Human respiratory syncytial virus N, P and M protein interactions in HEK-293T cells

Characterization of Human Respiratory Syncytial Virus (HRSV) protein interactions with host cell components is crucial to devise antiviral strategies. Viral nucleoprotein, phosphoprotein and matrix protein genes were optimized for human codon usage and cloned into expression vectors. HEK-293T cells were transfected with these vectors, viral proteins were immunoprecipitated, and co-immunoprecipitated cellular proteins were identified through mass spectrometry. Cell proteins identified with higher confidence scores were probed in the immunoprecipitation using specific antibodies. The results indicate that nucleoprotein interacts with arginine methyl-transferase, methylosome protein and Hsp70. Phosphoprotein interacts with Hsp70 and tropomysin, and matrix with tropomysin and nucleophosmin. Additionally, we performed immunoprecipitation of these cellular proteins in cells infected with HRSV, followed by detection of co-immunoprecipitated viral proteins. The results indicate that these interactions also occur in the context of viral infection, and their potential contribution for a HRSV replication model is discussed.

Highly expressed and alien genes of the Synechocystis genome

Comparisons of codon frequencies of genes to several gene classes are used to characterize highly expressed and alien genes on the Synechocystis PCC6803 genome. The primary gene classes include the ensemble of all genes (average gene), ribosomal protein (RP) genes, translation processing factors (TF) and genes encoding chaperone/degradation proteins (CH). A gene is predicted highly expressed (PHX) if its codon usage is close to that of the RP/TF/CH standards but strongly deviant from the average gene. Putative alien (PA) genes are those for which codon usage is significantly different from all four classes of gene standards. In Synechocystis, 380 genes were identified as PHX. The genes with the highest predicted expression levels include many that encode proteins vital for photosynthesis. Nearly all of the genes of the RP/TF/CH gene classes are PHX. The principal glycolysis enzymes, which may also function in CO2 fixation, are PHX, while none of the genes encoding TCA cycle enzymes are PHX. The PA genes are mostly of unknown function or encode transposases. Several PA genes encode polypeptides that function in lipopolysaccharide biosynthesis. Both PHX and PA genes often form significant clusters (operons). The proteins encoded by PHX and PA genes are described with respect to functional classifications, their organization in the genome and their stoichiometry in multi-subunit complexes.

Gene recognition via spliced sequence alignment.

Gene recognition is one of the most important problems in computational molecular biology. Previous attempts to solve this problem were based on statistics, and applications of combinatorial methods for gene recognition were almost unexplored. Recent advances in large-scale cDNA sequencing open a way toward a new approach to gene recognition that uses previously sequenced genes as a clue for recognition of newly sequenced genes. This paper describes a spliced alignment algorithm and software tool that explores all possible exon assemblies in polynomial time and finds the multiexon structure with the best fit to a related protein. Unlike other existing methods, the algorithm successfully recognizes genes even in the case of short exons or exons with unusual codon usage; we also report correct assemblies for genes with more than 10 exons. On a test sample of human genes with known mammalian relatives, the average correlation between the predicted and actual proteins was 99%. The algorithm correctly reconstructed 87% of genes and the rare discrepancies between the predicted and real exon-intron structures were caused either by short (less than 5 amino acids) initial/terminal exons or by alternative splicing. Moreover, the algorithm predicts human genes reasonably well when the homologous protein is nonvertebrate or even prokaryotic. The surprisingly good performance of the method was confirmed by extensive simulations: in particular, with target proteins at 160 accepted point mutations (PAM) (25% similarity), the correlation between the predicted and actual genes was still as high as 95%.

Mercuric ion reduction and resistance in transgenic Arabidopsis thaliana plants expressing a modified bacterial merA gene.

