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PubMed2001

Transcriptional analysis of the tet(P) operon from Clostridium perfringens.

The Clostridium perfringens tetracycline resistance determinant from the 47-kb conjugative R-plasmid pCW3 is unique in that it consists of two overlapping genes, tetA(P) and tetB(P), which mediate resistance by different mechanisms. Detailed transcriptional analysis has shown that the inducible tetA(P) and tetB(P) genes comprise an operon that is transcribed from a single promoter, P3, located 529 bp upstream of the tetA(P) start codon. Deletion of P3 or alteration of the spacing between the -35 and -10 regions significantly reduced the level of transcription in a reporter construct. Induction was shown to be mediated at the level of transcription. Unexpectedly, a factor-independent terminator, T1, was detected downstream of P3 but before the start of the tetA(P) gene. Deletion or mutation of this terminator led to increased read-through transcription in the reporter construct. It is postulated that the T1 terminator is an intrinsic control element of the tet(P) operon and that it acts to prevent the overexpression of the TetA(P) transmembrane protein, even in the presence of tetracycline.

Antiporters↗

PubMed2003

Novel polymorphisms influencing transcription of the human CHRM2 gene in airway smooth muscle.

Muscarinic receptors are a functionally important family of G-protein-coupled receptors. Using a combination of rapid amplification of 5' cDNA ends and reporter gene assays, we characterized the 5' untranslated region of the CHRM2 gene as expressed in human airway smooth muscle (HASM) cells. A splice site is present 46 bp upstream from the ATG start codon. Five exons with alternative splicing patterns are present upstream of this splice site, separated by introns ranging from 87 bp to > 145 kb. There is evidence for the gene being under the control of a TATA-less promoter with Sp1, GATA, and activator protein-2 binding sites. Multiple transcription start sites (TSSs) were identified. We identified a novel 0.5-kb hypervariable region located 648 bp upstream of the most 5' TSS, a multiallelic (CA) tandem repeat 96 bp downstream of the most 5' TSS, and a common C-->A SNP located 136 bp upstream of the most 5' TSS. Functional studies in primary HASM cells and the BEAS-2B cell line demonstrated highest promoter activity to be upstream of the most 3' TSS, with potential repressor elements operating in a cell type-dependent manner, located upstream of the most 5' TSS. We present functional data to show that the CA repeat may influence the transcription of the gene in HASM and BEAS-2B cells.

5' Untranslated Regions↗

PubMed1998

PepA, a secreted protein of Pseudomonas aeruginosa, is necessary for cytotoxicity and virulence.

Pseudomonas aeruginosa is an opportunistic pathogen and a leading cause of hospital-acquired pneumonia. We identified a 73kDa protein, designated Pseudomonas exoprotein A (PepA), that was secreted by P. aeruginosa strain PA103. PepA was necessary for in vitro killing of epithelial cells as well as virulence in a mouse model of acute pneumonia. Several properties of PepA suggested that it was secreted by a type III system. Secretion occurred without cleavage of a signal peptide and in low-calcium environments in the presence of a divalent cation chelator, as is the case for characterized P. aeruginosa type III secreted proteins. Secretion of PepA was absent from isogenic mutants with defective type III pathways. Finally, amino-terminal peptide sequence analysis indicated that the amino-terminal five residues of PepA were identical to those of ExoS and ExoT, two type III secreted proteins of P. aeruginosa. After secretion, PepA underwent cleavage at two sites, each with the sequence A-X-K-S, suggesting that the cleavage may be caused by a protease. The gene encoding PepA, designated pepA, was cloned and sequenced, and comparisons with the genetic database using BLAST alignments indicated that the nucleotide sequence of pepA and the inferred protein sequence of PepA had no homology to known sequences. A nucleotide sequence identical to the consensus element for binding of ExsA, a transcriptional activator of P. aeruginosa type III secretion genes, was located 84 bp 5' of the translational start codon. Analysis of transposon insertion mutants indicated that the carboxy terminus was required for cytotoxicity. Examination of respiratory clinical isolates demonstrated that pepA was a variable trait and probably acquired by horizontal transmission. Consistent with this hypothesis was the identification of a putative insertion element 94 bp 5' of the PepA translational start site. Analysis of G + C content of the PepA coding sequence and the adjacent insertion element suggested that they were acquired together from a different species. In summary, PepA is a secreted protein of P. aeruginosa that is necessary for epithelial cell cytotoxicity in vitro and virulence in a mouse model of pneumonia.

Amino Acid Sequence↗

PubMed1994

Genomic structure analysis of promoter sequence of a mouse mu opioid receptor gene.

