SEARCH · Search PubMed

Results for “Codon optimality”

Search indexed PubMed citations on genomics, clinical trials, systematic reviews and public health. Explore titles, authors and supplied subject terms, then open the PubMed record.

SourceResearch connectionFrom yearThrough year

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 1,171 records · Page 65Linked to original sources

PubMed2003

Correction/mutation of acid alpha-D-glucosidase gene by modified single-stranded oligonucleotides: in vitro and in vivo studies.

Deficiency in acid alpha-D-glucosidase results in Pompe's disease. Modified single-stranded oligonucleotide (ODN) was designed to correct the acid alpha-D-glucosidase gene with a C1935 --> A (Asp --> Glu) point mutation which causes a complete loss of enzymatic activity for glycogen digestion in the lysosome. The ODN vectors contained a stretch of normal oligonucleotide flanked by phosphorothioated sequences. The 25mer and 35mer ODNs were homologous to the target sequence, except for a mismatched base in the middle. The ODNs caused permanent and inheritable restoration of acid alpha-D-glucosidase activity in skin fibroblast cells carrying this mutation derived from a Pompe's disease patient. Gene correction was confirmed by amplification refractory mutation system-PCR (ARMS-PCR), restriction fragment length polymorphism (RFLP) and direct DNA cloning and sequencing. The increased acid alpha-D-glucosidase activity was detected using 4-MUG as the artificial substrate. The correction efficiency, ranging from 0.5 to 4%, was dependent on the length and polarity of the MSSOV used, the optimal design being a sense-strand 35mer ODNs. Repeated treatment of the mutant fibroblast cells with the ODNs substantially increased correction. We also constructed ODN vectors to trigger specific and in vivo nonsense mutation in the mouse acid alpha-D-glucosidase gene. The ODNs were in complex with YEEE-K(18), an asialoglycoprotein-receptor ligand tagged with polylysine and targeted to hepatocytes and renal cells in vivo through intravenous injection. The mutated genotype was detected in the liver and the kidney by ARMS-PCR and glycogen accumulation in the lysosome of the liver cells. The studies demonstrate the utility of single-stranded ODN to direct targeted gene correction or mutation in a human hereditary disease and in an animal model. Our data open the possibility of developing ODN vector as a therapeutic approach for treatment of human hereditary diseases caused by point mutation.

Animals↗

PubMed1996

High volumetric yields of functional dimeric miniantibodies in Escherichia coli, using an optimized expression vector and high-cell-density fermentation under non-limited growth conditions.

Functional bivalent miniantibodies, directed against the epidermal growth factor receptor, accumulated to more than 3 gl-1 in high-cell-density cultures of Escherichia coli RV308(pHKK) on a pilot scale. The miniantibodies consist of scFv fragments with a C-terminal hinge followed by a helix-turn-helix motif, which homodimerizes in vivo. The improved expression vector pHKK is characterized by the hoklsok suicide system, improving plasmid maintenance, and the inducible lac pl o promoter system with the very strong T7g10 Shine-Dalgarno sequence. The expression unit is flanked by terminators. The prototrophic RV308 cells were cultivated in glucose mineral salt medium and reached a cell density of 145 g dry biomass l-1 after 33 h. After induction, growth continued almost unchanged for a further 4 h with concomitant miniantibody formation. In the fedbatch phase, the concentration of glucose was kept almost constant at the physiological level of approximately 1.5 gl-1, using on-line flow injection analysis for control. Surprisingly, E. coli RV308(pHKK) did not accumulate significant amounts of the metabolic by-product acetate under these unlimited aerobic growth conditions.

Acetates↗

PubMed1995

Segmented structure of protein sequences and early evolution of genome by combinatorial fusion of DNA elements.

A theory of an early stage of genome evolution by combinatorial fusion of circular DNA units is suggested, based on protein sequence "fossil" evidence. The evidence includes preference of protein sequence lengths for certain sizes--multiples of 123 aa for eukaryotes and multiples of 152 aa for prokaryotes. At the DNA level these sizes correspond to 350-450 base pairs--the known optimal range for DNA ring closure. The methionine residues repeatedly appear along the sequences with the same period of about 120 aa (in eukaryotes), presumably marking the sites of insertion of the early genes--rings of protein-coding DNA. No torsional constraint in this DNA results in very sharp estimate of the helical periodicity of the early DNA, indistinguishable from the experimental mean value for extant DNA. According to the combinatorial fusion theory, based on the above evidence, in the pregenomic, prerecombinational stage the genes and the noncoding sequences existed in form of autonomously replicating DNA rings of close to standard size, randomly segregating between dividing cells, like modern plasmids do. In the recombinational early genomic stage the rings started to fuse, forming larger DNA molecules consisting of several unit genes connected in various combinations and forming long protein-coding sequences (combinatorial fusion). This process, which involved, perhaps, noncoding sequences as well, eventually resulted in the formation of large genomes. The dispersed circular DNA--or, rather, evolutionarily advanced derivatives thereof--may still exist in the form of various mobile DNA elements.