With global heavy metal contamination increasing, plants that can process heavy metals might provide efficient and ecologically sound approaches to sequestration and removal. Mercuric ion reductase, MerA, converts toxic Hg2+ to the less toxic, relatively inert metallic mercury (Hg0) The bacterial merA sequence is rich in CpG dinucleotides and has a highly skewed codon usage, both of which are particularly unfavorable to efficient expression in plants. We constructed a mutagenized merA sequence, merApe9, modifying the flanking region and 9% of the coding region and placing this sequence under control of plant regulatory elements. Transgenic Arabidopsis thaliana seeds expressing merApe9 germinated, and these seedlings grew, flowered, and set seed on medium containing HgCl2 concentrations of 25-100 microM (5-20 ppm), levels toxic to several controls. Transgenic merApe9 seedlings evolved considerable amounts of Hg0 relative to control plants. The rate of mercury evolution and the level of resistance were proportional to the steady-state mRNA level, confirming that resistance was due to expression of the MerApe9 enzyme. Plants and bacteria expressing merApe9 were also resistant to toxic levels of Au3+. These and other data suggest that there are potentially viable molecular genetic approaches to the phytoremediation of metal ion pollution.

Transgenic barley expressing a protein-engineered, thermostable (1,3-1,4)-beta-glucanase during germination.

The codon usage of a hybrid bacterial gene encoding a thermostable (1,3-1,4)-beta-glucanase was modified to match that of the barley (1,3-1,4)-beta-glucanase isoenzyme EII gene. Both the modified and unmodified bacterial genes were fused to a DNA segment encoding the barley high-pI alpha-amylase signal peptide downstream of the barley (1,3-1,4)-beta-glucanase isoenzyme EII gene promoter. When introduced into barley aleurone protoplasts, the bacterial gene with adapted codon usage directed synthesis of heat stable (1,3-1,4)-beta-glucanase, whereas activity of the heterologous enzyme was not detectable when protoplasts were transfected with the unmodified gene. In a different expression plasmid, the codon modified bacterial gene was cloned downstream of the barley high-pI alpha-amylase gene promoter and signal peptide coding region. This expression cassette was introduced into immature barley embryos together with plasmids carrying the bar and the uidA genes. Green, fertile plants were regenerated and approximately 75% of grains harvested from primary transformants synthesized thermostable (1,3-1,4)-beta-glucanase during germination. All three trans genes were detected in 17 progenies from a homozygous T1 plant.

Whole genome analysis reveals a high incidence of non-optimal codons in secretory signal sequences of Escherichia coli

Translational pausing may occur due to a number of mechanisms, including the presence of non-optimal codons, and it is thought to play a role in the folding of specific polypeptide domains during translation and in the facilitation of signal peptide recognition during see-dependent protein targeting. In this whole genome analysis of Escherichia coli we have found that non-optimal codons in the signal peptide-encoding sequences of secretory genes are overrepresented relative to the mature portions of these genes; this is in addition to their overrepresentation in the 5'-regions of genes encoding non-secretory proteins. We also find increased non-optimal codon usage at the 3' ends of most E. coli genes, in both non-secretory and secretory sequences. Whereas presumptive translational pausing at the 5' and 3' ends of E. coli messenger RNAs may clearly have a general role in translation, we suggest that it also has a specific role in sec-dependent protein export, possibly in facilitating signal peptide recognition. This finding may have important implications for our understanding of how the majority of non-cytoplasmic proteins are targeted, a process that is essential to all biological cells. (C) 2004 Elsevier Inc. All rights reserved.

Investigation of recombinant Schistosoma japonicum paramyosin fragments for immunogenicity and vaccine efficacy in mice

Schistosoma japonicum paramyosin, a 97 kDa myofibrillar protein, is a recognized vaccine candidate against schistosomiasis. To improve its expression and to identify protective epitopic regions on paramyosin, the published Chinese Schistosoma japonicum paramyosin cDNA sequence was redesigned using Pichia codon usage and divided into four overlapping fragments (fragments 1, 2, 3, 4) of 747, 651, 669 and 678 bp, respectively. These gene fragments were synthesized and expressed in Pichia pastoris (fragments 2 and 3) or E. coli (fragments 1 and 4). The recombinant proteins were produced at high level and purified using a two-step process involving Ni-NTA affinity chromatography and gel filtration. BALB/c mice were immunized subcutaneously three times at 2-week-intervals with the purified proteins formulated in adjuvant Quil A. The protein fragments were highly immunogenic, inducing high, though variable, ELISA antibody titres, and each was shown to resemble native paramyosin in terms of its recognition by the anti-fragment antibodies in Western blotting. The immunized mice were subjected to cercarial challenge 2 weeks after the final injection and promising protective efficacy in terms of significant reductions in worm burdens, worm-pair numbers and liver eggs in the vaccinated mice resulted. There was no apparent correlation between the antibody titres generated and protective efficacy, as all fragments produced effective but similar levels of protection.

