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PubMed1999

Allele-specific, nested, one tube PCR: application to Pfmdr1 polymorphisms in Plasmodium falciparum.

An allele-specific, one tube PCR for the sensitive and reliable detection of point mutations in Plasmodium falciparum DNA is described. Design of specific internal primers and optimization of the PCR is simple, and the procedure is robust and sensitive. Single nucleotide polymorphisms at codons 184, 1034, 1042 and 1246 of the P, falciparum multidrug resistance gene Pfmdr1, were examined in 6 laboratory isolates, to validate the technique.

Alleles↗

PubMed1990

Expression of human asparagine synthetase in Saccharomyces cerevisiae.

Human asparagine synthetase was expressed in the yeast Saccharomyces cerevisiae. The identity of the expressed protein was confirmed by immunoblotting and in vitro enzymatic activity. The recombinant enzyme was shown to have both the ammonia- and glutamine-dependent asparagine synthetase activity in vitro. In contrast to overproduction in Escherichia coli, the expressed protein was found to be soluble in the yeast cell. Furthermore, expression in yeast made it possible to isolate non-degraded human asparagine synthetase which had also the N-terminal methionine correctly processed. The yeast expression plasmid was constructed for optimal production of the recombinant enzyme. In addition, unique restriction enzyme sites that bracket the first five codons of the human asparagine synthetase gene were introduced. This will allow the use of oligonucleotide cassette mutagenesis to investigate the role of the N-terminal amino acids in asparagine synthetase enzymatic activity.

Amino Acid Sequence↗

PubMed1996

Controlled gene expression systems for Lactococcus lactis with the food-grade inducer nisin.

The kinetics, control, and efficiency of nisin-induced expression directed by the nisA promoter region were studied in Lactococcus lactis with transcriptional and translational fusions to the gusA reporter genes. In the nisin-producing L. lactis strain NZ9700, the specific beta-glucuronidase activity increased very rapidly after mid-exponential growth until the maximum level at the start of the stationary phase was reached. Expression of the gusA gene was also studied in L. lactis NZ9800, an NZ9700 derivative carrying a deletion in the structural nisA gene that abolishes nisin production, and in L. lactis NZ3900, an MG1363 derivative containing the regulatory nisRK genes integrated in the chromosome. In both strains, beta-glucuronidase activity was linearly dependent on the amount of nisin added to the medium. Without nisin, no beta-glucuronidase production was observed. To optimize translation initiation, an expression vector was constructed by fusing the gusA gene translationally to the start codon of the nisA gene. Use of the translational fusion vector yielded up to six times more beta-glucuronidase activity than the transcriptional fusion vector in these strains after induction by nisin. In this way, gene expression can be achieved in a dynamic range of more than 1,000-fold. The beta-glucuronidase activity was found to be up to 25-fold higher in extracts of strain NZ3900 than in extracts of strain NZ9800. This translational fusion vector was used for high-level production of aminopeptidase N, up to 47% of the total intracellular protein. These results clearly illustrate the potential of the nisin-inducible expression system for overproduction of desired proteins.

Aminopeptidases↗

PubMed2000

Identification of ciprofloxacin-resistant Campylobacter jejuni by use of a fluorogenic PCR assay.

Fluoroquinolones are one class of antimicrobial agents commonly used to treat severe Campylobacter jejuni infection. C. jejuni strains resistant to high levels of the fluoroquinolone ciprofloxacin (MIC >/=16 microg/ml) have been predominantly characterized with a C-->T transition in codon 86 of gyrA. The gyrA gene encodes one subunit of DNA gyrase, which is a primary target for fluoroquinolone antibiotics. This study establishes a rapid PCR-based TaqMan method for identifying ciprofloxacin-resistant C. jejuni strains that carry the C-->T transition in codon 86 of gyrA. The assay uses real-time detection, eliminating the need for gel electrophoresis. Optimization of the assay parameters using purified Campylobacter DNA resulted in the ability to detect femtogram levels of DNA. The method should be useful for monitoring the development of ciprofloxacin resistance in C. jejuni. Compiled nucleotide sequence data on the quinolone resistance-determining region of gyrA in Campylobacter indicate that sequence comparison of this region is a useful method for tentative identification of Campylobacter isolates at the species level.

Animals↗

PubMed2002

Transcription regulatory sequences and mRNA expression levels in the coronavirus transmissible gastroenteritis virus.

