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PubMed1998

Assessing protein coding region integrity in cDNA sequencing projects.

MOTIVATION: In cDNA sequencing projects, it is vital to know whether the protein coding region of a sequence is complete, or whether errors have occurred during library construction. Here we present a linear discriminant approach that predicts this completeness by estimating the probability of each ATG being the initiation codon. RESULTS: Because of the current shortage of full-length cDNA data on which to base this work, tests were performed on a non-redundant set of 660 initiation codon-containing DNA sequences that had been conceptually spliced into mRNA/cDNA. We also used an edited set of the same sequences that only contained the region following the initiation codon as a negative control. Using the criterion that only a single prediction is allowed for each sequence, a cut-off was selected at which discrimination of both positive and negative sets was equal. At this cut-off, 67% of each set could be correctly distinguished, with the correct ATG codon also being identified in the positive set. Reliability could be increased further by raising the cut-off or including homologues, the relative merits of which are discussed. AVAILABILITY: The prediction program, called ATGpr, and other data are available at http://www.hri.co.jp/atgpr CONTACT: swintech@hri.co.jp

Base Sequence↗

PubMed1995

Improved gene expression by a modified bicistronic retroviral vector.

We have previously described the construction of a bicistronic retroviral vector using the picornavirus internal ribosome entry site (IRES), which allows two genes expression simultaneously from a single transcript. This vector transcribes RNA efficiently; however, in some cases the levels of protein production are low. In this report, we further modified the bicistronic vector by abolishing the functional viral gag initiation codon that is retained in the vector at 5' to the first initiation codon of transduced gene. Five different genes, human interleukin 2 (hIL-2), human interleukin 4 (hIL-4), human granulocyte macrophage stimulating factor (hGM-CSF), herpes simplex virus thymidine kinase (HSV-tk) gene, and hepatitis C virus (HCV) core gene (C190), were tested on this modified vector for gene transfer and expression. Our results demonstrated that the new bicistronic vector greatly increased the protein levels when compared with the original one. As the RNA levels and splicing patterns from these two vectors remained similar, the improvement was most likely resulted from the increased translational efficiency.

Animals↗

PubMed1995

Genomic organization of the mouse fibroblast growth factor receptor 3 (Fgfr3) gene.

The fibroblast growth factor receptor 3 (Fgfr3) protein is a tyrosine kinase receptor involved in the signal transduction of various fibroblast growth factors. Recent studies suggest its important role in normal development. In humans, mutation in Fgfr3 is responsible for growth disorders such as achondroplasia, hypoachondroplasia, and thanatophoric dysplasia. Here, we report the complete genomic organization of the mouse Fgfr3 gene. The murine gene spans approximately 15 kb and consists of 19 exons and 18 introns. One major and one minor transcription initiation site were identified. Position +1 is located 614 nucleotides upstream from the ATG initiation codon. The translation initiation and termination sites are located in exons 2 and 19, respectively. Five Sp1 sites, two AP2 sites, one Zeste site, and one Krox 24 site were observed in the 5'-flanking region. The Fgfr3 promoter appears to be contained within a CpG island and, as is common in genes having multiple Sp1-binding sites, lacks a TATA box.

Animals↗

PubMed2004

Patterns of gene divergence and VL promoter activity in immunoglobulin light chain clusters of the channel catfish.