Incorporating a TEV cleavage site reduces the solubility of nine recombinant mouse proteins

Failure to express soluble proteins in bacteria is mainly attributed to the properties of the target protein itself, as well as the choice of the vector, the purification tag and the linker between the tag and protein, and codon usage. The expression of proteins with fusion tags to facilitate subsequent purification steps is a widely used procedure in the production of recombinant proteins. However, the additional residues can affect the properties of the protein; therefore, it is often desirable to remove the tag after purification. This is usually done by engineering a cleavage site between the tag and the encoded protein that is recognised by a site-specific protease, such as the one from tobacco etch virus (TEV). In this study, we investigated the effect of four different tags on the bacterial expression and solubility of nine mouse proteins. Two of the four engineered constructs contained hexahistidine tags with either a long or short linker. The other two constructs contained a TEV cleavage site engineered into the linker region. Our data show that inclusion of the TEV recognition site directly downstream of the recombination site of the Invitrogen Gateway vector resulted, in a loss of solubility of the nine mouse proteins. Our work suggests that one needs to be very careful when making modifications to expression vectors and combining different affinity and fusion tags and cleavage sites: (c) 2006 Elsevier Inc. All rights reserved.

Development of a novel platform for high-throughput gene design and artificial gene synthesis to produce large libraries of recombinant venom peptides for drug discovery

Tese de Doutoramento em Ciências Veterinárias na Especialidade de Ciências Biológicas e Biomédicas

Mitochondrial Inheritance, Mito-nuclear Coevolution, and Sex-associated Genes in Bivalve Molluscs: Transcriptomics and Molecular Evolution

Bivalvia represents an ancient taxon including around 25,000 living species that have adapted to a wide range of environmental conditions, and show a great diversity in body size, shell shapes, and anatomic structure. Bivalves are characterized by highly variable genome sizes and extremely high levels of heterozygosity, which obstacle complete and accurate genome assemblies and hinder further genomic studies. Moreover, some bivalve species presented a stable evolutionary exception to the strictly maternal inheritance of mitochondria, namely doubly uniparental inheritance (DUI), making these species a precious model to study mitochondrial biology. During my PhD, I focused on a DUI species, the Manila clam Ruditapes philippinarum, and my work was two-folded. First, taking advantage of a newly assembled draft genome and a large RNA-seq dataset from different tissues of both sexes, I investigated 1) the role of gene expression and alternative splicing in tissue differentiation; 2) the relationship across tissue specificity, regulatory network connectivity, and sequence evolution; 3) sexual contrasting genetic markers potentially associated with sexual differentiation. The detailed information for this part is in Chapter 2. Second, using the same RNA-seq data, I investigated how nuclear oxidative phosphorylation (OXPHOS) genes coordinate with two divergent mitochondrial genomes in DUI species (mito-nuclear coordination and coevolution). To address this question, I compared transcription, polymorphism, and synonymous codon usage in the mitochondrial and nuclear OXPHOS genes of R. philippinarum in Chapter 3. To my knowledge, this thesis represents the first study exploring the role of alternative splicing in tissue differentiation, and the first study analyzing both transcriptional regulation and sequence evolution to investigate the coordination of OXPHOS genes in bivalves.

Information and the evolution of codon bias

The informational properties of biological systems are the subject of much debate and research. I present a general argument in favor of the existence and central importance of information in organisms, followed by a case study of the genetic code (specifically, codon bias) and the translation system from the perspective of information. The codon biases of 831 Bacteria and Archeae are analyzed and modeled as points in a 64-dimensional statistical space. The major results are that (1) codon bias evolution does not follow canonical patterns, and (2) the use of coding space in organsims is a subset of the total possible coding space. These findings imply that codon bias is a unique adaptive mechanism that owes its existence to organisms' use of information in representing genes, and that there is a particularly biological character to the resulting biased coding and information use.

