDNAs coding for nucleolin, confirming the existence of several nucleolin genes in carp. Amino acid-derived sequence from carp cDNAs differ from mammal protein because they span additional acidic domains at the amino end, whose functional significance remains unclear. We performed amino acid sequence comparison and phylogenetic analyses showing that the three isoforms of carp nucleolin, which we describe herein, cluster in two groups. cNUC1 probably diverges from cNUC2 and cNUC3 as result of ancestral fish-specific genome duplication, indeed C. carpio is a tetraploid fish.

PnCcp, aPhytophthora nicotianaeprotein containing a single complement control protein module, is sorted into large peripheral vesicles in zoospores

Motile zoospores are instrumental in establishing infection in many species of Phytophthora and other pathogenic oomycetes. We have identified Phytophthora nicotianae complement control protein (PnCcp), a protein that occurs in the large peripheral vesicles in Phytophthora nicotianae zoospores. Phytophthora nicotianae PnCcp contains a single complement control protein module and has homologues in P. infestans, P. sojae and P. ramorum. Sequence analysis revealed that PnCcp is a single copy, intron-less gene. Unlike the promoters of other Phytophthora genes that have been described, the PnCcp promoter contains a putative non-canonical TATA box, a CT box-containing initiator element and a downstream promoter element. PnCcp was expressed at low levels in mycelia and germinated cysts butwas significantly upregulated in sporulating hyphae and zoospores. Immunolocalisation studies in zoospores and sporangia showed that PnCcp colocalised with LPV, a high molecular weight glycoprotein that is resident in the large peripheral vesicles. However, in mycelia arising from cysts that had germinated up to 3 days earlier, the two proteins did not colocalise, suggesting that the large peripheral vesicles may contain components that are processed separately during the asexual life cycle.

Cloning and sequence analysis ofarom gene fromSclerotinia sclerotiorum

Anarom gene was cloned from genomic DNA ofScleortinia sclerotiorum by inverse PCR. The evolutionary relationships ofS. sclerotiorum and other fungi inarom gene were studied. Results showed that thearom gene from ofS. sclerotiorum has a single open reading frame of 4773 bp and does not include any introns. The derived amino acid sequence consists of 1 590 residues, and it is homologous to all fungal AROM proteins studied so far. The theoretical isoelectric point (pI) and molecular weight (Mw) is 6.5 and 172.66 kD, respectively. GC percentage of thearom gene is 44.94. According to the results of searching from CDD and Prosite database, AROM protein ofS. sclerotiorum contains five conserve domains: 3-dehydroquinate synthase domain, 3-dehydroquinate dehydratase (3-dehydroquinase) domain, shikimate 5-dehydrogenase domain, shikimate kinase domain, and-enolpyruvylshikimate-3-phosphate synthase (EPSP sythase) domain, and four motifs: two EPSP synthase signatures, dehydroquinase class I active site, shikimate kinase signature. According to the PIR Site Rule PIRSR000514-1, four functionally important amino acid residues are found by alignment. Putative TATA box and CAAT box locate separately in −23 and −77 loci in 5′ un-translated region, and two loci found in downstreamarom gene are likely polyadenylation signals. In addition, phylogeny ofarom gene is analyzed.

Purification and properties of an extracellular endo-1,4-β-xylanase from Penicillium citrinum and characterization of the encoding gene

An extracellular endo-1,4-beta-xylanase was purified from the culture filtrate of a filamentous fungus Penicillium citrinum FERM P-15944 grown on birch-wood xylan. The purified enzyme showed a single band on SDS-PAGE with an apparent M(r) of 20,000 and had an isoelectric point below 3.5. Xylanase activity was optimal at pH 5.0 and 55 degrees C. The genomic DNA and cDNAs encoding this protein were cloned and sequenced. Southern blot analysis indicated that the xylanase gene (xynA) was present as a single copy in the genome. An open reading frame of 657 bp was interrupted by two introns of 65 and 55 bp, and encoded a presumed prepropeptide of 27 amino acids and a mature protein of 190 amino acids. Three distinct transcription start points were observed at positions -20 (A), -31 (A), and -36 (A) from the start codon. The 5'-noncoding region had a putative TATA box at nt -66 (TATAAA). The xynA cDNA was functionally expressed under the control of the alcohol oxidase I gene promoter in the methylotrophic yeast Pichia pastoris. A neighbor-joining tree showed that the P. citrinum enzyme is closely related to several other fungal xylanases belonging to the glycoside hydrolase family 11: Trichoderma reesei XYN2, Aspergillus niger xynNB, Penicillium funiculosum xynC, Penicillium sp. strain 40 xynA, Chaetomium gracile cgxB, and Aspergillus nidulans xlnA and xlnB.