We have isolated mouse mu opioid receptor genomic clones (termed MOR) containing the entire amino acid coding sequence corresponding to rat MOR-1 cDNA, including additional 5' flanking sequence. The mouse MOR gene is > 53 kb long, and the coding sequence is divided by three introns, with exon junctions in codons 95 and 213 and between codons 386 and 387. The first intron is > 26 kb, the second is 0.8 kb, and the third is > 12 kb. Multiple transcription initiation sites were observed, with four major sites confirmed by 5' rapid amplification of cDNA ends and RNase protection located between 291 and 268 bp upstream of the translation start codon. Comparison of the 5' flanking sequence with a transcription factor database revealed putative cis-acting regulatory elements for transcription factors affected by cAMP, as well as those involved in the action of gluco- and mineralocorticoids, cytokines, and immune-cell-specific factors.

Amino Acid Sequence↗

PubMed1993

Gene-specific translational control of the yeast GCN4 gene by phosphorylation of eukaryotic initiation factor 2.

Phosphorylation of the alpha subunit of eukaryotic initiation factor 2 (eIF-2 alpha) is one of the best-characterized mechanisms for down-regulating total protein synthesis in mammalian cells in response to various stress conditions. Recent work indicates that regulation of the GCN4 gene of Saccharomyces cerevisiae by amino acid availability represents a gene-specific case of translational control by phosphorylation of eIF-2 alpha. Four short open reading frames in the leader of GCN4 mRNA (uORFs) restrict the flow of scanning ribosomes from the cap site to the GCN4 initiation codon. When amino acids are abundant, ribosomes translate the first uORF and reinitiate at one of the remaining uORFs in the leader, after which they dissociate from the mRNA. Under conditions of amino acid starvation, many ribosomes which have translated uORF1 fail to reinitiate at uORFs 2-4 and utilize the GCN4 start codon instead. Failure to reinitiate at uORFs 2-4 in starved cells results from a reduction in the GTP-bound form of eIF-2 that delivers charged initiator tRNA(iMet) to the ribosome. When the levels of eIF-2.GTP.Met-tRNA(iMet) ternary complexes are low, many ribosomes will not rebind this critical initiation factor following translation of uORF1 until after scanning past uORF4, but before reaching GCN4. Phosphorylation of eIF-2 by the protein kinase GCN2 decreases the concentration of eIF-2.GTP.Met-tRNA(iMet) complexes by inhibiting the guanine nucleotide exchange factor for eIF-2, which is the same mechanism utilized in mammalian cells to inhibit total protein synthesis by phosphorylation of eIF-2.

Amino Acids↗

PubMed1991

Two types of TATA elements for the CYC1 gene of the yeast Saccharomyces cerevisiae.

Functional TATA elements in the 5' untranslated region of the CYC1 gene in the yeast Saccharomyces cerevisiae have been defined by transcriptional analysis of site-directed mutations. Five sites previously suggested to contain functional TATA elements were altered individually and in all possible combinations. The results indicated that only two elements are required for transcription at the normal level and the normal start sites. The two functional TATA elements are located at sites -178 and -123, where the A of the ATG start codon is assigned nucleotide position +1. They direct initiation within windows encompassing -70 to -46 and -46 to -28, respectively. Only when both of the upstream TATA sites were rendered nonfunctional were the third and fourth downstream TATA-like sequences activated, as indicated by the presence of low levels of transcription starting at -28. The two upstream functional TATA elements differed in sequence. The sequence of the most 5' one at site 1, denoted beta-type, was ATATATATAT, whereas that of the second one at site 2, denoted alpha-type, was TATATAAAA. The following rearrangements of the beta-type and alpha-type elements at two sites (1 and 2) were examined: site1 beta-site2 alpha; site 1 alpha-site 2 beta; site1 alpha-site2 alpha; and site1 beta-site2 beta. When different types were at different sites (site1 beta-site2 alpha and site1 alpha-site2 beta), both were used equally. In contrast, when the same type was present at both sites (site1 alpha-site2 alpha and site1 beta-site2 beta), only the upstream element was used. We suggest that the two TATA elements are recognized by different factors of the transcription apparatus.

Base Sequence↗

PubMed1988

Sequence and analysis of the DNA encoding protective antigen of Bacillus anthracis.