Biological Evolution↗

PubMed1999

Significantly lower entropy estimates for natural DNA sequences.

If DNA were a random string over its alphabet {A, C, G, T}, an optimal code would assign two bits to each nucleotide. DNA may be imagined to be a highly ordered, purposeful molecule, and one might therefore reasonably expect statistical models of its string representation to produce much lower entropy estimates. Surprisingly, this has not been the case for many natural DNA sequences, including portions of the human genome. We introduce a new statistical model (compression algorithm), the strongest reported to date, for naturally occurring DNA sequences. Conventional techniques code a nucleotide using only slightly fewer bits (1.90) than one obtains by relying only on the frequency statistics of individual nucleotides (1.95). Our method in some cases increases this gap by more than fivefold (1.66) and may lead to better performance in microbiological pattern recognition applications. One of our main contributions, and the principle source of these improvements, is the formal inclusion of inexact match information in the model. The existence of matches at various distances forms a panel of experts which are then combined into a single prediction. The structure of this combination is novel and its parameters are learned using Expectation Maximization (EM). Experiments are reported using a wide variety of DNA sequences and compared whenever possible with earlier work. Four reasonable notions for the string distance function used to identify near matches, are implemented and experimentally compared. We also report lower entropy estimates for coding regions extracted from a large collection of nonredundant human genes. The conventional estimate is 1.92 bits. Our model produces only slightly better results (1.91 bits) when considering nucleotides, but achieves 1.84-1.87 bits when the prediction problem is divided into two stages: (i) predict the next amino acid-based on inexact polypeptide matches, and (ii) predict the particular codon. Our results suggest that matches at the amino acid level play some role, but a small one, in determining the statistical structure of nonredundant coding sequences.

Algorithms↗

PubMed2005

Laser pressure catapulting (LPC): optimization LPC-system and genotyping of colorectal carcinomas.

Genotype analysis is becoming more and more useful in clinical practice, since specific mutations in tumors often correlate with prognosis and/or therapeutic response. Unfortunately, current molecular analytical techniques often require time-consuming and costly steps of analysis, thus making their routine clinical use difficult. Moreover, one of the most difficult problems arising during tumor research is that of their cell heterogeneity, which depends on their clear molecular heterogeneity. SSCP analysis discriminates by means of aberrant electrophoresis migration bands, mutated alleles which may represent as little as 15-20% of their total number. Nevertheless, in order to identify by sequencing the type of alteration revealed by this technique, only the mutated allele must be isolated. The advent of laser microdissection is a procedure which easily solves these problems of accuracy, costs, and time. The aims of this study were to perfect the system of laser pressure catapulting (LPC) laser microdissection for the assessment of the mutational status of p53 and k-ras genes in a consecutive series of 67 patients with colorectal carcinomas (CRC), in order to compare this technique with that involving hand-dissection and to demonstrate that since the LPC system guarantees more accurate biomolecular analyses, it should become part of clinical routine in this field. The LPC-system was perfected with the use of mineral oil and the LPC-membrane. To compare the techniques of hand- and LPC-microdissection, alcohol-fixed, paraffin-embedded tissue from 67 cases of CRC were both hand- and laser-microdissected. In either case, dissected samples were analyzed by SSCP/sequencing and direct sequencing for k-ras and p53 gene mutations. LPC-microdissection made it possible to pick up mutations by direct sequencing or SSCP/sequencing, whereas hand-microdissection mutations were identified only by means of SSCP followed by sequencing; direct sequencing did not reveal any mutation. In the 67 patients examined by either method, 36% (24/67) showed p53 mutations, 32 of which identified. Seventy-eight percent (25/32) were found in the conserved areas of the gene, while 12% (4/32) were in the L2 loop, 50% (16/32) were in the L3 loop, and 12% (4/32) in the LSH motif of the protein. Moreover, of the 67 cases examined, 40% (27/67) showed mutations in k-ras, with a total of 29 mutations identified. Of these, 14 (48%) were found in codon 12 and 15 (52%) in codon 13. The modifications which we brought to the LPC system led to a vast improvement of the technique, making it an ideal substitution for hand-microdissection and guaranteeing a considerable number of advantages regarding facility, accuracy, time, and cost. Furthermore, the data obtained from the mutational analyses performed confirm that the LPC system is more efficient and rapid than hand-microdissection for acquiring useful information regarding molecular profile and can therefore be used with success in clinical routine.

