Development of a New Generation of Vectors for Gene Expression, Gene Replacement, and Protein-Protein Interaction Studies in Mycobacteria

Escherichia coli-mycobacterium shuttle vectors are important tools for gene expression and gene replacement in mycobacteria. However, most of the currently available vectors are limited in their use because of the lack of extended multiple cloning sites (MCSs) and convenience of appending an epitope tag(s) to the cloned open reading frames (ORFs). Here we report a new series of vectors that allow for the constitutive and regulatable expression of proteins, appended with peptide tag sequences at their N and C termini, respectively. The applicability of these vectors is demonstrated by the constitutive and induced expression of the Mycobacterium tuberculosis pknK gene, coding for protein kinase K, a serine-threonine protein kinase. Furthermore, a suicide plasmid with expanded MCS for creating gene replacements, a plasmid for chromosomal integrations at the commonly used L5 attB site, and a hypoxia-responsive vector, for expression of a gene(s) under hypoxic conditions that mimic latency, have also been created. Additionally, we have created a vector for the coexpression of two proteins controlled by two independent promoters, with each protein being in fusion with a different tag. The shuttle vectors developed in the present study are excellent tools for the analysis of gene function in mycobacteria and are a valuable addition to the existing repertoire of vectors for mycobacterial research.

Distinctive contributions of the ribosomal P-site elements m(2)G966, m(5)C967 and the C-terminal tail of the S9 protein in the fidelity of initiation of translation in Escherichia coli

The accuracy of pairing of the anticodon of the initiator tRNA (tRNA(fMet)) and the initiation codon of an mRNA, in the ribosomal P-site, is crucial for determining the translational reading frame. However, a direct role of any ribosomal element(s) in scrutinizing this pairing is unknown. The P-site elements, m(2)G966 (methylated by RsmD), m(5)C967 (methylated by RsmB) and the C-terminal tail of the protein S9 lie in the vicinity of tRNA(fMet). We investigated the role of these elements in initiation from various codons, namely, AUG, GUG, UUG, CUG, AUA, AUU, AUC and ACG with tRNA(CAU)(fmet) (tRNA(fMet) with CAU anticodon); CAC and CAU with tRNA(GUG)(fme); UAG with tRNA(GAU)(fMet) using in vivo and computational methods. Although RsmB deficiency did not impact initiation from most codons, RsmD deficiency increased initiation from AUA, CAC and CAU (2- to 3.6-fold). Deletion of the S9 C-terminal tail resulted in poorer initiation from UUG, GUG and CUG, but in increased initiation from CAC, CAU and UAC codons (up to 4-fold). Also, the S9 tail suppressed initiation with tRNA(CAU)(fMet)lacking the 3GC base pairs in the anticodon stem. These observations suggest distinctive roles of 966/967 methylations and the S9 tail in initiation.

Local Structural and Environmental Factors Define the Efficiency of an RNA Pseudoknot Involved in Programmed Ribosomal Frameshift Process

In programmed -1 ribosomal frameshift, an RNA pseudoknot stalls the ribosome at specific sequence and restarts translation in a new reading frame. A precise understanding of structural characteristics of these pseudoknots and their PRF inducing ability has not been clear to date. To investigate this phenomenon, we have studied various structural aspects of a -1 PRF inducing RNA pseudoknot from BWYV using extensive molecular dynamics simulations. A set of functional and poorly functional forms, for which previous mutational data were available, were chosen for analysis. These structures differ from each other by either single base substitutions or base-pair replacements from the native structure. We have rationalized how certain mutations in RNA pseudoknot affect its function; e.g., a specific base substitution in loop 2 stabilizes the junction geometry by forming multiple noncanonical hydrogen bonds, leading to a highly rigid structure that could effectively resist ribosome-induced unfolding, thereby increasing efficiency. While, a CG to AU pair substitution in stem 1 leads to loss of noncanonical hydrogen bonds between stems and loop, resulting in a less stable structure and reduced PRF inducing ability, inversion of a pair in stem 2 alters specific base-pair geometry that might be required in ribosomal recognition of nucleobase groups, negatively affecting pseudoknot functioning. These observations illustrate that the ability of an RNA pseudoknot to induce -1 PRF with an optimal rate depends on several independent factors that contribute to either the local conformational variability or geometry

Is the cellular initiation of translation an exclusive property of the initiator tRNAs?