The structure and genomic organization of V, J, and C segments in multiple gene clusters of the G class of catfish light (L) chain were determined to study evolutionary patterns of cluster divergence and regulatory function. The results showed that the organizational pattern is conserved; two VL segments reside in opposite transcriptional orientation to a pseudogene JL segment (psiJ), a functional JL segment, and a CL segment. Structures within the central V-psiJ-J-C regions indicate that cluster duplication occurred after the V-V-psiJ-J-C organization became fixed. VL divergence in gene clusters subsequently occurred by mechanisms that principally targeted complementarity-determining regions. The sequence of the VL-flanking regions, which contained regulatory octamer, TATA, and Pax-5 (BSAP) binding site motifs, was conserved during G cluster duplication and within VL-flanking regions in divergent lineages of bony fish. Reporter assays of catfish B cells transfected with a 742-bp VL-flanking fragment showed promoter activity in the absence of enhancer elements. The promoter's activity doubled when coupled with the catfish IgH enhancer. A 136-bp fragment containing the motifs conserved in bony fish phylogeny and located between the leader initiation codon and the initiation sites of sterile transcripts served as a minimal promoter and provided the highest B-cell activity. These constructs, however, did not act as promoters in catfish non-lymphoid cells or mammalian BJA-B B cells or fibroblasts. These results indicate that the structure and function of VL promoter regions in the regulation and tissue specificity of L-chain gene expression evolved early in phylogeny.

Amino Acid Sequence↗

PubMed1990

Cloning and analysis of the 5'-flanking region of rat Na+/K(+)-ATPase alpha 1 subunit gene.

We cloned a 13.3 kilobase (kb) fragment of genomic DNA spanning at least the first two exons of the rat Na+/K(+)-ATPase alpha 1 subunit gene (NKAA1) and 1.5 kb of the 5'-flanking region. S1 nuclease mapping analysis of the 5' end of the Na+/K(+)-ATPase mRNA indicated that the transcription initiation site was located 262 base pairs (bp) upstream of the translation initiation codon. The transcription initiation site of the Na+/K(+)-ATPase alpha 1 subunit gene was identical among six tissues of adult rat (kidney, brain, heart, thyroid, liver and lung). A TATA-box-like sequence (at position -32), two Sp1 factor binding sequences (-137, -56), an active transcription factor consensus binding sequence (-71) and two glucocorticoid-responsive element half consensus sequences (-750, -481) were found in the 5'-flanking region. The sequence of the first exon and the 5'-flanking region of the rat NKAA1 was 63% homologous to that of the horse equivalent. Maximum homology (82%) between the two genes was observed in the region from 361 bp upstream of the translation initiation site to the 3' end of the first exon. The TATA-like box, Sp1 binding site and the active transcriptional factor (ATF) consensus site in this region were conserved in both rat and horse.

Animals↗

PubMed1994

Structure, promoter analysis and chromosomal assignment of the human APEX gene.

APEX nuclease is a mammalian DNA repair enzyme having apurinic/apyrimidinic endonuclease, 3'-5'-exonuclease, DNA 3' repair diesterase and DNA 3'-phosphatase activities. This report describes the organization of the gene (APEX gene) for human APEX nuclease. Human APEX gene was cloned using human APEX cDNA and a human leukocyte genomic library in bacteriophage vector EMBL-3. We proved that human APEX gene consists of 5 exons spanning 2.64 kilobases and suggested that the gene exists as a single copy in the haploid genome. The boundaries between exon and intron follow the GT/AG rule. The major transcription initiation site was assigned by primer extension analysis to C at 515 nucleotides upstream from the ATG initiation codon. The translation initiation and termination sites locate in the exon II and V, respectively. The 5' flanking region (0.89 kilobase) sequenced lacks typical TATA and CAAT boxes, but contains TATA- and CAAT-like sequences and putative cis-acting regulatory elements such as binding sites for Sp1, AP2 and ATF. A part of the 5' flanking region belongs to a CpG island, which extends to the intron II. The CpG island is thought to be a transcription regulatory region of APEX gene, a housekeeping gene. The promoter activity of the 5' upstream region was analyzed by introducing the region in HeLa cells in an expression construct containing luciferase gene as a reporter gene, and the region from position 130 bp upstream to position 205 bp downstream of the major transcription initiation site was shown to be enough for high promoter activity. Northern hybridization experiments suggested that the gene is expressed ubiquitously in human cells. The locus of APEX gene was mapped to human chromosome 14q11.2-q12 using the in situ hybridization technique.

Amino Acid Sequence↗

PubMed1995

Nucleotide sequence of the Staphylococcus aureus RNA polymerase rpoB gene and comparison of its predicted amino acid sequence with those of other bacteria.

