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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↗
PubMed2002

A cell-free protein synthesis system as an investigational tool for the translation stop processes.

Using Escherichia coli cell-free protein synthesis system and aminoacylated amber suppressor tRNA, we successfully inserted an unnatural amino acid S-(2-nitrobenzyl)cysteine into human erythropoietin. Three different types of translation stop suppression were observed and each of the three types was easily discerned with SDS-PAGE. Optimal conditions were established for correct stop and programmed suppressions. Since this system differentiates proteins produced by misreading of codons from those produced by programmed suppression, we conclude that this cell-free translation system that we describe in this paper will be of a great use for future investigations on translation stop processes.

Cell-Free System↗
PubMed1999

Recombination and chimeragenesis by in vitro heteroduplex formation and in vivo repair.

We describe a simple method for creating libraries of chimeric DNA sequences derived from homologous parental sequences. A heteroduplex formed in vitro is used to transform bacterial cells where repair of regions of non-identity in the heteroduplex creates a library of new, recombined sequences composed of elements from each parent. Heteroduplex recombination provides a convenient addition to existing DNA recombination methods ('DNA shuffling') and should be particularly useful for recombining large genes or entire operons. This method can be used to create libraries of chimeric polynucleotides and proteins for directed evolution to improve their properties or to study structure-function relationships. We also describe a simple test system for evaluating the performance of DNA recombination methods in which recombination of genes encoding truncated green fluorescent protein (GFP) reconstructs the full-length gene and restores its characteristic fluorescence. Comprising seven truncated GFP constructs, this system can be used to evaluate the efficiency of recombination between mismatches separated by as few as 24 bp and as many as 463 bp. The optimized heteroduplex recombination protocol is quite efficient, generating nearly 30% fluorescent colonies for recombination between two genes containing stop codons 463 bp apart (compared to a theoretical limit of 50%).

Cloning, Molecular↗
PubMed1997

Screening of a mutant plasmid with high expression efficiency of GC-rich leuB gene of an extreme thermophile, Thermus thermophilus, in Escherichia coli.

A mutant plasmid with elevated expression efficiency of GC-rich Thermus thermophilus leuB gene was screened in Escherichia coli. A wild-type plasmid pHB2 carrying T. thermophilus leuB gene was introduced into leuB-deficient E. coli C600 cells. During successive cultures of the transformant in leucine-free medium, the original plasmid was spontaneously replaced by a mutant plasmid. The expression efficiency of the leuB gene on the mutant plasmid was 4.8-fold higher than that of the wild-type plasmid. Sequencing of the mutant plasmid revealed that the open reading frame (ORF1) in front of the leuB gene was shortened from 822 to 306 bp. Several expression vectors were constructed to investigate the effect of the length of ORF1, and the optimal length for the expression of the following leuB gene was determined. It was also shown that the stop codon of ORF1 should be overlapped with the initiation codon of leuB gene for the highest efficiency.

Amino Acid Sequence↗
PubMed1999

The Mycobacterium tuberculosis katG promoter region contains a novel upstream activator.

An Escherichia coli-mycobacterial shuttle vector, pJCluc, containing a luciferase reporter gene, was constructed and used to analyse the Mycobacterium tuberculosis katG promoter. A 1.9 kb region immediately upstream of katG promoted expression of the luciferase gene in E. coli and Mycobacterium smegmatis. A smaller promoter fragment (559 bp) promoted expression with equal efficiency, and was used in all further studies. Two transcription start sites were mapped by primer extension analysis to 47 and 56 bp upstream of the GTG initiation codon. Putative promoters associated with these show similarity to previously identified mycobacterial promoters. Deletions in the promoter fragment, introduced with BAL-31 nuclease and restriction endonucleases, revealed that a region between 559 and 448 bp upstream of the translation initiation codon, designated the upstream activator region (UAR), is essential for promoter activity in E. coli, and is required for optimal activity in M. smegmatis. The katG UAR was also able to increase expression from the Mycobacterium paratuberculosis P(AN) promoter 15-fold in E. coli and 12-fold in M. smegmatis. An alternative promoter is active in deletion constructs in which either the UAR or the katG promoters identified here are absent. Expression from the katG promoter peaks during late exponential phase, and declines during stationary phase. The promoter is induced by ascorbic acid, and is repressed by oxygen limitation and growth at elevated temperatures. The promoter constructs exhibited similar activities in Mycobacterium bovis BCG as they did in M. smegmatis.

