Characterization of the molecular components and function of BARE-1, Hin-Mu and Mu transposition machineries

Transposable elements, transposons, are discrete DNA segments that are able to move or copy themselves from one locus to another within or between their host genome(s) without a requirement for DNA homology. They are abundant residents in virtually all the genomes studied, for instance, the genomic portion of TEs is approximately 3% in Saccharomyces cerevisiae, 45% in humans, and apparently more than 70% in some plant genomes such as maize and barley. Transposons plays essential role in genome evolution, in lateral transfer of antibiotic resistance genes among bacteria and in life cycle of certain viruses such as HIV-1 and bacteriophage Mu. Despite the diversity of transposable elements they all use a fundamentally similar mechanism called transpositional DNA recombination (transposition) for the movement within and between the genomes of their host organisms. The DNA breakage and joining reactions that underlie their transposition are chemically similar in virtually all known transposition systems. The similarity of the reactions is also reflected in the structure and function of the catalyzing enzymes, transposases and integrases. The transposition reactions take place within the context of a transposition machinery, which can be particularly complex, as in the case of the VLP (virus like particle) machinery of retroelements, which in vivo contains RNA or cDNA and a number of element encoded structural and catalytic proteins. Yet, the minimal core machinery required for transposition comprises a multimer of transposase or integrase proteins and their binding sites at the element DNA ends only. Although the chemistry of DNA transposition is fairly well characterized, the components and function of the transposition machinery have been investigated in detail for only a small group of elements. This work focuses on the identification, characterization, and functional studies of the molecular components of the transposition machineries of BARE-1, Hin-Mu and Mu. For BARE-1 and Hin-Mu transpositional activity has not been shown previously, whereas bacteriophage Mu is a general model of transposition. For BARE-1, which is a retroelement of barley (Hordeum vulgare), the protein and DNA components of the functional VLP machinery were identified from cell extracts. In the case of Hin-Mu, which is a Mu-like prophage in Haemophilus influenzae Rd genome, the components of the core machinery (transposase and its binding sites) were characterized and their functionality was studied by using an in vitro methodology developed for Mu. The function of Mu core machinery was studied for its ability to use various DNA substrates: Hin-Mu end specific DNA substrates and Mu end specific hairpin substrates. The hairpin processing reaction by MuA was characterized in detail. New information was gained of all three machineries. The components or their activity required for functional BARE-1 VLP machinery and retrotransposon life cycle were present in vivo and VLP-like structures could be detected. The Hin-Mu core machinery components were identified and shown to be functional. The components of the Mu and Hin-Mu core machineries were partially interchangeable, reflecting both evolutionary conservation and flexibility within the core machineries. The Mu core machinery displayed surprising flexibility in substrate usage, as it was able to utilize Hin-Mu end specific DNA substrates and to process Mu end DNA hairpin substrates. This flexibility may be evolutionarily and mechanistically important.

