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PubMed1998

Neurovirulent simian immunodeficiency virus incorporates a Nef-associated kinase activity into virions.

We have demonstrated that a molecular clone, SIV/17E-Fr, is neurovirulent in vivo and molecular analyses of this virus in primary macrophages and neuroendothelial cells mapped the domains critical for this phenotype to the transmembrane and Nef proteins. The Nef protein is crucial for virus replication and pathogenesis in SIV-infected rhesus macaques. In addition, both HIV and SIV require full-length Nef proteins for efficient virus replication in primary cells and optimal virion infectivity. To characterize further the contribution of Nef to enhanced infectivity and replication, we analyzed virus particles from a number of SIV recombinant clones. These clones contained nef genes derived from either a lymphocyte-tropic (SIVmac239) or neurovirulent (SIV/17E-Fr) virus or a nef gene with a premature stop codon or deletion. Immunoprecipitation of Nef from virus particles revealed that SIV Nef is incorporated into virions. Incorporation of the Nef protein was dependent on the presence of the N-terminal myristoylation sequence in the nef gene. In addition, enhanced replication and virion infectivity was associated only with viruses containing the full-length Nef protein. To investigate a potential mechanism of virion modification by Nef, in vitro kinase assays were performed on the virion-derived Nef protein. Nef-associated kinase activity was detected only in virions containing Nef sequences derived from the neurovirulent virus SIV/17E-Fr. Thus, these results suggest that selection for specific nef sequences occurs in vivo and has a significant effect on virus replication in specific cells and organs.

Amino Acid Sequence↗
PubMed1992

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

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

1,4-alpha-Glucan Branching Enzyme↗
PubMed2000

Early fixation of an optimal genetic code.

The evolutionary forces that produced the canonical genetic code before the last universal ancestor remain obscure. One hypothesis is that the arrangement of amino acid/codon assignments results from selection to minimize the effects of errors (e.g., mistranslation and mutation) on resulting proteins. If amino acid similarity is measured as polarity, the canonical code does indeed outperform most theoretical alternatives. However, this finding does not hold for other amino acid properties, ignores plausible restrictions on possible code structure, and does not address the naturally occurring nonstandard genetic codes. Finally, other analyses have shown that significantly better code structures are possible. Here, we show that if theoretically possible code structures are limited to reflect plausible biological constraints, and amino acid similarity is quantified using empirical data of substitution frequencies, the canonical code is at or very close to a global optimum for error minimization across plausible parameter space. This result is robust to variation in the methods and assumptions of the analysis. Although significantly better codes do exist under some assumptions, they are extremely rare and thus consistent with reports of an adaptive code: previous analyses which suggest otherwise derive from a misleading metric. However, all extant, naturally occurring, secondarily derived, nonstandard genetic codes do appear less adaptive. The arrangement of amino acid assignments to the codons of the standard genetic code appears to be a direct product of natural selection for a system that minimizes the phenotypic impact of genetic error. Potential criticisms of previous analyses appear to be without substance. That known variants of the standard genetic code appear less adaptive suggests that different evolutionary factors predominated before and after fixation of the canonical code. While the evidence for an adaptive code is clear, the process by which the code achieved this optimization requires further attention.

Amino Acids↗
PubMed2007

Multiexon skipping leading to an artificial DMD protein lacking amino acids from exons 45 through 55 could rescue up to 63% of patients with Duchenne muscular dystrophy.

Approximately two-thirds of Duchenne muscular dystrophy (DMD) patients show intragenic deletions ranging from one to several exons of the DMD gene and leading to a premature stop codon. Other deletions that maintain the translational reading frame of the gene result in the milder Becker muscular dystrophy (BMD) form of the disease. Thus the opportunity to transform a DMD phenotype into a BMD phenotype appeared as a new treatment strategy with the development of antisense oligonucleotides technology, which is able to induce an exon skipping at the pre-mRNA level in order to restore an open reading frame. Because the DMD gene contains 79 exons, thousands of potential transcripts could be produced by exon skipping and should be investigated. The conventional approach considers skipping of a single exon. Here we report the comparison of single- and multiple-exon skipping strategies based on bioinformatic analysis. By using the Universal Mutation Database (UMD)-DMD, we predict that an optimal multiexon skipping leading to the del45-55 artificial dystrophin (c.6439_8217del) could transform the DMD phenotype into the asymptomatic or mild BMD phenotype. This multiple-exon skipping could theoretically rescue up to 63% of DMD patients with a deletion, while the optimal monoskipping of exon 51 would rescue only 16% of patients.

Adolescent↗
PubMed2001

Vector design for optimal protein expression.

