SEARCH · PubMed Health

Results for “codon optimization”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 253 records · Page 14Linked to original sources

PubMed1995

Progress in genetic screening of multiple endocrine neoplasia type 2A: is calcitonin testing obsolete?

BACKGROUND: Recent identification of RET mutations in multiple endocrine neoplasia type 2A (MEN 2A) allows a DNA-based approach to diagnosis in lieu of calcitonin sampling. To prospectively evaluate the efficacy of mutational analysis, genetic screening was performed in 124 patients (53 male, 71 female; age, 1 month to 80 years) at risk for MEN 2A referred over 3 months. METHODS: Analysis used genomic DNA and a polymerase chain reaction-based denaturing gradient gel electrophoresis strategy for mutation detection at RET codons 609, 611, 618, 620, and 634. Ninety-three of 124 patients were from established MEN 2A kindreds (group A), and screening replaced calcitonin testing. Twenty-one of 124 patients (group B) represented index cases of medullary thyroid carcinoma (MTC), and DNA analysis was performed to distinguish sporadic from hereditary disease. Ten patients (group C) had modest calcitonin elevations or had undergone thyroidectomy without confirming pathologic results, and testing was undertaken to clarify status. RESULTS: Group A: RET mutations occurred in 29 (median age, 10 years) of 93 patients, 14 of whom underwent thyroidectomy. No false-positive results were observed. Group B: five (24%) of 21 patients with seemingly sporadic MTC had RET mutations at codons 618 (one), 620 (one), or 634 (three). Group C: Nine of 10 patients with alleged MEN 2A had genetically negative results. CONCLUSIONS: Denaturing gradient gel electrophoresis reliably detects MEN 2A. Modest calcitonin elevations may lead to a false-positive diagnosis of MTC. DNA testing is the optimal approach to evaluating MEN 2A. Index cases of sporadic MTC should also undergo DNA analysis.

Adolescent↗

PubMed1982

A convenient and adaptable package of DNA sequence analysis programs for microcomputers.

We describe a package of DNA data handling and analysis programs designed for microcomputers. The package is convenient for immediate use by persons with little or no computer experience, and has been optimized by trial in our group for a year. By typing a single command, the user enters a system which asks questions or gives instructions in English. The system will enter, alter, and manage sequence files or a restriction enzyme library. It generates the reverse complement, translates, calculates codon usage, finds restriction sites, finds homologies with various degrees of mismatch, and graphs amino acid composition or base frequencies. A number of options for data handling and printing can be used to produce figures for publication. The package will be available in ANSI Standard FORTRAN for use with virtually any FORTRAN compiler.

Amino Acid Sequence↗

PubMed1988

Disruption of the CHO1 gene encoding phosphatidylserine synthase in Saccharomyces cerevisiae.

A Saccharomyces cerevisiae mutant that lacked phosphatidylserine synthase [EC 2.7.8.8] (CDP-1,2-diacyl-sn-glycerol: L-serine O-phosphatidyltransferase) completely was constructed by disrupting its structural gene, CHO1. Over two-thirds of its coding region, from the starting to the 200th codon, was replaced with a LEU2 DNA fragment. This new cho1 mutant showed no detectable synthesis of phosphatidylserine but grew slowly in a medium that contained either ethanolamine or choline. These results indicate that phosphatidylserine synthase and most probably phosphatidylserine are dispensable in S. cerevisiae but necessary for its optimal growth. Additional supplementation with myo-inositol raised the cellular content of phosphatidylinositol and improved the growth of the mutant, suggesting the importance of the negative charges of the membrane surface. The CHO1-disrupted mutant, when grown on choline, accumulated phosphatidylethanolamine to a significant level even after extensive dilution of the initial culture. It segregated prototrophic revertants that could synthesize phosphatidylethanolamine without recovery of phosphatidylserine synthesis. These results imply the presence of a route(s) for the formation of ethanolamine or its phosphorylated derivative in S. cerevisiae.

Blotting, Southern↗

PubMed1989

Control of reovirus messenger RNA translation efficiency by the regions upstream of initiation codons.

