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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↗
PubMed2001

High guanine-cytosine content is not an adaptation to high temperature: a comparative analysis amongst prokaryotes.

The causes of the variation between genomes in their guanine (G) and cytosine (C) content is one of the central issues in evolutionary genomics. The thermal adaptation hypothesis conjectures that, as G:C pairs in DNA are more thermally stable than adenonine:thymine pairs, high GC content may he a selective response to high temperature. A compilation of data on genomic GC content and optimal growth temperature for numerous prokaryotes failed to demonstrate the predicted correlation. By contrast, the GC content of Structural RNAs is higher at high temperatures. The issue that we address here is whether more freely evolving sites in exons (i.e. codonic third positions) evolve in the same manner as genomic DNA as a whole, Showing no correlated response, or like structural RNAs showing a strong correlation. The latter pattern would provide strong support for the thermal adaptation hypothesis, as the variation in GC content between orthologous genes is typically most profoundly seen at codon third sites (GC3). Simple analysis of completely sequenced prokaryotic genomes shows that GC3, but not genomic GC, is higher on average in thermophilic species. This demonstrates, if nothing else, that the results from the two measures cannot be presumed to be the same. A proper analysis, however, requires phylogenetic control. Here, therefore, we report the results of a comparative analysis of GC composition and optimal growth temperature for over 100 prokaryotes. Comparative analysis fails to show, in either Archea or Eubacteria, any hint of connection between optimal growth temperature and GC content in the genome as a whole, in protein-coding regions or, more crucially at GC. Conversely, comparable analysis confirms that GC content of structural RNA is strongly correlated with optimal temperature. Against the expectations of the thermal adaptation hypothesis, within prokaryotes GC content in protein-coding genies, even at relatively freely evolving sites, cannot be considered an adaptation to the thermal environment.

Adaptation, Physiological↗
PubMed1997

The human cytomegalovirus UL97 protein is a protein kinase that autophosphorylates on serines and threonines.

The product of the human cytomegalovirus (CMV) UL97 gene, which controls ganciclovir phosphorylation in virus-infected cells, is homologous to known protein kinases but diverges from them at a number of positions that are functionally important. To investigate UL97, we raised an antibody against it and overexpressed it in baculovirus-infected insect cells. Recombinant baculovirus expressing full-length UL97 directed the phosphorylation of ganciclovir in insect cells, which was abolished by a four-codon deletion that confers ganciclovir resistance to CMV. When incubated with [gamma-32P]ATP, full-length UL97 was phosphorylated on serine and threonine residues. Phosphorylation was severely impaired by a point mutation that alters lysine-355 in a motif that aligns with subdomain II of protein kinases. However, phosphorylation was impaired much less severely by the four-codon deletion. A UL97 fusion protein expressed from recombinant baculovirus was purified to near homogeneity. It too was phosphorylated upon incubation with [gamma-32P]ATP in vitro. This phosphorylation, which was abolished by the lysine 355 mutation, was optimal at high NaCl and high pH. The activity required either Mn2+ or Mg2+, with a preference for Mn2+, and utilized either ATP or GTP as a phosphate donor, with Kms of 2 and 4 microM, respectively. The phosphorylation rate was first order with protein concentration, consistent with autophosphorylation. These data strongly argue that UL97 is a serine/threonine protein kinase that autophosphorylates and suggest that the four-codon deletion affects its substrate specificity.

Animals↗
PubMed1995

Purification and characterization of recombinant cytochrome P450TYR expressed at high levels in Escherichia coli.

The multifunctional tyrosine N-hydroxylase, cytochrome P450TYR (CYP79), from Sorghum bicolor catalyzing the conversion of tyrosine to p-hydroxyphenyl-acetaldoxime in the biosynthesis of the cyanogenic glucoside dhurrin, has been expressed in Escherichia coli using the isopropyl-beta-D-thiogalactopyranoside-inducible vector pSP19g10L, containing the cDNA encoding CYP79. The expression construct was optimized by reducing the length of the N-terminal hydrophobic core of the signal sequence of cytochrome P450TYR and by exchanging the first eight codons with the first eight codons of bovine P45017 alpha. The highest yielding construct provided 200-500 nmol P450TYR/liter cell culture. The recombinant P450TYR was gently and efficiently extracted from E. coli spheroblasts by temperature-induced phase partitioning of Triton X-114 in the presence of 30% glycerol and isolated by DEAE and reactive red chromatography. In reconstitution experiments using saturating amounts of sorghum NADPH-cytochrome P450 reductase, the Km and turnover rate for isolated recombinant P450TYR was 0.22 +/- 0.06 mM and 49.2 +/- 3.8 min-1, respectively, whereas a turnover rate as high as 350 min-1, was obtained using E. coli membranes. Addition of 3 mM glutathione stimulated the activity of reconstituted P450TYR and of sorghum microsomes although the effect was highly variable. Phenylalanine, the precursor of several cyanogenic glucosides, gave a type I binding spectrum, but was not metabolized by P450TYR, demonstrating the high substrate specificity of this P450. Administration of radioactively labeled p-hydroxyphenylacetaldoxime to E. coli cells, showed E. coli metabolized p-hydroxyphenylacetaldoxime independent of the expression of P450TYR.