Many DNA constructs are generated for protein expression studies. Translational properties and mRNA stability are crucial aspects that have to be accounted for during DNA construction. An optimized vector for protein overexpression studies is described considering elements in the mature mRNA that influence translatability and stability. Recommendations regarding vector construction for Xenopus laevis embryo injection are provided, based on literature and experimental data. The 5'untranslated region (5'UTR) should be non-regulated, short, unstructured, and without AUG codons. The sequence around the start codon should match the initiation context of the species studied (ACCAUGG, for vertebrates), and the open reading frame should be cloned with its own stop codon, followed by a G or A residue. Furthermore, the 3'UTR should be non-regulated, and a strong polyadenylation signal must be included in DNA vectors. In RNA template vectors, the presence of a poly(A) or AC tail is essential for stability, as well as for translation efficiency in mRNA injection experiments. These aspects result in high-level expression of exactly the desired protein. Easily obtainable examples of the sequences [5'UTR, 3'UTR, and poly(A) signal] are suggested.

3' Untranslated Regions↗
PubMed1999

Modulation of translational efficiency by contextual nucleotides flanking a baculovirus initiator AUG codon.

In a previous study of translational regulation of a baculovirus gene, we observed that translation initiated at an unexpectedly high efficiency from an AUG codon found in what was believed to be a poor context (M.-J. Chang and G. W. Blissard, 1997, J. Virol. 71, 7448-7460). In the current study, we examined the roles of nucleotides flanking a baculovirus AUG initiator codon in modulating translation initiation in lepidopteran insect cells. The roles of nucleotides flanking the AcMNPV gp64 initiator codon were examined by site-directed mutagenesis and functional assays in transfected Sf9 cells. To eliminate potential cis-acting sequences and effects, the gp64 initiator context was cloned in-frame with a chloramphenicol acetyl transferase reporter gene and under the control of a heterologous promoter. All possible single-nucleotide substitutions were generated in positions -6 to -1 and +4 to +6, relative to the A of the initiator AUG codon, which was designated +1. Constructs were transfected into lepidopteran cells and translation products were quantified by an enzyme-linked immunosorbent assay procedure. Substitutions of pyrimidines or other nucleotides at the -3 position resulted in little or no detectable effect on translation efficiency. In contrast, specific substitutions at the +4 and +5 positions resulted in approximately 2- to 3-fold increases in translation. Substitution of A in the +4 position resulted in an approximately 3-fold increase in translation, and substitution of any nucleotide for T in the +5 position resulted in approximately 1.9- to 2.8-fold increases. Substitutions at other positions (-6 to -1 and +6) resulted in no detectable increase or decrease in translation efficiency. These experimental results suggest an optimal initiator context of 5'-N N N N N N A U G A a/c/g N-3' for efficient translation initiation in lepidopteran cells. Consensus translation initiation contexts were generated from baculovirus genes and lepidopteran genes, then compared with the experimental results from the gp64 initiator context.

Animals↗
PubMed1997

Antibody humanization using monovalent phage display.

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

Amino Acid Sequence↗
PubMed1987

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

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

Algorithms↗
PubMed2000

Type I, II, III, IV, and V cystic fibrosis transmembrane conductance regulator defects and opportunities for therapy.

Recent advances in cellular and molecular biology have furthered the understanding of several genetic diseases, including cystic fibrosis. Mutations that cause cystic fibrosis are now understood in terms of the specific molecular consequences to the cystic fibrosis transmembrane conductance regulator (CFTR) protein expression and function. This knowledge has spawned interest in the development of therapies aimed directly at correcting the defective CFTR itself. In this article, we review the molecular defect underlying each recognized class of CFTR mutation and the potential therapies currently under investigation. Opportunities for protein-repair therapy appear to be vast and range from naturally occurring compounds, such as isoflavonoids, to pharmaceuticals already in clinical use, including aminoglycoside antibiotics, butyrate analogues, phosphodiesterase inhibitors, and adenosine nucleotides. Future therapies may resemble designer compounds like benzo[c]quinoliziniums or take the form of small peptide replacements. Given the heterogeneity and progressive nature of cystic fibrosis, however, optimal benefit from protein-repair therapy will most likely require the initiation of combined therapies early in the course of disease to avoid irreparable organ damage.

Codon, Terminator↗
PubMed1998

Pleiotropy and the preservation of perfection.

A mathematical model is presented in which a single mutation can affect multiple phenotypic characters, each of which is subject to stabilizing selection. A wide range of mutations is allowed, including ones that produce extremely small phenotypic changes. The analysis shows that, when three or more characters are affected by each mutation, a single optimal genetic sequence may become common. This result provides a hypothesis to explain the low levels of variation and low rates of substitution that are observed at some loci.

Animals↗
PubMed1998

Predictive factors in eradicating hepatitis C virus using a relatively small dose of interferon.

Interferon (IFN) can reduce hepatitis C virus load and even eliminate the virus in 30-40% of patients. Several predictive factors for eradication of the virus have been reported and a higher dose of IFN tends to result in elimination of the virus. However, a small dose of IFN sometimes is as effective as a large dose in eradicating the virus. The predictive factors for such a response are not well established. We retrospectively analysed 50 patients with chronic hepatitis C who were treated with relatively small amounts of IFN (equal or less than 252 million units). Eleven patients were responders (elimination of hepatitis C virus (HCV) and normalization of alanine amino transferase (ALT) for at least 6 months), but the remaining 39 were non-responders. Multivariate analysis showed that the pretreatment viral load and total dose of IFN per kilogram of bodyweight were significant predictive factors of response to therapy. We also assessed the amino acid substitutions in the IFN sensitivity determining region (ISDR), NS5A codon 2209-2248, of HCV in serum samples obtained from 31 patients with HCV genotype 1b. The presence of more than one amino acid substitution in the ISDR tended to correlate with HCV genotype 1b elimination. As IFN is expensive and has a number of serious side effects, our study suggests that the optimal dose of IFN may vary from one patient to another and that more stringent criteria should be used to select the optimal dose for therapy.

