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Results for “codon optimization”

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PubMed2001

Inhibition of hepadnaviral replication by polyethylenimine-based intravenous delivery of antisense phosphodiester oligodeoxynucleotides to the liver.

Antisense oligodeoxynucleotides (ODNs) appear as attractive anti-hepatitis B virus (HBV) agents. We investigated in vivo, in the duck HBV (DHBV) infection model, whether linear polyethylenimine (lPEI)-based intravenous delivery of the natural antisense phosphodiester ODNs (O-ODNs) can prevent their degradation and allow viral replication inhibition in the liver. DHBV-infected Pekin ducklings were injected with antisense O-ODNs covering the initiation codon of the DHBV large envelope protein, either in free form (O-ODN-AS2) or coupled to lPEI (lPEI/O-ODN-AS2). Following optimization of lPEI/O-ODN complex formulation, complete O-ODN condensation into a homogenous population of small (20-60 nm) spherical particles was achieved. Flow cytometry analysis showed that lPEI-mediated transfer allowed the intrahepatic delivery of lPEI/O-ODN-AS2 to increase three-fold as compared with the O-ODN-AS2. Following 9-day therapy the intrahepatic levels of both DHBV DNA and RNA were significantly decreased in the lPEI/O-ODN-AS2-treated group as compared with the O-ODN-AS2-treated, control lPEI/O-ODN-treated, and untreated controls. In addition, inhibition of intrahepatic viral replication by lPEI/O-ODN-AS2 was not associated with toxicity and was comparable with that induced by the phosphorothioate S-ODN-AS2 at a five-fold higher dose. Taken together, our results demonstrate that phosphodiester antisense lPEI/O-ODN complexes specifically inhibit hepadnaviral replication. Therefore we provide here the first in vivo evidence that intravenous treatment with antisense phosphodiester ODNs coupled to lPEI can selectively block a viral disease-causing gene in the liver.

Animals↗

PubMed2000

Growth factor-mediated stabilization of amyloid precursor protein mRNA is mediated by a conserved 29-nucleotide sequence in the 3'-untranslated region.

Using a cell-free translation system, we previously demonstrated that the turnover and translation of amyloid precursor protein (APP) mRNA was regulated by a 29-nucleotide instability element, located 200 nucleotides downstream from the stop codon. Here we have examined the regulatory role of this element in primary human capillary endothelial cells under different nutritional conditions. Optimal proliferation required a growth medium (endothelial cell growth medium) supplemented with epidermal, basic fibroblast, insulin-like, and vascular endothelial growth factors. In vitro transcribed mRNAs with the 5'-untranslated region (UTR) and coding region of beta-globin and the entire 3'-UTR of APP 751 were transfected into cells cultured in endothelial cell growth medium. Wild-type globin-APP mRNA containing an intact APP 3'-UTR and mutant globin-APP mRNA containing a mutated 29-nucleotide element decayed with identical half-lives (t 1/2 = 60 min). Removal of all supplemental growth factors from the culture medium significantly accelerated the decay of transfected wild-type mRNA (t 1/2 = 10 min), but caused only a moderate decrease in the half-life of transfected mutant mRNA (t 1/2 = 40 min). We therefore conclude that the 29-nucleotide 3'-UTR element is an mRNA destabilizer whose function can be inhibited by inclusion of the aforementioned mixture of growth factors in the culture medium.

3' Untranslated Regions↗

PubMed1999

Nucleotide analogs and new buffers improve a generalized method to enrich for low abundance mutations.

A high sensitivity method for detecting low level mutations is under development. A PCR reaction is performed in which a restriction site is introduced in wild-type DNA by alteration of specific bases. Digestion of wild-type DNA by the cognate restriction endonuclease (RE) enriches for products with mutations within the recognition site. After reamplification, mutations are identified by a ligation detection reaction (LDR). This PCR/RE/LDR assay was initially used to detect PCR error in known wild-type samples. PCR error was measured in low |Deltap K a| buffers containing tricine, EPPS and citrate, as well as otherwise identical buffers containing Tris. PCR conditions were optimized to minimize PCR error using perfect match primers at the Msp I site in the p53 tumor suppressor gene at codon 248. However, since mutations do not always occur within pre-existing restriction sites, a generalized PCR/RE/LDR method requires the introduction of a new restriction site. In principle, PCR with mismatch primers can alter specific bases in a sequence and generate a new restriction site. However, extension from 3' mismatch primers may generate misextension products. We tested conversion of the Msp I (CCGG) site to a Taq I site (TCGA). Conversion was unsuccessful using a natural base T mismatch primer set. Conversion was successful when modified primers containing the 6 H,8 H -3, 4-dihydropyrimido[4,5- c ][1,2]oxazine-7-one (Q6) base at 3'-ends were used in three cycles of preconversion PCR prior to conversion PCR using the 3' natural base T primers. The ability of the pyrimidine analog Q6 to access both a T-like and C-like tautomer appears to greatly facilitate the conversion.