The complete nucleotide sequence of the rpoB gene which encodes the beta subunit of S. aureus RNA polymerase has been determined. The RpoB protein, consists of 1182 amino acids and has a novel initiation codon UUG which initiates protein synthesis with methionine. There is a very strong Shine-Dalgarno complementarity and the -10 and -35 promoter hexameric sequences are TAATAT and CCGTTT, respectively. A rho-dependent termination site, CAATCAA, is present which overlaps the -35 promoter sequence of the adjacent rpoC gene a feature which may have a role in the co-ordinate expression of the two genes. A strong homology and conserved regions were found to exist over the predicted amino acid sequences coding for S. aureus rpoB and the equivalent proteins in Escherichia coli, Pseudomonas putida, Salmonella typhimurium, Chlamydia trachomatis, cyanobacterium Anabaena sp. strain PCC 7120.

Amino Acid Sequence↗

PubMed1995

Cloning, sequence analysis, and chromosomal assignment of the mouse Apex gene.

APEX nuclease (Apex gene product) is a mammalian multifunctional DNA repair enzyme possibly involved in the repair of apurinic/apyrimidinic (AP) sites and single-strand DNA breaks with 3' termini blocked by nucleotide fragments and also in transcriptional regulation via redox activation of the AP-1 transcription factors. We cloned a 15-kb DNA fragment containing the Apex gene from a mouse leukocyte genomic library and determined a 4-kb stretch of its nucleotide sequence, including the complete sequence of the mouse Apex gene. The gene consists of 5 exons and 4 introns spanning 2.21 kb, and the boundaries between exons and introns follow the GT/AG rule. Two major and one minor transcription initiation sites were assigned to positions +1 and +24 and position +14, respectively, by a combination of ribonuclease protection, primer extension, and 5' RACE analyses. Position +1 is located 312 nucleotides upstream from the ATG initiation codon. The translation initiation and termination sites are located in exon II and exon V, respectively. The sequenced 5' flanking region (1.32 kb) lacks a typical TATA box, but contains a CAAT box and putative binding sites for several transcription factors, such as ATF, NF-IL6, Sp1, and AP2. The 0.8-kb region from position -410 (5' flanking region) to position +386 (intron II) contains a CpG island. The Apex gene locus was mapped to mouse chromosome 14C2-D1 using in situ hybridization.

Animals↗

PubMed1997

Characterization of the double-stranded RNA genome segment S3 of avian reovirus.

The double-stranded RNA genome segment S3 of avian reovirus (ARV) S1133 was cloned following polyadenylation of both strands and cDNA synthesis of S3 RNA. The complete segment S3 nucleotide sequence was determined. S3 is 1196 base pairs long with one long open reading frame (ORF). The ORF possesses the AUG initiation codon in an optimum context for translation and starts at the first initiation codon (residue 24) and extends for 367 codons, sufficient to encode a protein of the same size as the known S3 gene product, protein sigmaB, one of the major outer capsid proteins of avian reovirus (Mr 41471). Protein sigmaB was subsequently expressed in Escherichia coli. The expressed protein sigmaB was indistinguishable from virion protein sigmaB as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, immunoblot assay, and N-terminal amino acid sequencing of several peptides generated by Staphyloccus aureus V8 protease digestion. ARV S3 genome segment possesses a pentanucleotide UCAUC at the 3'-terminus of its plus strand. The pentanucleotide sequence is common to the other genome segment S1 of ARV and to ten genome segments of mammalian reovirus at the 3'-terminus of their plus strands. Amino acid sequence analysis revealed that ARV sigmaB does not contain a repeated basic amino acid motif as do the three serotypes of mammalian reovirus. The results of amino acid sequencing suggest that the most susceptible cleavage sites of sigmaB to V8 protease are located in a hydrophilic area between amino acids 95 and 140.

Animals↗

PubMed1984

Nucleotide sequence encoding the flavoprotein and hydrophobic subunits of the succinate dehydrogenase of Escherichia coli.