Adult↗
PubMed1983

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

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

Base Sequence↗
PubMed1989

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

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

Amino Acid Sequence↗
PubMed2002

Identification of two SNPs in the 5' flanking region of the ACP1 gene and evaluation of disequilibrium among polymorphic sites.

We have studied the 5' flanking region of the ACP1 gene looking for new polymorphisms. Two SNPs, DdeI and FokI restricted, have been found in this region. We determined the genotype of DdeI and FokI SNPs, as well as of three other known SNPs, codon 43 (CfoI restricted), codon 41 and codon 105 (TaqI restricted), located respectively in exons 3, 4 and 6 in 62 unrelated subjects from the Italian population. Haplotype distribution for the ten possible pairs of loci were determined by a maximum likelihood procedure. Overall, statistically significant deviations from expected frequencies assuming equilibrium have been observed for the following pairs: FokI/codon 41, FokI/TaqI, codon 41/TaqI (complete association), DdeI/FokI, DdeI/codon 41 and DdeI/TaqI. The data suggest that the FokI area could include sequences operating in strict functional association with sequences included in the codon 41/TaqI area, possibly in order to regulate the F/S isoforms ratio of the A* and *B alleles. Since the ratio between the concentration of the two F and S isoforms is different for the three ACP1 alleles, we suggest that the ACP1 locus has been subjected to strong selective pressure to obtain an optimal alternative splicing mechanism of the *A and *B alleles. The *C variant, on the other hand, seems completely independent from sequences in the FokI/codon 41/TaqI areas, resulting in an inverted F/S ratio compared to that found for *A and *B alleles.

5' Flanking Region↗
PubMed2006

[Cloning of alpha-bungarotoxin gene and its prokaryotic expression as a non-fusion protein].

On the basis of the reported amino acid sequence of alpha-bungarotoxin (alpha-BGT), DNA sequence of alpha-BGT was deduced and fourteen partially complementary oligonucleotides were designed and synthesized. A plasmid carrying the coding region of alpha-BGT was obtained by primer extension, PCR and ligation with pMD-18-T. The target fragment was digested with Xba I and EcoR I, recovered and ligated with pET28a(+). The resultant expression vector was transformed into BL21 (DE3), BL21 (DE3) Codon plus, and BL21 (DE3) plysS, respectively. Recombinant alpha-BGT was expressed in BL21 (DE3) and was analyzed by 15% Tris/tricine SDS-PAGE. The result showed that the recombinant protein, mostly found in inclusion bodies, accounted for 11.98% of the total bacterial lysate. The expression capacity could be increased to 16.28% by optimizing expression conditions. Western blotting results showed that the expressed protein had similar immunogenicity with the natural alpha-BGT protein purified from the venom of Krait Bungarus spp. In vivo toxicity assay of purified and renatured proteins in mice showed that LD50 was about 1.28 microg/g.

Amino Acid Sequence↗
PubMed1995

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

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

Animals↗
PubMed1990

A generalized information function applied to the genetic code.

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

Amino Acids↗
PubMed1999

Generation of a novel A kinase anchor protein and a myristoylated alanine-rich C kinase substrate-like analog from a single gene.

A unique Drosophila gene encodes two novel signaling proteins. Drosophila A kinase anchor protein 200 (DAKAP200) (753 amino acids) binds regulatory subunits of protein kinase AII (PKAII) isoforms in vitro and in intact cells. The acidic DAKAP200 polypeptide (pI approximately 3.8) contains an optimal N-terminal myristoylation site and a positively charged domain that resembles the multifunctional phosphorylation site domain of vertebrate myristoylated alanine-rich C kinase substrate proteins. The 15-kilobase pair DAKAP200 gene contains six exons and encodes a second protein, DeltaDAKAP200. DeltaDAKAP200 is derived from DAKAP200 transcripts by excision of exon 5 (381 codons), which encodes the PKAII binding region and a Pro-rich sequence. DeltaDAKAP200 appears to be a myristoylated alanine-rich C kinase substrate analog. DAKAP200 and DeltaDAKAP200 are evident in vivo at all stages of Drosophila development. Thus, both proteins may play important physiological roles throughout the life span of the organism. Nevertheless, DAKAP200 gene expression is regulated. Maximal levels of DAKAP200 are detected in the pupal phase of development; DeltaDAKAP200 content is elevated 7-fold in adult head (brain) relative to other body parts. Enhancement or suppression of exon 5 excision during DAKAP200 pre-mRNA processing provides potential mechanisms for regulating anchoring of PKAII and targeting of cAMP signals to effector sites in cytoskeleton and/or organelles.

A Kinase Anchor Proteins↗