The transcription regulatory sequences (TRSs) of the coronavirus transmissible gastroenteritis virus (TGEV) have been characterized by using a helper virus-dependent expression system based on coronavirus-derived minigenomes to study the synthesis of subgenomic mRNAs. The TRSs are located at the 5' end of TGEV genes and include a highly conserved core sequence (CS), 5'-CUAAAC-3', that is essential for mediating a 100- to 1,000-fold increase in mRNA synthesis when it is located in the appropriate context. The relevant sequences contributing to TRS activity have been studied by extending the CS 5' upstream and 3' downstream. Sequences from virus genes flanking the CS influenced transcription levels from moderate (10- to 20-fold variation) to complete mRNA synthesis silencing, as shown for a canonical CS at nucleotide (nt) 120 from the initiation codon of the S gene that did not lead to the production of the corresponding mRNA. An optimized TRS has been designed comprising 88 nt from the N gene TRS, the CS, and 3 nt 3' to the M gene CS. Further extension of the 5'-flanking nucleotides (i.e., by 176 nt) decreased subgenomic RNA levels. The expression of a reporter gene (beta-glucuronidase) by using the selected TRS led to the production of 2 to 8 microg of protein per 10(6) cells. The presence of an appropriate Kozak context led to a higher level of protein expression. Virus protein levels were shown to be dependent on transcription and translation regulation.

3' Flanking Region↗

PubMedDate not supplied

[Evolution of the genetic code and earliest proteins. Reconstruction from the current sequences].

One would expect that present-day protein sequences have changed many times during their evolution, at every point, so that there is no chance to recognize in the sequences any traces of their ancient organization. It turns out to be not true. Massive analysis of complete genomes of bacteria allows one to derive, according to very specific predictions, distinct features of very early sequences and to outline the history of evolution protein. Modern proteins appear to have evolved from short peptides of mixed sequences of two alphabet types. They were then closed to sequences of optimal size from which modern folds/domains and multidomain proteins were formed. The reconstruction of amino acid and codon chronology is described. A specific idea on the nature and evolutionary significance of gene splicing is suggested. The gene splicing, while obeying the rules of basic structural organization of proteins, offers accessibility to regions of sequence space that could not be reached by mutational changes typical for prokaryotes.

Amino Acid Sequence↗

PubMed2002

A quality control pathway that down-regulates aberrant T-cell receptor (TCR) transcripts by a mechanism requiring UPF2 and translation.

Nonsense-mediated decay (NMD) is an RNA surveillance pathway that degrades mRNAs containing premature termination codons (PTC). T-cell receptor (TCR) and immunoglobulin (Ig) transcripts, which are encoded by genes that very frequently acquire PTCs during lymphoid ontogeny, are down-regulated much more dramatically in response to PTCs than are other known transcripts. Another feature unique to TCR, Ig, and a subset of other mRNAs is that they are down-regulated in response to nonsense codons in the nuclear fraction of cells. This is paradoxical, as the only well recognized entity that recognizes nonsense codons is the cytoplasmic translation apparatus. Therefore, we investigated whether translation is responsible for this nuclear-associated mechanism. We found that the down-regulation of TCR-beta transcripts in response to nonsense codons requires several features of translation, including an initiator ATG and the ability to scan. We also found that optimal down-regulation depends on a Kozak consensus sequence surrounding the initiator ATG and that it can be initiated by an internal ribosome entry site, neither of which has been demonstrated before for any other PTC-bearing mRNA. At least a portion of this down-regulatory response is mediated by the NMD pathway as antisense hUPF2 transcripts increased the levels of PTC-bearing TCR-beta transcripts in the nuclear fraction of cells. We conclude that a hUPF2-dependent RNA surveillance pathway with translation-like features operating in the nuclear fraction of cells prevents the expression of potentially deleterious truncated proteins encoded by non-productively rearranged TCR genes.

Adaptor Proteins, Signal Transducing↗

PubMed2001

Optimality of the genetic code with respect to protein stability and amino-acid frequencies.

BACKGROUND: The genetic code is known to be efficient in limiting the effect of mistranslation errors. A misread codon often codes for the same amino acid or one with similar biochemical properties, so the structure and function of the coded protein remain relatively unaltered. Previous studies have attempted to address this question quantitatively, by estimating the fraction of randomly generated codes that do better than the genetic code in respect of overall robustness. We extended these results by investigating the role of amino-acid frequencies in the optimality of the genetic code. RESULTS: We found that taking the amino-acid frequency into account decreases the fraction of random codes that beat the natural code. This effect is particularly pronounced when more refined measures of the amino-acid substitution cost are used than hydrophobicity. To show this, we devised a new cost function by evaluating in silico the change in folding free energy caused by all possible point mutations in a set of protein structures. With this function, which measures protein stability while being unrelated to the code's structure, we estimated that around two random codes in a billion (109) are fitter than the natural code. When alternative codes are restricted to those that interchange biosynthetically related amino acids, the genetic code appears even more optimal. CONCLUSIONS: These results lead us to discuss the role of amino-acid frequencies and other parameters in the genetic code's evolution, in an attempt to propose a tentative picture of primitive life.