The nucleotide sequence of a 3614 base-pair segment of DNA containing the sdhA gene, encoding the flavoprotein subunit of succinate dehydrogenase of Escherichia coli, and two genes sdhC and sdhD, encoding small hydrophobic subunits, has been determined. Together with the iron-sulphur protein gene (sdhB) these genes form an operon (sdhCDAB) situated between the citrate synthase gene (gltA) and the 2-oxoglutarate dehydrogenase complex genes (sucAB): gltA-sdhCDAB-sucAB. Transcription of the gltA and sdhCDAB gene appears to diverge from a single intergenic region that contains two pairs of potential promoter sequences and two putative CRP (cyclic AMP receptor protein)-binding sites. The sdhA structural gene comprises 1761 base-pairs (587 codons, excluding the initiation codon, AUG) and it encodes a polypeptide of Mr 64268 that is strikingly homologous with the flavoprotein subunit of fumarate reductase (frdA gene product). The FAD-binding region, including the histidine residue at the FAD-attachment site, has been identified by its homology with other flavoproteins and with the flavopeptide of the bovine heart mitochondrial succinate dehydrogenase. Potential active-site cysteine and histidine residues have also been indicated by the comparisons. The sdhC (384 base-pairs) and sdhD (342 base-pairs) structural genes encode two strongly hydrophobic proteins of Mr 14167 and 12792 respectively. These proteins resemble in size and composition, but not sequence, the membrane anchor proteins of fumarate reductase (the frdC and frdD gene products).

Amino Acid Sequence↗

PubMed1988

Nucleotide sequence of the gene encoding the GMP reductase of Escherichia coli K12.

(1) The nucleotide sequence of a 1991 bp segment of DNA that expresses the GMP reductase (guaC) gene of Escherichia coli K12 was determined. (2) This gene comprises 1038 bp, 346 codons (including the initiation codon but excluding the termination codon), and it encodes a polypeptide of Mr 37,437 which is in good agreement with previous maxicell studies. (3) The sequence contains a putative promoter 102 bp upstream of the translational start codon, and this is immediately followed by a (G + C)-rich discriminator sequence suggesting that guaC expression may be under stringent control (4) The GMP reductase exhibits a high degree of sequence identity (34%) with IMP dehydrogenase (the guaB gene product) indicative of a close evolutionary relationship between the salvage pathway and the biosynthetic enzymes, GMP reductase and IMP dehydrogenase, respectively. (5) A single conserved cysteine residue, possibly involved in IMP binding to IMP dehydrogenase, was located within a region that possesses some of the features of a nucleotide binding site. (6) The IMP dehydrogenase polypeptide contains an internal segment of 123 amino acid residues that has no counterpart in GMP reductase and may represent an independent folding domain flanked by (alanine + glycine)-rich interdomain linkers.

Base Sequence↗

PubMed1991

Potential oncogenic effects of basic fibroblast growth factor requires cooperation between CUG and AUG-initiated forms.

Normal adult bovine aortic endothelial cells were infected with various recombinant retroviruses expressing one, two, or three human basic fibroblast growth factor (bFGF) proteins normally synthesized by an alternative use of translation initiation codons. We show here that the constitutive expression of the AUG-initiated from (18 kDa) leads the transfected cells to form colonies in soft agar. The expression of the high molar weight (HMW) forms (22.5 and 21 kDa) initiated at one of the two CUG initiation codons allows cell immortalization, whereas the tumorigenic potential is reached when the three forms are constitutively expressed. Furthermore, we provide evidence that constitutive expression of (HMW) bFGF forms has a down-regulation effect on bFGF synthesis from the gene naturally active in parental endothelial cells.

Animals↗

PubMed1983

Intergenic DNA sequences flanking the pseudo alpha globin genes of human and chimpanzee.