Base Sequence↗

PubMed1992

Nucleotide sequence of aminoglycoside 6'-N-acetyltransferase [AAC(6')] determinant from Serratia sp. 45.

Gene for aminoglycoside 6'-N-acetyltransferase [AAC(6')] from Serratia sp. 45 was cloned into E. coli. The enzyme produced in E. coli carrying the recombinant plasmid was compared to the Serratia enzyme. Both enzymes acetylated the 6'-C position of amikacin, dibekacin, tobramycin, sisomicin, gentamicin C1a and kanamycin but effected gentamicin C1, gentamicin C2 and micronomycin minimally. No significant difference in optimal pH, isoelectric point or molecular weight was detected. The nucleotide sequence of the gene was determined. Initiating with a GTG codon for methionine, it was composed of 552 base pair coding for 184 amino acids. The molecular weight of the enzyme was about 20418. Comparison of the amino acid sequence of this AAC(6') with the amino acid sequence of aacA4 gene from Serratia marcescens (G. Tran Van Nhieu and E. Collatz, J. Bacteriol., 169, 5708(1987)) showed 98.3% homology.

Acetyltransferases↗

PubMed1997

Strategies for optimal synthesis and secretion of heterologous proteins in the methylotrophic yeast Pichia pastoris.

Numerous heterologous proteins have been produced at greater than gram per liter levels in the methylotrophic yeast, Pichia pastoris, using the methanol oxidase promoter. The factors that drastically influence protein production in this system include: copy number of the expression cassette, site and mode of chromosomal integration of the expression cassette, mRNA 5'- and 3'-untranslated regions (UTR), translational start codon (AUG) context, A+T composition of cDNA, transcriptional and translational blocks, nature of secretion signal, endogenous protease activity, host strain physiology, media and growth conditions, and fermentation parameters. All these factors should be considered in designing an optimal production system. The inherent ability of P. pastoris to convert the zymogen (pro-enzyme) form of matrix metalloproteinases (MMP) into active mature forms (which tend to self-degrade, and in some instances also cause damage to cells), largely limits the use of this system for the production of MMP. However, this problem can be partly alleviated by co-expression of tissue inhibitor of MMP (TIMP-1).

Cloning, Molecular↗

PubMed1999

Effects of DksA and ClpP protease on sigma S production and virulence in Salmonella typhimurium.

Salmonella typhimurium responds to a variety of environmental stresses by accumulating the alternative sigma factor sigmaS. The repertoire of sigmaS -dependent genes that are subsequently expressed confers tolerance to a variety of potentially lethal conditions including low pH and stationary phase. The mechanism(s) responsible for triggering sigmaS accumulation are of considerable interest, because they help to ensure survival of the organism during encounters with suboptimal environments. Two genes associated with regulating sigmaS levels in S. typhimurium have been identified. The first is clpP, encoding the protease known to be responsible for degrading sigmaS in Escherichia coli. The second is dksA, encoding a protein of unknown function not previously associated with regulating sigmaS levels. As predicted, clpP mutants accumulated large amounts of sigmaS even in log phase. However, dksA mutants failed to accumulate sigmaS in stationary phase and exhibited lower accumulation during acid shock in log phase. DksA appears to be required for the optimal translation of rpoS based upon dksA mutant effects on rpoS transcriptional and translational lacZ fusions. The region of rpoS mRNA between codons 8 and 73 is required to see the effects of dksA mutations. This distinguishes the role of DksA from that of HF-I (hfq ) in rpoS translation, as the HF-I target area occurs well upstream of the rpoS start codon. DksA appears to be involved in the expression of several genes in addition to rpoS based on two-dimensional SDS-PAGE analysis of whole-cell proteins. As a result of their effects on gene expression, mutations in clpP and dksA decreased the virulence of S. typhimurium in mice, consistent with a role for sigmaS in pathogenesis.

