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PubMed2001

Amino acid translation program for full-length cDNA sequences with frameshift errors.

Here we present an amino acid translation program designed to suggest the position of experimental frameshift errors and predict amino acid sequences for full-length cDNA sequences having phred scores. Our program generates artificial insertions into artificial deletions from low-accuracy positions of the original sequence, thereby generating many candidate sequences. The validity of the most probable sequence (the likelihood that it represents the actual protein) is evaluated by using a score (V(a)) that is calculated in light of the Kozak consensus, preferred codon usage, and position of the initiation codon. To evaluate the software, we have used a database in which, out of 612 cDNA sequences, 524 (86%) carried 773 frameshift errors in the coding sequence. Our software detected and corrected 48% of the total frameshift errors in 62% of the total cDNA sequences with frameshift errors. The false positive rate of frameshift correction was 9%, and 91% of the suggested frameshifts were true.

Base Composition↗
PubMed1990

Cap-independent translation of encephalomyocarditis virus RNA: structural elements of the internal ribosomal entry site and involvement of a cellular 57-kD RNA-binding protein.

Translation of encephalomyocarditis virus (EMCV) mRNA occurs by ribosomal internal entry into the 5'-nontranslated region (5' NTR) rather than by ribosomal scanning. The internal ribosomal entry site (IRES) in the EMCV 5' NTR was determined by in vitro translation with RNAs that were generated by in vitro transcription of EMCV cDNAs containing serial deletions from either the 5' or 3' end of the EMCV 5' NTR. Regions downstream of nucleotide 403 and upstream of nucleotide 811 of EMCV were required for efficient translation. Site-directed mutagenesis revealed that a stem-loop structure (400 nucleotides upstream of the initiation codon) was essential for IRES function. We discovered a 57-kD cellular protein whose specific interaction with this stem-loop appears to be prerequisite for IRES function. A A pyrimidine-rich stretch proximal to the initiation codon was also crucial for efficient translation of EMCV mRNA. We propose that ribosomes bind directly to the initiating AUG without scanning.

Animals↗
PubMed1989

Unusual transcriptional and translational features of the aminoglycoside phosphotransferase gene (aph) from Streptomyces fradiae.

The aminoglycoside phosphotransferase gene (aph) from the neomycin producer Streptomyces fradiae encodes an enzyme (APH) that phosphorylates, and thereby inactivates, the antibiotic neomycin. Two promoters were identified upstream of and oriented toward the aph coding sequence. One promoter (aphp1) initiated transcription at the A of the ATG translational initiation codon, or one to two bases upstream. Mutations made in this promoter region identified functionally important nucleotides and verified that the aphp1 transcript was translated to yield the APH protein, despite the lack of a conventional ribosome binding site. A second aph promoter, aphp2, initiated transcription 315 bp upstream of the translational initiation codon but gave transcripts that appeared to terminate before reaching the coding sequence. Multiple transcriptional initiation sites (pA1-pA5) were identified also in the aph regulatory region oriented in the opposite direction to aph transcription. Promoters for the pA2 and pA4 transcripts overlap with aphp1 such that down-promoter mutations in aphp1 also reduce transcription from the overlapping pA promoters.

Amino Acid Sequence↗
PubMed1985

Messenger RNA structure participating in the initiation of synthesis of cucumber mosaic virus coat protein.

The sequence of the 5'-terminal 106 nucleotides of cucumber mosaic virus (strain Y) RNA 4, the mRNA coding for viral coat protein, has been determined. The first AUG was located at 77 nucleotides from the 5'-terminus and was confirmed to be an initiation codon by analysis of the N-terminal amino acid sequence of the protein. The nucleotide sequence (positions 77-106) beyond the AUG codon predicted the sequence of ten amino acids corresponding to the N-terminal region of the protein, which exactly matched the determined amino acid sequence containing an acetyl methionine as the N-terminal amino acid. The distance of the initiation codon AUG from the cap structure was 76 nucleotides and the longest among the mRNAs for coat protein of plant viruses so far reported (9-36 nucleotides). This noncoding region is rich in U residues (40%) and the number of G residues (21 nucleotides) is the largest among these mRNAs (usually 1 or 2 residues). A possible secondary structure is postulated for the region, which might be implicated in efficient translation of the RNA 4 in vivo.