Translation of mRNAs is the primary function of the ribosomal machinery. Although cells allow for a certain level of translational errors/mistranslation (which may well be a strategic need), maintenance of the fidelity of translation is vital for the cellular function and fitness. The P-site bound initiator tRNA selects the start codon in an mRNA and specifies the reading frame. A direct P-site binding of the initiator tRNA is a function of its special structural features, ribosomal elements, and the initiation factors. A highly conserved feature of the 3 consecutive G:C base pairs (3GC pairs) in the anticodon stem of the initiator tRNAs is vital in directing it to the P-site. Mutations in the 3GC pairs diminish/abolish initiation under normal physiological conditions. Using molecular genetics approaches, we have identified conditions that allow initiation with the mutant tRNAs in Escherichia coli. During our studies, we have uncovered a novel phenomenon of in vivo initiation by elongator tRNAs. Here, we recapitulate how the cellular abundance of the initiator tRNA, and nucleoside modifications in rRNA are connected with the tRNA selection in the P-site. We then discuss our recent finding of how a conserved feature in the mRNA, the Shine-Dalgarno sequence, influences tRNA selection in the P-site.

Genotypes, Recombinant Forms, and Variants of Norovirus GII.4 in Gipuzkoa (Basque Country, Spain), 2009-2012

Background: Noroviruses (NoVs) are genetically diverse, with genogroup II-and within it-genotype 4 (GII.4) being the most prevalent cause of acute gastroenteritis worldwide. The aim of this study was to characterize genogroup II NoV causing acute gastroenteritis in the Basque Country (northern Spain) from 2009-2012. Methods: The presence of NoV RNA was investigated by reverse transcriptase-polymerase chain reaction (RT-PCR) in stool specimens from children younger than 15 years old with community-acquired acute gastroenteritis, and from hospitalized adults or elderly residents of nursing homes with acute gastroenteritis. For genotyping, the open reading frames ORF1 (encoding the polymerase) and ORF2 (encoding the major capsid protein) were partially amplified and sequenced. Recombinant strains were confirmed by PCR of the ORF1/ORF2 junction region. Results: NoV was detected in 16.0% (453/2826) of acute gastroenteritis episodes in children younger than 2 years, 9.9% (139/1407) in children from 2 to 14 years, and 35.8% (122/341) in adults. Of 317 NoVs characterized, 313 were genogroup II and four were genogroup I. The GII.4 variants Den Haag-2006b and New Orleans-2009 predominated in 2009 and 2010-2011, respectively. In 2012, the New Orleans-2009 variant was partially replaced by the Sydney-2012 variant (GII.Pe/GII.4) and New Orleans-2009/Sydney-2012 recombinant strains. The predominant capsid genotype in all age groups was GII.4, which was the only genotype detected in outbreaks. The second most frequent genotype was GII.3 (including the recently described recombination GII.P16/GII.3), which was detected almost exclusively in children. Conclusion: Nine different genotypes of NoV genogroup II were detected; among these, intergenotype recombinant strains represented an important part, highlighting the role of recombination in the evolution of NoVs. Detection of new NoV strains, not only GII.4 strains, shortly after their first detection in other parts of the world shows that many NoV strains can spread rapidly.

Identification and characterization of novel reptile cathelicidins from elapid snakes

Three cDNA sequences coding for elapid cathelicidins were cloned from constructed venom gland cDNA libraries of Naja atra, Bungarus fasciatus and Ophiophagus hannah. The open reading frames of the cloned elapid cathelicidins were all composed of 576 bp an

Identification and characterization of hypoxia-induced genes in Carassius auratus blastulae embryonic cells using suppression subtractive hybridization

Organisms living in water are inevitably exposed to periods of hypoxia. Environmental hypoxia has been an important stressor having manifold effects on aquatic life. Many fish species have evolved behavioral, physiological, biochemical and molecular adaptations that enable them to cope with hypoxia. However, the molecular mechanisms of hypoxia tolerance in fish, remain unknown. in this study, we used suppression subtractive hybridization to examine the differential gene expression in CAB cells (Carassius auratus blastulae embryonic cells) exposed to hypoxia for 24 h. We isolated 2100 clones and identified 211 differentially expressed genes (e-value <= 5e-3; Identity > 45%). Among the genes whose expression is modified in cells, a vast majority involved in metabolism, signal transduction, cell defense, angiogenesis, cell growth and proliferation. Twelve genes encoding for ERO1-L, p53, CPO, HO-1, MKP2, PFK-2, cystatin B, GLUT1, BTG1, TGF beta 1, PGAM1, hypothetical protein F1508, were selected and identified to be hypoxia-induced using semi-quantitive RT-PCR and real-time PCR. Among the identified genes, two open reading frames (ORFs) encoding for CaBTG1 and Cacystatin B were obtained. The deduced amino acid sequence of CaBTG1 had 94.1%, 72.8%, 72.8%, 72.8%, 68.6% identity with that of DrBTG1, HsBTG1, BtBTG1, MmBTG1 and XIBTG1. Comparison of Cacystatin B with known cystatin B, the molecules exhibited 49.5 to 76.0% identity overall. These results may provide significant information for further understanding of the adaptive mechanism by which C. auratus responds to hypoxia. (c) 2008 Elsevier Inc. All rights reserved.

