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PubMed2001

The level and landscape of optimization in the origin of the genetic code.

We consider a model of the origin of genetic code organization incorporating the biosynthetic relationships between amino acids and their physicochemical properties. We study the behavior of the genetic code in the set of codes subject both to biosynthetic constraints and to the constraint that the biosynthetic classes of amino acids must occupy only their own codon domain, as observed in the genetic code. Therefore, this set contains the smallest number of elements ever analyzed in similar studies. Under these conditions and if, as predicted by physicochemical postulates, the amino acid properties played a fundamental role in genetic code organization, it can be expected that the code must display an extremely high level of optimization. This prediction is not supported by our analysis, which indicates, for instance, a minimization percentage of only 80%. These observations can therefore be more easily explained by the coevolution theory of genetic code origin, which postulates a role that is important but not fundamental for the amino acid properties in the structuring of the code. We have also investigated the shape of the optimization landscape that might have arisen during genetic code origin. Here, too, the results seem to favor the coevolution theory because, for instance, the fact that only a few amino acid exchanges would have been sufficient to transform the genetic code (which is not a local minimum) into a much better optimized code, and that such exchanges did not actually take place, seems to suggest that, for instance, the reduction of translation errors was not the main adaptive theme structuring the genetic code.

Algorithms↗
PubMed1999

Effects of various amino acid 256 mutations on sarcoplasmic/endoplasmic reticulum Ca2+ ATPase function and their role in the cellular adaptive response to thapsigargin.

Upon direct selection of mammalian cells for resistance to thapsigargin (TG), a potent inhibitor of the sarcoplasmic/endoplasmic reticulum Ca2+ transport ATPase (SERCA), the ATPase can acquire specific mutations at amino acid position 256 (aa256). In particular, Phe256 --> Leu and Phe256 --> Ser substitutions can occur upon TG selection, with each substitution resulting in a SERCA that is 4- to 5-fold resistant to TG inhibition (M. Yu et al., J. Biol. Chem. 273, 3542-3546, 1998). We have now identified a third substitution, i.e., Phe256 --> Val, that occurs when the Chinese hamster lung fibroblast cell line DC-3F is selected for TG resistance. Although the Phe256 --> Val substitution at codon 256 results in a SERCA whose enzymological properties in terms of Ca2+ transport and ATP hydrolysis are essentially similar to that of wild-type (wt) SERCA, the mutant enzyme is more than 40-fold resistant to TG inhibition. To analyze further the role of aa256 in TG-SERCA interactions, mutational analysis of this particular residue was also carried out. Of all the mutations introduced, only the Phe256 --> Glu substitution interferes with expression of the ATPase. The Phe256 --> Arg substitution does not interfere with SERCA expression, but the resulting enzyme is totally inactive. In terms of sensitivity of the various mutants to TG, maximal reduction in the ATPase's affinity for TG occurs with amino acid substitutions containing branched side chains, i.e. with the Phe256 --> Val, Phe256 --> Ile, and Phe256 --> Thr mutants. Since a corresponding Phe is conserved in the Na+, K+-ATPase which is not sensitive to TG, our findings suggest that this amino acid provides stabilization of the stalk segment with respect to the membrane interface, thereby optimizing specific interactions of TG with neighboring S3 residues (L. Zhong and G. Inesi, J. Biol. Chem. 273, 12994-12998, 1998). It is likely that a relatively high frequency of codon 256 mutations favor the aa256 mutants as a specific adaptive response to TG selection.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed1993

Expression of polypeptides of human immunodeficiency virus-1 reverse transcriptase in Escherichia coli.

We have prepared a plasmid, pRC-RT, for expression of HXB2 HIV-1 reverse transcriptase (RT) in Escherichia coli (Becerra et al., Biochemistry 30, 11707-11719, 1991). Here we describe the optimization of RT overexpression and its purification. In pRC-RT, the precise RT coding region of HXB2 proviral DNA is flanked by start and stop codons, and expression is driven by the phage lambda pL promoter in a temperature-inducible system. The 64,484-Da RT polypeptide (termed p66) is expressed as approximately 10% of total cell protein after 2 h of induction, and the RT is readily solubilized and purified free of DNA Pol I and to near homogeneity as a homodimer of p66 or as a heterodimer of p66 and p51, resembling the natural enzyme. After achieving appropriate expression of the full-length p66 RT, we next created vectors to express multiple individual segments of the p66 polypeptide. These segments are: a 51,000-Da peptide, representing C-terminal truncation of p66, and several peptides representing consecutive N-terminal, central, and C-terminal segments of p66. The latter peptide, corresponding to the RNase H domain of RT, has been purified in large quantities and is currently under study for solution of its structure by NMR. This peptide is devoid of enzyme activity and of substrate-binding capacity, but exists in solution as a folded globular protein with structure resembling that of E. coli ribonuclease H and that of a similar HIV-1 RT RNase H domain peptide examined by X-ray crystallography (Becerra et al., FEBS Lett. 270, 67-80, 1990). Various other RT peptides described here should prove to be similarly useful for structural studies, as well as other approaches.