Plexin B2 in mouse and zebrafish development

Plexins (plxn) are receptors of semaphorins (sema), which were originally characterized as axon guidance cues. Semaphorin-plexin signalling has now been implicated in many other developmental and pathological processes. In this thesis, my first aim was to study the expression of plexins during mouse development. My second aim was to study the function of Plexin B2 in the development of the kidney. Thirdly, my objective was to elucidate the evolutionary conservation of Plexin B2 by investigating its sequence, expression and function in developing zebrafish. I show by in situ hybridisation that plexins are widely expressed also in the non-neuronal tissues during mouse development. Plxnb1 and Plxnb2, for example, are expressed also in the ureteric epithelium, developing glomeruli and undifferentiated metanephric mesenchyme of the developing kidney. Plexin B2-deficient (Plxnb2-/-) mice die before birth and have severe defects in the nervous system. I demonstrate that they develop morphologically normal but hypoplastic kidneys. The ureteric epithelium of Plxnb2-/- kidneys has fewer branches and a lower rate of proliferating cells. 10% of the embryos show unilateral double ureters and kidneys. The defect in the branching is intrinsic to the epithelium as the isolated ureteric epithelium grown in vitro fails to respond to Glial-cell-line-derived neurotrophic factor (Gdnf). We prove by co-immunoprecipitation that Plexin B2 interacts with the Gdnf-receptor Ret. Sema4C, the Plexin B2 ligand, increases branching of the ureteric epithelium in controls but not in Plxnb2-/- kidney explants. These results suggest that Sema4C-Plexin B2 signalling modulates ureteric branching in a positive manner, possibly through directly regulating the activation of Ret. I cloned the zebrafish orthologs of Plexin B2, Plexin B2a and B2b. The corresponding proteins contain the conserved domains the B-subfamily plexins. Especially the expression pattern of plxnb2b recapitulates many aspects of the expression pattern of Plxnb2 in mouse. Plxnb2a and plxnb2b are expressed, for example, in the pectoral fins and at the midbrain-hindbrain region during zebrafish development. The nearly complete knockdown of Plexin B2a alone or together with the 45% knockdown of Plexin B2b did not interfere with the normal development of the zebrafish. In conclusion, my thesis reveals that plexins are broadly expressed during mouse embryogenesis. It also shows that Sema4C-Plexin B2 signalling modulates the branching of the ureteric epithelium during kidney development, perhaps through a direct interaction with Ret. Finally, I show that the sequence and expression of Plexin B2a and B2b are conserved in zebrafish. Their knockdown does not, however, result in the exencephaly phenotype of Plxnb2-/- mice.

Functional Significance of an Evolutionarily Conserved Alanine (GCA) Resume Codon in tmRNA in Escherichia coli

Occasionally, ribosomes stall on mRNAs prior to the completion of the polypeptide chain. In Escherichia coli and other eubacteria, tmRNA-mediated trans-translation is a major mechanism that recycles the stalled ribosomes. The tmRNA possesses a tRNA-like domain and a short mRNA region encoding a short peptide (ANDENYALAA in E. coli) followed by a termination codon. The first amino acid (Ala) of this peptide encoded by the resume codon (GCN) is highly conserved in tmRNAs in different species. However, reasons for the high evolutionary conservation of the resume codon identity have remained unclear. In this study, we show that changing the E. coli tmRNA resume codon to other efficiently translatable codons retains efficient functioning of the tmRNA. However, when the resume codon was replaced with the low-usage codons, its function was adversely affected. Interestingly, expression of tRNAs decoding the low-usage codon from plasmid-borne gene copies restored efficient utilization of tmRNA. We discuss why in E. coli, the GCA (Ala) is one of the best codons and why all codons in the short mRNA of the tmRNA are decoded by the abundant tRNAs.

A multi-level multi-scale approach to study essential genes in Mycobacterium tuberculosis

Background: The set of indispensable genes that are required by an organism to grow and sustain life are termed as essential genes. There is a strong interest in identification of the set of essential genes, particularly in pathogens, not only for a better understanding of the pathogen biology, but also for identifying drug targets and the minimal gene set for the organism. Essentiality is inherently a systems property and requires consideration of the system as a whole for their identification. The available experimental approaches capture some aspects but each method comes with its own limitations. Moreover, they do not explain the basis for essentiality in most cases. A powerful prediction method to recognize this gene pool including rationalization of the known essential genes in a given organism would be very useful. Here we describe a multi-level multi-scale approach to identify the essential gene pool in a deadly pathogen, Mycobacterium tuberculosis. Results: The multi-level workflow analyses the bacterial cell by studying (a) genome-wide gene expression profiles to identify the set of genes which show consistent and significant levels of expression in multiple samples of the same condition, (b) indispensability for growth by using gene expression integrated flux balance analysis of a genome-scale metabolic model, (c) importance for maintaining the integrity and flow in a protein-protein interaction network and (d) evolutionary conservation in a set of genomes of the same ecological niche. In the gene pool identified, the functional basis for essentiality has been addressed by studying residue level conservation and the sub-structure at the ligand binding pockets, from which essential amino acid residues in that pocket have also been identified. 283 genes were identified as essential genes with high-confidence. An agreement of about 73.5% is observed with that obtained from the experimental transposon mutagenesis technique. A large proportion of the identified genes belong to the class of intermediary metabolism and respiration. Conclusions: The multi-scale, multi-level approach described can be generally applied to other pathogens as well. The essential gene pool identified form a basis for designing experiments to probe their finer functional roles and also serve as a ready shortlist for identifying drug targets.