We have determined the sequence of 2400 base pairs upstream from the human pseudo alpha globin (psi alpha) gene, and for comparison, 1100 base pairs of DNA within and upstream from the chimpanzee psi alpha gene. The region upstream from the promoter of the psi alpha gene shows no significant homology to the intergenic regions of the adult alpha 2 and alpha 1 globin genes. The chimpanzee gene has a coding defect in common with the human psi alpha gene, showing that the product of this gene, if any, was inactivated before the divergence of human and chimpanzee. However the chimpanzee gene contains a normal ATG initiation codon in contrast to the human gene which has GTG as the initiation codon. The psi alpha genes of both human and chimpanzee are flanked by the same Alu family member. The structure and position of this repeat have not been altered since the divergence of human and chimpanzee, and it is at least as well conserved as its immediate flanking sequence. Comparing human and chimpanzee, the 300 bp Alu repeat has accumulated only two base substitutions and one length mutation; the adjacent 300 bp flanking region has accumulated five base substitutions and twelve length mutations.

Animals↗

PubMed1986

Complete nucleotide sequence of the Escherichia coli recB gene.

The complete nucleotide sequence of the Escherichia coli recB gene which encodes a subunit of the ATP-dependent DNase, Exonuclease V, has been determined. The proposed coding region for the RecB protein is 3543 nucleotides long and would encode a polypeptide of 1180 amino acids with a calculated molecular weight of 133,973. The start of the recB coding sequence overlaps the 3' end of the upstream ptr gene, and the recB termination codon overlaps the initiation codon of the downstream recD gene, suggesting that these genes may form an operon. No sequences which reasonably fit the consensus for an E. coli promoter could be identified upstream of the proposed recB translational start. The predicted RecB amino acid sequence contains regions of homology with ATPases, DNA binding proteins and DNA repair enzymes.

Amino Acid Sequence↗

PubMed1996

Molecular cloning of the Corynebacterium glutamicum ('Brevibacterium lactofermentum' AJ12036) odhA gene encoding a novel type of 2-oxoglutarate dehydrogenase.

The Corynebacterium glutamicum ('Brevibacterium lactofermentum' AJ12036) odhA gene, encoding 2-oxoglutarate dehydrogenase (E1o subunit of the 2-oxoglutarate dehydrogenase complex), has been isolated and identified as an homologous counterpart of the Escherichia coll sucA and Bacillus subtilis odhA genes. The nucleotide sequence of a 4394 bp chromosomal fragment containing the C. glutamicum odhA gene was determined. The odhA gene comprised 3771 bp (1257 codons, including the initiation codon) and a molecular mass of 138656 Da was predicted for the OdhA polypeptide. Northern blot analysis revealed a 3.9 kb transcript. The size of the transcript, together with the presence of a rho-independent terminator-like structure, suggests that C. glutamicum odhA is monocistronic. Cells harbouring plasmids carrying C. glutamicum odhA showed a threefold increase in specific 2-oxoglutarate dehydrogenase complex activity and expression of a protein with an apparent molecular mass of 136 kDa, in good agreement with the predicted size of the OdhA polypeptide. The C-terminal region of the C. glutamicum OdhA protein shows strong sequence similarity to E1os from other organisms. C. glutamicum OdhA has an N-terminal extension not found in previously reported E1os. The amino acid sequence of this extension shows similarity to that of the C-terminal region of dihydrolipoamide S-succinyltransferase (E2o) subunits of 2-oxoglutarate dehydrogenase complexes and dihydrolipoamide S-acetyltransferase (E2p) subunits of pyruvate dehydrogenase complexes. It suggests that the C. glutamicum odhA gene might encode a novel bifunctional protein with E1o and E2o activities.

Amino Acid Sequence↗

PubMed1983

The pyruvate dehydrogenase complex of Escherichia coli K12. Nucleotide sequence encoding the pyruvate dehydrogenase component.