Adenosine Triphosphatases↗

PubMed1998

A decision tree system for finding genes in DNA.

MORGAN is an integrated system for finding genes in vertebrate DNA sequences. MORGAN uses a variety of techniques to accomplish this task, the most distinctive of which is a decision tree classifier. The decision tree system is combined with new methods for identifying start codons, donor sites, and acceptor sites, and these are brought together in a frame-sensitive dynamic programming algorithm that finds the optimal segmentation of a DNA sequence into coding and noncoding regions (exons and introns). The optimal segmentation is dependent on a separate scoring function that takes a subsequence and assigns to it a score reflecting the probability that the sequence is an exon. The scoring functions in MORGAN are sets of decision trees that are combined to give a probability estimate. Experimental results on a database of 570 vertebrate DNA sequences show that MORGAN has excellent performance by many different measures. On a separate test set, it achieves an overall accuracy of 95 %, with a correlation coefficient of 0.78, and a sensitivity and specificity for coding bases of 83 % and 79%. In addition, MORGAN identifies 58% of coding exons exactly; i.e., both the beginning and end of the coding regions are predicted correctly. This paper describes the MORGAN system, including its decision tree routines and the algorithms for site recognition, and its performance on a benchmark database of vertebrate DNA.

Algorithms↗

PubMed1988

Mutational analysis of the HIS4 translational initiator region in Saccharomyces cerevisiae.

We have mutated various features of the 5' noncoding region of the HIS4 mRNA in light of established Saccharomyces cerevisiae and mammalian consensus translational initiator regions. Our analysis indicates that insertion mutations that introduce G + C-rich sequences in the leader, particularly those that result in stable stem-loop structures in the 5' noncoding region of the HIS4 message, severely affect translation initiation. Mutations that alter the length of the HIS4 leader from 115 to 39 nucleotides had no effect on expression, and sequence context changes both 5' and 3' to the HIS4 AUG start codon resulted in no more than a twofold decrease of expression. Changing the normal context at HIS4 5'-AAUAAUGG-3' to the optimal sequence context proposed for mammalian initiator regions 5'-CACCAUGG-3' did not result in stimulation of HIS4 expression. These studies, in conjunction with comparative and genetic studies in S. cerevisiae, support a general mechanism of initiation of protein synthesis as proposed by the ribosomal scanning model.

Alleles↗

PubMed1997

Two genes encoding an endoglucanase and a cellulose-binding protein are clustered and co-regulated by a TTA codon in Streptomyces halstedii JM8.

Streptomyces halstedii JM8 Cel2 is an endoglucanase of 28 kDa that is first produced as a protein of 42 kDa (p42) and is later processed at its C-terminus. Cel2 displays optimal activity towards CM-cellulose at pH6 and 50 degrees C and shows no activity against crystalline cellulose or xylan. The N-terminus of p42 shares similarity with cellulases included in family 12 of the beta-glycanases and the C-terminus shares similarity with bacterial cellulose-binding domains included in family II. This latter domain enables the precursor to bind so tightly to Avicel that it can only be eluted by boiling in 10% (w/v) SDS. Another open reading frame (ORF) situated 216 bp downstream from the p42 ORF encodes a protein of 40 kDa (p40) that does not have any clear hydrolytic activity against cellulosic or xylanosic compounds, but shows high affinity for Avicel (crystalline cellulose). The p40 protein is processed in old cultures to give a protein of 35 kDa that does not bind to Avicel. Translation of both ORFs is impaired in Streptomyces coelicolor bldA mutants, suggesting that a TTA codon situated at the fourth position of the first ORF is responsible for this regulation. S1 nuclease protection experiments demonstrate that both ORFs are co-transcribed.

Amino Acid Sequence↗

PubMed1987

Endosymbiotic origin and codon bias of the nuclear gene for chloroplast glyceraldehyde-3-phosphate dehydrogenase from maize.