The 10 species of reovirus messenger RNA are translated in vivo with efficiencies/frequencies that differ by as much as 100-fold. The s1 mRNA, which is translated 10 times less efficiently than the s4 mRNA but 10 times more efficiently than the/1 and m1 mRNAs, has a unique BamH1 cleavage site located immediately downstream of its initiation codon. Because the reovirus mRNAs have been cloned, this provides the opportunity for placing modified and altered sequences upstream of its coding sequence. The translation efficiencies of the variant mRNAs, transcribed via the SP6 in vitro transcription system, can then be measured in the rabbit reticulocyte lysate in vitro translation system. Using this system it was found that replacing the 5'-upstream sequence of the s1 mRNA with that of the s4 mRNA increases its in vitro translation efficiency by 4-fold; that the trinucleotide immediately upstream of the s1 initiation codon renders it very weak, and that it is only slightly superior to the weakest Kozak consensus sequence; that the nature of the nucleotides further upstream than position -3 can profoundly affect translation efficiency; that the nature of this effect is in turn markedly modified by the nature of nucleotides in positions -1 to -3; and that there is a minimum optimal 5'-upstream sequence length of about 14 nucleotides. We also investigated the effect of secondary structure involvement on the ability of 5'-upstream sequences to promote translation. Two effects were noted. First, being part of moderately stable stem loops (delta G, -18 kcal/mol) decreased translation efficiency about 3-fold; second, mRNA in which only three 5'-terminal nucleotides were unpaired were translated five times less efficiently than mRNA in which six nucleotides were unpaired. Accessibility of the 5'-cap as well as secondary structure of the 5'-upstream sequences are therefore factors that affect translation efficiency. Finally, we showed that the m1 mRNA, which is transcribed very poorly in vivo, is translated very efficiently in vitro; and that its 5'-upstream sequence is as effective in increasing protein sigma 1 formation as that of s4 mRNA. Since both m1 mRNA and protein mu 2 are stable in infected cells, the reason why m1 mRNA is translated so inefficiently in vivo therefore remains unexplained.

Codon↗

PubMed1995

In vitro antibody maturation. Improvement of a high affinity, neutralizing antibody against IL-1 beta.

Abs as therapeutic antagonists should be of relatively high affinity to effectively neutralize their target Ag. Typically, Abs with nanomolar affinities to protein Ags can be obtained in vivo, however, this may not be the upper limit of affinity because the biologic process attempts to optimize Ab function, of which affinity is only one component. SK48, a high affinity neutralizing murine Fab against human IL-1 beta was used to explore the nature of Ab-Ag interactions and the potential for further affinity improvement in vitro using mutagenesis and selection via phage display. The codons of six amino acids in the third complementarity-determining region of the heavy chain (CDR3-H) were both individually and combinatorialy randomized and the resultant libraries were screened for IL-1 binding phenotype. Mutations that reduced affinity suggested that both the backbone conformation of the CDR3 loop and certain side chains are essential for binding, yet alterations to the canonical salt bridge residues at the base of the loop had minimal effect on affinity. Four rounds of selection of the phage Ab libraries on immobilized IL-1 beta gave predominantly the wild-type sequence, indicating efficient affinity maturation of this CDR in vivo. However, a twofold improvement in affinity was observed for both single and double amino acid changes at two positions in the middle of the CDR. Moreover, a 10-fold increase in affinity for IL-1 beta was achieved by combining two of the phage-selected single amino acid substitutions in CDR3-H, thereby demonstrating that a significant improvement in affinity can be achieved through CDR mutagenesis, even in a matured Ab. The increased affinity of this Fab did not, however, enhance its neutralizing activity in vitro.

Amino Acid Sequence↗

PubMedDate not supplied

[Use of the hygromycin phosphotransferase gene as the dominant selective marker for Chlamydomonas reinhardtii transformation].

The hygromycin phosphotransferase gene (hpt) from E. coli under the control of the SV40 early promoter was used as a dominant selectable marker for transformation of Chlamydomonas reinhardtii. Cells were transformed by electroporation (pulse length, 2 ms, field strength, 1 kV/cm). The culture growth phase was a crucial parameter for transformation (optimal density approximately 10(6) cells/ml). It was possible to obtain approximately 10(3) Hyg-resistant colonies under these conditions. Foreign DNA integrated into the Chlamydomonas genome was maintained for at least 8 months but the Hyg-resistant phenotype of the transformed clones was unstable. The frequency of codon usage in the hpt gene was compared with the one in Chlamydomonas nuclear genes. It is supposed that highly biased codon usage in Chlamydomonas does not preclude expression. Advantages of this selection system for studying Chlamydomonas transformation by heterologous genes are discussed.

Animals↗

PubMed1992

The glgB gene from the thermophile Bacillus caldolyticus encodes a thermolabile branching enzyme.

