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PubMed1982

Cloning and nucleotide sequence analysis of human embryonic zeta-globin cDNA.

Clones of human embryonic alpha-like zeta-globin cDNA were isolated, by detection using cross-hybridization to human alpha-globin cDNA probes, from a cDNA library derived from the mRNA of the human erythroleukemia cell line K562. Nucleotide sequence analysis of these cDNA clones revealed a coding sequence that corresponds perfectly to the independently derived amino acid sequence of the human zeta-globin chain. Comparison of the nucleotide sequence of human zeta-globin cDNA with that of human alpha-globin cDNA confirmed previous estimates of very distant evolutionary divergence between the human zeta- and alpha-globin genes. Nevertheless, the human zeta-globin cDNA sequence shares a remarkable similarity to that of the alpha-globin gene in its codon usage, high G + C base composition, and lack of bias against usage of CG dinucleotides.

Base Composition↗
PubMed2005

Describing ancient horizontal gene transfers at the nucleotide and gene levels by comparative pathogenicity island genometrics.

MOTIVATION: Lateral gene transfer is a major mechanism contributing to bacterial genome dynamics and pathovar emergence via pathogenicity island (PAI) spreading. However, since few of these genomic exchanges are experimentally reproducible, it is difficult to establish evolutionary scenarios for the successive PAI transmissions between bacterial genera. Methods initially developed at the gene and/or nucleotide level for genomics, i.e. comparisons of concatenated sequences, ortholog frequency, gene order or dinucleotide usage, were combined and applied here to homologous PAIs: we call this approach comparative PAI genometrics. RESULTS: YAPI, a Yersinia PAI, and related islands were compared with measure evolutionary relationships between related modules. Through use of our genometric approach designed for tracking codon usage adaptation and gene phylogeny, an ancient inter-genus PAI transfer was oriented for the first time by characterizing the genomic environment in which the ancestral island emerged and its subsequent transfers to other bacterial genera.

Chromosome Mapping↗
PubMed1987

Cloning and characterization of the gene coding for cytoplasmic seryl-tRNA synthetase from Saccharomyces cerevisiae.

We have screened a Saccharomyces cerevisiae expression library with antibodies against seryl-tRNA synthetase (SerRS) from baker's yeast. In this way we obtained clones which contain serS, the structural gene for seryl-tRNA synthetase. Genomic Southern blots show that the serS gene resides on a 5.0 kb SalI fragment. Nucleotide sequence analysis of the genes revealed a single open reading frame from which we deduced the amino acid sequence of the enzyme consistent with that of two peptides isolated from SerRS. The enzyme is comprised of 462 amino acids consistent with earlier determinations of its molecular weight. The codon usage of serS is typical of abundant yeast proteins. Nuclease S1 analysis of serS mRNA defined the RNA initiation site 20-40 bases downstream from an AT rich sequence containing the TATA box and 21-39 nucleotides upstream of the translation initiation codon. Yeast strains transformed with the cloned gene overproduce seryl-tRNA synthetase in vivo.

Amino Acid Sequence↗
PubMed1988

Cloning and expression in Escherichia coli of two additional amylase genes of a strictly anaerobic thermophile, Dictyoglomus thermophilum, and their nucleotide sequences with extremely low guanine-plus-cytosine contents.