Carcinoma↗

PubMed2005

Optimization of BLyS production and purification from Escherichia coli.

B lymphocyte stimulator (BLyS) is a member of the tumor necrosis factor superfamily of cytokines. When the 152 amino acids of the C-terminus are associated into a homotrimer, this protein exhibits the ability to stimulate B cell proliferation and differentiation. Since numerous potential therapeutic indications have been identified for BLyS and other BLyS-derived products, large quantities of the protein are needed to further basic research and clinical trials. In this work, we have developed a high yield recombinant expression system that utilizes Escherichia coli as the host organism. Recombinant soluble BLyS (rsBLyS) production was achieved through the use of the phoA promoter system. This expression system, coupled to a semi-defined fermentation process, resulted in final purified yields of 435 mg/L of properly folded, trimeric, biologically active rsBLyS. This level of production is an 11-fold increase in volumetric yields compared to the process currently being used for clinical production. Furthermore, the increased rsBLyS production obtained from this process enabled the development of a conventional purification scheme that eliminated the use of a BLyS-affinity resin.

Amino Acid Sequence↗

PubMed1995

An improved phage display antibody cloning system using newly designed PCR primers optimized for Pfu DNA polymerase.

An improved combinatorial library system to raise mouse monoclonal antibodies was constructed. PCR primers have been newly designed to optimize the reaction for Pfu DNA polymerase, which has proofreading activity. The phagemid vector (pPDS) is designed to accommodate VH and Vk cDNAs, which had previously been assembled by PCR either in single chain fragment of variable regions (scFv) or Fab form. Antibody cloned in scFv form can be converted to Fab form by substituting the scFv linker of (Gly4Ser)3 with a fragment containing murine CH1 cDNA. This vector will produce soluble Fab in non-amber suppressor cells and allow the shuffling of light chains against a heavy chain. Hybridoma cell lines producing anti-human procollagenase monoclonal antibodies were used as the source of antibody mRNA. Antigen-binding ability of both scFv- and Fab-displaying phage was confirmed by ELISA against human procollagenase. They were also analyzed by DNA sequencing to verify the fidelity of Pfu DNA polymerase and to identify the primer incorporated. The mutation rate was considerably reduced compared to the mutation rate achieved by Taq DNA polymerase. Primers are incorporated into target sequences in most cases.

Amino Acid Sequence↗

PubMed1989

A strategy to optimize translation initiation in recombinant mRNA: application to the Rop gene.

Using the Escherichia coli Rop gene, we demonstrate a strategy that could be applied generally to optimize the initiation of translation of recombinant genes in E. coli. This involves cloning of the gene encoding the protein of interest in a suitable expression vector between an "efficient" ribosome binding site and the gene for the alpha-peptide of beta-galactosidase. By oligonucleotide-directed deletion mutagenesis, the two coding sequences are then fused in the correct frame. A second oligonucleotide is then used to place the initiator AUG (or GUG) at the correct distance from the Shine-Dalgarno sequence. In this step, however, we use an oligonucleotide that has a degenerate sequence. That is, on the basis of the "efficient" ribosome binding site sequence, we introduce random substitutions at various positions, both upstream and downstream from the initiator ATG, to obtain, after the mutagenesis experiment, a collection of random ribosome binding sites fused to the coding sequence. The development of blue colonies on indicator plates permits selection of clones in which an efficient ribosome binding site has been created for the specific gene of interest. We discuss the results obtained by applying the method to the Rop gene.

Bacterial Proteins↗

PubMed2000

Inferring regulatory elements from a whole genome. An analysis of Helicobacter pylori sigma(80) family of promoter signals.