Bacterial Proteins↗
PubMed1997

Tracking and quantitation of retroviral-mediated transfer using a completely humanized, red-shifted green fluorescent protein gene.

We have developed murine retroviral vectors (RVs) containing an optimized green fluorescent protein (GFP) gene to study retroviral gene transfer and expression in living cells. We used the codon "humanized", "red-shifted" GFP gene, hGFP-S65T, a gain of function variant of the wild-type GFP from the jellyfish Aequorea victoria. We cloned the hGFP-S65T gene into the RV plasmid pLNCX (pLNChG65T). A stable amphotropic RV-producer cell line (VPC), designated LNChG65T VPC, was generated that exhibited bright fluorescence in greater than 95% of the cells. Human A375 melanoma cells and IGROV ovarian carcinoma cells transduced from LNCh-G65T VPC demonstrated high levels of fluorescence. The expression of a single integrated hGFP-S65T gene in eukaryotic cells provides a powerful tool to study gene transfer, expression and functional studies in vitro and in vivo.

Animals↗
PubMed1993

Genotypic classification of colorectal adenocarcinoma. Biologic behavior correlates with K-ras-2 mutation type.

BACKGROUND: New measures enabling better prediction of biologic behavior of large bowel cancer are highly desirable. One hundred ninety-four consecutive primary, recurrent, and metastatic colorectal adenocarcinomas, accessioned during 1991 at Rhode Island Hospital, were classified according to the presence and specific type of K-ras-2 point mutation. METHODS: An integrated histopathologic-genetic approach was used to detect mutations starting with minute, topographically selected, tissue samples from formaldehyde-fixed, paraffin-embedded specimens. RESULTS: Each colorectal adenocarcinoma exhibited either no or only one of seven specific types of K-ras-2 mutation. The mutation type of each primary tumor was present consistently in its metastatic deposits. Thirty-five percent of primary colorectal adenocarcinomas were found to be mutated (42 of 119). A significantly higher mutation rate (65%) was seen in lymphogenous-hematogenous metastases as a group (35 of 54; P < 0.005). By contrast, 22% of anastomotic recurrences and transcoelomic metastasis were mutated (4 of 18). Twenty-eight percent of adenocarcinomas with invasion limited to muscularis propria (Tis, T1, T2) were mutated (16 of 57), compared to 41% for more deeply invasive tumors (T3, T4; 26 of 63). When colorectal adenocarcinomas were analyzed by specific K-ras-2 mutation type, it was found that codon 13 mutated tumors did not progress to local or distant metastasis (P < 0.01). Tumors having a codon 12 valine substitution did not metastasize beyond pericolonic-perirectal lymph nodes. In contrast, colorectal cancers with codon 12 aspartic acid substitutions accounted for most of the distant hematogenous deposits (P < 0.01). Tumors with normal K-ras-2 accounted for most intraperitoneal deposits. CONCLUSIONS: Genotyping of colorectal adenocarcinoma by K-ras-2 status can identify subsets of patients likely to pursue indolent and aggressive forms of disease. The integrated histopathologic-genetic approach outlined is feasible for use in diagnostic pathology, providing information that together with clinicopathologic staging may individualize and optimize treatment.

Adenine↗
PubMed1997

Line probe assay for rapid detection of drug-selected mutations in the human immunodeficiency virus type 1 reverse transcriptase gene.