We have cloned the structural gene for the Bacillus caldolyticus glycogen branching enzyme (glgB) in Escherichia coli. The glgB gene consisted of a 1998 bp open reading frame (ORF) encoding a 78,087 Da protein, which was highly similar to the Bacillus stearothermophilus branching enzyme. The 5' end of a second gene that encoded a protein with extensive similarity to E. coli ADP-glucose pyrophosphorylase (ADPGP) partly overlapped the 3' end of the glgB gene. A putative promoter recognized by Bacillus subtilis RNA polymerase containing the sigma factor H (E-sigma H) preceded the genes. These data suggest that in contrast to the situation observed in B. stearothermophilus, the genes involved in glycogen synthesis in B. caldolyticus are clustered on the chromosome, and are presumably coordinately expressed during the early stages of sporulation. An incomplete third gene started upstream of B. caldolyticus glgB. This gene was highly similar to a gene found directly upstream of B. stearothermophilus glgB, which encodes a putative membrane protein with unknown function. The B. caldolyticus glgB gene was expressed in E. coli and B. subtilis. Surprisingly, the branching enzyme appeared to be thermolabile, the temperature of optimal activity being only 39 degrees C.

1,4-alpha-Glucan Branching Enzyme↗

PubMed1997

Antibody humanization using monovalent phage display.

Antibody humanization often requires the replacement of key residues in the framework regions with corresponding residues from the parent non-human antibody. These changes are in addition to grafting of the antigen-binding loops. Although guided by molecular modeling, assessment of which framework changes are beneficial to antigen binding usually requires the analysis of many different antibody mutants. Here we describe a phage display method for optimizing the framework of humanized antibodies by random mutagenesis of important framework residues. We have applied this method to humanization of the anti-vascular endothelial growth factor murine monoclonal antibody A4.6.1. Affinity panning of a library of humanized A4.6.1 antibody mutants led to the selection of one variant with greater than 125-fold enhanced affinity for antigen relative to the initial humanized antibody with no framework changes. A single additional mutation gave a further 6-fold improvement in binding. The affinity of this variant, 9.3 nM, was only 6-fold weaker than that of a murine/human chimera of A4.6.1. This method provides a general means of rapidly selecting framework mutations that improve the binding of humanized antibodies to their cognate antigens and may prove an attractive alternative to current methods of framework optimization based on cycles of site-directed mutagenesis.

Amino Acid Sequence↗

PubMed1987

A computer program for the design of optimal synthetic oligonucleotide probes for protein coding genes.

A computer program has been written in FORTRAN 77 to locate on a protein sequence a region with optimum length and limited degeneracy in order to design artificial oligonucleotide probes for use in molecular cloning. In addition the program checks for regions of homology between this probe and any other base sequence found in nucleotide sequence data banks. There are options in the program to eliminate rare codons or to make preferential choices of bases in order to minimize the degeneracy of probes.

Algorithms↗

PubMed1983

Nucleotide sequence of a cDNA clone encoding the entire glycoprotein from the New Jersey serotype of vesicular stomatitis virus.

The nucleotide sequence of the mRNA encoding the glycoprotein from the New Jersey serotype of vesicular stomatitis virus (VSV) was determined from a cDNA clone containing the entire coding region. The sequence of 12 5'-terminal noncoding nucleotides present in the mRNA but not in the cDNA clone was determined from a primer extended to the 5' terminus of the mRNA. The mRNA is 1,573 nucleotides long (excluding polyadenylic acid) and encodes a protein of 517 amino acids. Only six nucleotides occur between the translation termination codon and the polyadenylic acid. Short homologies between the untranslated termini of this mRNA and the mRNAs of the Indiana serotype were found. The predicted protein sequence was compared with that of the glycoprotein of the Indiana serotype of VSV and with the glycoprotein of rabies virus, using a computer program which determines optimal alignment. An amino acid identity of 50.9% was found for the two VSV serotypes. Approximately 20% identity was found between the rabies virus and VSV New Jersey glycoproteins. The positions and sizes of the transmembrane domains, the signal sequences, and the glycosylation sites are identical in both VSV serotypes. Two of five serine residues which were possible esterification sites for palmitate in the glycoprotein from the Indiana serotype are changed to glycine residues in the glycoprotein from the New Jersey serotype. Because the glycoprotein of the New Jersey serotype does not contain esterified palmitate, we suggest that one or both of these residues are the probable esterification sites in the glycoprotein from the Indiana serotype.

Base Sequence↗

PubMed1989

Mature apolipoprotein AI and its precursor proApoAI: influence of the sequence at the 5' end of the gene on the efficiency of expression in Escherichia coli.