An obligately anaerobic and extremely thermophilic bacterium, Dictyoglomus thermophilum, produces multiple extracellular amylases. In addition to one of the amylase genes, amyA, which we previously cloned and characterized, we have cloned two additional genes, amyB and amyC, coding for amylases of this thermophile, into Escherichia coli and determined their nucleotide sequences. The two amylase genes were expressed under the control of E. coli promoters. Almost all activity was detected in the intracellular fraction in the E. coli cells. The molecular mass and NH2-terminal amino acid sequence of the AmyB enzyme, which was purified from an E. coli transformant containing the amyB gene, confirmed that the reading frame of amyB consisted of 562 amino acids (Mr 67,000). The molecular mass of the AmyC enzyme, estimated by activity staining of a crude extract of E. coli containing amyC, confirmed that AmyC consisted of 498 amino acids (Mr 59,000). The optimal temperatures for AmyB and AmyC activities on soluble starch were 80 degrees C and 70 degrees C, respectively. Both AmyB and AmyC showed a pH optimum of 5.5. AmyB and AmyC showed a different pattern of starch hydrolysis when examined by thin-layer chromatography. Some homology in the amino acid sequences with the functional regions of Taka-amylase A was found in both AmyB and AmyC. The codon usage in the amyA, amyB and amyC genes was highly biased, which reflects the fact that the guanine-plus-cytosine (G + C) content of DNA of D. thermophilum is 29 mol%. The distribution of G and C at each position of the codons was non-random; the G + C content of the first position of codons is significantly high, whereas that of the third position is somewhat low. In addition, codons consisting only of A and T were preferentially used in this thermophile.

Amino Acid Sequence↗
PubMed1986

Cloning and characterization of the gene for beta-tubulin from a benomyl-resistant mutant of Neurospora crassa and its use as a dominant selectable marker.

We cloned the beta-tubulin gene of Neurospora crassa from a benomyl-resistant strain and determined its nucleotide sequence. The gene encodes a 447-residue protein which shows strong homology to other beta-tubulins. The coding region is interrupted by six introns, five of which are within the region coding for the first 54 amino acids of the protein. Intron position comparisons between the N. crassa gene and other fungal beta-tubulin genes reveal considerable positional conservation. The mutation responsible for benomyl resistance was determined; it caused a phenylalanine-to-tyrosine change at position 167. Codon usage in the beta-tubulin gene is biased, as has been observed for other abundantly expressed N. crassa genes such as am and the H3 and H4 histone genes. This bias results in pyrimidines in the third positions of 96% of the codons in codon families in which there is a choice between purines and pyrimidines in this position. Bias is also evident by the absence of 19 of the 61 sense codons. We demonstrated that benomyl resistance is due to the cloned beta-tubulin gene of strain Bml511(r)a and that this gene can be used as a dominant selectable marker in N. crassa transformation.

Amino Acid Sequence↗
PubMed2005

Are stop codons recognized by base triplets in the large ribosomal RNA subunit?

The precise mechanism of stop codon recognition in translation termination is still unclear. A previously published study by Ivanov and colleagues proposed a new model for stop codon recognition in which 3-nucleotide Ter-anticodons within the loops of hairpin helices 69 (domain IV) and 89 (domain V) in large ribosomal subunit (LSU) rRNA recognize stop codons to terminate protein translation in eubacteria and certain organelles. We evaluated this model by extensive bioinformatic analysis of stop codons and their putative corresponding Ter-anticodons across a much wider range of species, and found many cases for which it cannot explain the stop codon usage without requiring the involvement of one or more of the eight possible noncomplementary base pairs. Involvement of such base pairs may not be structurally or thermodynamically damaging to the model. However, if, according to the model, Ter-anticodon interaction with stop codons occurs within the ribosomal A-site, the structural stringency which that site imposes on sense codon.tRNA anticodon interaction should also extend to stop codon.Ter-anticodon interactions. Moreover, with Ter-tRNA in place of an aminoacyl-tRNA, for each of the various Ter-anticodons there is a sense codon that can interact with it preferentially by complementary and wobble base-pairing. Both these considerations considerably weaken the arguments put forth previously.

Base Pairing↗
PubMed1991

Cloning, characterization, and physical location of the rplY gene which encodes ribosomal protein L25 in Escherichia coli K12.

The rplY gene of Escherichia coli K12 encoding ribosomal protein L25 was cloned from the ordered clone bank and located at coordinate 2,291 kb on the physical map of E. coli. Determination of the nucleotide sequence indicated that the coding region contains 285 nucleotide pairs including a translational initiator and terminator. The amino acid sequence of the protein deduced from the nucleotide sequence matched completely the sequence determined for ribosomal protein L25. The coding region was found to be preceded by a typical promoter-like sequence and was followed by a DNA region capable of forming a secondary structure characteristic of a transcriptional terminator. Thus, the gene was concluded to constitute a transcriptional unit (operon). A preliminary analysis by Northern blot supported this conclusion. The codon usage pattern of the rplY gene is characteristic of the ribosomal protein genes in E. coli.