The nucleotide sequence of a 3780-base-pair segment of DNA containing the aceE gene encoding the pyruvate dehydrogenase component (E1) of the pyruvate dehydrogenase complex of Escherichia coli, has been determined by the dideoxy chain-termination method. The aceE structural gene comprises 2655 base pairs (885 codons, excluding the initiation codon AUG), it is preceded by a good ribosome binding site and several potential RNA polymerase binding sites. Its polarity and location in the restriction map of the corresponding segment of DNA are consistent with it being the proximal gene in the ace operon, as defined in previous genetic and post-infection labelling studies. The relative molecular mass (99474), composition (885 amino acids), amino-terminal residue and carboxy-terminal sequence predicted from the nucleotide sequence are in excellent agreement with published information obtained from studies with the purified pyruvate dehydrogenase component (E1). The nucleotide sequence also contains a second gene (gene A) situated upstream of the aceE gene. It appears to be an independent gene containing 708 base pairs (236 codons) and encoding a weakly expressed product (protein A; Mr = 27049) of unknown function.

Amino Acids↗

PubMed1983

The pyruvate dehydrogenase complex of Escherichia coli K12. Nucleotide sequence encoding the dihydrolipoamide acetyltransferase component.

The nucleotide sequence of the aceF gene, which encodes the dihydrolipoamide acetyltransferase component (E2) of the pyruvate dehydrogenase complex of Escherichia coli K12, has been determined using the dideoxy chain-termination method. The aceF gene comprises 1887 base pairs (629 codons excluding the initiation codon AUG); it is preceded by a short intercistronic segment of 14 base pairs containing a good ribosomal binding site, and it is followed closely by a potential rho-independent terminator. The results extend by 1980 base pairs the previously sequenced segment of 3780 base pairs containing the structural gene (aceE) of the pyruvate dehydrogenase component (E1) and they confirm that aceE and aceF are the proximal and distal genes of the ace operon. The amino terminus, carboxy-terminal sequence and amino acid composition of the acetyltransferase subunit predicted from the nucleotide sequence are in excellent agreement with previous studies with the purified protein. The predicted molecular weight (Mr = 65959) confirms experimental values derived from sedimentation equilibrium analysis and indicates that the higher values (78000-89000) that have been reported are due to unusual features of the protein that lead to anomalous mobilities during sodium dodecyl sulphate/polyacrylamide gel electrophoresis and in gel filtration. The primary structure fully supports conclusions, based on limited tryptic proteolysis, that the acetyltransferase subunit possesses two heterologous domains: the lipoyl domain and the subunit binding and catalytic domain. The lipoyl domain corresponds to the amino-terminal segment of the protein. It is acidic and contains three remarkably homologous repeating units of approximately 100 amino acids, each possessing a potential lipoyl binding site and a region that is characteristically rich in alanine and proline residues. The subunit binding and catalytic domain occupies most of the residual polypeptide in the carboxy-terminal segment.

Acetyltransferases↗

PubMed1984

Nucleotide sequence of the sucA gene encoding the 2-oxoglutarate dehydrogenase of Escherichia coli K12.

The nucleotide sequence of a 3180-base-pair segment of DNA, containing the sucA gene encoding the 2-oxoglutarate dehydrogenase component (E1o) of the 2-oxoglutarate dehydrogenase complex of Escherichia coli, has been determined by the dideoxy chain-termination method. The sucA structural gene contains 2796 base pairs (932 codons, excluding the initiation codon AUG) and encodes a polypeptide having a glutamine residue at the amino terminus, a glutamate residue at the carboxy-terminus and a calculated Mr = 104905. The predicted amino acid composition is in good agreement with published information obtained by hydrolysis of the purified enzyme. There is a striking lack of sequence homology between the 2-oxoglutarate dehydrogenase (E1o) and the corresponding pyruvate dehydrogenase (E1p), which suggests that the two components are not closely related in evolutionary terms. The location and polarity of the sucA gene, relative to the restriction map of the corresponding segment of DNA, are consistent with it being the proximal gene of the suc operon, as defined in previous genetic and post-infection labelling studies, but it could also form part of a more complex regulatory unit. The sucA gene is preceded by a segment of DNA that contains many substantial regions of hyphenated dyad symmetry including an IS-like sequence of the type that is thought to function as an intercistronic regulatory element. This segment also contains three putative RNA polymerase binding sites and a good ribosome binding site.

Amino Acid Sequence↗