Apolipoprotein AI (ApoAI) plays a central role in the regulation of lipid metabolism. Initial attempts to express human apoAI cDNA in Escherichia coli did not yield detectable levels of the mature protein. By analyzing the efficiency of expression of apoAI-lacZ gene fusions, we have been able to show that the sequence at the 5' end of the ApoAI-coding region is a critical parameter. Indeed, silent changes in the codons for the first 8 residues of ApoAI, which did not alter the amino acid sequence, affected expression dramatically. Analysis of the corresponding mRNA steady-state levels suggested a role for differential mRNA stability in the control of apoAI expression in this system. Among all the possible alternative sequences, we have identified an optimal sequence which, when reinserted in the original expression plasmid, yields high level production of mature ApoAI. This procedure has been extended to the production of the natural variant ApoAI-Milano and the precursor proApoAI. Availability of these recombinant molecules would allow the investigation of their structural and biological features. In addition, the methodology used to optimize ApoAI expression is of general interest in assuring high expression of heterologous proteins in E. coli.

Amino Acid Sequence↗

PubMed1995

Kinetics of translation of gamma B crystallin and its circularly permutated variant in an in vitro cell-free system: possible relations to codon distribution and protein folding.

Analysis of nascent gamma B-crystallin peptides accumulating during in vitro translation in a rabbit reticulocyte lysate cell-free system was carried out. As a consequence of the irregular distribution of rare codons along the polypeptide chain of gamma B-crystallin, translation of the two-domain protein is a non-uniform process characterized by specific pauses. One of the major delays occurs during the translation of the connecting peptide between the domains. Comparing the kinetics of translation of natural gamma B-crystallin and its circularly permutated variant (with the order of the N- and C-terminal domains exchanged) reveals that the natural N-terminal domain is translated faster than the C-terminal one. Since the N-terminal domain in natural gamma B-crystallin is known to be more stable and to fold faster than the C-terminal one [E.-M. Mayr et al. (1994) J. Mol. Biol. 235, 84-88], the present data suggest that the translation rates are optimized to tune the synthesis and folding of the nascent polypeptide chain. In this connection, the pause in the linker region between the domains provides a delay allowing the correct folding of the N-terminal domain and its subsequent assistance in the stabilization of the C-terminal one.

Animals↗

PubMed1990

A generalized information function applied to the genetic code.

The problem of the partitioning of the degeneracy of the codons in the genetic code is considered in the framework of a generalized information function IG = c sigma kpk(ln pk + G(Ek] where k represents the number of codons in a specific degeneracy class and G(Ek) is an arbitrary real valued function. For G(Ek) = 0 the Shannon information function is recovered. For a particular choice of G(Ek) that takes the dominance of even degeneracies into account, it is found by direct numerical calculations that the correct degeneracy partitioning appears as optimal values of the Ig function. This results is also supported by optimization calculations in which the generalized information function is regarded as a continuous function in the degeneracy variables.

Amino Acids↗

PubMed1992

Detection of hepatitis B pre-core mutant by allele specific polymerase chain reaction.

AIM: Development of a specific polymerase chain reaction (PCR) assay for detection of the pre-core, stop codon, mutant of hepatitis B virus (HBV). METHODS: PCR primers, specific at the 3'-end for nucleotide 1896 of either the pre-core, stop codon, mutant or wild type HBV, were synthesised using published sequence data. Positive control templates for both types of virus were synthesised by the PCR, incorporating sequences specific for each virus type at the appropriate position. These templates were used to optimise the specificity of the procedure. Formalin fixed, paraffin wax embedded human tissue from acute or fulminant HBV hepatitis from Hong Kong or Oxford was then investigated for presence of mutant or wild type virus. The HBV DNA was amplified from this tissue using a two step procedure, with an initial amplification phase followed by a second diagnostic phase on optimally diluted target DNA. RESULTS: Specific detection of mutant or wild type HBV was achieved. An important factor in determining specificity was the temperature of annealing, 70 degrees C proving to be highly specific. To overcome the inherent variation of target copy number in clinical samples and to provide an intrinsic positive control, it was important to generate and standardise the amount of target HBV used for the specific PCR. Two cases of fulminant hepatitis and four cases of acute hepatitis from Hong Kong, and one case of fulminant hepatitis from Oxford, contained only wild type HBV, with no evidence of a mutant virus. CONCLUSION: This method can be applied to FFPE tissues. It is rapid, non-radioactive, and specific for the stop codon mutation at nucleotide 1896 of HBV. Preliminary investigation of a small number of cases of fulminant hepatitis from Oxford and Hong Kong showed only wild type virus. The result differs from results published from Japan and Israel.

