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PubMed1983

A complex attenuator regulates inducible resistance to macrolides, lincosamides, and streptogramin type B antibiotics in Streptococcus sanguis.

Macrolide-lincosamide-streptogramin B resistance specified by Streptococcus sanguis plasmid pAM77 involves an adenine methylase, whose synthesis, demonstrable both phenotypically and by analysis of methionine-labeled proteins made in Bacillus subtilis minicells, is inducible by erythromycin, lincomycin, and streptogramin type B antibiotics. Localization of the methylase structural gene, including its control region in DNA fragments obtained with restriction endonucleases, has been deduced from DNA blot experiments with characterized target and probe DNAs from other streptococci, combined with DNA sequence analysis and comparison of the putative streptococcal methylase sequence with that of a cognate methylase in staphylococcal plasmid pE194. The streptococcal methylase migrates electrophoretically in polyacrylamide gels with the mobility of a 29,000-dalton protein. The sequence organization of the putative streptococcal methylase mRNA leader sequence partially resembles its staphylococcal counterpart and can support a similar mechanism of secondary structure rearrangement leading to methylase synthesis. The deduced 5' leader sequence preceding the pAM77 methylase structural gene sequence comprises approximately 155 nucleotides within which one can identify a putative control peptide 36 amino acid residues in length (in contrast to 19 in the pE194 peptide) and at least 14 possible classes of overlapping inverted complementary repeat sequences (in contrast to 3 in the pE194 control region), one of which can sequester the sequence AGGAG 7 nucleotides upstream from the putative (methionine) start codon of the streptococcal methylase. Comparison of the pAM77 and pE194 methylase amino acid sequences and their respective nucleotide sequences shows 51% conservation of amino acid residues (124 of 244) and 59% conservation of nucleotide residues (433 of 738), which suggests a common origin for the two methylase structural gene sequences. Differences in mRNA base composition associated with conserved amino acid residues occur mostly in the third nucleotide ("wobble") position of codons and may reflect adaptation of methylase genes to optimal expression in host cells with differing codon use patterns.

Amino Acid Sequence↗

PubMed2001

Cassettes for PCR-mediated construction of green, yellow, and cyan fluorescent protein fusions in Candida albicans.

We have developed a set of plasmids containing fluorescent protein cassettes for use in PCR-mediated gene tagging in Candida albicans. We engineered YFP and CFP variants of the GFP sequence optimized for C. albicans codon usage. The fluorescent protein sequences, linked to C. albicans auxotrophic marker sequences, were amplified by PCR and transformed directly into yeast. Gene-specific sequence was incorporated into the PCR primers, such that the tag-cassette integrates by homologous recombination at the 3'-end of the gene of interest. This technique was used to tag Cdc3 and Tub1 with GFP, YFP and CFP, which were readily visualized by fluorescence microscopy and localized as expected. In addition, Tub1-YFP and Cdc3-CFP were visualized in the same cells. Thus, this technique directs one-step construction of multiple fluorescent protein fusions, facilitating the study of protein co-expression and co-localization in C. albicans cells in vivo.

Bacterial Proteins↗

PubMed2005

Molecular pathogenesis of MEN2-associated tumors.

Although the gene responsible for multiple endocrine neoplasia type 2 (MEN2) was discovered many years ago, the exact mechanisms of tumor development in patients affected with RET germline mutations remain unknown. In vitro studies have certain pitfalls, one of which is the use of cell culture systems such as the NIH3T3 cells, in which RET usually is not expressed in contrast to the in vivo situation. Recent data suggest that an overrepresentation of mutant RET as a 'second hit' event might trigger tumorigenesis. However, alterations in other genes might contribute to this overrepresentation of RET or impact on MEN 2-related tumor development through completely different mechanisms and pathways. The final goal of further elucidating the natural history and pathogenesis of MEN2-related tumors should be the chance to offer patients with RET germline mutations an optimal cancer prevention (e.g. codon specific recommendations for prophylactic thyroidectomy) and treatment program, especially for metastatic medullary thyroid carcinoma for which presently no effective therapy other than surgery exists.

Cell Culture Techniques↗

PubMedDate not supplied

[A new Escherichia coli strain producing human tumor necrosis factor].