Amino Acid Substitution↗

PubMed1995

Nucleotide sequence and structural analysis of the rat RT1.Eu and RT1.Aw3l genes, and of genes related to RT1.O and RT1.C.

A cDNA library was constructed using mRNA isolated from the R21 strain of rats which have the major histocompatibility complex (MHC) haplotype RT1.AlBlDlEu and the growth and reproduction complex (grc) genotype grc+. The cDNA clones that hybridized with the class I probes pAG64c and pARI.5 and were 1.3-1.7 kilobases were selected. Full-length clones were identified by sequencing partially the 5' and 3' ends of each clone, by the presence of a start codon at the 5' end, and by a polyadenylation sequence at the 3' end. The full-length cDNA clones were examined for in vitro transcription by transfection into human CIR cells using electroporation, and expression was detected by flow cytometry using monoclonal antibodies specific to the heavy chains and polyclonal antibody to beta 2-microglobulin. The RT1.Eu gene was transcribed and expressed optimally, and its nucleotide and deduced amino acid sequences differed significantly from the RT1.Aa, RT1.A(l), RT.Au, LW2, and 11/3R genes but only slightly from the RT1.K gene. The high level of sequence similarity between RT1.Eu and RT1.K suggests that the two genes may have originated from a common ancestral gene. In addition, three new genes (RT1.Aw3l, RT1.C-type, and RT1.O-type) were identified. The RT1.Aw3l gene is almost identical to RT1.A(l) with the exception of an in frame deletion of 21 nucleotides in exon 2 leading to a 7 amino acid deletion in the alpha 1 domain of the deduced amino acid sequence and 11 nucleotide substitutions and insertions in the rest of the sequence. It transcribed optimally, but no significant expression was detected. The RT1.C-type gene 119 is very similar (97%) to the LW2 gene in the 3' untranslated region, which suggests that it is in the RT1.C region. It transcribed optimally, but no significant expression was detected. The RT1.O-type gene 149 has all the features of a class Ib gene, but a premature stop codon in the alpha 1 domain causes incomplete translation. Its in vitro transcription was very low, and no expression was detected. These studies, combined with previous work, indicate that in the MHC of the R21 strain three class Ia genes (Eu, A(l), Aw3l) and three class Ib genes (C-type, O-type, N) are transcribed but only two class Ia genes (Eu, A(l)) are expressed.

Amino Acid Sequence↗

PubMed2026

Robust error-minimization in the genetic code across physicochemical metrics and variant codes: A graph-theoretic analysis in GF(2)6.

The standard genetic code reduces the impact of point mutations, but the robustness of this property across physicochemical metrics, naturally occurring variant codes, and codon-reassignment mechanisms remains incompletely quantified. Embedding the 64 codons in GF(2)6 represents the hypercube Q6 as a coordinate-dependent subgraph of the encoding-independent single-nucleotide mutation graph H(3,4), and enables continuous ρ-interpolation between the two. Under a quartet-pattern shuffle null (n=10,000), the standard code is significantly low-cost across four established, code-independent physicochemical distance metrics with partially overlapping content (Grant ham p=0.0062; Miyata p<0.001; Woese polar requirement p=0.003; Kyte-Doolittle hydropathy p=0.001), and the signal strengthens monotonically as ρ moves Q6→H(3,4). A structure-aware sensitivity analysis under the alignment-derived ProtSub matrix (Jia & Jernigan 2021) yields the most extreme percentile of any measure tested (p=0.0004; all five p-values pass Bonferroni at α=0.05). Across the 27 NCBI translation tables, near-optimality is preserved: 11 of 12 informative-distance variants retain top-5% placement after BH-FDR correction. Natural codon reassignments avoid disrupting codon-family connectivity: under the encoding-independent H(3,4) adjacency, observed events are topology-breaking at relative risk 0.32 versus the candidate landscape (permutation p≤10-4). The H(3,4) result is stable by construction; the Q6 decomposition is representation-specific and fails to show depletion under 8 of 24 base-to-bit encodings, so we report H(3,4) as the primary test and Q6 as a sensitivity. Event-level conditional-logit modelling shows that topology avoidance and local physicochemical cost provide complementary, only weakly correlated signal (rs=0.15), and that topology adds explanatory value beyond physicochemistry under both Q6 and encoding-independent H(3,4) adjacency. Retrospective reanalysis of nine genome-recoding datasets is consistent with codon-family topology operating as an evolutionary-trajectory constraint distinct from acute engineering fitness. The contribution is the second axis: code evolution is jointly constrained by physicochemical smoothness and codon-family topological integrity, and these two constraints are partly independent.