Deregulated MHC class II transactivator expression leads to a strong Th2 bias in CD4+ T lymphocytes.

The MHC class II (MHC-II) transactivator (CIITA) is the master transcriptional regulator of genes involved in MHC-II-restricted Ag presentation. Fine tuning of CIITA gene expression determines the cell type-specific expression of MHC-II genes. This regulation is achieved by the selective usage of multiple CIITA promoters. It has recently been suggested that CIITA also contributes to Th cell differentiation by suppressing IL-4 expression in Th1 cells. In this study, we show that endogenous CIITA is expressed at low levels in activated mouse T cells. Importantly CIITA is not regulated differentially in murine and human Th1 and Th2 cells. Ectopic expression of a CIITA transgene in multiple mouse cell types including T cells, does not interfere with normal development of CD4(+) T cells. However, upon TCR activation the CIITA transgenic CD4(+) T cells preferentially differentiate into IL-4-secreting Th2-type cells. These results imply that CIITA is not a direct Th1-specific repressor of the IL-4 gene and that tight control over the expression of CIITA and MHC-II is required to maintain the normal balance between Th1 and Th2 responses.

HIV-1 coreceptor usage and CXCR4-specific viral load predict clinical disease progression during combination antiretroviral therapy

Background: Although combination antiretroviral therapy (cART) dramatically reduces rates of AIDS and death, a minority of patients experience clinical disease progression during treatment. <p>Objective: To investigate whether detection of CXCR4(X4)-specific strains or quantification of X4-specific HIV-1 load predict clinical outcome. Methods: From the Swiss HIV Cohort Study, 96 participants who initiated cART yet subsequently progressed to AIDS or death were compared with 84 contemporaneous, treated nonprogressors. A sensitive heteroduplex tracking assay was developed to quantify plasma X4 and CCR5 variants and resolve HIV-1 load into coreceptor-specific components. Measurements were analyzed as cofactors of progression in multivariable Cox models adjusted for concurrent CD4 cell count and total viral load, applying inverse probability weights to adjust for sampling bias. Results: Patients with X4 variants at baseline displayed reduced CD4 cell responses compared with those without X4 strains (40 versus 82 cells/mu l; P= 0.012). The adjusted multivariable hazard ratio (HR) for clinical progression was 4.8 [95% confidence interval (Cl) 2.3-10.0] for those demonstrating X4 strains at baseline. The X4-specific HIV-1 load was a similarly independent predictor, with HR values of 3.7(95%Cl, 1.2-11.3) and 5.9 (95% Cl, 2.2-15.0) for baseline loads of 2.2-4.3 and > 4.3 log(10)copies/ml, respectively, compared with < 2.2 log(10)copies/ml. Conclusions: HIV-1 coreceptor usage and X4-specific viral loads strongly predicted disease progression during cART, independent of and in addition to CD4 cell count or total viral load. Detection and quantification of X4 strains promise to be clinically useful biomarkers to guide patient management and study HIV-1 pathogenesis.

Transferring control demands across incidental learning tasks - Stronger sequence usage in serial reaction task after shortcut option in letter string checking

After incidentally learning about a hidden regularity, participants can either continue to solve the task as instructed or, alternatively, apply a shortcut. Past research suggests that the amount of conflict implied by adopting a shortcut seems to bias the decision for vs. against continuing instruction-coherent task processing. We explored whether this decision might transfer from one incidental learning task to the next. Theories that conceptualize strategy change in incidental learning as a learning-plus-decision phenomenon suggest that high demands to adhere to instruction-coherent task processing in Task 1 will impede shortcut usage in Task 2, whereas low control demands will foster it. We sequentially applied two established incidental learning tasks differing in stimuli, responses and hidden regularity (the alphabet verification task followed by the serial reaction task, SRT). While some participants experienced a complete redundancy in the task material of the alphabet verification task (low demands to adhere to instructions), for others the redundancy was only partial. Thus, shortcut application would have led to errors (high demands to follow instructions). The low control demand condition showed the strongest usage of the fixed and repeating sequence of responses in the SRT. The transfer results are in line with the learning-plus-decision view of strategy change in incidental learning, rather than with resource theories of self-control.