Tenebrio molitor antifreeze protein gene identification and regulation

The yellow mealworm, Tenebrio molitor, is a freeze susceptible, stored product pest. Its winter survival is facilitated by the accumulation of antifreeze proteins (AFPs), encoded by a small gene family. We have now isolated 11 different AFP genomic clones from 3 genomic libraries. All the clones had a single coding sequence, with no evidence of intervening sequences. Three genomic clones were further characterized. All have putative TATA box sequences upstream of the coding regions and multiple potential poly(A) signal sequences downstream of the coding regions. A TmAFP regulatory region, B1037, conferred transcriptional activity when ligated to a luciferase reporter sequence and after transfection into an insect cell line. A 143 bp core promoter including a TATA box sequence was identified. Its promoter activity was increased 4.4 times by inserting an exotic 245 bp intron into the construct, similar to the enhancement of transgenic expression seen in several other systems. The addition of a duplication of the first 120 bp sequence from the 143 bp core promoter decreased promoter activity by half. Although putative hormonal response sequences were identified, none of the five hormones tested enhanced reporter activity. These studies on the mechanisms of AFP transcriptional control are important for the consideration of any transfer of freeze-resistance phenotypes to beneficial hosts.

Use of a restriction endonuclease cytotoxicity assay to identify inducible GAL1 promoter variants with reduced basal activity

Inducible promoter fusions are commonly employed to study the biological functions of genes as well as to investigate mechanisms of transcription regulation. A concern for many studies of heterologous gene expression is that steady state transcription may be too high under non-inducing conditions, producing undesired phenotypes prior to induction. Fusions containing the galactose-inducible GAL1 promoter joined to PvuII, a bacterial DNA endonuclease gene, are toxic to yeast cells even under non-inducing conditions, i.e., in glucose media. This toxicity was utilized in conjunction with PCR-based mutagenesis of the GAL1 regulatory region to isolate mutant promoters that retained high inducibility but exhibited reduced basal level expression. The Mig1 repressor binding and putative TATA box regions were unchanged among four mutant promoters examined in detail. However, each promoter contained one or more mutations within previously identified binding sites for the Gal4 activator protein. Genetic assays developed to monitor GAL1p::I-SceI endonuclease-induced recombination demonstrated that basal expression from two of the new promoters (designated GAL1-V4 and GAL1-V10) was strongly reduced. These experiments and additional quantitative luciferase reporter gene assays demonstrate the utility of the approach for identifying promoters that permit more tightly controlled gene expression.

Molecular Cloning and Characterization of the Human AKT1 Promoter Uncovers Its Up-regulation by the Src/Stat3 Pathway

Akt1, also known as protein kinase B (PKB) alpha, is frequently activated in human cancers and has been implicated in many cell processes by phosphorylation of downstream molecules. However, transcriptional regulation of Akt1 has not been documented. Here, we report the isolation and characterization of the human AKT1 promoter and demonstrate transcriptional up-regulation of AKT1 by the Src/Stat3 pathway. Protein and mRNA levels of AKT1 are elevated in cells expressing constitutively active Stat3 as well as in v-Src-transformed NIH3T3 cells. Knockdown of Stat3 reduces AKT1 expression induced by v-Src. Although the 4.2-kb region upstream of the transcription start site of the AKT1 promoter contains five putative Stat3-binding motifs, the promoter failed to be induced by Stat3 and/or Src. Further analysis reveals that major Stat3 response elements are located within exon 1 and intron 1 regions of the AKT1 gene, which is upstream of the AKT1 translation initiation site. In addition, ectopic expression of wild type AKT1 in Stat3(-/-) MEF cells largely rescues serum starvation-induced cell death. These findings indicate that the AKT1 promoter comprises exon 1 and intron 1, in addition to the sequence upstream of transcriptional start site. Our data further show that AKT1 is a direct target gene of Stat3 and contributes to Stat3 anti-apoptotic function.

