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PubMed1998

Evidence for in vivo ribosome recycling, the fourth step in protein biosynthesis.

Ribosome recycling factor (RRF) catalyzes the fourth step of protein synthesis in vitro: disassembly of the post-termination complex of ribosomes, mRNA and tRNA. We now report the first in vivo evidence of RRF function using 12 temperature-sensitive Escherichia coli mutants which we isolated in this study. At non-permissive temperatures, most of the ribosomes remain on mRNA, scan downstream from the termination codon, and re-initiate translation at various sites in all frames without the presence of an initiation codon. Re-initiation does not occur upstream from the termination codon nor beyond a downstream initiation signal. RRF inactivation was bacteriostatic in the growing phase and bactericidal during the transition between the stationary and growing phase, confirming the essential nature of the fourth step of protein synthesis in vivo.

Alleles↗

PubMed1993

Effects of consecutive AGG codons on translation in Escherichia coli, demonstrated with a versatile codon test system.

A system for testing the effects of specific codons on gene expression is described. Tandem test and control genes are contained in a transcription unit for bacteriophage T7 RNA polymerase in a multicopy plasmid, and nearly identical test and control mRNAs are generated from the primary transcript by RNase III cleavages. Their coding sequences, derived from T7 gene 9, are translated efficiently and have few low-usage codons of Escherichia coli. The upstream test gene contains a site for insertion of test codons, and the downstream control gene has a 45-codon deletion that allows test and control mRNAs and proteins to be separated by gel electrophoresis. Codons can be inserted among identical flanking codons after codon 13, 223, or 307 in codon test vectors pCT1, pCT2, and pCT3, respectively, the third site being six codons from the termination codon. The insertion of two to five consecutive AGG (low-usage) arginine codons selectively reduced the production of full-length test protein to extents that depended on the number of AGG codons, the site of insertion, and the amount of test mRNA. Production of aberrant proteins was also stimulated at high levels of mRNA. The effects occurred primarily at the translational level and were not produced by CGU (high-usage) arginine codons. Our results are consistent with the idea that sufficiently high levels of the AGG mRNA can cause essentially all of the tRNA(AGG) in the cell to become sequestered in translating peptidyl-tRNA(AGG) -mRNA-ribosome complexes stalled at the first of two consecutive AGG codons and that the approach of an upstream translating ribosome stimulates a stalled ribosome of frameshift, hop, or terminate translation.

Arginine↗

PubMed1984

In vivo and in vitro transcription of the Escherichia coli glutaminyl-tRNA synthetase gene.

We have characterized the in vivo and in vitro transcription products of the Escherichia coli glnS gene which codes for the enzyme glutaminyl-tRNA synthetase. The in vivo glnS transcript is about 1.9 kilobases long. Sequence analysis of the 5'- and 3'-ends of glnS mRNA showed that transcription initiates approximately 30 bases upstream from the translation initiation codon AUG and terminates approximately 230 bases downstream from the termination codon UAA. Characterization of the in vitro transcripts of glnS revealed similar transcription initiation and termination sites as were found in the glnS mRNA produced in vivo. These results indicate that the Pribnow box structure upstream and the dyad symmetry terminator structure downstream of the glnS structural region are regulatory signals used for glnS expression. In vitro transcription of glnS is not autogenously regulated by glutaminyl-tRNA synthetase and glutamine; it is also not affected by the presence of tRNA.

Amino Acyl-tRNA Synthetases↗

PubMed1997

[Regulation of translation of the distal lacZ gene in polycistronic mRNA by the ribosome stream from the proximal gene].

Four series of plasmids (pNSI, pNSII, pNLI, and pNLII) with artificial polycistrons containing the lacZ test gene were constructed. These plasmids coded for polycistronic mRNAs with two different types of cistron (orfZ and lacZ) coupling: in pNSI and pNLI, the orfZ termination codon and the lacZ initiation codon overlapped (type I); in pNSII and pNLII, the orfZ termination codon, was located upstream of the lacZ SD sequence. The length of the orfZ cistron was 60 bp in pNSI and pNSII or 300 bp in pNLI and pNLII. Plasmids with the same type of cistron coupling contained the same lacZ translation initiation region, whereas the structure of the orfZ translation initiation region varied, thereby providing varying efficiency of the orfZ gene translation. The effect of these variations on the efficiency of the lacZ gene translation was evaluated by direct measurement of the beta-galactosidase activity in Escherichia coli cells transformed with the corresponding plasmids. We found that the level of translation of the distal lacZ gene depended on the ribosome stream from the proximal gene and was maximal at the optimal ribosome stream level, which, in turn, depended on the type of cistron coupling.