The nuclei of plant cells harbor genes for two types of glyceraldehyde-3-phosphate dehydrogenases (GAPDH) displaying a sequence divergence corresponding to the prokaryote/eukaryote separation. This strongly supports the endosymbiotic theory of chloroplast evolution and in particular the gene transfer hypothesis suggesting that the gene for the chloroplast enzyme, initially located in the genome of the endosymbiotic chloroplast progenitor, was transferred during the course of evolution into the nuclear genome of the endosymbiotic host. Codon usage in the gene for chloroplast GAPDH of maize is radically different from that employed by present-day chloroplasts and from that of the cytosolic (glycolytic) enzyme from the same cell. This reveals the presence of subcellular selective pressures which appear to be involved in the optimization of gene expression in the economically important graminaceous monocots.

Amino Acid Sequence↗

PubMed1984

Genetic code and optimal resistance to the effects of mutations.

This paper deals with the notion of resistance of the genetic code to the effects of mutations. We measure the resistance of a group of t codons as the number of pairs of those which differ from each other in only one of their three bases. We find for each value of t the maximum possible value of the resistance and we describe some groups of codons giving this value. Important examples of such configurations are found in the genetic code, among these are the groups of synonymous codons, as observed elsewhere, and the cluster of codons which have an hydrophobic amino acid for translation.

Base Sequence↗

PubMed1994

Optimally parsing a sequence into different classes based on multiple types of evidence.

We consider the problem of parsing a sequence into different classes of subsequences. Two common examples are finding the exons and introns in genomic sequences and identifying the secondary structure domains of protein sequences. In each case there are various types of evidence that are relevant to the classification, but none are completely reliable, so we expect some weighted average of all the evidence to provide improved classifications. For example, in the problem of identifying coding regions in genomic DNA, the combined use of evidence such as codon bias and splice junction patterns can give more reliable predictions than either type of evidence alone. We show three main results: 1. For a given weighting of the evidence a dynamic programming algorithm returns the optimal parse and any number of sub-optimal parses. 2. For a given weighting of the evidence a dynamic programming algorithm determines the probability of the optimal parse and any number of sub-optimal parses under a natural Boltzmann-Gibbs distribution over the set of possible parses. 3. Given a set of sequences with known correct parses, a dynamic programming algorithm allows one to apply gradient descent to obtain the weights that maximize the probability of the correct parses of these sequences.

Algorithms↗

PubMed1995

Many combinations of amino acid sequences in a conserved region of the D1 protein satisfy photosystem II function.

The putative de helix of the D1 protein is located at the acceptor side of photosystem II (PS II) and serves as an indispensable part of a niche that binds the secondary plastoquinone QB. Combinatorial mutagenesis was applied to a stretch of four residues in a highly conserved region of this putative helix in order to reveal amino acid combinations that are able to support PS II function. An obligate photoheterotrophic mutant of the cyanobacterium Synechocystis sp. PCC 6803, missing four residues (delta YFGR254-7) in the de helix, was transformed with a D1-coding sequence carrying fully degenerate combinations of codons at the site of the deletion. Upon selection for photoautotrophy, 25 mutants with functional PS II were isolated. All mutants showed different codon combinations at positions 254 to 257; none was identical to the wild-type sequence, and none of the conserved residues was found to be mandatory for PS II function. However, 24 of the mutants contained Tyr of Phe at position 254 while at the other three positions many different amino acid combinations could be functionally accommodated. Most sequences maintained an amphiphilic arrangement of the helix that may align Tyr254 facing the QB binding pocket. This residue is proposed to be functionally analogous to Phe216 of the L subunit in purple bacteria which contributes to binding of QB. Most of the PS II properties were similar in the mutants compared to wild-type. Noticeable modifications in the mutants concerned the semiquinone equilibrium of electron transfer between QA and QB, and the affinity of PS II inhibitors. Differential effects on the semiquinone equilibrium were observed between two distinct quinones occupying the QB site (plastoquinone versus 2,5-dichloro-p-benzo- quinone), implying that residues in this domain are involved, directly or indirectly, with different binding determinants of the quinones. Even though many different combinations of amino acids in positions 254 to 257 of the D1 protein may satisfy the primary function of PS II, complex requirements need to be combined for optimized performance of the QB binding niche.

Amino Acid Sequence↗

PubMed1999

Local activation and inactivation of thyroid hormones: the deiodinase family.