An Escherichia coli strain producing human tumor necrosis factor (TNF-alpha) was obtained using a semisynthetic gene partially optimized in respect of codon composition and a phage T7 promoter. The expression product was accumulated in cells as inclusion bodies in a yield of 50-70 mg/l of culture medium. The recombinant TNF-alpha in the form of inclusion bodies was used for immunization of rats to give a polyclonal antiserum. The resulting antibodies were specific under the immunoblotting conditions to the antigen used for the immunization. A dilution-based refolding procedure was developed; it provided a yield of soluble protein exceeding 85%.

Amino Acid Sequence↗

PubMed2004

Effects of two cis-acting mutations on the regulation and expression of release factor one in Escherichia coli.

Together with release factor (RF) 2, RF1 recognises the stop codons and triggers the hydrolysis of the nascent peptide from peptidyl-tRNA during translation termination. prfA, the gene that codes for RF1, is located at 27 min on the Escherichia coli map as the second gene in the hemA-operon. The concentration of RF1 has been shown to increase with increased growth rate, but it is not known where and how this control is exerted. In this study we show that the growth rate regulation of RF1, at least in part, is controlled at P(hemA1), one of two promoters preceding the hemA gene. We have also characterised two mutations, asuA1 and asuA2, that are antisuppressors to the tRNA suppressor Su2. Our data indicate that the antisuppressor phenotype is caused by an increased amount of RF1. The asuA2 mutation is a G to an A change just downstream of the -10 region of P(hemA1), it leads to a higher concentration of RF1 in the cell and abolishes the growth rate regulation. This indicates that the sequence between the -10 region and the transcription start site is important for growth rate control. The increase in concentration of RF1 caused by asuA1 is most likely at the translational level. The efficiency of translation initiation of prfA is low due to a long distance between the start codon and the Shine-Dalgarno (SD) sequence. The asuA1 mutation creates a new start codon with a more optimal distance to the SD sequence. This leads to an increased expression of RF1, probably due to increased initiation efficiency.

Base Sequence↗

PubMed2006

Visualizing cytoskeleton dynamics in mammalian cells using a humanized variant of monomeric red fluorescent protein.

Fluorescent proteins are versatile tools for live cell imaging studies. In particular, recent progress was achieved in the development of monomeric red fluorescent proteins (mRFPs) that show improved properties in respect to maturation and intracellular fluorescence. mRFPmars, a red fluorescent protein designed especially for the use in Dictyostelium, proved to be a brilliant label for different cytoskeletal elements. Here we report on the synthesis of a humanized version of a monomeric RFP, mRFPruby, which differs in sequence from mRFPmars in four amino acids and has a codon usage that is optimized for the application in mammalian cells. In order to demonstrate the usefulness of this new mRFP variant, mRFPruby fused to beta-actin was expressed in different mouse cell lines and used to visualize actin cytoskeleton dynamics by live cell microscopy.

3T3 Cells↗

PubMed2005

Gene synthesis, expression, purification, and characterization of human Jagged-1 intracellular region.

Notch signaling plays a key role in cell differentiation and is very well conserved from Drosophila to humans. Ligands of Notch receptors are type I, membrane spanning proteins composed of a large extracellular region and a 100-150 residue cytoplasmic tail. We report here, for the first time, the expression, purification, and characterization of the intracellular region of a Notch ligand. Starting from a set of synthetic oligonucleotides, we assembled a synthetic gene optimized for Escherichia coli codon usage and encoding the cytoplasmic region of human Jagged-1 (residues 1094-1218). The protein containing a N-terminal His(6)-tag was over-expressed in E. coli, and purified by affinity and reversed phase chromatography. After cleavage of the His(6)-tag by a dipeptidyl aminopeptidase, the protein was purified to homogeneity and characterized by spectroscopic techniques. Far-UV circular dichroism, fluorescence emission spectra, fluorescence anisotropy measurements, and (1)H nuclear magnetic resonance spectra, taken together, suggest that the cytoplasmic tail of human Jagged-1 behaves as an intrinsically unstructured domain in solution. This result was confirmed by the high susceptibility of the recombinant protein to proteolytic cleavage. The significance of this finding is discussed in relation to the recently proposed role of the intracellular region of Notch ligands in bi-directional signaling.