Genes↗

PubMed1988

Synthesis of functional mouse cytochromes P-450 P1 and chimeric P-450 P3-1 in the yeast Saccharomyces cerevisiae.

Mouse liver cytochrome P-450 P1 was produced in the yeast Saccharomyces cerevisiae transformed by various expression vectors. The relative efficiency of the phosphoglycerate kinase and GAL10-CYC1 promoters to direct the P-450 P1 mRNA synthesis was determined. The level of protein synthesis was found to be dependent on the amount of the 5'-noncoding sequence of the original cDNA removed during the construction. Yeast-synthesised P-450 P1 was found to be integrated into the microsomal membrane in a fully functional form, as judged by Western blotting, optical spectra and enzymatic activities. The amount of P-450 reached up to 0.6% of the microsomal protein level. A nucleotide sequence coding for a chimeric enzyme in which 40 N-terminal codons of P-450 P1 were replaced by 36 N-terminal codons of P-450 P3 was constructed and expressed in yeast. The resulting protein retained full P-450 P1 activity and was produced with a similar efficiency suggesting that the P-450 N-terminal sequence is not involved in structures critical for the substrate specificities of the P1 isoenzyme.

Amino Acid Sequence↗

PubMed1980

The sequence at the 3' terminus of mouse immunoglobulin secreted mu chain messenger RNA determined from cloned cDNA.

The 3' terminal nucleotide sequence of two clones containing DNA complementary to mu chain mRNA of IgM-secreting cells has been determined. The sequence shows a termination codon (UGA) adjacent to the terminal tyrosine codon for the secreted protein and a 3' non-coding region of at least 106 bases. The primary translation product of this mu chain mRNA seems to terminate at the tyrosine of the secreted protein.

Animals↗

PubMed1989

An efficient and site-specific gene trimming method.

Trimming a DNA strand into a precisely determined fragment can be carried out efficiently by an improved method involving a site-specific trim-primer and a single-stranded DNA template which is generated from a multifunctional vector, pTZ18R, and linearized by using an Eco RI-pTZ18R splinter. A complementary DNA strand is synthesized by DNA polymerase I (Klenow), and the 3'-end of the template upstream from the annealed primer is trimmed by subsequent T4 DNA polymerase reaction. An ATG translation initiator codon or a termination codon can be incorporated into the trim-primer, providing versatility to this single-stranded DNA-initiated gene trimming method that can be applied to subcloning and expression of any DNA fragment with known terminal sequences.

Biotechnology↗

PubMed1993

Molecular cloning and sequencing of a canine tracheobronchial mucin cDNA containing a cysteine-rich domain.

To date the complete sequence of only one mammalian mucin cDNA, MUC1, has been reported, although several mucin proteins have been partially characterized. Here we report the nucleotide sequence of a canine tracheal mucin cDNA containing two potential translation initiation codons, one translation termination codon and a poly(A) tail. A lambda gt11 cDNA library prepared from canine tracheal epithelial cells was screened with polyclonal anti-apo-canine tracheal mucin antibodies with the aim of obtaining the deduced amino acid sequence of the mucin core protein. Antibody-positive clones containing overlapping inserts of various lengths were purified and used for nucleotide sequencing. Based on the sequencing data, synthetic oligonucleotide primers were constructed and both ends (5' and 3') of the cDNA were determined. The complete sequence was 3.7 kb and included an open reading frame with coding capacity for 1118 aa, two translation initiation ATG codons in context with Kozak consensus sequences, one polyadenylylation site, and a poly(A) stretch. The protein was rich in Thr, Pro, Ser, Gly, and Ala and poor in Tyr, Phe, and Trp. Although tandem repeats of amino acids were absent in the deduced canine tracheal mucin sequence, motifs TPTPTP and TTTTPV appeared 13 and 19 times, respectively. The C-terminal region contained a Cys-rich domain (although a few Cys residues were also present in the middle of the protein) as has been reported for bovine submaxillary mucin, porcine submaxillary mucin, rat intestinal mucin, human intestinal mucin, and frog skin mucin. This suggested that a broad group of mucins contain such a Cys-rich domain whose functional significance is yet to be understood. Three potential N-glycosylation sites were present in canine tracheal mucin and the amino acid sequence showed homology with both human tracheal and intestinal mucins. The N-terminal domain showed more flexibility (probably due to a high number of Pro residues in this region) when analyzed by the University of Wisconsin Genetics Computer Group program package to determine the predicted secondary structure. Evaluation of the transcripts using the canine mucin cDNA as a probe indicated a polydisperse message with total RNA.