Upon prolonged treatment with various antiretroviral nucleoside analogs such as 3'-azido-3'-deoxythymidine, 2',3'-dideoxyinosine, 2',3'-dideoxycytidine, (-)- beta-L-2', 3'dideoxy-3'thiacytidine and 2',3'-didehydro-3'-deoxythymidine, selection of human immunodeficiency virus type 1 (HIV-1) strains with mutations in the reverse transcriptase (RT) gene has been reported. We designed a reverse hybridization line probe assay (LiPA) for the rapid and simultaneous characterization of the following variations in the RT gene: M41 or L41; T69, N69, A69, or D69; K70 or R70; L74 or V74; V75 or T75; M184, I184, or V184; T215, Y215, or F215; and K219, Q219, or E219. Nucleotide polymorphisms for codon L41 (TTG or CTG), T69 (ACT or ACA), V75 (GTA or GTG), T215 (ACC or ACT), and Y215 (TAC or TAT) could be detected. In addition to the codons mentioned above, several third-letter polymorphisms in the direct vicinity of the target codons (E40, E42, K43, K73, D76, Q182, Y183, D185, G213, F214, and L214) were found, and specific probes were selected. In total, 48 probes were designed and applied to the LiPA test strips and optimized with a well-characterized and representative reference panel. Plasma samples from 358 HIV-infected patients were analyzed with all 48 probes. The amino acid profiles could be deduced by LiPA hybridization in an average of 92.7% of the samples for each individual codon. When combined with changes in viral load and CD4+ T-cell count, this LiPA approach proved to be useful in studying genetic resistance in follow-up samples from antiretroviral agent-treated HIV-1-infected individuals.

Acquired Immunodeficiency Syndrome↗
PubMed2002

Cloning and high expression of hbFGF with a new strategy.

Computer program DNASIS v2.5 was used to help designing the site-directed mutations for optimizing the expression of hbFGF in E. coli. The secondary structure of the translation initiation region (TIR) is a determinant factor for translation initiation rate, meanwhile, codon preference plays an important role, too. According to the two principles, 4 sites in 5' end of hbFGF cDNA were definitely changed, and another 4 sites randomly changed. These mutations will lead to potential variation in the secondary structure of TIR. Then computer program DNASIS v2.5 was utilized to analyse the total 32 TIR sequences resulted from the combination of the 4 randomly mutated sites. Ten sequences with highest free formation energy (delta G0) were chosen for subsequent cloning. By PCR using synthetic primers containing the 8 changed sites described above, ten hbFGF cDNA were amplified and cloned to pET-3c respectively. E. coli strain BL21 (DE3) was transformed and induced to express recombinant hbFGF. Two high-expression clones were obtained by SDS-PAGE and MTT assay, indicating that computer program-aided design for optimizing expression of foreign genes in E. coli is useful.

Cloning, Molecular↗
PubMed2003

AGenDA: homology-based gene prediction.

We present a www server for homology-based gene prediction. The user enters a pair of evolutionary related genomic sequences, for example from human and mouse. Our software system uses CHAOS and DIALIGN to calculate an alignment of the input sequences and then searches for conserved splicing signals and start/stop codons around regions of local sequence similarity. This way, candidate exons are identified that are used, in turn, to calculate optimal gene models. The server returns the constructed gene model by email, together with a graphical representation of the underlying genomic alignment.

Algorithms↗
PubMed1998

Coding sequences enhance internal initiation of translation by hepatitis A virus RNA in vitro.

Hepatitis A virus (HAV), unlike other picornaviruses, has a slow-growth phenotype in permissive cell lines and in general does not induce host cell cytopathology. Although there are no published reports of productive infection of HeLa cells by HAV, HAV RNA appears to be readily translated in HeLa cells when transcribed by T7 RNA polymerase provided by a recombinant vaccinia virus. The 5' noncoding region of HAV was fused to poliovirus (PV) coding sequences to determine the effect on translation efficiency in HeLa cell extracts in vitro. Conditions were optimized for utilization of the HAV internal ribosome entry segment (IRES). Transcripts from chimeric constructs fused precisely at the initiation codon were translated very poorly. However, chimeric RNAs which included 114 or more nucleotides from the HAV capsid coding sequences downstream of the initiation codon were translated much more efficiently than those lacking these sequences, making HAV-directed translation efficiency similar to that directed by the PV IRES. Sixty-six nucleotides were insufficient to confer increased translation efficiency. The most 5'-terminal HAV 138 nucleotides, previously determined to be upstream of the IRES, had an inhibitory effect on translation efficiency. Constructs lacking these terminal sequences, or those in which the PV 5'-terminal sequences replaced those from HAV, translated three- to fourfold better than those with the intact HAV 5'-terminal end.