Calcium-Binding Proteins↗

PubMed2003

Progress toward an expanded eukaryotic genetic code.

Expanding the eukaryotic genetic code to include unnatural amino acids with novel properties would provide powerful tools for manipulating protein function in eukaryotic cells. Toward this goal, a general approach with potential for isolating aminoacyl-tRNA synthetases that incorporate unnatural amino acids with high fidelity into proteins in Saccharomyces cerevisiae is described. The method is based on activation of GAL4-responsive HIS3, URA3, or lacZ reporter genes by suppression of amber codons in GAL4. The optimization of GAL4 reporters is described, and the positive and negative selection of active Escherichia coli tyrosyl-tRNA synthetase (EcTyrRS)/tRNA(CUA) is demonstrated. Importantly, both selections can be performed on a single cell and with a range of stringencies. This method will facilitate the isolation of a range of aminoacyl-tRNA synthetase (aaRS)/tRNA(CUA) activities from large libraries of mutant synthetases.

Amino Acids↗

PubMed1999

[Construction of DNA vaccine including a chimeric gene encoding Cysticercus cellulosae antigen and porcine interleukin-4].

AIM: To construct a fusion expression vector for DNA vaccine including porcine interleukin-4(IL-4) and antigen cC1 to enhance the protective immunity of Cysticercus cellulosae antigen cC1. METHODS: The cDNA fragments encoding porcine IL-4 and cC1 were amplified respectively by PCR and the fused. The obtained chimeric gene IL-4cC1 contained a synthetic linker of ten amino acids and the sequence surrounding its 5' AUG initiatory codon was changed to optimized translational initiation. RESULTS: Identified by restriction enzyme analysis, an insert fragment of 1.5 kb was demonstrated. It had the same sequence as reported and designed by DNA sequencing analysis. CONCLUSION: A fusion expression plasmid containing porcine IL-4 and cC1 was constructed.

Animals↗

PubMed1992

Optimizing nucleotide mixtures to encode specific subsets of amino acids for semi-random mutagenesis.

In random mutagenesis, synthesis of an NNN triplet (i.e. equiprobable A, C, G, and T at each of the three positions in the codon) could be considered an optimal nucleotide mixture because all 20 amino acids are encoded. NN(G,C) might be considered a slightly more intelligent "dope" because the entire set of amino acids is still encoded using only half as many codons. Using a general algorithm described herein, it is possible to formulate more complex doping schemes which encode specific subsets of the twenty amino acids, excluding others from the mix. Maximizing the equiprobability of amino acid residues contributing to such a subset is suggested as an optimal basis for performing semi-random mutagenesis. This is important for reducing the nucleotide complexity of combinatorial cassettes so that "sequence space" can be searched more efficiently. Computer programs have been developed to provide tables of optimized dopes compatible with automated DNA synthesizers.

Amino Acid Sequence↗

PubMed1991

Influence of the codon following the initiation codon on the expression of the lacZ gene in Saccharomyces cerevisiae.

A set of 32 different codons were introduced in a lacZ expression vector (pPTK400) immediately 3' from the AUG initiation codon. Expression of the lacZ gene was determined in Saccharomyces cerevisiae by measuring the amount of beta-galactosidase fusion protein using immuno-gel electrophoresis. A 5.3-fold difference in expression was found among the various constructs. It was found that there was no preference for a certain nucleotide in any position of the second codon and there was no distinct correlation between the level of tRNA corresponding to any particular second codon and expression. No correlation could be found between the local secondary structure and expression. When the overall codon usage in yeast and the codon usage in the second position of the mRNA is compared, there is no obvious significant difference in preference. This indicates that in yeast, in contrast to Escherichia coli, the codon choice at the beginning of the mRNA does not deviate from the one further downstream and is determined by the requirements for optimal translation elongation. Important determinants of the optimal context for an initiation codon in yeast therefore must be located mainly 5' from this codon.

Amino Acid Sequence↗

PubMed1997

Evolution of codon usage bias in Drosophila.