Amino Acid Sequence↗
PubMed2002

ORF-FINDER: a vector for high-throughput gene identification.

We have developed a simple and efficient system (ORF-FINDER) for selecting open reading frames (ORFs) from randomly fragmented genomic DNA fragments. The ORF-FINDER vectors are plasmids that contain a translational start site out of frame with respect to the gene for green fluorescent protein (GFP). Insertion of DNA fragments that bring the initiating ATG in frame with GFP and that contain no stop codons (that is, ORFs) results in the expression of ORF-GFP fusion proteins. In addition, we have developed software (GeneWorks and GenomeAnalyzer) to predict the optimal insert size for maximizing the number of gene-coding ORFs and minimizing unintentionally selected non-coding ORFs. To demonstrate the feasibility of using the ORF-FINDER system to screen genomes for ORFs, we cloned yeast genomic DNA and succeeded in enriching for ORFs by 25-fold. Furthermore, we have shown that the vector can effectively isolate ORFs from the more complex genomes of eukaryotic parasites. We envision that ORF-FINDER will have several applications including genome sequencing projects, gene building from oligonucleotides and construction of expression libraries enriched for ORFs.

Animals↗
PubMed1999

Rapid, phenotypic HIV-1 drug sensitivity assay for protease and reverse transcriptase inhibitors.

BACKGROUND: Development of drug resistance is one of the major reasons for the failure of antiretroviral therapy of HIV-1 infection. Knowing the drug sensitivity-resistance profile of viruses present in a patient prior to treatment or change in treatment could help to optimize therapy. OBJECTIVE: Development of a rapid standardized phenotypic HIV-1 drug sensitivity assay for protease (PR) and reverse transcriptase (RT) inhibitors. DESIGN: The PR gene (codons 1-99) and the 5' part of the RT gene (codons 1-300) of HIV-1 is amplified from the plasma of infected individuals by RT-PCR and ligated into a proviral clone of HIV-1 containing a deletion of the PR gene and the 5' part of the RT gene. Bacteria are transformed with the ligation product and plasmid DNA is prepared from a library of transformed bacteria. The plasmid DNA is transfected into 293 T cells and recombinant virus is harvested from the supernatant of the transfected cells 2 days after transfection. The sensitivity of the recombinant virus is determined with the help of a sensitive indicator cell line. RESULTS: Recombinant viruses were generated with high efficiency. Determination of the drug sensitivity of the recombinant viruses with an indicator cell line was highly reproducible. The recombinant viruses accurately reflected the sensitivity-resistance profile of the parental viruses. The phenotypic drug sensitivity determined by this assay correlated well with the treatment history of patients. CONCLUSION: This assay system should allow rapid, high-throughput analyses of phenotypic HIV-1 drug sensitivity for PR and RT inhibitors. Due to the efficient generation of recombinant viruses, propagation of the recombinant viruses in cell culture is not required prior to the determination of the sensitivity of the recombinant viruses. The risk of selecting fitter non-resistant viruses due to culture conditions is minimized.

Anti-HIV Agents↗
PubMed1997

In vitro suppression as a tool for the investigation of translation initiation.

An in vitro protein synthesizing system that employs rabbit reticulocyte lysates has been employed for protein production from mRNAs containing nonsense (UAG) codons in the presence of misacylated suppressor tRNAs.The system includes a misacylated Escherichia coli tRNAAlaCUA that functions at least as efficiently as any suppressor tRNA transcript reported to date and which has been shown not to be a substrate for (re)activation by alanyl-tRNA synthetase. Application of the optimized system for preparation of dihydrofolate analogs has also permitted analysis of competing mechanisms that control the sites(s) of translation initiation.

Amino Acid Sequence↗
PubMed2000

Second codon positions of genes and the secondary structures of proteins. Relationships and implications for the origin of the genetic code.