Codon reassignment↗

PubMed1991

Restructuring the translation initiation region of the human parathyroid hormone gene for improved expression in Escherichia coli.

Overexpression of native human parathyroid hormone in Escherichia coli was achieved by a modification of the 5' end of the genomic gene sequence, thereby adapting this part of the translation initiation region to the bacterial host. Some simple rules abstracted from optimization studies of translation initiation of a beta-interferon gene were applied. These included (a) extending complementarity of the mRNA to the anticodon loop of tRNAfMet by use of a codon with a purine nucleotide directly following the ATG, (b) avoidance of stable secondary structure in the mRNA by use of synonymous A/U-rich codons, (c) elimination of a potential second Shine-Dalgarno sequence. The appropriate silent changes led to a 20-fold increase in parathyroid hormone production resulting in 4.3% of total soluble protein. This result proves the validity of our simple approach for optimization of foreign gene expression in E. coli.

Base Sequence↗

PubMed1987

Inducible expression vectors incorporating the Escherichia coli atpE translational initiation region.

New expression vectors were constructed for use in strains of Escherichia coli. Their most important feature is a polylinker system that facilitates the insertion of a gene in an optimal relationship to the highly efficient E. coli atpE translational initiation region (from nucleotide -50 to the start codon). Three ATG-containing restriction endonuclease sites can be used for the insertion of the 5' end of a gene at, or near to, its translational initiation codon. These sites may alternatively be used for the creation of a suitable translational start codon. Transcription is started by the bacteriophage lambda major promoters pR and pL in tandem and terminated by the bacteriophage fd terminator. Transcriptional initiation is very effectively repressed at 28-30 degrees C by the product of the bacteriophage lambda cIts857 gene, which is also present on the vectors. Full induction is achieved by shifting the incubation temperature to 42 degrees C. The combination of highly efficient transcriptional and translational signals on these vectors allowed high-level expression of sequences encoding human interferon beta and interleukin 2 and of the E. coli atpA, sucC and sucD genes.

DNA Restriction Enzymes↗

PubMed1989

On the information content of the genetic code.

In living organisms 20 amino acids along with the terminator value(s) are encoded by 64 codons giving a degeneracy of the codons as described by the genetic code. A basic theoretical problem of genetic codes is to explain the particular distribution of degeneracies of partitions involved in the codes. In this work the degeneracy problem is considered in the framework of information theory. It is shown by direct numerical evaluation of a certain degeneracy information function associated with the genetic code that the degeneracy of the codes is observed to be related to the optimization of this function.

Amino Acids↗

PubMed1999

Clinical value of K-ras codon 12 analysis and endobiliary brush cytology for the diagnosis of malignant extrahepatic bile duct stenosis.

Extrahepatic biliary stenosis can be caused by benign and malignant disorders. In most cases, a tissue diagnosis is needed for optimal management of patients, but the sensitivity of biliary cytology for the diagnosis of a malignancy is relatively low. The additional diagnostic value of K-ras mutational analysis of endobiliary brush cytology was assessed. Endobiliary brush cytology specimens obtained during endoscopic retrograde cholangiopancreaticography were prospectively collected from 312 consecutive patients with extrahepatic biliary stenosis. The results of conventional light microscopic cytology and K-ras codon 12 mutational analysis were compared and evaluated in view of the final diagnosis made by histological examination of the stenotic lesion and/or patient follow-up. The sensitivities of cytology and mutational analysis to detect malignancy were 36 and 42%, respectively. When both tests were combined, the sensitivity increased to 62%. The specificity of cytology was 98%, and the specificity of the mutational analysis and of both tests combined was 89%. Positive predictive values for cytology, mutational analysis, and both tests combined were 98, 92, and 94%, whereas the corresponding negative predictive values were 34, 34, and 44%, respectively. The sensitivity of K-ras mutational analysis was 63% for pancreatic carcinomas compared to 27% for bile duct, gallbladder, and ampullary carcinomas. K-ras mutational analysis can be considered supplementary to conventional light microscopy of endobiliary brush cytology to diagnose patients with malignant extrahepatic biliary stenosis, particularly in the case of pancreatic cancer. The presence of a K-ras codon 12 mutation in endobiliary brush cytology per se supports a clinical suspicion of malignancy, even when the conventional cytology is negative or equivocal.