The nucleotide sequence of the protective antigen (PA) gene from Bacillus anthracis and the 5' and 3' flanking sequences were determined. PA is one of three proteins comprising anthrax toxin; and its nucleotide sequence is the first to be reported from B. anthracis. The open reading frame (ORF) is 2319 bp long, of which 2205 bp encode the 735 amino acids of the secreted protein. This region is preceded by 29 codons, which appear to encode a signal peptide having characteristics in common with those of other secreted proteins. A consensus TATAAT sequence was located at the putative -10 promoter site. A Shine-Dalgarno site similar to that found in genes of other Bacillus sp. was located 7 bp upstream from the ATG start codon. The codon usage for the PA gene reflected its high A + T (69%) base composition and differed from those of genes for bacterial proteins from most other sequences examined. The TAA translation stop codon was followed by an inverted repeat forming a potential termination signal. In addition, a 192-codon ORF of unknown significance, theoretically encoding a 21.6-kDa protein, preceded the 5' end of the PA gene.

Amino Acid Sequence↗

PubMed2000

Structure and location of a ferritin gene of the yellow fever mosquito Aedes aegypti.

We have isolated and sequenced a genomic clone encoding the 24- and 26-kDa ferritin subunits in the mosquito Aedes aegypti (Rockefeller strain). The A. aegypti gene differs from other known ferritin genes in that it possesses an additional intron and an unusually large second intron. The additional intron is located within the 5' untranslated region, between the CAP site and the start codon. The second intron contains numerous putative transposable elements. In addition, unlike the human and rat ferritin genes, the A. aegypti ferritin gene is a single copy gene, located at 88.3% FLpter on the q-arm of chromosome 1. Primer extension analysis indicates that the A. aegypti ferritin gene has multiple transcriptional start sites. A differential usage of these sites is observed with varied cellular iron concentrations.

5' Untranslated Regions↗

PubMed1999

Internal ribosome entry site biology and its use in expression vectors.

Internal ribosome entry sites (IRESs) are cis-acting elements that recruit the small ribosomal subunits to an internal initiator codon in the mRNA with the help of cellular trans-acting factors. The recent discovery of the IRES recognition site of the eIF4G initiation factor is beginning to shed some light into how IRES elements are recognized by the translational machinery. Additionally, the progress made in the understanding of the parameters that influence start codon selection will be instrumental in establishing the rational design of bicistronic expression vectors.

Animals↗

PubMed1991

ms2i6A deficiency enhances proofreading in translation.

The hypermodified base 2-methylthio-N6-isopentenyladenosine (ms2i6A) at position 37 occurs frequently in tRNAs that read codons starting with uridine. Here we have studied how ms2i6A affects the accuracy of poly(U) translation in vitro. Deficiency leads to a higher rejection rate of tRNA4(Leu) by more aggressive proofreading on the wild-type ribosome, but with the initial selection step unchanged. Our data indicate that ms2i6A has no effect on codon-anticodon interactions on wild-type ribosomes as long as aminoacyl-tRNA is in ternary complex with EF-Tu and GTP. ms2i6A deficiency in the cognate poly(U) reader tRNA(Phe) leads to increased misreading when the near-cognate competitor tRNA4(Leu) is wild-type. ms2i6A deficiency in tRNA4(Leu) gives a decreased error level in competition with wild-type tRNA(Phe).

Codon↗

PubMed1989

Regulation of the RAD2 gene of Saccharomyces cerevisiae.

Regulation of the DNA damage-inducible RAD2 gene was investigated in yeast cells transformed with centromeric plasmids containing RAD2-lacZ fusion constructs. Deletion analysis defined several regions in the 350bp region upstream of the translational start codon which are required for induction of beta-galactosidase activity. No deletions resulted in constitutively enhanced expression. We therefore conclude that induction of RAD2 by DNA-damaging agents is positively regulated. Two domains required for induction have a similar sequence and are located approximately 70 and approximately 140bp upstream of the major transcriptional start site. Four other sequence domains required for induction contain uninterrupted poly(dA) poly(dT) stretches 9-13bp long. Deletion of some of these AT-rich domains also affects constitutive expression of RAD2. Expression of RAD2 is not cell-cycle-regulated in mitotic cells. However, meiosis is accompanied by increased steady-state levels of RAD2 mRNA in the absence of DNA damage. This enhanced transcription is not dependent on the presence of upstream sequences required for regulation of induction by DNA damage. Increased steady-state levels of RAD2 mRNA are induced by cycloheximide in asynchronously dividing populations of cells, but not in non-replicating cells arrested in G1 phase of the cell cycle. Following exposure to u.v. irradiation induction is also dramatically reduced in non-replicating cells.

Base Sequence↗

PubMed1996

The downstream box: an efficient and independent translation initiation signal in Escherichia coli.