Identifying functionally important cis-peptide containing segments in proteins and their utility in molecular function annotation

Cis-peptide embedded segments are rare in proteins but often highlight their important role in molecular function when they do occur. The high evolutionary conservation of these segments illustrates this observation almost universally, although no attempt has been made to systematically use this information for the purpose of function annotation. In the present study, we demonstrate how geometric clustering and level-specific Gene Ontology molecular-function terms (also known as annotations) can be used in a statistically significant manner to identify cis-embedded segments in a protein linked to its molecular function. The present study identifies novel cis-peptide fragments, which are subsequently used for fragment-based function annotation. Annotation recall benchmarks interpreted using the receiver-operator characteristic plot returned an area-under-curve >0.9, corroborating the utility of the annotation method. In addition, we identified cis-peptide fragments occurring in conjunction with functionally important trans-peptide fragments, providing additional insights into molecular function. We further illustrate the applicability of our method in function annotation where homology-based annotation transfer is not possible. The findings of the present study add to the repertoire of function annotation approaches and also facilitate engineering, design and allied studies around the cis-peptide neighborhood of proteins.

Bioengineering of AAV2 Capsid at Specific Serine, Threonine, or Lysine Residues Improves Its Transduction Efficiency in Vitro and in Vivo

We hypothesized that the AAV2 vector is targeted for destruction in the cytoplasm by the host cellular kinase/ubiquitination/proteasomal machinery and that modification of their targets on AAV2 capsid may improve its transduction efficiency. In vitro analysis with pharmacological inhibitors of cellular serine/threonine kinases (protein kinase A, protein kinase C, casein kinase II) showed an increase (20-90%) on AAV2-mediated gene expression. The three-dimensional structure of AAV2 capsid was then analyzed to predict the sites of ubiquitination and phosphorylation. Three phosphodegrons, which are the phosphorylation sites recognized as degradation signals by ubiquitin ligases, were identified. Mutation targets comprising eight serine (S) or seven threonine (T) or nine lysine (K) residues were selected in and around phosphodegrons on the basis of their solvent accessibility, overlap with the receptor binding regions, overlap with interaction interfaces of capsid proteins, and their evolutionary conservation across AAV serotypes. AAV2-EGFP vectors with the wild-type (WT) capsid or mutant capsids (15 S/T -> alanine A] or 9 K -> arginine R] single mutant or 2 double K -> R mutants) were then evaluated in vitro. The transduction efficiencies of 11 S/T -> A and 7 K -> R vectors were significantly higher (similar to 63-90%) than the AAV2-WT vectors (similar to 30-40%). Further, hepatic gene transfer of these mutant vectors in vivo resulted in higher vector copy numbers (up to 4.9-fold) and transgene expression (up to 14-fold) than observed from the AAV2-WT vector. One of the mutant vectors, S489A, generated similar to 8-fold fewer antibodies that could be cross-neutralized by AAV2-WT. This study thus demonstrates the feasibility of the use of these novel AAV2 capsid mutant vectors in hepatic gene therapy.

Structural and mechanistic motifs in membrane proteins: the three-dimensional modelling of rhodopsin, band 3, and the nicotinic acetylcholine receptor

Because so little is known about the structure of membrane proteins, an attempt has been made in this work to develop techniques by which to model them in three dimensions. The procedures devised rely heavily upon the availability of several sequences of a given protein. The modelling procedure is composed of two parts. The first identifies transmembrane regions within the protein sequence on the basis of hydrophobicity, β-turn potential, and the presence of certain amino acid types, specifically, proline and basic residues. The second part of the procedure arranges these transmembrane helices within the bilayer based upon the evolutionary conservation of their residues. Conserved residues are oriented toward other helices and variable residues are positioned to face the surrounding lipids. Available structural information concerning the protein's helical arrangement, including the lengths of interhelical loops, is also taken into account. Rhodopsin, band 3, and the nicotinic acetylcholine receptor have all been modelled using this methodology, and mechanisms of action could be proposed based upon the resulting structures.