Tissue-specific activation and inactivation of ligands of nuclear receptors which belong to the steroid retinoid-thyroid hormone superfamily of transcription factors represents an important principle of development- and tissue-specific local modulation of hormone action. Recently, several enzyme families have been identified which act as 'guardians of the gate' of ligand-activated transcription modulation. Three monodeiodinase isoenzymes which are involved in activation the 'prohormone' L-thyroxine (T4), the main secretory product of the thyroid gland, have been identified, characterized, and cloned. Both, type I and type II 5'-deiodinase generate the thyromimetically active hormone 3,3',5-triiodothyronine (T3) by reductive deiodination of the phenolic ring of T4. Inactivation of T4 and its product T3 occurs by deiodination of iodothyronines at the tyrosyl ring. This reaction is catalyzed both the type III 5-deiodinase and also by the type I enzyme, which has a broader substrate specificity. The three deiodinases appear to constitute a newly discovered family of selenocysteine-containing proteins and the presence of selenocysteine in the protein is critical for enzyme activity. Whereas the selenoenzyme characteristics of the type I and type III deiodinases are definitively established some controversy still exists for the type II 5'-deiodinase in mammals. The mRNA probably encoding the type II 5'-deiodinase subunit is markedly longer than those of the two other deiodinases and its selenocysteine-insertion element is located more than 5 kB downstream of the UGA-codon in the 3'-untranslated region. The three deiodinase isoenzymes show a distinct development- and tissue-specific pattern of expression, operate at individual optimal substrate levels, are differently regulated and modulated by hormones, cytokines, signaling pathways, natural factors, and pharmaceuticals. Whereas circulating T3 mainly originates from hepatic production via the type I 5'-deiodinase, the local cellular thyroid hormone concentration in various tissues including the central nervous system is controlled by complex para-, auto-, and intracrine interactions of all three deiodinases. Local thyroid hormone availability is further modulated by conjugation reactions of the phenolic 4'-OH-group of iodothyronines, which also inactivate the thyroid hormones.

Animals↗

PubMed1992

Rapid and direct detection of the most frequent Mediterranean beta-thalassemic mutations by multiplex allele-specific enzymatic amplification.

A rapid nonradioactive method for the diagnosis of the most frequent Mediterranean beta-thalassemic mutations is described based on a multiplex allele-specific polymerase chain reaction (PCR). This method allows direct detection of normal or mutated alleles on genomic DNA. We have used this approach to detect the most frequent Mediterranean mutations: IVS-1 nt 110 (G----A) and 39 nonsense (C----T). For each mutation three allele-specific oligonucleotides were used: one common upstream primer and two downstream primers differing in their terminal 3' nucleotide (one specific for the normal allele and one for the mutant allele). For each sample two PCR reactions were performed in parallel using in one case IVS-1 nt 110 and codon 39 normal primers and in the second case using the corresponding mutated primers. In both cases the different PCR fragments were visualized. After optimization these primers directed only amplification of their complementary allele. A single blind study was performed on the DNA of 18 individuals who were homozygous or heterozygous for these mutations. In comparison with a parallel investigation, using oligonucleotide probes, all the results were unambiguous. This diagnosis method, which is rapid, easy, direct, and inexpensive, allows the screening of a population group, including heterozygotes, which is required from an epidemiological and anthropological point of view. It could be extended to the large series screening of haplotypes before targeted diagnosis of various genetic diseases.

Algeria↗

PubMedDate not supplied

[Cloning of the gene for thermostable Thermus aquaticus YT1 DNA polymerase and its expression in Escherichia coli].