The downstream box (DB) was originally described as a translational enhancer of several Escherichia coli and bacteriophage mRNAs located just downstream of the initiation codon. Here, we introduced nucleotide substitutions into the DB and Shine-Dalgarno (SD) region of the highly active bacteriophage T7 gene 10 ribosome binding site (RBS) to examine the possibility that the DB has an independent and functionally important role. Eradication of the SD sequence in the absence of a DB abolished the translational activity of RBS fragments that were fused to a dihydrofolate reductase reporter gene. In contrast, an optimized DB at various positions downstream of the initiation codon promoted highly efficient protein synthesis despite the lack of a SD region. The DB was not functional when shifted upstream of the initiation codon to the position of the SD sequence. Nucleotides 1469-1483 of 16S rRNA ('anti-downstream box') are complementary to the DB, and optimizing this complementarity strongly enhanced translation in the absence and presence of a SD region. We propose that the stimulatory interaction between the DB and the anti-DB places the start codon in close contact with the decoding region of 16S rRNA, thereby mediating independent and efficient initiation of translation.

Bacteriophage T7↗

PubMed1978

DNA sequence from the histidine operon control region: seven histidine codons in a row.

The DNA sequence of 250 base pairs preceding the first structural gene of the histidine operon of Salmonella typhimurium was determined by the dideoxy chain-termination method. Single-stranded DNA template was provided by an M13-histidine transducing phage constructed for the purpose by in vitro recombination. The termination site for the histidine leader RNA is identified by analogy with the trp operon leader termination sequence, and is 47 nucleotides before the start codon of the first structural gene G. Beginning 150 nucleotides before the end of the presumed leader RNA is a possible short protein-coding region with seven histidine codons in a row. It is proposed that the major mechanism of histodine operon control must involve a ribosome arrested at this run of histidine codons when histidine is limiting.

Base Sequence↗

PubMed1988

Organization and nucleotide sequence of genes at both junctions between the two inverted repeats and the large single-copy region in the rice chloroplast genome.

We describe the isolation and organization, at the nucleotide sequence level, of genes located at the two junctions of the large single-copy region (LSCR) and the two inverted repeats (IRA and IRB) in the rice chloroplast genome. This is the first example where the two junctions are precisely located in a monocot. In rice, a ribosomal protein gene cluster, rpl23-rpl2-rps19, which codes for the ribosomal proteins L23 (rpl23), L2 (rpl2) and S19 (rps19), lies at the ends of the two IRs near the LSCR. The inverted repeats end 45 bp from the translation stop codon of rps19. The gene for the 32-kDa photosystem II protein, psbA, is located at the extremity of the LSCR near IRA, and transcribed towards IRA. The translation stop codon of psbA is 68 bp from the right-hand junction (JLA). Thus, JLA is located within the intergenic sequence of the two genes, rps19 and psbA. Around the left-hand junction (JLB), there is a typical ribosomal protein gene cluster, rpl23-rpl2-rps19-rpl22 (rpl22 for the ribosomal protein L22). The translation start codon of rpl22 is located in the LSCR 25 bp from JLB. Therefore, JLB is located within the intergenic sequence between rps19 and rpl22.

Chloroplasts↗

PubMed2006

Characterization of the human PRIP-1 gene structure and transcriptional regulation.

The PRIP [phospholipase C related, but catalytically inactive protein] family has been isolated as a novel inositol 1,4,5-trisphosphate binding protein with a domain organization similar to phospholipase C-delta but lacking the enzyme activity, comprising PRIP-1 and PRIP-2. The PRIP-1 gene is expressed predominantly in the brain, while PRIP-2 exhibits a relatively ubiquitous expression in rats and mice. We also found that PRIP-1 plays an important role in type A receptor signaling for gamma-aminobutyric acid in the brain. In this study, we investigated PRIP-1 gene structure and the possible mechanisms involved in the expression. The tissue distribution pattern of PRIP gene expression in humans was similar to that in rodents. 5'RACE (rapid amplification of cDNA ends) analysis using PRIP-1 gene specific primers with human brain mRNA revealed the presence of three new exons, indicating that the PRIP-1 gene is organized into 8 exons intervened by 7 introns. Although three transcripts resulting from the alternative splicing of exon 2 and/or 3 were detected, a transcript lacking exons 2 and 3 was predominantly expressed in humans, suggesting that the translation start codon of human PRIP-1 exists in exon 1. To characterize the human PRIP-1 promoter, transient luciferase assay was carried out with luciferase constructs including various lengths of the 5' flanking region of the PRIP-1 gene. The results indicated that the positive regulatory region is located -237 to -108 bp upstream from the transcription start site. Gel shift assay revealed the specific binding of some nuclear proteins to this region, suggesting that the existence of transcription factors contributes to the positive regulation of PRIP-1 gene expression. Mutation analyses revealed that the binding of a transcription factor, MAZ to the regulatory site leads to the promoter activity, indicating that MAZ is involved in the expression regulation of the human PRIP-1 gene.