Amino Acid Sequence↗
PubMed2006

Gene activation in plastids by the CRE site-specific recombinase.

We developed a novel system for gene activation in plastids that uses the CRE/loxP site-specific recombination system to create a translatable reading frame by excision of a blocking sequence. To test the system, we introduced an inactive gfp* gene into the tobacco plastid genome downstream of the selectable spectinomcyin resistance (aadA) marker gene. The aadA gene is the blocking sequence, and is flanked by directly oriented loxP sites for excision by the CRE. In the non-activated state, gfp* is transcribed from the aadA promoter, but the mRNA is not translated due to the lack of an AUG translation initiation codon. Green Fluorescent Protein (GFP) expression is activated by excision of the aadA coding segment to link up the gfp* coding region with the translation initiation codon of aadA. Tobacco plants that carry the inactive gfp* gene do not contain detectable levels of GFP. However, activation of gfp* resulted in GFP accumulation, proving the utility of CRE-induced protein expression in tobacco chloroplasts. The gene activation system described here will be useful to probe plastid gene function and for the production of recombinant proteins in chloroplasts.

Base Sequence↗
PubMed1992

Genes encoding a histone H3.3-like variant in Arabidopsis contain intervening sequences.

Two genes encoding a particular H3 histone variant were isolated from Arabidopsis thaliana. These genes differ from the H3 genes previously cloned from Arabidopsis and other plants by several interesting properties: (1) the two genes are located close to each other; (2) their coding regions are interrupted by two or three small introns, the two closest to the initiation codon being located at the same place in the two genes; (3) another, long intron is located in the 5'-untranslated region just before the initiation codon of gene I as deduced from the sequence of several corresponding cDNAs, and very likely also of gene II; (4) these genes do not show preferential expression in organs containing meristematic tissues contrary to the classical intronless replication-dependent histone genes, thus suggesting that their expression is not replication-dependent; (5) the protein encoded by both genes is the same and corresponds to a minor H3 variant highly conserved among all the plant species studied up to now. All these characteristics are common with the animal replication-independent H3.3 histone genes and it is assumed that the genes described here are the first example of the equivalent H3.3 gene family in plants. Interestingly, the promoter regions of the two genes have the same general structure as the Arabidopsis intronless genes. Possible implications on the regulation of H3 genes expression are discussed.

Amino Acid Sequence↗
PubMed1993

Sequence analysis of the zebrafish hox-B5/B6 region.

The region between the zebrafish homeobox genes hox-B5 and hox-B6 was sequenced, and searched for consensus binding sites of retinoic acid receptors and other transcription factors. A continuous sequence of 7.2 kb covering the zebrafish hox-B5/B6 genes was then compared to the corresponding region of the mouse Hox-B complex. Except for the open reading frames, the only highly conserved regions that could be found were stretches extending 0.3 kb upstream from the initiation codons. Within the conserved upstream regions of hox-B5/B6, we identified a common 10 bp sequence, which is also present close to the initiation codons of several other Hox genes and which therefore may be implicated in the control of their expression.

Animals↗
PubMed1999

Cloning and characterization of ethanol-regulated esterase genes in Acetobacter pasteurianus.

The esterase encoding genes, est1 and est2, were cloned from Acetobacter pasteurianus. Nucleotide sequence analysis of est1 revealed a gene of 954 bp, and est1 coded for an arylesterase with a molecular weight of 34863 Da consisting of 317 amino acids. The est2 gene contained an open reading frame composed of 1221 bp encoding an esterase with a molecular weight of 43389 Da consisting of 406 amino acids. The est1 gene showed some similarity, but the est2 gene showed no significant homology to other esterases reported in various microorganisms. Northern blot analysis of total RNA from A. pasteurianus revealed that transcription of the est1 gene was induced only when the cells were grown in a medium containing ethanol, and suggested that the est1 transcript is monocistronic. In contrast, transcription of the est2 gene was repressed in the presence of ethanol. In the absence of ethanol, expression of the est2-mRNA, capable of encoding a multiple number of proteins, was revealed by Northern blot analysis. In addition, deletion analysis indicated that the 5'-region of the est2 gene contained a cis-acting domain for est2 transcriptional regulation. Analysis of the est1 promoter using the chloramphenicol acetyltransferase gene as a reporter gene showed that the promoter within the 305-bp fragment upstream of the ATG initiation codon was responsible for the transcription in cells grown in the presence of ethanol. Primer extension analysis of est1-mRNA showed that the transcription initiation site was 49 bp upstream from the ATG initiation codon. The results of a gel mobility shift assay indicated that there is a regulatory protein related to est1 regulation, which may have some relation to the ethanol resistance of Acetobacter sp.