The nucleotide frequencies in the second codon positions of genes are remarkably different for the coding regions that correspond to different secondary structures in the encoded proteins, namely, helix, beta-strand and aperiodic structures. Indeed, hydrophobic and hydrophilic amino acids are encoded by codons having U or A, respectively, in their second position. Moreover, the beta-strand structure is strongly hydrophobic, while aperiodic structures contain more hydrophilic amino acids. The relationship between nucleotide frequencies and protein secondary structures is associated not only with the physico-chemical properties of these structures but also with the organisation of the genetic code. In fact, this organisation seems to have evolved so as to preserve the secondary structures of proteins by preventing deleterious amino acid substitutions that could modify the physico-chemical properties required for an optimal structure.

Chemical Phenomena↗
PubMed1999

Imaging cells in the developing nervous system with retrovirus expressing modified green fluorescent protein.

To visualize the movements of cells and their processes in developing vertebrates, we constructed replication-incompetent retroviral vectors encoding green fluorescent protein (GFP) that can be detected as a single integrated copy per cell. To optimize GFP expression, the CMV enhancer and avian beta-actin promoter were incorporated within a retrovirus construct to drive transcription of redshifted (F64L, S65T) and codon-modified GFP (EGFP), EGFP tagged with GAP-43 sequences targeting the GFP to the cell membrane, or EGFP with additional mutations that increase its ability to fold properly at 37 degrees C (S147P or V163A, S175G). We have used these viruses to efficiently mark and follow the developmental progression of a large population of cells in rat neocortex and whole avian embryos. In the chick embryo, the migration and development of GFP-marked neural crest cells were monitored using time-lapse videomicroscopy. In the neocortex, GFP clearly delineates the morphology of a variety of neuronal and glial phenotypes. Cells expressing GFP display normal dendritic morphologies, and infected cells persist into adulthood. Cortical neurons appear to form normal local axonal and long-distance projections, suggesting that the presence of cytoplasmic or GAP-43-tagged GFP does not significantly interfere with normal development.

Actins↗
PubMed1993

Neutral adaptation of the genetic code to double-strand coding.

We lay new foundations to the hypothesis that the genetic code is adapted to evolutionary retention of information in the antisense strands of natural DNA/RNA sequences. In particular, we show that the genetic code exhibits, beyond the neutral replacement patterns of amino acid substitutions, optimal properties by favoring simultaneous evolution of proteins encoded in DNA/RNA sense-antisense strands. This is borne out in the sense-antisense transformations of the codons of every amino acid which target amino acids physicochemically similar to each other. Moreover, silent mutations in the sense strand generate conservative ones in its antisense counterpart and vice versa. Coevolution of proteins coded by complementary strands is shown to be a definite possibility, a result which does not depend on any physical interaction between the coevolving proteins. Likewise, the degree to which the present genetic code is dedicated to evolutionary sense-antisense tolerance is demonstrated by comparison with many randomized codes. Double-strand coding is quantified from an information-theoretical point of view.

Adaptation, Biological↗
PubMed1984

Efficient expression in Escherichia coli of a mature and a modified human interferon-beta 1.

Ten recombinant plasmids were constructed which direct the synthesis of a mature human interferon-beta 1 (IFN-beta 1) under the control of the Escherichia coli tryptophan (trp) promoter. The spacing between the Shine-Dalgarno sequence of the trpL and the ATG initiation codon of the interferon gene was varied from 6 to 23 nucleotides by utilizing a Cla I site located within the spacer region of the plasmids. The optimal spacing for expression of IFN-beta 1 was determined to be 8-13 nucleotides from the results of interferon assay. The E. coli lipoprotein (lpp) promoter was also used for expression of IFN-beta 1 in E. coli. The results with an expression vector carrying a lpp-lac promoter showed that a modified IFN-beta 1 containing an additional 7 amino acids at the amino-terminus might be less active than the mature molecule.

Amino Acid Sequence↗
PubMedDate not supplied

Novel mutations in the APC gene and clinical features in Swedish patients with polyposis coli.

The adenomatous polyposis coli (APC) gene was investigated in Swedish patients with familial adenomatous polyposis (FAP). A combination of analyses including single stranded conformation polymorphism (SSCP), heteroduplex (HD), protein truncation test (PTT) and direct sequencing was used to enable optimal mutation detection. Three novel mutations in the gene were identified, i.e. nt2644C- > T (giving an Arg876Stop mutation), nt4025del173 (leading to premature truncation of the protein at codon 1337) and nt3526insG (giving truncation at codon 1178). In addition, one previously described mutation, i.e. the 5-bp-deletion nt3942del5(AAAGA) in codon 1309 (giving a premature termination of the protein at codon 1314) was detected. All four mutations were located in the 5'-half of exon 15. The two latter mutations were associated with the CHRPE (congenital hypertrophy of retina pigment epithelium) phenotype (CHRPE was not examined in the other two cases). The patients with mutations in codon 1309 and 1336 had a more severe FAP phenotype.