Helicobacter pylori is adapted to life in a unique niche, the gastric epithelium of primates. Its promoters may therefore be different from those of other bacteria. Here, we determine motifs possibly involved in the recognition of such promoter sequences by the RNA polymerase using a new motif identification method. An important feature of this method is that the motifs are sought with the least possible assumptions about what they may look like. The method starts by considering the whole genome of H. pylori and attempts to infer directly from it a description for a family of promoters. Thus, this approach differs from searching for such promoters with a previously established description. The two algorithms are based on the idea of inferring motifs by flexibly comparing words in the sequences with an external object, instead of between themselves. The first algorithm infers single motifs, the second a combination of two motifs separated from one another by strictly defined, sterically constrained distances. Besides independently finding motifs known to be present in other bacteria, such as the Shine-Dalgarno sequence and the TATA-box, this approach suggests the existence in H. pylori of a new, combined motif, TTAAGC, followed optimally 21 bp downstream by TATAAT. Between these two motifs, there is in some cases another, TTTTAA or, less frequently, a repetition of TTAAGC separated optimally from the TATA-box by 12 bp. The combined motif TTAAGCx(21+/-2)TATAAT is present with no errors immediately upstream from the only two copies of the ribosomal 23 S-5 S RNA genes in H. pylori, and with one error upstream from the only two copies of the ribosomal 16 S RNA genes. The operons of both ribosomal RNA molecules are strongly expressed, representing an encouraging sign of the pertinence of the motifs found by the algorithms. In 25 cases out of a possible 30, the combined motif is found with no more than three substitutions immediately upstream from ribosomal proteins, or operons containing a ribosomal protein. This is roughly the same frequency of occurrence as for TTGACAx(15-19)TATAAT (with the same maximum number of substitutions allowed) described as being the sigma(70 )promoter sequence consensus in Bacillus subtilis and Escherichia coli. The frequency of occurrence of the new motif obtained, TTAAGCx(19-23)TATAAT, remains high when all protein genes in H. pylori are considered, as is the case for the TTGACAx(15-19)TATAAT motif in B. subtilis but not in E. coli.

Algorithms↗

PubMed2002

Conformational preferences of anticodon 3'-adjacent hypermodified nucleic acid base cis-or trans-zeatin and its 2-methylthio derivative, cis-or trans- ms(2)zeatin.

Conformational preferences of the hypermodified nucleic acid bases N6-(Delta(2)-cis-hydroxyisopentenyl)adenine, cis-io(6)Ade also known as cis-zeatin, and N(6)-(Delta(2)-trans-hydroxyisopentenyl)adenine, trans-io(6)ade or trans-zeatin, and 2-methylthio derivatives of these cis-ms(2)io(6)Ade or cis-ms(2)zeatin, and trans-ms(2)io6Ade or trans-ms(2)zeatin have been investigated theoretically by the quantum chemical Perturbative Configuration Interaction with Localized Orbitals (PCILO) method. Automated geometry optimization using quantum chemical MNDO, AM1 and PM3 methods has also been made to compare the salient features. The predicted most stable conformation of cis-io(6)Ade, trans-io(6)Ade, cis-ms(2)io(6)Ade and trans-ms(2)io(6)Ade are such that in each of these molecules the isopentenyl substituent spreads away (has "dista" conformation) from the five membered ring imidazole moiety of the adenine. The atoms N(6), C(10) and C(11) remain coplanar with the adenine ring in the predicted preferred conformation for each of these molecules. In cis-io(6)Ade as well as cis-ms(2)io(6)Ade the hydroxyl oxygen may participate in intramolecular hydrogen bonding with the H-C(10)-H group. In trans-io(6)Ade the hydroxyl group is oriented towards the H-C(2) instead. This orientation is retained in trans-ms(2)io(6)Ade, possible O-H...S hydrogen bonding may be a stabilizing factor. In all these four modified adenines C(11)-H is favourably placed to participate in intramolecular hydrogen bonding with N(1). In cis-ms(2)io(6)Ade as well as trans-ms(2)io(6)Ade the 2-methylthio group preferentially orients on the same side as C(2)-N(3) bond, due to this non-obstrusive placing, orientation of the hydroxyisopentenyl substituent remains unaffected by 2-methylthiolation. Thus the N(1) site remains shielded irrespective of the 2-methylthiolation status in these various cis-and trans-zeatin analogs alike. Firmly held orientation of hydroxyisopentenyl substituent in zeatin isomers and derivatives, in contrast to adaptable orientation of isopentenyl substituent in i(6)Ade and ms(2)i(6)Ade, may account for the increased efficiency of suppressor tRNA and reduced codon context sensitivity accompanied with the occurrence of ms(2)-zeatin (ms(2)io(6)Ade) modification.