Amino Acid Sequence↗

PubMed1995

Cloning and sequencing of the catechol 2,3-dioxygenase gene of Alcaligenes sp. KF711.

The catechol 2,3-dioxygenase is an aromatic ring-fission enzyme catalyzing the conversion of catechol to 2-hydroxymuconic semialdehyde. A catechol 2,3-dioxygenase gene has been cloned from chromosomal DNA of Alcaligenes sp. KF711, and its sequence was determined. The catechol 2,3-dioxygenase gene was consisted of 927 nucleotides with ATG initiation codon and TGA termination codon, which can encode a polypeptide of molecular weight 35 kDa containing 308 amino acid residues. G+C content of the gene was 58 mol%, and a putative ribosome-binding sequence was identified at about 10 nucleotides upstream from the ATG initiation codon. The sequence of catechol 2,3-dioxygenase from Alcaligenes sp. KF711 exhibited 81-92% homology at nucleotide level and 84-92% homology at amino acid level with those of corresponding enzymes encoded in xylE of TOL plasmid, nahH of NAH7 plasmid, and dmpB of Pseudomonas CF600.

Alcaligenes↗

PubMed1995

Fine structure of the human ceruloplasmin gene.

We characterized the genomic region corresponding to the human ceruloplasmin cDNA previously reported. Using PCR-direct sequencing methods, we determined precise intron/exon boundaries and intron-exon composition of the gene in the region. The gene region spanned about 50 kb and was composed of 19 exons and 18 introns. The lengths of exons and introns range from 107 to over 267 bp and from 0.44 to 10.0 kb, respectively. The translation initiation codon and the termination codon were located in exons 1 and 19, respectively. The nucleotide sequences of the introns were also determined in the region around the intron/exon boundaries for 24-220 bp. All the sequences around the intron/exon boundaries were consistent with the 5' and 3' consensus sequences for splice junctions of transcribed genes. Putative lariat sequences were identified between -17 and -42 nucleotides from the 3' splice junction for all 18 introns.

Alternative Splicing↗

PubMed1996

Structure of catechol 2,3-dioxygenase gene encoded in chromosomal DNA of Pseudomonas putida KF715.

A catechol 2,3-dioxygenase gene in chromosomal DNA of P. putida KF715 was cloned and its nucleotide sequence analyzed. The enzyme gene was composed of 924 base pairs with ATG initiation codon and TGA termination codon, which can encode a polypeptide of molecular weight 35 kDa containing 307 amino acids. A promoter-like sequence and a ribosome-binding sequence were identified upstream the enzyme gene. A deduced amino acid sequence of the catechol 2,3-dioxygenase exhibited 94% identity with that of corresponding enzyme in TOL plasmid and 25% identity with that of 2,3-dihydroxybiphenyl 1,2-dioxygenase from the same strain. Furthermore, sequence comparison of the catechol 2,3-dioxygenase with other extradiol-type dioxygenases has led to identify evolutionally conserved amino acid residues whose possible catalytic roles are proposed.

Amino Acid Sequence↗

PubMed1998

Genetic structure of the bphG gene encoding 2-hydroxymuconic semialdehyde dehydrogenase of Achromobacter xylosoxidans KF701.

2-Hydroxymuconic semialdehyde dehydrogenase catalyzes the conversion of 2-hydroxymuconic semialdehyde (HMS) to an enol form of 4-oxalocrotonate which is a step in the catechol meta-cleavage pathway. A bphG gene encoding HMS dehydrogenase of A. xylosoxidans KF701, a soil bacterium degrading biphenyl, was identified at between catechol 2,3-dioxygenase gene and HMS hydrolase gene, and its sequence was analyzed. An open reading frame (ORF) corresponding to bphG gene was consisted of 1461 nucleotides with ATG initiation codon and TGA termination codon. The ORF exhibited 66% of G + C content, and a putative ribosome-binding sequence, AGAGA, was identified at about 10 nucleotides upstream initiation codon of the bphG gene. The bphG gene can encode a polypeptide of molecular weight 52 kDa containing 486 amino acid residues. A deduced amino acid sequence of HMS dehydrogenase encoded in bphG gene from A. xylosoxidans KF701 exhibited the highest 94% homology with that of corresponding enzyme encoded in xylG from P. putida mt-2, 63% to 90% homology with those of other reported HMS dehydrogenases, and 29% to 42% homology with those of betaine aldehyde dehydrogenase, 5-carboxy-HMS dehydrogenase, aldehyde dehydrogenase, indole-3-acetaldehyde dehydrogenase, succinic semialdehyde dehydrogenase, methylmalonate semialdehyde dehydrogenase, and succinylglutamate 5-semialdehyde dehydrogenase. From an alignment of amino acid sequence of HMS dehydrogenase from A xylosoxidans KF701 with other reported dehydrogenases, putative cofactor NAD(+)-binding regions and catalytic residues were identified.