Specific residues in the rhodopsin and iodopsin sequences were identified, which may regulate the proteins' wavelength selectivities. A hinge-like motion of helices M3, M4, and M5 with respect to the rest of the protein was proposed to result in the activation of transducin, the G-protein associated with rhodopsin. A similar mechanism is also proposed for signal transduction by the muscarinic acetylcholine and β-adrenergic receptors.

The nicotinic acetylcholine receptor was modelled with four trans-membrane helices per subunit and with the five homologous M2 helices forming the cation channel. Putative channel-lining residues were identified and a mechanism of channel-opening based upon the concerted, tangential rotation of the M2 helices was proposed.

Band 3, the anion exchange protein found in the erythrocyte membrane, was modelled with 14 transmembrane helices. In general the pathway of anion transport can be viewed as a channel composed of six helices that contains a single hydrophobic restriction. This hydrophobic region will not allow the passage of charged species, unless they are part of an ion-pair. An arginine residue located near this restriction is proposed to be responsible for anion transport. When ion-paired with a transportable anion it rotates across the barrier and releases the anion on the other side of the membrane. A similar process returns it to its original position. This proposed mechanism, based on the three-dimensional model, can account for the passive, electroneutral, anion exchange observed for band 3. Dianions can be transported through a similar mechanism with the additional participation of a histidine residue. Both residues are located on M10.

Interactions of chromatin context, binding site sequence content, and sequence evolution in stress-induced p53 occupancy and transactivation.

Cellular stresses activate the tumor suppressor p53 protein leading to selective binding to DNA response elements (REs) and gene transactivation from a large pool of potential p53 REs (p53REs). To elucidate how p53RE sequences and local chromatin context interact to affect p53 binding and gene transactivation, we mapped genome-wide binding localizations of p53 and H3K4me3 in untreated and doxorubicin (DXR)-treated human lymphoblastoid cells. We examined the relationships among p53 occupancy, gene expression, H3K4me3, chromatin accessibility (DNase 1 hypersensitivity, DHS), ENCODE chromatin states, p53RE sequence, and evolutionary conservation. We observed that the inducible expression of p53-regulated genes was associated with the steady-state chromatin status of the cell. Most highly inducible p53-regulated genes were suppressed at baseline and marked by repressive histone modifications or displayed CTCF binding. Comparison of p53RE sequences residing in different chromatin contexts demonstrated that weaker p53REs resided in open promoters, while stronger p53REs were located within enhancers and repressed chromatin. p53 occupancy was strongly correlated with similarity of the target DNA sequences to the p53RE consensus, but surprisingly, inversely correlated with pre-existing nucleosome accessibility (DHS) and evolutionary conservation at the p53RE. Occupancy by p53 of REs that overlapped transposable element (TE) repeats was significantly higher (p<10-7) and correlated with stronger p53RE sequences (p<10-110) relative to nonTE-associated p53REs, particularly for MLT1H, LTR10B, and Mer61 TEs. However, binding at these elements was generally not associated with transactivation of adjacent genes. Occupied p53REs located in L2-like TEs were unique in displaying highly negative PhyloP scores (predicted fast-evolving) and being associated with altered H3K4me3 and DHS levels. These results underscore the systematic interaction between chromatin status and p53RE context in the induced transactivation response. This p53 regulated response appears to have been tuned via evolutionary processes that may have led to repression and/or utilization of p53REs originating from primate-specific transposon elements.

HOX genes: seductive science, mysterious mechanisms

HOX genes are evolutionarily highly conserved. The HOX proteins which they encode are master regulators of embryonic development and continue to be expressed throughout postnatal life. The 39 human HOX genes are located in four clusters (A-D) on different chromosomes at 7p15, 17q21 [corrected] 12q13, and 2q31 respectively and are assumed to have arisen by duplication and divergence from a primordial homeobox gene. Disorders of limb formation, such as hand-foot-genital syndrome, have been traced to mutations in HOXA13 and HOXD13. Evolutionary conservation provides unlimited scope for experimental investigation of the functional control of the Hox gene network which is providing important insights into human disease. Chromosomal translocations involving the MLL gene, the human homologue of the Drosophila gene trithorax, create fusion genes which exhibit gain of function and are associated with aggressive leukaemias in both adults and children. To date 39 partner genes for MLL have been cloned from patients with leukaemia. Models based on specific translocations of MLL and individual HOX genes are now the subject of intense research aimed at understanding the molecular programs involved, and ultimately the design of chemotherapeutic agents for leukaemia. Investigation of the role of HOX genes in cancer has led to the concept that oncology may recapitulate ontology, a challenging postulate for experimentalists in view of the functional redundancy implicit in the HOX gene network.