Alternative Splicing↗

PubMed2002

A gene fusion method to screen for regulatory effects on gene expression: application to the LDL receptor.

We have evaluated the feasibility of using fusion genes to link transcriptional promoters of biomedical interest to reporter genes to screen for pharmacological or genetic regulatory effects. Using gene targeting, we generated two lines of embryonic stem (ES) cells expressing human alpha-fetoprotein (hAFP) under control of the endogenous promoter for the LDL receptor (LDLR). In one line, hAFP was introduced into the first intron after the translational start codon; in the other, hAFP was positioned in the 3'-untranslated region leaving the potential for expression of LDLR intact. In both lines, an internal ribosome entry site (IRES) was included to facilitate translation. Readily measurable levels of hAFP were found in the medium with both targeted ES cell lines, compared with undetectable levels with the starting cell line. The expectation that the level of hAFP would reflect the steady-state level of mRNA for the fusion transcript and secondarily transcriptional control of LDLR was confirmed by correlating hAFP levels with the abundance of LDLR and fusion transcripts. We also generated mice carrying the LDLR-hAFP fusion in the 3'-untranslated region and these mice produced detectable levels of hAFP in serum. Levels of hAFP in culture medium and in serum were increased by simvastatin, a drug known to up-regulate LDLR. These ES cell clones and mice are suitable for pharmacological and genetic screening to detect effects on expression of LDLR. The data demonstrate the feasibility of using gene fusions to screen for drugs and genetic factors that affect expression of a wide variety of genes of interest.

3' Untranslated Regions↗

PubMed1995

Activity of mycobacterial promoters during intracellular and extracellular growth.

pUS933, a bifunctional Mycobacterium-Escherichia coli translational fusion vector containing an amino-terminally truncated E. coli lacZ reporter gene, was constructed. Derivatives of pUS933, containing the promoter, RBS and start codon of the Mycobacterium bovis BCG hsp60 gene, the Mycobacterium leprae 28 kDa gene and the M. leprae 18 kDa gene were constructed and introduced into E. coli, Mycobacterium smegmatis and M. bovis BCG. beta-Galactosidase activity was measured for mycobacteria grown in liquid culture. Primer-extension analysis was used to determine the transcriptional start point for the 18 kDa promoter in M. smegmatis. Murine macrophages were infected with recombinant BCG containing the pUS933 derivatives and expression levels were examined, by fluorescence microscopy and fluorometry, during intracellular growth of BCG. Both the BCG hsp60 gene promoter and the M. leprae 28 kDa gene promoter gave high levels of beta-galactosidase expression in all situations examined. In contrast, the M. leprae 18 kDa promoter fragment gave very low levels of expression in M. smegmatis and BCG grown in liquid culture, but in BCG growing within macrophages it was induced to levels almost as high as the other promoters. This indicated that the 18 kDa gene is specifically activated during intracellular growth and may therefore be involved in survival of M. leprae within macrophages. This pattern of regulation may be useful for controlling expression of foreign genes in recombinant BCG strains.

Animals↗

PubMed1994

Characterization of the mnp2 gene encoding manganese peroxidase isozyme 2 from the basidiomycete Phanerochaete chrysosporium.

The nucleotide (nt) sequence of a gene (mnp2) encoding manganese peroxidase isozyme 2 (MnP-2) from Phanerochaete chrysosporium was determined. The sequence of 3297 bp includes 1287 bp of 5'-flanking sequence and 490 bp 3' to the stop codon. Comparison of cDNA and genomic sequences indicates seven introns varying in size from 50-55 bp. The 5' upstream region of the mnp2 gene contains a TATAA element, three inverted CCAAT elements (ATTGG), six putative heat-shock elements (HSE) and three putative metal response elements (MRE) (TGCRCNC), all located within 1100-bp upstream from the start codon. The positions of the putative HSE and MRE in the promoter region of mnp2 are compared with the corresponding sequences in the mnp1 gene promoter. A Northern blot probed with a fragment specific for the mnp2 gene suggests that the transcription of mnp2 is regulated by Mn ions.

Amino Acid Sequence↗