Amino Acid Sequence↗
PubMed1989

The expression of a highly expressed Bacillus subtilis gene is not reduced by introduction of multiple codons normally not present in such genes.

Four serine or threonine codons were introduced into a highly expressed Bacillus subtilis gene. The introduced codons were ones either common in highly expressed B. subtilis genes, or never used in such genes. Strikingly, the level and rate of expression of the modified genes containing either type of extra codons was identical. This suggests that in B. subtilis codon usage patterns may play little or no role in effecting the level of gene expression.

Bacillus subtilis↗
PubMed1985

Sequence of the viral replicase gene from foot-and-mouth disease virus C1-Santa Pau (C-S8).

The nucleotide sequence of the region including the viral replicase gene, the carboxy terminus of protein P18, and the 3'-extracistronic region of foot-and-mouth disease virus (FMDV) type C1-Santa Pau (C-S8) has been determined from previously cloned cDNA fragments [Villanueva et al., Gene 23 (1983) 185-194]. The comparison with the corresponding gene segments of FMDV of serotypes A or O shows base substitutions in 7.2-8.6% of residues in the replicase gene with no insertions or deletions. This is about fourfold lower variation than found for the region encoding capsid protein VP1 of the corresponding viruses. Intermediate variability (substitution at 16.1-23.6% positions) exists in the 3'-extracistronic region, including point mutations, insertions and deletions. The predicted amino acid sequence of the replicase gene indicates that 75.5-82.6% of mutations are silent and that 93.4% of amino acids are conserved in the four FMDV replicases. The frequency of certain types of silent mutations and of rare codon usage is significantly lower for the replicase gene than for the protein VP1 coding region.

Amino Acid Sequence↗
PubMed1987

The nucleotide sequence of a streptomycin streptomycin phosphotransferase (streptomycin kinase) [corrected] gene from a streptomycin producer.

The nucleotide sequence of the DNA fragment containing the streptomycin phosphotransferase (streptomycin kinase) [corrected] gene from the streptomycin-producer Streptomyces griseus strain HUT 6037 was determined. Analysis of the sequence revealed an open reading frame which could encode 325 amino acid residues. A biased codon usage pattern, reflecting the high G + C composition (approximately 74%) of Streptomyces DNA, was observed in the gene.

Amino Acid Sequence↗
PubMed1986

Evolutionary aspects of trypanosomes: analysis of genes.

The genes for four glycolytic enzymes of Trypanosoma brucei have been analyzed. The proteins encoded by these genes show 38-57% identity with their counterparts in other organisms, whether pro- or eukaryotic. These data are consistent with a phylogenetic tree in which trypanosomes diverged very early from the main branch of the eukaryotic lineage. No definite conclusion can be drawn yet about the evolutionary origin of glycosomes, the microbodies of trypanosomes which contain most enzymes of the glycolytic pathway. A bias could be observed in the codon usage of the glycolytic genes and genes for other housekeeping proteins, indicating that trypanosomes may have selected a nucleotide sequence that enables efficient translation. However, the genes for variant surface glycoproteins (VSGs) do not show such a bias. This lack of preference for special codons is explained by the high evolutionary rate that could be observed for VSG genes.

Animals↗
PubMed2005

Strategies for efficient production of heterologous proteins in Escherichia coli.

In recent years, the number of recombinant proteins used for therapeutic applications has increased dramatically. Production of these proteins has a remarkable demand in the market. Escherichia coli offers a means for the rapid and economical production of recombinant proteins. These advantages, coupled with a wealth of biochemical and genetic knowledge, have enabled the production of such economically therapeutic proteins such as insulin and bovine growth hormone. These demands have driven the development of a variety of strategies for achieving high-level expression of protein, particularly involving several aspects such as expression vectors design, gene dosage, promoter strength (transcriptional regulation), mRNA stability, translation initiation and termination (translational regulation), host design considerations, codon usage, and fermentation factors available for manipulating the expression conditions, which are the major challenges is obtaining the high yield of protein at low cost.