Anticodon↗

PubMed1997

Cloning, bacterial expression and biological characterization of recombinant human granulocyte chemotactic protein-2 and differential expression of granulocyte chemotactic protein-2 and epithelial cell-derived neutrophil activating peptide-78 mRNAs.

Human osteosarcoma cells secrete a novel C-X-C chemokine called granulocyte chemotactic protein-2 (GCP-2), which was previously identified by amino acid sequencing of the purified natural protein. In order to understand the role of this new protein in inflammatory reactions, we cloned GCP-2 DNA sequences to generate recombinant protein and specific DNA probes and primers. By means of PCR on cloned cDNA of osteosarcoma cells induced by interleukin-1 beta and fibroblasts induced by lipopolysaccharide plus dsRNA, the complete coding domain of GCP-2 was isolated. This sequence was cloned into the bacterial expression vector pHEN1 and, after induction, GCP-2 was secreted into the periplasm of Escherichia coli. Recombinant GCP-2 (rGCP-2) was purified and characterized by SDS/PAGE as a monomeric 6.5-kDa protein and by amino-terminal sequencing. The chemoattractive potency of GCP-2 for neutrophilic granulocytes was about 10-times less than that of interleukin-8 and the minimal effective dose was 10 ng/ml. However, at optimal dose (100 ng/ml) the maximal chemotactic response was comparable with that of interleukin-8. Both characteristics correspond with those of natural GCP-2. In addition, intracellular calcium release in neutrophils by recombinant GCP-2 was achieved with as little as 10 ng/ml. Quantitation studies using reverse transcriptase and the polymerase chain reaction revealed higher GCP-2 mRNA production in normal fibroblasts than in tumor cells. When compared with epithelial-cell-derived neutrophil-activating peptide-78 (ENA-78) mRNA, the GCP-2 mRNA levels were higher in all cell lines tested. In addition, GCP-2 and ENA-78 expression seem to be differentially regulated in that phorbol ester and lipopolysaccharide have opposing effects on their mRNA induction in diploid fibroblasts and epithelial cells, respectively. Interleukin-1 was demonstrated to be a general inducer for both chemokines, while interferon-gamma down-regulates their mRNA expression. The availability of recombinant GCP-2 together with the quantitation studies on mRNA expression will help to further elucidate the biological role of GCP-2 during the inflammatory response.

Amino Acid Sequence↗

PubMed2026

Genomic characterization of avian metapneumovirus subtypes A and B in United States poultry by targeted amplicon sequencing.

Avian metapneumovirus (aMPV) subtypes A and B emerged in United States poultry in late 2023 and early 2024, prompting genome-scale surveillance from clinical samples. Here, we developed and optimized targeted amplicon sequencing (TAS) assays for both subtypes and applied them to 104 subtype-positive clinical samples collected from chicken and turkey farms across nine US states between early 2024 and early 2026. TAS recovered 91 genomes suitable for comparative analysis, including 44 aMPV-A and 47 aMPV-B sequences, with successful recovery extending to Ct values of 34.6 for aMPV-A and 31.2 for aMPV-B. Recovered genomes showed near-complete breadth and high mapping efficiency. Phylogenetic analyses of both G-gene and whole-genome datasets showed that US aMPV-A field strains formed a distinct monophyletic lineage within group IV and resolved into three closely related clusters. Cluster 2, first recognized in North Carolina and later detected in Ohio, spread across 30 turkey and chicken farms. Cluster 2 genomes were defined by a concentrated G-protein hotspot within residues 209-275, and most North Carolina Cluster 2 genomes carried 10-11 nonsynonymous substitutions in this region, including multiple proline substitutions suggestive of local structural change. Missouri Cluster 3 remained cohesive but distinct from both Cluster 1 and Cluster 2 in the G-gene and whole-genome trees. In contrast, US aMPV-B field strains remained highly homogeneous across hosts and states, with more than 99% nucleotide identity by both G-gene and WGS analyses. We also identified 12 vaccine-derived genomes on both vaccinated and nonvaccinated farms. These included six genomes related to aMPV-A vaccine and six related to aMPV-B vaccines (VCO3/50 and 1062), all of which retained vaccine-defining markers together with additional substitutions consistent with continued circulation after vaccine use in the field. Selection analyses showed that the G gene had the highest gene-wise dN/dS ratio in both subtypes. Additional elevated signal was observed in SH and M2, and candidate positively or episodically selected codons were concentrated in the subtype A Cluster 2 G-gene hotspot. These findings show that TAS supports direct-from-sample aMPV genomic surveillance and provides genomic context for field clusters, vaccine-derived lineages, and continued adaptive change in aMPV in US poultry.