Characterization of complete genome sequence of the spring viremia of carp virus isolated from common carp (Cyprinus carpio) in China

The complete genome of spring viraemia of carp virus (SVCV) strain A-1 isolated from cultured common carp (Cyprinus carpio) in China was sequenced and characterized. Reverse transcription-polymerase chain reaction (RT-PCR) derived clones were constructed and the DNA was sequenced. It showed that the entire genome of SVCV A-1 consists of 11,100 nucleotide base pairs, the predicted size of the viral RNA of rhabdoviruses. However, the additional insertions in bp 4633-4676 and bp 4684-4724 of SVCV A-1 were different from the other two published SVCV complete genomes. Five open reading frames (ORFs) of SVCV A-1 were identified and further confirmed by RT-PCR and DNA sequencing of their respective RT-PCR products. The 5 structural proteins encoded by the viral RNA were ordered 3'-N-P-M-G-L-5'. This is the first report of a complete genome sequence of SVCV isolated from cultured carp in China. Phylogenetic analysis indicates that SVCV A-1 is closely related to the members of the genus Vesiculovirus, family Rhabdoviridae.

Complete genome sequence of lymphocystis disease virus isolated from China

Lymphocystis diseases in fish throughout the world have been extensively described. Here we report the complete genome sequence of lymphocystis disease virus isolated in China (LCDV-C), an LCDV isolated from cultured flounder (Paralichthys olivaceus) with lymphocystis disease in China. The LCDV-C genome is 186,250 bp, with a base composition of 27.25% G+C. Computer-assisted analysis revealed 240 potential open reading frames (ORFs) and 176 nonoverlapping putative viral genes, which encode polypeptides ranging from 40 to 1,193 amino acids. The percent coding density is 67%, and the average length of each ORF is 702 bp. A search of the GenBank database using the 176 individual putative genes revealed 103 homologues to the corresponding ORFs of LCDV-1 and 73 potential genes that were not found in LCDV-1 and other iridoviruses. Among the 73 genes, there are 8 genes that contain conserved domains of cellular genes and 65 novel genes that do not show any significant homology with the sequences in public databases. Although a certain extent of similarity between putative gene products of LCDV-C and corresponding proteins of LCDV-1 was revealed, no colinearity was detected when their ORF arrangements and coding strategies were compared to each other, suggesting that a high degree of genetic rearrangements between them has occurred. And a large number of tandem and overlapping repeated sequences were observed in the LCDV-C genome. The deduced amino acid sequence of the major capsid protein (MCP) presents the highest identity to those of LCDV-1 and other iridoviruses among the LCDV-C gene products. Furthermore, a phylogenetic tree was constructed based on the multiple alignments of nine MCP amino acid sequences. Interestingly, LCDV-C and LCDV-1 were clustered together, but their amino acid identity is much less than that in other clusters. The unexpected levels of divergence between their genomes in size, gene organization, and gene product identity suggest that LCDV-C and LCDV-1 shouldn't belong to a same species and that LCDV-C should be considered a species different from LCDV-1.

Microarray Analyses of Gene Expression during the Tetrahymena thermophila Life Cycle

Background: The model eukaryote, Tetrahymena thermophila, is the first ciliated protozoan whose genome has been sequenced, enabling genome-wide analysis of gene expression. Methodology/Principal Findings: A genome-wide microarray platform containing the predicted coding sequences (putative genes) for T. thermophila is described, validated and used to study gene expression during the three major stages of the organism's life cycle: growth, starvation and conjugation. Conclusions/Significance: Of the,27,000 predicted open reading frames, transcripts homologous to only,5900 are not detectable in any of these life cycle stages, indicating that this single-celled organism does indeed contain a large number of functional genes. Transcripts from over 5000 predicted genes are expressed at levels >5x corrected background and 95 genes are expressed at >250x corrected background in all stages. Transcripts homologous to 91 predicted genes are specifically expressed and 155 more are highly up-regulated in growing cells, while 90 are specifically expressed and 616 are up-regulated during starvation. Strikingly, transcripts homologous to 1068 predicted genes are specifically expressed and 1753 are significantly up-regulated during conjugation. The patterns of gene expression during conjugation correlate well with the developmental stages of meiosis, nuclear differentiation and DNA elimination. The relationship between gene expression and chromosome fragmentation is analyzed. Genes encoding proteins known to interact or to function in complexes show similar expression patterns, indicating that co-ordinate expression with putative genes of known function can identify genes with related functions. New candidate genes associated with the RNAi-like process of DNA elimination and with meiosis are identified and the late stages of conjugation are shown to be characterized by specific expression of an unexpectedly large and diverse number of genes not involved in nuclear functions.