Journal Article↗
PubMed2004

Severe hemochromatosis in a Portuguese family associated with a new mutation in the 5'-UTR of the HAMP gene.

Juvenile hereditary hemochromatosis is a genetically heterogeneous disorder transmitted as an autosomal recessive trait. It is most often caused by mutations in the HJV gene and rarely in the HAMP gene. Hepcidin is considered to constitute a negative regulator of iron absorption, and its production is increased in inflammatory states and iron overload. We report the detection of a new mutation in the HAMP gene leading to juvenile hemochromatosis in 2 members of a Portuguese family. The mutation lies in the 5'-UTR (untranslated region) of the gene and creates a new initiation codon in the context of a Kozak sequence. We found no trace of hepcidin protein in the patients' urine, suggesting that ribosomes select the mutant initiation codon for translation. The decrease of hepcidin production would thus lead to increased iron absorption, resulting in iron deposition in parenchymal tissues. Phlebotomy therapy of the 2 patients resulted in impressive clinical improvement.

5' Untranslated Regions↗
PubMed1991

Chemically-synthesized gene encoding modified human superoxide dismutase: its construction, expression and properties of the product.

The gene encoding modified human superoxide dismutase (h-SOD) with 153 amino acid residues was constructed by chemical synthesis using the phosphoramidite method. The gene was designed so as to use bacterial codons for expression in prokaryotes and to introduce several unique restriction sites for further mutagenesis by the cassette exchange method. The distance between Shine-Dalgarno sequence and initiation codon was adjusted to maximum expression by using synthesized oligonucleotide. In addition, Cys 6 of h-SOD was changed to Ala to improve instability of native h-SOD. Synthesized structural gene of h-SOD was expressed in E. coli after induction of isopropyl beta-D-thiogalactoside by inserting the gene into the expression vector pKK223-3 having tac promoter. The gene that has 10 base pairs between Shine-Dalgarno sequence and initiation codon showed the most efficient expression. The gene produced three active SOD isomers as revealed by chromatofocusing. The main isomer was purified to homogeneity and characterized. The h-SOD-Ala6 showed similar properties to those of native h-SOD with respect to molecular weight, subunit structure, absorption spectrum, but the modified SOD was more resistant to heat denaturation than was native h-SOD; half-denaturing temperature was shifted by 10 degrees C. Thus, the exchange of Cys 6 to Ala of h-SOD increased a stability of the enzyme.

Amino Acid Sequence↗
PubMed1993

Expression of bacteriophage T4 gene 25 is regulated via RNA secondary structure in the translational initiation region.

Analysis of the nucleotide sequence in the 5' flanking region of bacteriophage T4 gene 25 revealed three potential Shine and Dalgarno sequences, SD1, SD2 and SD3, with a spacing of 8, 17 and 27 nucleotides from the initiation codon of this gene, respectively. Results of our experiments in the bacteriophage T7 expression system clearly demonstrate that the SD3 sequence is required for efficient expression of gene 25. We propose the existence of a stem-loop structure that includes SD1 and SD2 sequences and brings the SD3 sequence to a favourable spacing with the initiation codon of gene 25. Since the predicted secondary structure in the translational initiation region of gene 25 is relatively unstable and the SD3 sequence, GAGG, is more typical than the SD1 sequence, GAG, we suggest that this structure could control the level of gene expression.

Bacteriophage T4↗
PubMed2005

Cloning and expression of superoxide dismutase from Mycobacterium bovis BCG.