Animals↗

PubMedDate not supplied

Next generation DNA vaccines for HIV-1.

We studied the effects of first generation HIV-1 plasmid vaccines in 167 individuals. The vaccines were very well tolerated and induced helper T cell responses in most vaccine recipients. However, the CTL responses were below a 20% response rate. Improvement in vaccine potency is an important goal of this technology and a central focus of our laboratory. To improve on these response rates, we used RNA optimized constructs pGag and pEnv). These vaccines express 20-100 fold better than first generation vectors. However, our studies support that additional enhancements are needed to further boost the immune response. We report that we can significantly enhance the induced CD8 effector cell response by including engineered B7 costimulatory molecules. We observed that B7.2 was more effective at driving cellular immune responses than B7.1 as a plasmid vaccine. We developed gene swaps and deletions between these two molecules. This manipulation resulted in a dramatically enhanced cellular immune response as measured by CTL, or ICC or Elispot. We have also explored the use of cytokines as plasmid vaccine adjuvants. We observed that IL-12 and IL-15 were effective as plasmid vaccine adjuvants. Interestingly, IL-15 appeared to allow T cell expansion in the absence of significant T cell help. Improvement of the immune response induced by plasmid vaccines can be engineered in multiple ways. Our studies show that both costimulation as well as cytokine signals can be harnessed for more potent vaccine development. These results have important implications for the design of vaccines for prophylaxis and therapy.

AIDS Vaccines↗

PubMed1991

Two-drug combinations of zidovudine, didanosine, and recombinant interferon-alpha A inhibit replication of zidovudine-resistant human immunodeficiency virus type 1 synergistically in vitro.

Optimal management of human immunodeficiency virus type 1 (HIV-1) infections may require combinations of anti-HIV-1 agents. Zidovudine (AZT, 3'-azido-3'-deoxythymidine), didanosine (ddI, 2',3'-dideoxyinosine), and recombinant interferon-alpha A (rIFN-alpha A) were evaluated in two-drug regimens against replication of AZT-resistant HIV-1 in vitro. AZT-sensitive and AZT-resistant isolate pairs derived from two individuals before and after extended AZT monotherapy were studied. Drug interactions using peripheral blood mononuclear cells infected with HIV-1 were evaluated mathematically. Synergistic interactions were seen among AZT, ddI, and rIFN-alpha A in two-drug regimens against AZT-resistant HIV-1 in vitro, even when AZT was included in the treatment regimen. Mixtures of wild-type and mutant reverse transcriptase genes were found in one of the late-AZT therapy isolates, suggesting that the mechanism of synergy of AZT-containing regimens may involve inhibition of AZT-sensitive viruses in the viral pool. These studies suggest that AZT may be useful in drug combination regimens, even when AZT-resistant viruses are isolated in vitro.

Base Sequence↗

PubMed2004

Psi-Phi: exploring the outer limits of bacterial pseudogenes.

Because bacterial chromosomes are tightly packed with genes and were traditionally viewed as being optimized for size and replication speed, it was not surprising that the early annotations of sequenced bacterial genomes reported few, if any, pseudogenes. But because pseudogenes are generally recognized by comparisons with their functional counterparts, as more genome sequences accumulated, many bacterial pathogens were found to harbor large numbers of truncated, inactivated, and degraded genes. Because the mutational events that inactivate genes occur continuously in all genomes, we investigated whether the rarity of pseudogenes in some bacteria was attributable to properties inherent to the organism or to the failure to recognize pseudogenes. By developing a program suite (called Psi-Phi, for Psi-gene Finder) that applies a comparative method to identify pseudogenes (attributable both to misannotation and to nonrecognition), we analyzed the pseudogene inventories in the sequenced members of the Escherichia coli/Shigella clade. This approach recovered hundreds of previously unrecognized pseudogenes and showed that pseudogenes are a regular feature of bacterial genomes, even in those whose original annotations registered no truncated or otherwise inactivated genes. In Shigella flexneri 2a, large proportions of pseudogenes are generated by nonsense mutations and IS element insertions, events that seldom produce the pseudogenes present in the other genomes examined. Almost all (>95%) pseudogenes are restricted to only one of the genomes and are of relatively recent origin, suggesting that these bacteria possess active mechanisms to eliminate nonfunctional genes.