Bile Duct Neoplasms↗

PubMed2001

Cooperative effects by the initiation codon and its flanking regions on translation initiation.

The purine-rich Shine-Dalgarno (SD) sequence located a few bases upstream of the mRNA initiation codon supports translation initiation by complementary binding to the anti-SD in the 16S rRNA, close to its 3' end. AUG is the canonical initiation codon but the weaker UUG and GUG codons are also used for a minority of genes. The codon sequence of the downstream region (DR), including the +2 codon immediately following the initiation codon, is also important for initiation efficiency. We have studied the interplay between these three initiation determinants on gene expression in growing Escherichia coli. One optimal SD sequence (SD(+)) and one lacking any apparent complementarity to the anti-SD in 16S rRNA (SD(-)) were analyzed. The SD(+) and DR sequences affected initiation in a synergistic manner and large differences in the effects were found. The gene expression level associated with the most efficient of these DRs together with SD(-) was comparable to that of other DRs together with SD(+). The otherwise weak initiation codon UUG, but not GUG, was comparable with AUG in strength, if placed in the context of two of the DRs. The +2 codon was one, but not the only, determinant for this unexpectedly high efficiency of UUG.

Base Sequence↗

PubMed1993

Folding of the MS2 coat protein in Escherichia coli is modulated by translational pauses resulting from mRNA secondary structure and codon usage: a hypothesis.

Possible translational pauses within the coat protein of the RNA bacteriophage MS2 were located on the basis of a distribution plot of rare codons and RNA secondary structure. It appeared that the position of certain codon pauses corresponds with the size of some nascent polypeptide intermediates, which have been isolated from MS2-infected cells. Other accumulated polypeptide intermediates seemed to be related to RNA regions, where double-stranded secondary structures occur, which probably impede the movement of ribosomes during chain elongation. We assume that a discontinuous translation rate is designed to allow optimal folding of this (and other) polypeptide(s).

Capsid↗

PubMed1997

Rapid evolution of translational control mechanisms in RNA genomes.

We have introduced 13 base substitutions into the coat protein gene of RNA bacteriophage MS2. The mutations, which are clustered ahead of the overlapping lysis cistron, do not change the amino acid sequence of the coat protein, but they disrupt a local hairpin, which is needed to control translation of the lysis gene. The mutations decreased the phage titer by four orders of magnitude but, upon passaging, the virus accumulated suppressor mutations that raised the fitness to almost wild-type level. Analysis of the pseudorevertants showed that the disruption of the local hairpin, controlling expression of the lysis gene, had apparently been so complete that its restoration by chance mutations could not be achieved. Instead, alternative foldings initiated by the starting mutations were further stabilized and optimized. Strikingly, in the pseudorevertants analyzed, translational control of the lysis gene had been restored. This feat was accomplished by, on average, four suppressor mutations that generally occurred at codon wobble positions. We also introduced 11 mutations in a hairpin more upstream in the coat protein gene and not implicated in lysis control. Here the titer dropped by three logs, but pseudorevertants with a fitness close to wild-type were soon generated. These pseudorevertants again were the result of the optimization of alternative foldings induced by the mutations. The transition of the secondary structure from wild-type to pseudorevertant could be visualized by structure probing. Our study shows that the folding of the RNA is an important phenotypic property of RNA viruses. However, its distortion can easily be overcome by optimizing alternative base-pairings. These new structures are not qualitatively equivalent to the original one, since they do not successfully compete with the wild-type.

Base Sequence↗

PubMed1995

Regulation of T-cell antigen receptor (TCR) alpha-chain expression by TCR beta-chain transcripts.

The TCR is an alpha beta heterodimer, a part of the multimeric structure through which physiological T-cell activation occurs. The expression of TCR alpha chain is greatly diminished in a beta-chain-deficient mutant Jurkat cell line (J.RT3-T3.5). The relationship between the expression of the TCR alpha and beta chains has been examined by stable transfection of a series of TCR beta-chain mutant constructs into this mutant cell line. The level of alpha-chain transcript was dramatically upregulated by the expression of the beta chain and specifically by a transcript of the beta-chain variable region alone, including a transcript in which the ATG start codon was mutated. The downregulation of the endogenous alpha-chain transcripts in mutants cells lacking complete beta-chain transcripts occurred primarily at the posttranscriptional level. This evidence for a regulatory function of the TCR beta-chain gene represents an unusual regulatory pathway in which the transcript of one gene is required for the optimal expression of another gene.

Amino Acid Sequence↗