Molecular characterization of the insulin-like growth factor-I ( IGF-I) gene in channel catfish ( Ictalurus punctatus)

The insulin-like growth factor I (IGF-I) gene was characterized in channel catfish. Partial cDNA sequence, missing exon 1 and part of exon 2, was obtained in 5′- and 3′-RACE experiments. Direct sequencing of two bacterial artificial chromosome clones revealed gene structure and provided sequence from 640 bp upstream of the initiator methionine to 136 bp beyond the polyadenylation site. Genomic sequence contained a putative TATA box 506 bp upstream of the initiator methionine. The 477-bp reading frame within five exons encoded a 159-amino acid (aa) pre-propeptide highly similar to IGF-I in higher vertebrates. The sequence encoding the signal peptide was unique in catfish and contained 70% G + C content with the potential for a stable stem–loop structure. Full-length cDNA was only maintained in recombination-deficient (DH10B) strain E. coli. Levels of IGF-I mRNA were highest in liver, followed by brain and muscle, then heart and kidney ( P < 0.05). A CT/GA dinucleotide microsatellite in intron 1 was highly polymorphic in commercial channel catfish, and permitted placement of the IGF-I gene on the catfish genetic map. However, specific IGF-I alleles were not correlated with differences in growth rate from 100 to 130 days post-hatch in USDA103 line catfish.

Cloning and Characterization of an Agglutinin Gene from Arisaema lobatum

A novel agglutinin gene was cloned from Arisaema lobatum using SMART RACE-PCR technology. The full-length cDNA of Arisaema lobatum agglutinin (ala) was 1078 bp and contained a 774 bp open reading frame encoding a lectin precursor (proproprotein) of 258 amino acid residues with a 23 aa signal peptide. ALA contained three mannose-binding sites (QXDXNXVXY) with two-conserved domains of 45% identity, ALA-DOM1 and ALA-DOM2. The three-dimensional structure of ALA was very similar to that of GNA (Galanthus nivalis agglutinin). ALA shared varying identities, ranging from 40% to 85%, with mannose-binding lectins from other species of plant families, such as Araceae, Alliaceae, Iridaceae, Lillaceae, Amaryllidaceae and Bromeliaceae. Genomic sequence of ala was also cloned using genomic walker technology, and it was found to contain three putative TATA boxes and eight possible CAAT boxes in the 5'-flanking region. No intron was found within the region of genomic sequence. Southern blot analysis indicated that the ala belonged to a multi-copy gene family. Expression pattern analysis revealed that the ala preferentially expressed in the tissues with the higher expression being found in spadix, bud, leaf, spathe and tuber. The cloning of the ala gene not only provides a basis for further investigation of its structure, expression and regulation mechanism, but also enables us to test its potential role in controlling pests and fungal diseases by transferring the gene into plants in the future.

Genomic organisation and expression of the natural killer cell enhancing factor (NKEF) gene in channel catfish, Ictalurus punctatus (Rafinesque)

The gene encoding Natural Killer Cell Enhancing Factor (NKEF) was identified in clones from a gynogenetic channel catfish BAC library. NKEF gene sequence (5.2 kb) was obtained by direct sequencing of BAC clones. The catfish NKEF gene contains one non-coding exon, five coding exons and five introns. A putative TATA box and other transcription factor binding sites were identified in the promoter region. The NKEF amino acid sequence is highly conserved between fish and mammals. Gene expression, measured by real-time quantitative PCR, was detected in all major tissues with the highest level of expression in stomach and heart and lowest levels in gonad and pituitary gland. Catfish NKEF mRNA levels were slightly upregulated 8 and 24 h after injection of lipopolysaccharide. A TAA-repeat microsatellite was identified in a BAC clone containing the NKEF gene, and this locus contained at least 12 alleles in a random-bred catfish population. Multipoint linkage analysis in two reference families placed the NKEF gene on linkage group U18, 5.1 cM from locus IpCG0177 ( r = 0.05; LOD = 45.8), 6.0 cM from a novel immune type receptor, syntenic (40 cM) with T-cell receptor α, and not linked with MHC loci.