Amino Acid Sequence↗
PubMed1990

The production, purification, and bioactivity of recombinant bovine trophoblast protein-1 (bovine trophoblast interferon).

Bovine trophoblast protein-1 (bTP-1) is a 172-amino acid interferon- alpha that has a role in maternal recognition of pregnancy in cattle. Here we describe production of bTP-1 by recombinant procedures in Escherichia coli. A bTP-1 gene was constructed which lacked the codons representing the signal sequence and provided a Met initiation codon ahead of the TGT codon encoding Cys1 of the mature protein. This construct was placed under the control of the Trp promoter within the expression vector pTrp2. Expression occurred optimally in E. coli D112 in the absence of tryptophan and in the presence of 0.5% acid-hydrolyzed casein (casamino acids) when 0.5 mM indole acetic acid was included in the medium. The bTP-1 was deposited in inclusion bodies and accounted for as much as 27% of the total cellular protein. The inclusion bodies were isolated by differential centrifugation and washed. The bTP-1 was solubilized by use of guanidinium-HCI and 2-mercaptoethanol and allowed to renature in air. Final purification was achieved by anion exchange chromatography on DEAE-cellulose. The yield of purified product, which had an antiviral activity greater than 10(8) international reference units/mg, was approximately 20 mg/liter. The recombinant bTP-1 was relatively stable to freeze-thawing and frozen storage, and could induce the production of an acidic protein of 70,000 mol wt in cultured explants of endometrium prepared from ewes on day 13 of the estrous cycle. The latter protein is a characteristic product of interferon-alpha action on uterine tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
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↗
PubMedDate not supplied

RET oncogene mutations in 75 cases of familial medullary thyroid carcinoma in Japan.

The familial form of medullary thyroid carcinoma (MTC) is caused by mutations of the RET protooncogene. We registered 60 multiple endocrine neoplasia (MEN) 2A patients, 12 familial non-MEN medullary carcinoma (FMTC) patients, and three MEN2B patients with a confirmed RET germline mutation. All 60 MEN2A patients had RET mutations in a cysteine-rich domain. Seven of the FMTC patients had a mutation in cysteine-rich domain, and the other five had a mutation in codon 768, which encodes a tyrosine-kinase domain. Two of the MEN2B patients had a mutation in codon 918, and one patient had a double mutation, one in codon 804 and the other in codon 806, both of which are all encoded tyrosine-kinase domain. The genotype-phenotype correlations of our data will allow individualized recommendations for the optimal timing of prophylactic surgery.

Carcinoma, Medullary↗
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↗
PubMed1983

Optimal sequence alignments.

Current theory is adequate to the task of finding an optimal alignment between two character strings such as nucleic acids. Most algorithms currently in use must fail to find the homologous alignment between a set of codons for the chicken alpha- and beta-hemoglobin sequence when it is in fact discoverable by a more general treatment of gaps. Fundamental reasons for this are discussed.

Journal Article↗
PubMed2006

Efficient incorporation of a nonnatural amino acid into a protein in an insect cell-free translation system.

Recently, we have succeeded in incorporating various nonnatural amino acids into proteins by using four-base codon-anticodon pairs in Spodoptera frugiperda 21 (Sf21) insect cell-free protein synthesizing system. Here, the reaction was conducted under various conditions in order to optimize the incorporation efficiency. The optimal concentration of aminoacyl-tRNA, reaction temperature, and reaction time were 2 nM, 25 degrees C, and 1.5 hr, respectively.

Amino Acids↗
PubMed2005

Design of protective and therapeutic DNA vaccines for the treatment of allergic diseases.

The DNA vaccine revolution has opened a vast scope of novel approaches for protective and therapeutic treatments of type I allergy. This review gives an overview on the current status of allergy DNA vaccines and presents advances in the design of vaccine constructs. An immense number of concurring studies have proven the stimulation of Th1 cells and the induction of a balanced Th1/Th2 cytokine milieu as the fundamental mechanisms underlying the anti-allergic effects of DNA vaccines. Basic vaccine formulations thus can be optimized by improving the cellular immunogenicity via co-administration of cytokines, co-expression or co-application of immunostimulatory DNA sequences or adapting the codon usage. The latter is a frequent and major reason for impaired vaccine expression (e.g. translation of plant allergen genes in mammal cells). Because of unwanted side effects during conventional specific immunotherapy with allergen extracts, safety is increasingly demanded for both, protein and DNA vaccines for allergy treatment. We discuss the creation of hypoallergenic DNA vaccines based on deliberate allergen gene fragmentation, the use of mutations and the routine production of hypoallergenic DNA vaccines by forced ubiquitination. Furthermore, allergen-expressing DNA replicon vaccines are introduced, which enable a drastic reduction of the vaccine dose without loss of anti-allergic efficacy. Finally, the development of DNA multi vaccines and fusion vaccines for protective and therapeutic applications against certain groups of allergens is addressed.

Allergens↗
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↗