Using the phasmid vector pSL5, the genomic DNA fragment of T. aquaticus YT1 which contained the thermostable DNA polymerase (Taq-polymerase) gene was cloned. The BglII fragment of this genome locus was subcloned in the BamHI site of the pUC19 plasmid. To optimize the Taq-polymerase gene expression in E. coli cells, the gene was cloned in the correct reading frame regarding the initiation ATG codon of the pPR-TGATG-1 expression vector. The gene expression in this vector was controlled by the phage lambda PR promoter and the temperature-sensitive phage lambda repressor. We used PCR to amplify the short 5'-end fragment of the Taq-polymerase gene coding for the part into which an artificial SacI site was introduced. This site has been used for cloning the PCR product into the pPR-TGATG-1 vector, and the missing gene part was cloned into the KpnI site of the PCR product from the natural cloned gene. The cells of the E. coli PVG-A1 strain, which was obtained in the end, expressed efficiently the Taq-polymerase gene at the nonpermissive temperature. The content of the recombinant Taq-polymerase in the cells was about 1-2% of total proteins. The purified nearly homogeneous Taq-polymerase amplified efficiently in the PCR DNA fragments up to 5.5 kb long and was useful in DNA sequencing the by Sanger method. The half-life of the purified Taq polymerase was about 60 min at 95 degrees C, it was active for at least 65 standard PCR circles. The specific activity of recombinant enzyme preparations was about 180-200,000 units per mg of protein. The E. coli PVG-A1 strain enables one to isolate up to 500,000 units of purified enzyme from 2 l of bacterial culture.

Bacteriophage lambda↗

PubMed2001

Efficient translation initiation is required for replication of bovine viral diarrhea virus subgenomic replicons.

An internal ribosome entry site (IRES) mediates translation initiation of bovine viral diarrhea virus (BVDV) RNA. Studies have suggested that a portion of the N(pro) open reading frame (ORF) is required, although its exact function has not been defined. Here we show that a subgenomic (sg) BVDV RNA in which the NS3 ORF is preceded only by the 5' nontranslated region did not replicate to detectable levels following transfection. However, RNA synthesis and cytopathic effects were observed following serial passage in the presence of a noncytopathic helper virus. Five sg clones derived from the passaged virus contained an identical, silent substitution near the beginning of the NS3 coding sequence (G400U), as well as additional mutations. Four of the reconstructed mutant RNAs replicated in transfected cells, and in vitro translation showed increased levels of NS3 for the mutant RNAs compared to that of wild-type (wt) MetNS3. To more precisely dissect the role of these mutations, we constructed two sg derivatives: ad3.10, which contains only the G400U mutation, and ad3.7, with silent substitutions designed to minimize RNA secondary structure downstream of the initiator AUG. Both RNAs replicated and were translated in vitro to similar levels. Moreover, ad3.7 and ad3.10, but not wt MetNS3, formed toeprints downstream of the initiator AUG codon in an assay for detecting the binding of 40S ribosomal subunits and 43S ribosomal complexes to the IRES. These results suggest that a lack of stable RNA secondary structure(s), rather than a specific RNA sequence, immediately downstream of the initiator AUG is important for optimal translation initiation of pestivirus RNAs.

Amino Acid Sequence↗

PubMed2000

Use of mitogenomic information in teleostean molecular phylogenetics: a tree-based exploration under the maximum-parsimony optimality criterion.

We explored the phylogenetic utility and limits of the individual and concatenated mitochondrial genes for reconstructing the higher-level relationships of teleosts, using the complete (or nearly complete) mitochondrial DNA sequences of eight teleosts (including three newly determined sequences), whose relative phylogenetic positions were noncontroversial. Maximum-parsimony analyses of the nucleotide and amino acid sequences of 13 protein-coding genes from the above eight teleosts, plus two outgroups (bichir and shark), indicated that all of the individual protein-coding genes, with the exception of ND5, failed to recover the expected phylogeny, although unambiguously aligned sequences from 22 concatenated transfer RNA (tRNA) genes (stem regions only) recovered the expected phylogeny successfully with moderate statistical support. The phylogenetic performance of the 13 protein-coding genes in recovering the expected phylogeny was roughly classified into five groups, viz. very good (ND5, ND4, COIII, COI), good (COII, cyt b), medium (ND3, ND2), poor (ND1, ATPase 6), and very poor (ND4L, ND6, ATPase 8). Although the universality of this observation was unclear, analysis of successive concatenation of the 13 protein-coding genes in the same ranking order revealed that the combined data sets comprising nucleotide sequences from the several top-ranked protein-coding genes (no 3rd codon positions) plus the 22 concatenated tRNA genes (stem regions only) best recovered the expected phylogeny, with all internal branches being supported by bootstrap values >90%. We conclude that judicious choice of mitochondrial genes and appropriate data weighting, in conjunction with purposeful taxonomic sampling, are prerequisites for resolving higher-level relationships in teleosts under the maximum-parsimony optimality criterion.

Animals↗