Adenomatous Polyposis Coli↗
PubMed1996

Rate of translation of natural mRNAs in an optimized in vitro system.

We report results on in vitro translation of an mRNA coding for elongation factor TuB which was in vitro transcribed from the tufB gene from Escherichia coli. Translation occurs at a rate of about 10 codons per second, which is close to the in vivo rate. Protein elongation obeys Michaelis-Menten kinetics with respect to the concentrations of the elongation factors EF-Tu and EF-G in the translation system. The measured K(m) values for EF-Tu and EF-G are 10 and 0.25 microM, respectively. The obtained k(cat) and K(m) values were used to estimate the average k(cat)/K(m) of about 24 x 10(6) s-1 M-1 for the interaction of individual EF-Tu*GTP*aa-tRNA complexes with ribosomes. The estimated k(cat)/K(m) value for EF-G is 36 x 10(6) s-1 M-1. We have also studied translation with a "hyperaccurate" ribosome variant that is pseudodependent on streptomycin (SmP). We have found that SmP ribosomes translate the TuB mRNA significantly slower than wild-type ribosomes do. This is mainly due to a threefold lower k(cat)/K(m) for the interaction of EF-Tu*GTP*aa-tRNA complexes with SmP ribosomes.

Animals↗
PubMed1998

Optimized non-radioactive protein truncation test for mutation analysis of the adenomatous polyposis coli (APC) gene.

Germline mutations in the adenomatous polyposis coli gene cause familial adenomatous polyposis, a colon cancer predisposition syndrome. More than 95% of the identified mutations result in the generation of stop codons or reading frame shifts and encode a truncated gene product, a mutation profile also found in other tumor predisposition genes such as the breast cancer or the hereditary non-polyposis coli. Therefore the protein truncation test is ideally suited for screening of mutations in these genes, starting from simple blood samples. Gene segments of interest are amplified from genomic DNA or mRNA, thereby incorporating a T7 promoter at the 5'-end. After in vitro transcription and translation of the PCR products, the resulting protein is analysed by gel electrophoresis. Truncated translation products indicate the presence of a stop mutation. We have developed a non-radioactive protein truncation test that uses a biotinylated Lys-t-RNA to label the translation products and allows a chemiluminescent detection instead of the standard radioactive method. This generic protein truncation test kit was then used to develop a parameter-specific protein truncation test for adenomatous polyposis coli. The adenomatous polyposis coli gene was divided in 5 overlapping segments, and primers were optimized to produce distinct bands with very low background in the protein truncation test. The assay was tested on 20 familial adenomatous polyposis patient samples, where 18 mutations were found, demonstrating the efficiency of this method.

Adenomatous Polyposis Coli↗
PubMed1997

[A promoter responsible for over-expression of cholera toxin B subunit in cholera toxin A subunit structure gene].

A promoter sequence, which promotes the transcription of cholera toxin B subunit gene, was found in cholera toxin A subunit structure gene. The transcription starts at the adenine Located at +833, that is 456bp upstream to the A of the initiation codon ATG of cholera toxin B gene. Under the control of the promoter, cholera toxin B subunit was over-expressed as high as 200 mg/L at an optimized culture condition. The chloramphenicol acetyl transferase gene and beta-galactosidase could also be efficiently expressed under the direction of the promoter. This promoter may be responsible for the 6 fold and 7 fold higher expression level of cholera toxin B subunit than cholera toxin A subunit in V. cholerae and Escheria coli respectively. The over-expression of CTB may be useful in preparing vaccine against cholera and facilitating the construction of peptide-bearing immunogenic hybrid proteins.

Base Sequence↗
PubMed2003

Allele-specific rpoB PCR assays for detection of rifampin-resistant Mycobacterium tuberculosis in sputum smears.