5′ Diversity of human hepatic PXR (NR1I2) transcripts and identification of the major transcription initiation site

The human pregnane X receptor (PXR) is a crucial regulator of the genes encoding several major cytochrome P450 enzymes and transporters, such as CYP3A4 and MDR1, but its own transcriptional regulation remains unclear. To elucidate the transcriptional mechanisms of human PXR gene, we first endeavored to identify the transcription initiation site of human PXR using 5'-RACE. Five types of 5'-variable transcripts (a, b, c, d, and e) with common exon 2 sequence were found, and comparison of these sequences with the genomic sequence suggested that their 5' diversity is derived from initiation by alternative promoters and alternative splicing. None of the exons found in our study contain any new in-frame coding regions. Newly identified introns IVS-a and IVS-b were found to have CT-AC splice sites that do not follow the GT-AG rule of conventional donor and acceptor splice sites. Of the five types of 5' variable transcripts identified, RT-PCR showed that type-a was the major transcript type. Four transcription initiation sites (A-D) for type-a transcript were identified by 5'-RACE using GeneRacer RACE Ready cDNA (human liver) constructed by the oligo-capping method. Putative TATA boxes were located approximately 30 bp upstream from the transcriptional start sites of the major transcript (C) and the longest minor transcript (A) expressed in the human liver. These results indicate that the initiation of transcription of human PXR is more complex than previously reported.

Characterization of a rice class II metallothionein gene: Tissue expression patterns and induction in response to abiotic factors

Data mining the complete rice genome sequences revealed a genomic fragment encoding a characteristic metallothionein (MT) protein, and its full-length cDNA was isolated from rice developing seeds by RT-PCR. This cDNA, designated OsMT-II-1a, contains an open reading frame of 264 bp encoding a protein of 87 amino acid residues. The predicted amino acid sequence was shown to have structural features characteristic of plant class II MT proteins. By sequence analysis of its 5'-flanking region, one putative TATA box, four putative CAAT boxes, and several short sequences homologous to previously reported regulatory cis-elements were identified. Northern blot analysis showed that accumulation of OsMT-II-1a mRNA is specifically abundant in developing seeds and 2-day glumes after pollination, and OsMT-II-1a transcription can markedly be induced by H2O2, paraquat, SNP, ethephon, ABA and SA, but barely by metal ions or other exogenous abiotic factors such as low temperature and PEG. These results coincide with the prediction of existing regulatory cis-elements in its 5'-flanking region. Taken together, the above results suggest that the processes of pollination and seed development might be mediated, at least in part, by expression of the OsMT-II-1a gene that is regulated by several abiotic factors.

Role of cis -Acting Sites NorL , a TATA Box, and AflR1 in nor-1 Transcriptional Activation in Aspergillus parasiticus

The transcription factor AflR is required for up-regulation of specific pathway genes involved in aflatoxin biosynthesis in the filamentous fungus Aspergillus. nor-1 encodes an early aflatoxin pathway enzyme; its promoter contains a consensus AflR binding site (AflR1). Proteins in Aspergillus parasiticus cell extracts and AflR expressed in Escherichia coli do not bind to A. parasiticus AflR1 in vitro, so it was not clear if this site was required for nor-1 expression or if other transcription factors contributed to gene regulation. In this study we defined the role of AflR1 in nor-1 expression in A. parasiticus and identified additional cis-acting sites required for maximum nor-1 transcriptional activation. Deletion and substitution of AflR1 in the nor-1 promoter in A. parasiticus nor-1::GUS reporter strains showed that this site is required for nor-1 transcriptional activation in vivo. Substitution of a putative TATA box in the nor-1 promoter resulted in nondetectable beta-glucuronidase (GUS) activity, demonstrating that this TATA box is functional in vivo. We also identified a novel cis-acting site, designated NorL, between residues -210 and -238 that was required for maximum nor-1 transcriptional activation in A. parasiticus grown in liquid medium and on solid medium. Using an electrophoretic mobility shift assay, we identified a specific NorL-dependent DNA-protein complex that relies on a functional AflR, either directly or indirectly, for maximum binding capacity. Because the NorL site appears only once in the aflatoxin gene cluster, its association with the nor-1 promoter may have important implications for the overall regulatory scheme for the aflatoxin pathway.