We have previously purified the superoxide dismutase (SOD) of Mycobacterium bovis bacillus Calmette-Guerin (BCG), and there is no signal peptide necessary for protein exportation [S.K. Kang, Y.J. Jung, C.H. Kim, C.Y. Song, Extracellular and cytosolic iron superoxide dismutase from Mycobacterium bovis BCG, Clin. Diagn. Lab. Immunol. 5 (1998) 784-789]. In the present study, SOD gene of M. bovis BCG was cloned and expressed in Escherichia coli, and its complete nucleotide sequence and deduced amino acid composition were determined. The open reading frame from the GTG initiation codon was 621 base pair (bp) in length for the SOD structural gene. The ribosomal-binding sequences (GGAAGG) were 6-12 bp upstream from the initiation codon. The amino acid sequence, deduced from the nucleotide sequence, revealed that the SOD consists of 207 amino acids residues with a molecular weight of 22.8 kDa. The N-terminal amino acid sequence predicted from the nucleotide sequence showed that the structural gene of the SOD is not preceded by leader sequences. There were no cysteine residues in the deduced amino acid composition, indicating that the SOD does not consist of disulfide bonds. Analyses of both nucleotide and amino acid sequences of the SOD showed significant similarity to other pathogenic mycobacterial SODs. Furthermore, the results of fractionation and two-dimensional electrophoresis showed that SOD is also associated with cell membrane, suggesting that there might be a specific mechanism for exportation of SOD in M. bovis BCG as well as other pathogenic mycobacteria. Overexpressed SOD in E. coli was purified from the inclusion bodies, and the histidine tag was removed from the protein using enterokinase. Enzyme activity was then determined by gel staining analysis.

Amino Acid Sequence↗
PubMed1986

Synthesis of bovine prolactin in Escherichia coli.

Transformation of Escherichia coli cells with a recombinant plasmid (pESP4) containing a modified bovine prolactin cDNA clone in a pEMBL vector resulted in efficient expression of prolactin. The cDNA was modified by removal of a 5' nontranslated sequence as well as the sequence that specified the signal peptide of preprolactin. To achieve a high level of synthesis, a sequence of 30 nucleotides in the cDNA, which included the ATG initiation codon and the first 7 codons of mature bovine prolactin, was replaced by a chemically synthesized oligonucleotide duplex. The sequence of this duplex was chosen from the consensus sequence around the initiation codon of E. coli genes and by the amino acid sequence of the protein. Prolactin, a single-chain polypeptide of molecular weight 24,000, was identified by Coomassie Blue staining of NaDodSO4-polyacrylamide gels of total protein from transformed E. coli cells, and by reaction with specific antibody. Increased levels of expression of the hormone, corresponding to the form secreted from the pituitary, were observed in the presence of isopropyl-beta-D-thiogalactopyranoside (IPTG).

Animals↗
PubMed1997

Beta-thalassemia and beta[A] globin gene haplotypes in Mexican mestizos.

B-globin haplotypes of 20 beta-thalassemia (beta-thal) and 87 beta(A) Mexican mestizo chromosomes were analyzed to ascertain the origin of the beta-thal alleles and the frequencies and distribution of the beta(A) haplotypes among northwestern Mexican mestizos. Sixteen beta-thal chromosomes carried six Mediterranean alleles [five codon 39 C-->T; two IVS1:1 G-->A; two IVS1:5 G-->A; three IVS1:110 G(A; one codon 11 (-T) and three (deltabeta)zero-thal]; the remaining four were linked to three rare alleles (two -28 A-->C and one each: -87 C-->T and initiation codon ATG-->GTG). Among the 87 beta(A) chromosomes, 17 different 5' haplotypes with frequencies for 1, 3, 2 and 5 of 39.0%, 17. 2%, 9.2% and 6.9%, respectively, were observed. The beta-haplotype analysis showed that 13 out of 16 Mediterranean chromosomes could easily be explained by gene migration; however, one codon 39 associated with haplotype 4 (----+ +-), one IVS1:1 with haplotype 1(+----++) and one IVS1:5 G-->A, may represent separate mutational events. Analysis of the rare alleles showed that the -28 A-->C mutation was associated with the commonest beta(A) haplotype in Mexican mestizos, Mediterraneans and the total world population; therefore an independent origin cannot be ruled out. The -87 C-->T and initiation codon ATG-->GTG were found with beta-haplotypes different from the reported ones, suggesting an indigenous origin.

Alleles↗
PubMed2004

The DNA beta satellite component associated with ageratum yellow vein disease encodes an essential pathogenicity protein (betaC1).