We first review what is known about patterns of codon usage bias in Drosophila and make the following points: (i) Drosophila genes are as biased or more biased than those in microorganisms. (ii) The level of bias of genes and even the particular pattern of codon bias can remain phylogenetically invariant for very long periods of evolution. (iii) However, some genes, even very tightly linked genes, can change very greatly in codon bias across species. (iv) Generally G and especially C are favored at synonymous sites in biased genes. (v) With the exception of aspartic acid, all amino acids contribute significantly and about equally to the codon usage bias of a gene. (vi) While most individual amino acids that can use G or C at synonymous sites display a preference for C, there are exceptions: valine and leucine, which prefer G. (vii) Finally, smaller genes tend to be more biased than longer genes. We then examine possible causes of these patterns and discount mutation bias on three bases: there is little evidence of regional mutation bias in Drosophila, mutation bias is likely toward A+T (the opposite of codon usage bias), and not all amino acids display the preference for the same nucleotide in the wobble position. Two lines of evidence support a selection hypothesis based on tRNA pools: highly biased genes tend to be highly and/or rapidly expressed, and the preferred codons in highly biased genes optimally bind the most abundant isoaccepting tRNAs. Finally, we examine the effect of bias on DNA evolution and confirm that genes with high codon usage bias have lower rates of synonymous substitution between species than do genes with low codon usage bias. Surprisingly, we find that genes with higher codon usage bias display higher levels of intraspecific synonymous polymorphism. This may be due to opposing effects of recombination.

Animals↗

PubMed1986

Codon usage in yeast: cluster analysis clearly differentiates highly and lowly expressed genes.

Codon usage data has been compiled for 110 yeast genes. Cluster analysis on relative synonymous codon usage revealed two distinct groups of genes. One group corresponds to highly expressed genes, and has much more extreme synonymous codon preference. The pattern of codon usage observed is consistent with that expected if a need to match abundant tRNAs, and intermediacy of tRNA-mRNA interaction energies are important selective constraints. Thus codon usage in the highly expressed group shows a higher correlation with tRNA abundance, a greater degree of third base pyrimidine bias, and a lesser tendency to the A+T richness which is characteristic of the yeast genome. The cluster analysis can be used to predict the likely level of gene expression of any gene, and identifies the pattern of codon usage likely to yield optimal gene expression in yeast.

Base Composition↗

PubMed1995

A combinatorial method for constructing libraries of long peptides displayed by filamentous phage.

We describe the construction and screening of a random peptide library displayed by filamentous phage. The peptides are expressed in multiple copies on the filamentous phage M13 as amino-terminal fusions with the major coat protein, the product of gene VIII. These libraries are efficiently screened for reactive peptides, using a combination of panning in solution followed by a plaque lift assay. Advantages of this system are that both high- and low-affinity phage clones are simultaneously identified and the analysis of non-reactive phage is minimized. The vector system utilized to construct this library enables it to be used for the construction of peptide libraries employing a combinatorial cloning strategy. This feature makes it especially suitable for construction of peptide libraries using codon-based oligonucleotide synthesis. The vectors also allow rapid optimization and modification of lead peptides by codon-based mutagenesis. A 20-amino acid long random peptide library of 1 x 10(9) members was constructed and screened for peptides that bound to (i) a monoclonal antibody recognizing the amino-terminus of beta-endorphin; (ii) a monoclonal antibody recognizing a peptide epitope derived from the v-ros oncogene product; and (iii) the constant region of murine IgG2b. The approach described here provides a means for the construction of customized libraries that can be screened with a variety of target molecules.

Amino Acid Sequence↗

PubMed1994

Premature translation termination mutations are efficiently suppressed in a highly conserved region of yeast Ste6p, a member of the ATP-binding cassette (ABC) transporter family.

The requirements for efficient translation termination are incompletely understood. Since the local context surrounding stop codons can influence the efficiency of translation termination, premature termination codons introduced by random mutation may not always terminate at the optimal efficiencies expected of naturally occurring stop codons. To investigate whether this could result in physiologically significant levels of read through, we examined the suppression of premature translation termination mutations within a sequence motif of the yeast Ste6 protein (Ste6p) that is highly conserved among members of the ATP-binding cassette (ABC) transporter family. The human cystic fibrosis transmembrane conductance regulator (CFTR), which is defective in individuals with the disease cystic fibrosis, is also a member of this protein family. The mutations examined in Ste6p were chosen because a premature termination codon at the corresponding residue of CFTR has previously been reported to cause less severe pulmonary involvement than some missense mutations, suggesting that low level suppression of this stop codon could be occurring. Our results indicate that these premature stop codons in Ste6p can be suppressed at frequencies as high as 10%. Characterization of this phenomenon using a beta-galactosidase read through assay system showed that a limited sequence context surrounding this site contained information that was sufficient to cause suppression of translation termination. Amino acid sequence analysis of the full-length translation products produced by read through of an amber codon demonstrated that termination suppression was mediated by near-cognate tRNA mispairing that resulted in the insertion of tyrosine, lysine, or tryptophan.