Alcaligenes↗

PubMed1985

The mitochondrial DNA molecular of Drosophila yakuba: nucleotide sequence, gene organization, and genetic code.

The sequence of the 16,019 nucleotide-pair mitochondrial DNA (mtDNA) molecule of Drosophila yakuba is presented. This molecule contains the genes for two rRNAs, 22 tRNAs, six identified proteins [cytochrome b, cytochrome c oxidase subunits I, II, and III (COI-III), and ATPase subunits 6 and 8] and seven presumptive proteins (URF1-6 and URF4L). Replication originates within a region of 1077 nucleotides that is 92.8% A + T and lacks any open reading frame larger than 123 nucleotides. An equivalent to the sequence found in all mammalian mtCDNAs that is associated with initiation of second-strand DNA synthesis is not present in D. yakuba mtDNA. Introns are absent from D. yakuba mitochondrial genes and there are few (0-31) intergenic nucleotides. The genes found in D. yakuba and mammalian mtDNAs are the same, but there are differences in their arrangement and in the relative proportions of the complementary strands of the molecule that serve as templates for transcription. Although the D. yakuba small and large mitochondrial rRNA genes are exceptionally low in G and C and are shorter than any other metazoan rRNA genes reported, they can be folded into secondary structures remarkably similar to the secondary structures proposed for mammalian mitochondrial rRNAs. D. yakuba mitochondrial tRNA genes, like their mammalian counterparts, are more variable in sequence than nonorganelle tRNAs. In mitochondrial protein genes ATG, ATT, ATA, and in one case (COI) ATAA appear to be used as translation initiation codons. The only termination codon found in these genes is TAA. In the D. yakuba mitochondrial genetic code, AGA, ATA, and TGA specify serine, isoleucine, and tryptophan, respectively. Fifty-nine types of sense condon are used in the D. yakuba mitochondrial protein genes, but 93.8% of all codons end in A or T. Codon-anticodon interactions may include both G-A and C-A pairing in the wobble position. Evidence is summarized that supports the hypothesis that A and T nucleotides are favored at all locations in the D. yakuba mtDNA molecule where these nucleotides are compatible with function.

Adenosine Triphosphatases↗

PubMed1988

DNA sequence analysis of the ade6 gene of Schizosaccharomyces pombe. Wild-type and mutant alleles including the recombination host spot allele ade6-M26.

The gene ade6 is located on chromosome III of the fission yeast Schizosaccharomyces pombe. It codes for the enzyme phosphoribosylaminoimidazole carboxylase involved in purine biosynthesis. A DNA fragment of 3043 nucleotides has been sequenced. It complements ade6 mutations when present on plasmids. An uninterrupted open reading frame of 552 amino acid residues was identified. A method for the cloning of chromosomal mutations by repair of gapped replication vectors in vivo has been developed. Twelve ade6 mutant alleles have been isolated. The sequence alterations of four mutant alleles have been determined. Among them are the ade6-M26 recombination hot spot mutation and the nearby ade6-M375 control mutation. Both are G to T base substitutions, converting adjacent glycine codons to TGA termination codons. They are suppressed by defined tRNA nonsense suppressors of the UGA type. The ade6-M26 mutation leads to a tenfold increase of the occurrence of conversion tetrads in comparison with other ade6 mutations. Possible explanations for the M26-induced increase of recombination frequency are discussed in relation to specific features of the nucleotide sequence identified in the region of the M26 mutation.

Alleles↗

PubMed1987

The nucleotide sequence and gene organization of the gerA spore germination operon of Bacillus subtilis 168.