Ageratum yellow vein disease (AYVD) is caused by the geminivirus ageratum yellow vein virus (AYVV) and an associated DNA beta satellite. We have mapped a DNA beta transcript to a highly conserved open reading frame (betaC1 ORF). The most abundant transcript 5'-terminus is located 8 bases upstream of the betaC1 ORF putative initiation codon while the transcript terminates at multiple sites downstream from the putative termination codon. Disruption of betaC1 protein expression by the introduction of an internal nonsense codon prevented infection of the AYVV-satellite complex in ageratum and altered the phenotype in Nicotiana benthamiana to that produced by AYVV alone although the mutant was maintained in systemically infected tissues. Modification of the putative initiation codon to a nonsense codon produced an intermediate phenotype in N. benthamiana and a mild yellow vein phenotype in ageratum, suggesting that betaC1 protein expression could be initiated from an alternative site. N. benthamiana plants containing a dimeric DNA beta transgene produced severe developmental abnormalities, vein-greening, and cell proliferation in the vascular bundles. Expression of betaC1 protein from a potato virus X (PVX) vector also induced abnormal plant growth. Our results demonstrate that the satellite encodes at least one protein that plays a major role in symptom development and is essential for disease progression in ageratum, the natural host of the AYVD complex.

Ageratum↗
PubMed1993

The influence of viral coding sequences on the efficiency of internal initiation of translation of cardiovirus RNAs.

Since internal initiation of translation of cardiovirus RNAs requires the approximately 450-nt segment of the viral genome immediately upstream of the authentic initiation codon for viral polyprotein synthesis, the question arises as to whether the immediately adjacent sequences, the start of the polyprotein coding region, also influence the efficiency of internal initiation. Therefore, a variety of constructs derived from encephalomyocarditis virus and Theiler's murine encephalomyelitis virus retaining various lengths of viral coding sequence were translated in rabbit reticulocyte lysates. Efficient internal initiation showed no requirement specifically for viral coding sequences, but on the other hand certain motifs, notably G-rich sequences, located immediately downstream of the initiation codon were highly inhibitory. These results suggest a possible explanation for the evolution of cardiovirus polyproteins lacking N-terminal myristylation signals and are also pertinent to the design of constructs in which the cardiovirus internal ribosome entry signal is used to drive the expression of a reporter cistron.

Base Sequence↗
PubMed1991

Relationship between utilization of dual translational initiation signals and protein processing in Streptomyces.

Two sets of the Shine-Dalgarno sequence and the initiation codon (ATG) for translation of a gene encoding the protein SSI (Streptomyces subtilisin inhibitor) were studied in vivo by site-directed mutagenesis. The result shows that each ATG can function as an initiator of translation in either Streptomyces lividans 66 or Escherichia coli. The choice of initiation codon seems dependent on the host strain and is closely related to the processing mechanism of pre-SSI protein. The upstream ATG is presumed to be utilized preferentially giving two cleavage sites in pre-SSI in S. albogriseolus S-3253, the original SSI producer strain.

Amino Acid Sequence↗
PubMed1994

Expression in Escherichia coli of a TetK determinant from Staphylococcus aureus.

The expression in Escherichia coli of a tet(K) gene, which originated in Staphylococcus aureus, was studied. The minimum inhibitory concentration (MIC) of tetracycline (TC) for E. coli cells that carried the tet(K) gene was only slightly higher than that for the recipient cells. This result indicated that the level of expression of the tet(K) gene in E. coli was very low. Insertion of a lac promoter into the upstream region of the tet(K) gene resulted in a slight increase in the MIC, from 12.5 to 50 micrograms/ml in the presence of isopropyl-beta-D-thiogalactopyronoside. An altered tet(K) gene, in which the initiation codon and the ribosome-binding site (RBS) were changed from TTG to ATG and from GAGG to GGAGG, respectively, and in which the distance between the RBS and the initiation codon was increased from 4 to 11 bases, was associated with high-level resistance to TC, with a MIC of 200 micrograms/ml. The MIC resembles that associated with expression of the tetA(B) gene of E. coli. These results indicate that the barrier to expression of the tet(K) gene in E. coli is located at the initiation of translation.

Base Sequence↗