A Natural CYP2B6 TATA Box Polymorphism (–82T→ C) Leading to Enhanced Transcription and Relocation of the Transcriptional Start Site

We investigated the impact of promoter polymorphisms on transcription of the human CYP2B6 gene. In total, 98 DNA samples from white persons from a previously characterized liver bank were sequenced throughout 2.3 kilobases of upstream sequence and haplotype structures were determined using additional coding sequence information. HepG2 cells and primary rat and human hepatocytes were transfected with luciferase reporter gene constructs driven by 2033 base pairs (bp) of the most frequent promoter variants. The novel haplotype *22 (-1848C--> A, -801G--> T, -750T--> C, and -82T--> C) showed 3- to 9-fold enhanced transcriptional activity in all transfected cells. Constructs containing single mutations surprisingly revealed -82T--> C, predicted to disrupt a putative TATA box, to be alone responsible for this effect. In silico analysis and electrophoretic mobility shift assay demonstrated conversion of the putative TATA box into a functional CCAAT/enhancer-binding protein binding site. Analysis of transcriptional start sites showed the mutant promoter to be transcribed from a start site located approximately 30 bp downstream of the wild-type start site, consistent with the use of a noncanonical TATA box at -55 bp. Median CYP2B6 mRNA expression and bupropion hydroxylase activity as a selective marker of CYP2B6 catalytic activity were approximately 2-fold higher in livers genotyped -82TC as in those genotyped -82TT (20.4 versus 9.8 arbitrary units, p = 0.007, and 201.8 versus 106.7 pmol/mg/min, p = 0.042, respectively). This promoter polymorphism thus contributes to CYP2B6 functional variability and represents a novel mechanism by which mutations can enhance transcription. Furthermore, a detailed interspecies comparison of CYP2B promoters and transcriptional start sites provided novel insights into evolutionary relationships.

Identification and Characterization of a Functional TATA Box Polymorphism of the UDP Glucuronosyltransferase 1A7 Gene

UDP glucuronosyltransferases (UGT) detoxify bilirubin and therapeutic drugs, a process influenced by single nucleotide polymorphisms (SNPs) in their structural genes and promoter elements. UGT1A1*28 is a functional UGT promoter polymorphism associated with Gilbert's disease and severe irinotecan toxicity, which also occurs in the absence of UGT1A1*28. The aim of this study was to identify and characterize UGT promoter variants relevant for irinotecan detoxification. Recombinant UGT1A proteins were analyzed for irinotecan metabolite glucuronidation by UGT activity assays. In 427 healthy blood donors and 71 homozygous UGT1A1*28 carriers, the 5'-untranslated region of the UGT1A7 gene locus was studied. An SNP was detected by allelic discrimination and characterized by reporter gene experiments. A novel -57 T--> G SNP with a gene frequency of 0.39 in healthy blood donors was identified in the putative TATA box of the UGT1A7 gene, reducing promoter activity to 30%. It is in linkage dysequilibrium with a variant of the UGT1A7 first exon that is present in the reduced-activity UGT1A7*3 and UGT1A7*4 alleles. Homozygous UGT1A1*28 carriers simultaneously carried this variant in 97%. We identified a novel reduced-function TATA box SNP of the UGT1A7 gene that catalyzes irinotecan metabolite detoxification. Its association with variants of the UGT1A1 promoter and UGT1A7 gene may influence irinotecan metabolism. Our finding emphasizes the importance of combinations of structural and regulatory gene polymorphisms that may be useful as markers of drug toxicity.