The nucleotide sequence of the second and third genes in the Bacillus subtilis spore germination locus, gerA, has been determined and the amino acid (aa) sequence was derived. Two open reading frames (ORFs), corresponding to genes II and III, encode 364-aa residue and 373-aa residue polypeptides, respectively. The gene II product, Mr 41,257, would contain long stretches of hydrophobic aa residues and may be a membrane protein; the gene III product, Mr 42,363, is relatively hydrophilic but possesses an apparent signal peptide for transfer across, and perhaps localisation on, a membrane. The ORFs for genes I and II overlap by eleven codons and the termination codon of gene II overlaps the initiation codon of gene III. Insertional inactivation experiments using integrational plasmids have indicated that the gerA locus is a single transcriptional unit. The expression of the gerA genes has been studied using a lacZ transcriptional fusion; they constitute a developmentally regulated operon.

Amino Acid Sequence↗

PubMed1991

A new single base substitution in a Japanese phenylketonuria (PKU) patient.

DNA analysis on phenylalanine hydroxylase (PAH) gene was performed in four Japanese PKU patients. By the Southern analysis, three patients were confirmed to be homozygous for haplotype 4, and the other one was homozygous for haplotype 2. Sequence analysis on the mutant PAH gene of this haplotype 2 patient, who was born to first-cousin parents, disclosed it to be a C-to-T transition in exon 7. This transition causes the substitution of Arg261 codon to a termination codon.

Base Sequence↗

PubMed1998

Mutations that alter RNA splicing of the human HPRT gene: a review of the spectrum.

The human HPRT gene contains spans approximately 42,000 base pairs in genomic DNA, has a mRNA of approximately 900 bases and a protein coding sequence of 657 bases (initiation codon AUG to termination codon UAA). This coding sequence is distributed into 9 exons ranging from 18 (exon 5) to 184 (exon 3) base pairs. Intron sizes range from 170 (intron 7) to 13,075 (intron 1) base pairs. In a database of human HPRT mutations, 277 of 2224 (12.5%) mutations result in alterations in splicing of the mRNA as analyzed by both reverse transcriptase mediated production of a cDNA followed by PCR amplification and cDNA sequencing and by genomic DNA PCR amplification and sequencing. Mutations have been found in all eight 5' (donor) and 3' (acceptor) splice sequences. Mutations in the 5' splice sequences of introns 1 and 5 result in intron inclusion in the cDNA due to the use of cryptic donor splice sequences within the introns; mutations in the other six 5' sites result in simple exon exclusion. Mutations in the 3' splice sequences of introns 1, 3, 7 and 8 result in partial exon exclusion due to the use of cryptic acceptor splice sequences within the exons; mutations in the other four 3' sites result in simple exon exclusion. A base substitution in exon 3 (209G-->T) creates a new 5' (donor) splice site which results in the exclusion of 110 bases of exon 3 from the cDNA. Two base substitutions in intron 8 (IVS8-16G-->A and IVS8-3T-->G) result in the inclusion of intron 8 sequences in the cDNA due to the creation of new 3' (acceptor) splice sites. Base substitution within exons 1, 3, 4, 6 and 8 also result in splice alterations in cDNA. Those in exons 1 and 6 are at the 3' end of the exon and may directly affect splicing. Those within exons 3 and 4 may be the result of the creation of nonsense codons, while those in exon 8 cannot be explained by this mechanism. Lastly, many mutations that affect splicing of the HPRT mRNA have pleiotropic effects in that multiple cDNA products are found.

Animals↗

PubMed1985

Molecular cloning of a full-length cDNA for human alcohol dehydrogenase.

We have cloned a full-length cDNA coding for human alcohol dehydrogenase (ADH; alcohol:NAD+ oxidoreductase, EC 1.1.1.1) from a human liver cDNA library constructed in phage lambda gt11. The library was screened by using a rabbit antibody against human ADH as a first probe, by the modified method of Young and Davis [Young, R. A. & Davis, R. W. (1983) Proc. Natl. Acad. Sci. USA 80, 1194-1198]. Mixed 14-mer synthetic oligonucleotides encoding Asp-Asp-His-Val-Val and Gln-Cys-Gly-Lys-Cys were used as a second probe. These amino acid sequences are considered to be common in all three subunits (alpha, beta, and gamma) controlled by the ADH1, ADH2, and ADH3 loci. Ten lambda gt11 recombinants of 35 positive plaques obtained by antibody screening contained inserted cDNAs of 1.5-2.4 kilobase pairs and were found to exhibit positive signals by hybridization with synthetic probes. One of them, with an inserted cDNA of 1631 base pairs, contained a sequence that encodes 374 amino acid residues of the human beta 1 subunit, a chain initiation codon, a chain termination codon, and additional 3' and 5' untranslated regions. A complete amino acid sequence of the human beta 1 subunit was deduced from the cDNA.

Alcohol Dehydrogenase↗