Codon↗
PubMed1983

Evidence that natural selection acts on silent mutation.

Analysis of nucleic acid sequence data of mammalian hemoglobin, yeast cytochrome c, and human interferon reveals strong biases in favor of specific codons. These biases do not appear to dissipate over time, suggesting that an indirect form of selection acts on silent mutations. The data are compatible with the "bootstrapping" hypothesis that silent mutations which alter the rate of evolution can hitchhike with traits whose appearance they facilitate. Selection involving modulating effects of codon usage on gene expression may also be involved, but the data appear to exclude simple maximization of gene expression.

Animals↗
PubMed1996

Location and sequence analysis of a 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase-encoding gene (bpdF) of the biphenyl/polychlorinated biphenyl degradation pathway in Rhodococcus sp. M5.

The 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HOPD) hydrolase-encoding gene (bpdF) in the biphenyl (BP)/polychlorinated biphenyl (PCB)-degrading bacterium, Rhodococcus sp. M5 (M5), was found to be located within a 4.5-kb HindIII-BamHI genomic DNA that was 5.4 kb downstream from the bpdC1C2BADE gene cluster. The deduced amino acid (aa) sequence of bpdF revealed that the hydrolase contains 297 aa (32679 Da) that was verified by expression in the Escherichia coli T7 RNA polymerase/promoter system. Unlike previously known HOPD hydrolases, the aa sequence of BpdF appears unique. Interestingly, all HOPD hydrolases and related proteins from the phenol and toluene/xylene degradation pathways, were found to have a bias in the codon usage in the catalytic Ser within the conserved VGNS(M/F)GG motif.

Amino Acid Sequence↗
PubMed2004

Plasmid encoding papillomavirus Type 16 (HPV16) DNA constructed with codon optimization improved the immunogenicity against HPV infection.

Human papillomavirus Type 16 (HPV16) infections can cause neoplasia, which is thought to be closely associated with the development of cervical cancers. In the study, we attempted to construct a DNA plasmid encoding a HPV16 capsid protein (L1) and a HPV16 oncoprotein (E7), which was capable of preventing HPV16 infection and eliminating HPV16-infected cells. A plasmid, L1E7hpSCA1, encoding the L1 and E7 genes with the codon usage optimized for mammalian cell expression, was constructed. Mutations were introduced into the E7 gene sequence for reducing its oncogenicity. C57BL/6 mice were intramuscularly immunized at tibialis anterior (TA) muscles with the newly constructed L1E7hpSCA1 plasmid. The immune responses induced by the L1E7hpSCA1 plasmid (with codon optimization) and a control L1E7pSCA1 plasmid (without codon optimization) were compared. It is shown that the L1E7hpSCA1 was able to induce much stronger immune responses than the L1E7pSCA1. Sera obtained from immunized animals were found to contain anti-HPV16 antibodies as detected by ELISA and hemagglutination inhibition (HAI) assays. Cytotoxicity and interferon-gamma assays showed that spleenocytes from immunized animals were able to recognize and lyze E7 expressing tumor TC-1 cells. Moreover, the growth of E7 expressing tumor mass was inhibited in vaccinated mice. In vivo tumor protection test indicated that tumor formation was prevented in the experimental animals (67%) after vaccination with L1E7hpSCA1, while for the control group injected with L1E7pSCA1 only and the animal group injected with pSCA1 only, tumor formation was observed in all experimental animals. Our results suggest that the L1E7h gene (with codon optimization) is more effective against HPV16 than the L1E7 gene (without codon optimization). The L1E7hpSCA1 plasmid was able to provide protection against E7 expressing tumor, and it might have the potential to be a vaccine candidate for HPV prevention.

Animals↗
PubMed1989

Nucleotide sequence and transcriptional analysis of the Streptomyces griseus gene (afsA) responsible for A-factor biosynthesis.