Characterization of the rolB promoter on mikimopine-type pRi1724 T-DNA

The promoter activity of the rolB gene encoded on mikimopine-type pRi1724 ( 1724rolB) was elucidated. Reverse transcription-polymerase chain reaction (RT-PCR) demonstrated that 1724rolB mRNA was highly accumulated in roots of 1724rolB-transformed tobacco plants. A β-glucuronidase (GUS) fluorometric assay using a 1724rolB promoter-GUS construct also showed high promoter activity in the roots. A GUS histochemical assay revealed that 1724rolB was expressed in the vicinity of root apical meristematic cells of the vascular cylinder in the main roots, in lateral root primordial and meristematic cells, in veins of cotyledons, and faintly in the shoot apex. Since the product of 1724rolB has a N-terminal stretch 17 amino acid longer than the rolB of agropine-type pRi1855 ( 1855rolB), primer extension analysis was used to show that the transcription initiation point of 1724rolB was located beyond the putative TATA box of 1855rolB, suggesting that the locations of the minimal promoters of the two rolB genes differed from each other. 1724rolB promoter analysis using deletion constructs indicated that the minimal region necessary for this promoter activity was, at most, −215 bp upstream of the translation initiation point. A predominant auxin-responsive sequence was located at position −313 to −216 upstream of the transcription initiation point, containing the Nicotiana tabacum rolB domain B factor 1 (NtBBF1) binding motif ACTTTA.

Cloning and Characterization of Partial Promoter of HMGCoA Reductase from Andrographis paniculata (Burm.f.) Wall.ex Nees — A Tropical Medicinal Plant

Hydroxymethylglutaryl-CoA (HMGCoA) reductase, a key enzyme of the mevalonate pathway is known to play a key role in the biosynthesis of terpenoids. Here we describe, the cloning and characterization of hmgr1 promoter from Andrographis paniculata. A 710 bp partial promoter fragment including a putative TATA box, 5’ UTR and 5’ coding sequence of the gene was cloned by amplifying the upstream sequences using PCR with appropriate primers. Analysis of the promoter sequence against PLACE (Plant cis-acting Regulatory DNA Elements) database showed the presence of certain putative light dependent regulatory cis-elements in the sequence along with TATA and CAAT boxes. RT-PCR detected GUS transcripts in transgenic plants.

Identification, Expression and Phylogenetic Analysis of the Anticarsia gemmatalis multicapsid nucleopolyhedrovirus (AgMNPV) Helicase

The helicase gene from Anticarsia gemmatalis multicapsid nucleopolyhedrovirus (AgMNPV) was identified and localized in the 58.85-65.90 m.u. of the viral genomic map. This gene encodes a putative polypeptide of 1221 amino acids containing motifs homologous to those found in the helicase superfamily. Expression of the AgMNPV helicase was observed as early as 4h post-infection (p.i.) up until 10 h p.i. A unique early transcription initiation site was observed upstream a putative TATA box. Phylogenetic analysis of the helicase genes of 23 baculoviruses indicated that the AgMNPV helicase is closely related to the helicase genes from Epiphyas postvitanna multicapsid nucleopolyhedrovirus and Choristoneura fumiferana defective nucleopolyhedrovirus.

Characterization of the chicken inward rectifier K+ channel IRK1/Kir2.1 gene

BackgroundInward rectifier potassium channels (IRK) contribute to the normal function of skeletal and cardiac muscle cells. The chick inward rectifier K+ channel cIRK1/Kir2.1 is expressed in skeletal muscle, heart, brain, but not in liver; a distribution similar but not identical to that of mouse Kir2.1. We set out to explore regulatory domains of the cIRK1 promoter that enhance or inhibit expression of the gene in different cell types.ResultsWe cloned and characterized the 5'-flanking region of cIRK1. cIRK1 contains two exons with splice sites in the 5'-untranslated region, a structure similar to mouse and human orthologs. cIRK1 has multiple transcription initiation sites, a feature also seen in mouse. However, while the chicken and mouse promoter regions share many regulatory motifs, cIRK1 possesses a GC-richer promoter and a putative TATA box, which appears to positively regulate gene expression. We report here the identification of several candidate cell/tissue specific cIRK1 regulatory domains by comparing promoter activities in expressing (Qm7) and non-expressing (DF1) cells using in vitro transcription assays.ConclusionWhile multiple transcription initiation sites and the combinatorial function of several domains in activating cIRK1 expression are similar to those seen in mKir2.1, the cIRK1 promoter differs by the presence of a putative TATA box. In addition, several domains that regulate the gene's expression differentially in muscle (Qm7) and fibroblast cells (DF1) were identified. These results provide fundamental data to analyze cIRK1 transcriptional mechanisms. The control elements identified here may provide clues to the tissue-specific expression of this K+ channel.