Codon, Nonsense↗

PubMed2006

Modulation of the immune response induced by gene electrotransfer of a hepatitis C virus DNA vaccine in nonhuman primates.

Induction of multispecific, functional CD4+ and CD8+ T cells is the immunological hallmark of acute self-limiting hepatitis C virus (HCV) infection in humans. In the present study, we showed that gene electrotransfer (GET) of a novel candidate DNA vaccine encoding an optimized version of the nonstructural region of HCV (from NS3 to NS5B) induced substantially more potent, broad, and long-lasting CD4+ and CD8+ cellular immunity than naked DNA injection in mice and in rhesus macaques as measured by a combination of assays, including IFN-gamma ELISPOT, intracellular cytokine staining, and cytotoxic T cell assays. A protocol based on three injections of DNA with GET induced a substantially higher CD4+ T cell response than an adenovirus 6-based viral vector encoding the same Ag. To better evaluate the immunological potency and probability of success of this vaccine, we have immunized two chimpanzees and have compared vaccine-induced cell-mediated immunity to that measured in acute self-limiting infection in humans. GET of the candidate HCV vaccine led to vigorous, multispecific IFN-gamma+CD8+ and CD4+ T lymphocyte responses in chimpanzees, which were comparable to those measured in five individuals that cleared spontaneously HCV infection. These data support the hypothesis that T cell responses elicited by the present strategy could be beneficial in prophylactic vaccine approaches against HCV.

Animals↗

PubMed2002

Complexities in ETS-domain transcription factor function and regulation: lessons from the TCF (ternary complex factor) subfamily. The Colworth Medal Lecture.

The ETS-domain transcription factor family can be divided into a series of subfamilies. Elk-1 represents the founding member of the ternary complex factor (TCF) subfamily. By focusing on the TCF subfamily, we can demonstrate the complexities that exist in the function and regulation of ETS-domain transcription factors. This article focuses on Elk-1 in detail and summarizes the functions of other TCFs. The key themes covered include the domain structure of the TCFs, the mechanisms of complex formation with serum response factor, regulation of TCFs by mitogen-activated protein kinase cascades, and transcriptional regulatory properties of the TCFs. Finally, the emerging role of the TCFs in vivo is discussed. A picture is developing indicating that, while these proteins exhibit significant sequence and functional conservation, key differences in their structure and regulation are being identified which may relate to unique functions of these proteins in vivo.

Amino Acid Sequence↗

PubMed2000

Optimization of a simple and rapid single-strand conformation analysis for detection of mutations in the PROS1 gene: identification of seven novel mutations and three novel, apparently neutral, variants.

Anticoagulant protein S (PS) deficiency is a known risk factor for thrombophilia. The structure and high allelic heterogeneity of the PS gene (PROS1), together with the presence of a 97% homologous pseudogene, complicates PROS1 analysis. We have optimized a simple, fast, and non-isotopic Single-Strand Conformation Analysis (SSCA or SSCP) method for PROS1 mutation detection. This is accomplished through the analysis of the single-stranded and heteroduplex DNA fragments corresponding to 15 PCR segments that include part of the 5'-upstream region and the 15 PROS1 exons with their intron boundaries. To standardize the method, 13 known PROS1 mutations or allele variants in 10 different fragments were analyzed under different electrophoretic conditions. The results indicated that, using a combination of two different electrophoretic settings, all the allele variants could be detected as a single-strand band shift and/or by the presence of a heteroduplex. This method was used to analyze the PROS1 gene in 31 propositi with different types of PS deficiency and thrombosis. Ten different cosegregating mutations, seven of which are novel (143C->G, L-27H, G96X, M599T, P626L, 1418delA, and 1877delT), were identified in the five families suffering from type I or quantitative PS deficiency and in four of the nine families with coexistence of type I and type III phenotypes. No clearly co-segregating PROS1 mutations were identified in any of the 17 type III propositi analyzed, although eight of them were heterozygotes for the uncommon P460 allele of the S/P460 variant. Furthermore, five apparently neutral allelic variants, three of which are novel (-296C->T, 182G->C and T57S), were identified in a normal control, two type I/III and two type III PS-deficient pedigrees.

Alleles↗