ATP-Binding Cassette Transporters↗

PubMed2006

[Design and implementation of DB sequence optimization software].

TIR (Translation Initiation Region) efficiency is very important in prokaryotic expression. The TIR's efficiency is highly dependent on SD (Shine-Dalgarno) sequence, distance between SD sequence and start codon, DB (Downstream Box) sequence, TIR's second structure, codon adaptation and so on. In this paper, we designed and implemented the software to optimize DB sequence and 5' rare codons. It generated some optimization sequences by analyzing the target sequence and comparing it with 16S RNA. And the optimization sequences is sorting by number of base pairing, location of base pairing and codon adaptation. We drew up the algorithm and the core of code in this paper.

Base Sequence↗

PubMed1989

Context effects and inefficient initiation at non-AUG codons in eucaryotic cell-free translation systems.

The context requirements for recognition of an initiator codon were evaluated in vitro by monitoring the relative use of two AUG codons that were strategically positioned to produce long (pre-chloramphenicol acetyl transferase [CAT]) and short versions of CAT protein. The yield of pre-CAT initiated from the 5'-proximal AUG codon increased, and synthesis of CAT from the second AUG codon decreased, as sequences flanking the first AUG codon increasingly resembled the eucaryotic consensus sequence. Thus, under prescribed conditions, the fidelity of initiation in extracts from animal as well as plant cells closely mimics what has been observed in vivo. Unexpectedly, recognition of an AUG codon in a suboptimal context was higher when the adjacent downstream sequence was capable of assuming a hairpin structure than when the downstream region was unstructured. This finding adds a new, positive dimension to regulation by mRNA secondary structure, which has been recognized previously as a negative regulator of initiation. Translation of pre-CAT from an AUG codon in a weak context was not preferentially inhibited under conditions of mRNA competition. That result is consistent with the scanning model, which predicts that recognition of the AUG codon is a late event that occurs after the competition-sensitive binding of a 40S ribosome-factor complex to the 5' end of mRNA. Initiation at non-AUG codons was evaluated in vitro and in vivo by introducing appropriate mutations in the CAT and preproinsulin genes. GUG was the most efficient of the six alternative initiator codons tested, but GUG in the optimal context for initiation functioned only 3 to 5% as efficiently as AUG. Initiation at non-AUG codons was artifactually enhanced in vitro at supraoptimal concentrations of magnesium.

Animals↗

PubMed1985

Synthetic oligonucleotide probes deduced from amino acid sequence data. Theoretical and practical considerations.

Synthetic probes deduced from amino acid sequence data are widely used to detect cognate coding sequences in libraries of cloned DNA segments. The redundancy of the genetic code dictates that a choice must be made between (1) a mixture of probes reflecting all codon combinations, and (2) a single longer "optimal" probe. The second strategy is examined in detail. The frequency of sequences matching a given probe by chance alone can be determined and also the frequency of sequences closely resembling the probe and contributing to the hybridization background. Gene banks cannot be treated as random associations of the four nucleotides, and probe sequences deduced from amino acid sequence data occur more often than predicted by chance alone. Probe lengths must be increased to confer the necessary specificity. Examination of hybrids formed between unique homologous probes and their cognate targets reveals that short stretches of perfect homology occurring by chance make a significant contribution to the hybridization background. Statistical methods for improving homology are examined, taking human coding sequences as an example, and considerations of codon utilization and dinucleotide frequencies yield an overall homology of greater than 82%. Recommendations for probe design and hybridization are presented, and the choice between using multiple probes reflecting all codon possibilities and a unique optimal probe is discussed.

Amino Acid Sequence↗