Molecular identification of 16 Porphyra lines using sequence-related amplified polymorphism markers

Sequence-related amplified polymorphism (SRAP) is a novel molecular marker technique designed to amplify open reading frames (ORFs). The SRAP analytic system was set up and applied to Porphyra germplasm identification in this study for the first time. Sixteen Porphyra lines were screened by SRAP technique with 30 primer combinations. In the analysis, 14 primer combinations produced stable and reproducible amplification patterns in three repetitive experiments. Among the total 533 amplified fragments, 522 (98%) were polymorphic, with an average of 38 fragments for each primer combination, ranging in size from 50 to 500 bp. The 533 fragments were visually scored one by one and then used to develop a dendrogram with Unweighted Pair-Group Method Arithmetic Average (UPGMA), and the 16 Porphyra lines were divided into two major groups at the 0.68 similarity level. From the total 533 fragments, I I amplified by two primer combinations, ME1/EM1 and ME4/EM6, were used to develop the DNA fingerprints of the 16 Porphyra lines. The DNA fingerprints were then converted into binary codes, with I and 0 representing presence and absence of the corresponding amplified fragment, respectively. In the DNA fingerprints, each of the 16 Porphyra lines has its unique binary code and can be easily distinguished from the others. This is the first report on the development of SRAP technique and its utilization in germplasm identification of seaweeds. The results demonstrated that SRAP is a simple, stable, polymorphic and reproducible molecular marker technique for the classification and identification of Porphyra lines. (c) 2007 Elsevier B.V. All rights reserved.

Hypoxia-inducible factor 1 alpha cDNA cloning and its mRNA and protein tissue specific expression in domestic yak (Bos grunniens) from Qinghai-Tibetan plateau

Adaptation to hypoxia is regulated by hypoxia-inducible factor 1 (HIF-1), a heterodimeric transcription factor consisting of an oxygen-regulated a-subunit and a constitutively expressed beta-subunit. How animals living on Qinghai-Tibetan plateau adapt to the extreme hypoxia environment is known indistinctly. In this study, the Qinghai yak which has been living at 3000-5000 m attitude for at least two millions of years was selected as the model of high hypoxia-tolerant adaptation species. The HIF-1 alpha ORFs (open reading frames) encoding for two isoforms of HIF-1 alpha have been cloned from the brain of the domestic yak. Its expression of HIF-1 alpha was analyzed at both mRNA and protein levels in various tissues. Both its HIF-1 alpha mRNA and protein are tissue specific expression. Its HIF-1 alpha protein's high expression in the brain, lung, and kidney showed us that HIF-1 alpha protein may play an important role in the adaptation to hypoxia environment. (c) 2006 Elsevier Inc. All rights reserved.

Confirmation of Data Mining Based Predictions of Protein Function

King, R. D. and Wise, P. H. and Clare, A. (2004) Confirmation of Data Mining Based Predictions of Protein Function. Bioinformatics 20(7), 1110-1118

Predicting gene function in Saccharomyces cerevisiae

Clare, A. and King R.D. (2003) Predicting gene function in Saccharomyces cerevisiae. 2nd European Conference on Computational Biology (ECCB '03). (published as a journal supplement in Bioinformatics 19: ii42-ii49)

The characterization of twenty sequenced human genomes.

We present the analysis of twenty human genomes to evaluate the prospects for identifying rare functional variants that contribute to a phenotype of interest. We sequenced at high coverage ten "case" genomes from individuals with severe hemophilia A and ten "control" genomes. We summarize the number of genetic variants emerging from a study of this magnitude, and provide a proof of concept for the identification of rare and highly-penetrant functional variants by confirming that the cause of hemophilia A is easily recognizable in this data set. We also show that the number of novel single nucleotide variants (SNVs) discovered per genome seems to stabilize at about 144,000 new variants per genome, after the first 15 individuals have been sequenced. Finally, we find that, on average, each genome carries 165 homozygous protein-truncating or stop loss variants in genes representing a diverse set of pathways.