The nucleotide sequence of the Streptomyces griseus afsA gene, possibly encoding a key enzyme for A-factor (2-isocapryloyl-3R-hydroxymethyl-gamma-butyrolactone) biosynthesis, was determined. The translational initiation codon was identified by introducing out-of-frame mutations at appropriate positions by oligonucleotide-directed mutagenesis. The afsA gene was thus found to code for a protein of 301 amino acid residues and 32.6 kilodaltons whose codon usage pattern was in agreement with the general tendency of Streptomyces genes with an extremely high guanine-plus-cytosine content. High-resolution S1 nuclease mapping indicated that the transcriptional start point was the A residue, the first position of the ATG translational initiation codon.

4-Butyrolactone↗
PubMed1986

Structure of the Saccharomyces cerevisiae HO gene and analysis of its upstream regulatory region.

The HO gene product of Saccharomyces cerevisiae is a site-specific endonuclease that initiates mating type interconversion. We have determined the nucleotide sequence of a 3,129-base-pair (bp) segment containing HO. The segment contains a single long open reading frame encoding a polypeptide of 586 amino acids, which has unusual (unbiased) codon usage and is preceded by 762 bp of upstream region. The predicted HO protein is basic (16% lysine and arginine) and is calculated to have a secondary structure that is 30% helical. The corresponding transcript is initiated approximately 50 nucleotides prior to the presumed initiation codon. Insertion of an Escherichia coli lacZ gene fragment into the putative HO coding segment inactivated HO and formed a hybrid HO-lacZ gene whose beta-galactosidase activity was regulated by the mating type locus in the same manner as HO (repressed by a 1-alpha 2). Upstream regions of 1,360 and 762 bp conferred strong repression; 436 bp led to partial constitutivity and 301 bp to full constitutivity. Thus, DNA sequences that confer repression of HO by a1-alpha 2 are at least 250 nucleotides upstream of the transcription start point and are within 436 nucleotides of the HO initiation codon. The progressive loss of repression suggests that both the -762 to -436 and the -436 to -301 intervals contain sites for regulation by a1-alpha 2. The HO gene contains two distinct regions that promote autonomous replication of plasmids in S. cerevisiae. These regions contain sequences that are homologous to the two conserved sequences that are associated with ARS activity.

Amino Acid Sequence↗
PubMed2004

Intragenic codon bias in a set of mouse and human genes.

To better conceptualize the mechanism underlying the evolution of synonymous codons, we have analysed intragenic codon usage in chosen "regions" of some mouse and human genes. We divided a given gene into two regions: one consisting of a trinucleotide repeat (TNR) and the other consisting of the "rest of the coding region" (RCR). Usually, a TNR is composed of a repetitive single codon, which may reflect its frequency in a gene. In contrast, a non-random frequency of a codon in the RCR versus TNR (or vice versa) of a gene should indicate a bias for that codon within the TNR. We examined this scenario by comparing codon frequency between the RCR and the cognate TNR(s) for a set of human and mouse genes. A TNR length of six amino acids or more was used to identify genes from the Genbank database. Twenty nine human and twenty one mouse genes containing TNRs coding for nine different amino acid runs were identified. The ratio of codon frequency in a TNR versus the corresponding RCR was expressed as "fold change" which was also regarded as a measure of codon bias (defined as preferential use either in TNR or in RCR). Chi-square values were then determined from the distribution of codon frequency in a TNR vs. the cognate RCR. At p<0.001, 22% and 27%, respectively, of human and mouse TNRs showed codon bias. Greater than 40% of the TNRs (29 out of 69 in human, and 18 of 42 in mouse) showed codon bias at p<0.05. In addition, we identify eight single-codon TNRs in mouse and ten in human genes. Thus, our results show intragenic codon bias in both mouse and human genes expressed in diverse tissue types. Since our results are independent of the Codon Adaptation Index (CAI) and starvation CAI, and since the tRNA repertoire in a cell or in a tissue is constant, our data suggest that other constraints besides tRNA abundance played a role in creating intragenic codon bias in these genes.

Amino Acids↗