Cell Extracts↗
PubMed2001

RNA editing by adenosine deaminases that act on RNA.

ADARs are RNA editing enzymes that target double-stranded regions of nuclear-encoded RNA and viral RNA. These enzymes are particularly abundant in the nervous system, where they diversify the information encoded in the genome, for example, by altering codons in mRNAs. The functions of ADARs in known substrates suggest that the enzymes serve to fine-tune and optimize many biological pathways, in ways that we are only starting to imagine. ADARs are also interesting in regard to the remarkable double-stranded structures of their substrates and how enzyme specificity is achieved with little regard to sequence. This review summarizes ongoing investigations of the enzyme family and their substrates, focusing on biological function as well as biochemical mechanism.

3' Untranslated Regions↗
PubMed1982

[Origin and evolution of the genetic code].

We propose a quantitative model which suggests that the present genetic code appeared under the influence of mutations, while optimizing its own resistance against their effects. Its evolution was realized by successive steps in which the number of translated codons grew, whereas the number of terminators decreased. The main constraint of this model is selection against nonsense mutations: the competition among many primitive codes gives advantage to those which resist best to the occurrence of nonsense mutations. The structures of the selected systems converge towards that of the present genetic code. This one appears then as built so as to resist to errors, information noise, mutations.

Biological Evolution↗
PubMed1999

Codons and hypercycles.

Several hypotheses on the origin of codon assignments imply that the present protein synthesizing machinery was already in place when the assignments were made. These are examined by computer modeling. The results do not suggest that assignments were optimized for resistance to reading and mutation errors, nor that the assignments are random. It is improbable that the number of species of amino acids increased in the course of evolution. An originally ambiguous dictionary is likely to have been subject to error catastrophe and is improbable. A relation between amino acid properties and their codons exists, and suggests that the codon assignments were established at the time of origin of the hypercycle, i.e. a system of aminoacyl synthetases which attaches amino acids to tRNA, and before the present protein synthesizing machinery was in place. The origin of a hypercycle is only possible if the system began with components which were catalytically active even when they did not form a self-replicating system. A model of such a system is proposed.

Amino Acids↗
PubMed1998

Nucleotide structure and characterization of the murine gene encoding anticoagulant protein C.

The 15,160 bp murine gene encoding anticoagulation protein C (PC) was cloned and sequenced, including 414 bp upstream of exon 1 and 80 bp downstream of the translation stop codon. Nine exons and eight introns were identified. The first exon was untranslated and contained the major transcriptional start site, the surrounding nucleotide sequence of which matched reasonably well with the consensus eukaryotic Cap element sequence. The translational initiator methionine residue was located in exon 2. The other introns were positioned as splices between the major domain units of the protein. The 5' untranslated region contained two possible CCAAT sequences and GC boxes, but no TATA box was obvious within the optimal range of distances from the transcription start site. The 3'-flanking nucleotides included a probable polyadenylation site (ATTAAA), beginning 80 nucleotides downstream of the translation stop codon, and a downstream consensus sequence (AGTGTTTC) required for the efficient formation of a 3' terminus of mRNA. Several high probability transcription factor recognition sequences, including proteins that are enriched in, or specific to, the liver, such as C/EBP alpha, C/EBP beta, HNF1, and HNF3 beta, have been located in the 5' region of the gene. These results indicate that all elements are present for liver-based transcription of the gene for murine PC.

Amino Acid Sequence↗