We describe an allele-specific PCR assay to detect mutations in three codons of the rpoB gene (516, 526, and 531) in Mycobacterium tuberculosis strains; mutations in these codons are reported to account for majority of M. tuberculosis clinical isolates resistant to rifampin (RIF), a marker of multidrug-resistant tuberculosis (MDR-TB). Three different allele-specific PCRs are carried out either directly with purified DNA (single-step multiplex allele-specific PCR), or with preamplified rpoB fragment (nested allele-specific PCR [NAS-PCR]). The method was optimized and validated following analysis of 36 strains with known rpoB sequence. A retrospective analysis of the 287 DNA preparations from epidemiologically unlinked RIF-resistant clinical strains from Russia, collected from 1996 to 2002, revealed that 247 (86.1%) of them harbored a mutation in one of the targeted rpoB codons. A prospective study of microscopy-positive consecutive sputum samples from new and chronic TB patients validated the method for direct analysis of DNA extracted from sputum smears. The potential of the NAS-PCR to control for false-negative results due to lack of amplification was proven especially useful in the study of these samples. The developed rpoB-PCR assay can be used in clinical laboratories to detect RIF-resistant and hence MDR M. tuberculosis in the regions with high burdens of the MDR-TB.

Alleles↗
PubMed1998

On error minimization in a sequential origin of the standard genetic code.

Distances between amino acids were derived from the polar requirement measure of amino acid polarity and Benner and co-workers' (1994) 74-100 PAM matrix. These distances were used to examine the average effects of amino acid substitutions due to single-base errors in the standard genetic code and equally degenerate randomized variants of the standard code. Second-position transitions conserved all distances on average, an order of magnitude more than did second-position transversions. In contrast, first-position transitions and transversions were about equally conservative. In comparison with randomized codes, second-position transitions in the standard code significantly conserved mean square differences in polar requirement and mean Benner matrix-based distances, but mean absolute value differences in polar requirement were not significantly conserved. The discrepancy suggests that these commonly used distance measures may be insufficient for strict hypothesis testing without more information. The translational consequences of single-base errors were then examined in different codon contexts, and similarities between these contexts explored with a hierarchical cluster analysis. In one cluster of codon contexts corresponding to the RNY and GNR codons, second-position transversions between C and G and transitions between C and U were most conservative of both polar requirement and the matrix-based distance. In another cluster of codon contexts, second-position transitions between A and G were most conservative. Despite the claims of previous authors to the contrary, it is shown theoretically that the standard code may have been shaped by position-invariant forces such as mutation and base content. These forces may have left heterogeneous signatures in the code because of differences in translational fidelity by codon position. A scenario for the origin of the code is presented wherein selection for error minimization could have occurred multiple times in disjoint parts of the code through a phyletic process of competition between lineages. This process permits error minimization without the disruption of previously useful messages, and does not predict that the code is optimally error-minimizing with respect to modern error. Instead, the code may be a record of genetic process and patterns of mutation before the radiation of modern organisms and organelles.

Amino Acids↗
PubMed2000

Cloning and Optimized Expression of Human Angiogenin in E.coli.

Angiogenin(ANG) is an important factor of angiogenesis during different stage of tumor development and exists widely in various tumors. To study the biological funcption and find the antagonistic drugs of angiogenin, the angiogenin was allowed to be expressed by E.coli. By the aid of computer, the sequence around the start codon of angiogenin gene was modified according to local secondary structure. The modified human ang gene was amplified by reverse-transcription polymerase chain reaction from the human lung cancer cell line A549, and inserted into the prokaryotic expression vector pLDH99. After screening, high expression recombinants were obtained, and the expression level of the hANG was about 30% of total bacteria protein by SDS-PAGE. Biological assays indicated that the rhANG could induce new blood vessel formation in CAM in vitro. Our data showed that the recombinant hANG was active and the optimized expression of ang gene was practicable.

Journal Article↗
PubMed1998

Bacterial adhesion pili are heterologous assemblies of similar subunits.

P-pili on uropathogenic bacteria are 68-A-diameter rods typically 1 microm in length. These structures project from the outer membrane of Escherichia coli, and contain on their distal tip a thin fibrillum, 25 A in diameter and 150 A long, displaying an adhesin protein responsible for the binding of the bacterium to the surface of epithelial cells lining the urinary tract. Operationally, it is possible to identify three morphologically distinct states of the 68-A-diameter P-pili rods, based on the degree of curvature each can adopt. These states are designated "straight," "curved," and "highly curved." The rods can also be unwound to form thin "threads" that are very similar to the tip fibrillae. Electron microscope data are used to distinguish among these four morphological states and to define limits on the shapes of the pilus proteins. The mechanical properties of the PapA polymers are assessed, and implications of rod polymorphism for pilus function are discussed. A wide variety of data are considered in light of the possibility that all pilins are similar in molecular architecture, with specific differences designed to optimize their specialized functions in the pilus assembly.

Amino Acid Sequence↗