Alleles↗

PubMed1990

Nucleotide sequence of the Synechococcus sp. PCC7942 branching enzyme gene (glgB): expression in Bacillus subtilis.

The nucleotide sequence of the Synechococcus sp. PCC7942 glgB gene has been determined. The gene contains a single open reading frame (ORF) of 2322 bp encoding a polypeptide of 774 amino acids (aa) with an Mr of 89,206. Extensive sequence similarity exists between the deduced aa sequence of the Synechococcus sp. glgB gene product and that of the Escherichia coli branching enzyme in the middle portions of the proteins (62% identical aa). In contrast, the N-terminal portions shared little homology. The sequenced region which follows glgB contains an ORF encoding 79 aa of the N terminus of a polypeptide that shares extensive sequence similarity (41% identical aa) with human and rat uroporphyrinogen decarboxylase. This suggests that the region downstream from glgB contains the hemE gene and, therefore, that the organization of genes involved in glycogen biosynthesis in Synechococcus sp. is different from that described for E. coli. A fusion gene was constructed between the 5' end of the Bacillus licheniformis penP gene and the Synechococcus sp. glgB gene. The fusion gene was efficiently expressed in the Gram+ micro-organism Bacillus subtilis and specified a branching enzyme with an optimal temperature for activity similar to the wild-type enzyme.

1,4-alpha-Glucan Branching Enzyme↗

PubMed1996

Optimized heterologous expression of the polymorphic human glutathione transferase M1-1 based on silent mutations in the corresponding cDNA.

An expression clone for large-scale production of the polymorphic human glutathione transferase (GST) M1-1 has been developed. Heterologous expression in Escherichia coli afforded a yield of 100 mg of GST M1-1 per 3 liters of culture medium, corresponding to an approximately 10-fold increased yield compared to the parental expression construct. Overproduction of the enzyme was dependent on the codon usage in the 5' region of the DNA sequence encoding glutathione transferase M1-1. High-level expression clones were generated by a combination of random silent mutations in selected wobble positions in the coding sequence and immunoselection of clones from the library of random mutants. The strategy used is generally applicable for the production of recombinant proteins provided that a suitable selection procedure is available for identifying the desired mutants.

Amino Acid Sequence↗

PubMed1989

A general approach to isolating Plasmodium falciparum genes using non-redundant oligonucleotides inferred from protein sequences of other organisms.

We have constructed a number of oligonucleotide probes and tested their utility in identifying various genes in Plasmodium falciparum. The probe sequences were based on known conserved regions of proteins from other organisms, coupled with an analysis of the codon usage of the parasite. By using long single oligonucleotides, we have successfully isolated the DHFR-TS gene, two actin genes and two tubulin genes from the K1 (Thailand) isolate of P. falciparum. We compare these single probes to multiply-redundant short oligonucleotide probes and to heterologous probes. We also present a detailed quantitative analysis of optimal probe design, and of how this approach can best be implemented as a general method of isolating plasmodial genes.

Animals↗

PubMed1994

A novel cis element essential for stimulated transcription of the p41 promoter of human herpesvirus 6.

The p41 DNA-binding protein of human herpesvirus 6 is an apparent processivity factor important for viral DNA replication. The p41 promoter was characterized to understand how this processivity factor is regulated. A single transcription start site and a functional TATA box are located 48 and 74 bp, respectively, upstream of the start codon. A reporter construct containing 1,027 bp of the sequence upstream of the p41 start codon was inactive in uninfected T cells but functioned as a strong promoter in human herpesvirus 6-infected cells. Mutational analysis identified a 21-bp element (the EA site) which is located at -73 to -52 bp relative to the transcription start site and is essential for promoter activity. The ability of the EA site to stimulate transcription optimally appears to be strictly dependent upon its distance from the p41 basal promoter. The EA site contains three overlapping sequences, a CAAT-enhancer-binding protein (C/EBP) transcription factor recognition site and two repeat elements. Mobility shift assays using the EA site identified four binding activities (C1 to C4). C1 and C2 are present in both uninfected and infected cells and do not contain C/EBP factors. In infected cells, point mutation of the EA site abrogates C1 and C2 binding activities and destroys transcriptional activity of the p41 promoter. C3 and C4 are present in uninfected cells only and were found to contain C/EBP factors. These findings indicate that in infected cells, transcriptional stimulation of the p41 promoter by the EA site requires C1 and C2 binding activities. These results further suggest that transcriptional activity may also depend upon the elimination of C3 and C4 binding activities.

Amino Acid Sequence↗