Differential expression of new LTA splice variants upon lymphocyte activation

Lymphotoxin alpha (LTA) is a member of the TNF cytokine superfamily, produced principally by lymphocytes. It plays an important role in immune and inflammatory responses. Many TNF superfamily members have functionally important isoforms generated by alternative splicing but alternative splicing of LTA has never been studied. The known LTA protein is encoded by a transcript containing four exons. Here we report seven new LTA splice variants, three of them evolutionary conserved. We demonstrate their presence in cytoplasmic RNA suggesting that they could be translated into new LTA isoforms. We observed that their expression is differentially regulated upon activation of peripheral blood mononuclear cells and lymphocyte subpopulations (CD4+, CD8+, and CD19+). Our data suggest that the new LTA splice variants might play a role in the regulation of the immune response. (C) 2007 Elsevier Ltd. All rights reserved.

Conserved tissue expression signatures of intronic noncoding RNAs transcribed from human and mouse loci

It has been postulated that noncoding RNAs (ncRNAs) are involved in the posttranscriptional control of gene expression, and may have contributed to the emergence of the complex attributes observed in mammalians. We show here that the complement of ncRNAs expressed from intronic regions of the human and mouse genomes comprises at least 78,147 and 39,660 transcriptional units, respectively. To identify conserved intronic sequences expressed in both humans and mice, we used custom-designed human cDNA microarrays to separately interrogate RNA from mouse and human liver, kidney, and prostate tissues. An overlapping tissue expression signature was detected for both species, comprising 198 transcripts; among these, 22 RNAs map to intronic regions with evidence of evolutionary conservation in humans and mice. Transcription of selected human-mouse intronic ncRNAs was confirmed using strand-specific RT-PCR. Altogether, these results support an evolutionarily conserved role of intronic ncRNAs in human and mouse, which are likely to be involved in the fine tuning of gene expression regulation in different mammalian tissues. (C) 2008 Elsevier Inc. All rights reserved.

Functional XPB/RAD25 redundancy in Arabidopsis genome: characterization of AtXPB2 and expression analysis

The xeroderma pigmentosum complementation group B (XPB) protein is involved in both DNA repair and transcription in human cells. It is a component of the transcription factor IIH (TFIIH) and is responsible for DNA helicase activity during nucleotide (nt) excision repair (NER). Its high evolutionary conservation has allowed identification of homologous proteins in different organisms, including plants. In contrast to other organisms, Arabidopsis thaliana harbors a duplication of the XPB orthologue (AtXPB1 and AtXPB2), and the proteins encoded by the duplicated genes are very similar (95% amino acid identity). Complementation assays in yeast rad25 mutant strains suggest the involvement of AtXPB2 in DNA repair, as already shown for AtXPB1, indicating that these proteins may be functionally redundant in the removal of DNA lesions in A. thaliana. Although both genes are expressed in a constitutive manner during the plant life cycle, Northern blot analyses suggest that light modulates the expression level of both XPB copies, and transcript levels increase during early stages of development. Considering the high similarity between AtXPB1 and AtXPB2 and that both of predicted proteins may act in DNA repair, it is possible that this duplication may confer more flexibility and resistance to DNA damaging agents in thale cress. (C) 2004 Elsevier B.V. All rights reserved.

Laminin-binding epitope on gp43 from Paracoccidioides brasiliensis is recognized by a monoclonal antibody raised against Staphylococcus aureus laminin receptor

Adhesion is regarded as an important step in the pathogenesis of several microorganisms. Thus, the ability to recognize extracellular matrix proteins, such as laminin or fibronectin, has been correlated with invasiveness. Studying the already characterized laminin-binding protein of Paracoccidioides brasiliensis, the 43 kDa glycoprotein (gp43), we evaluated whether MAb 1.H12, raised against the laminin-binding protein from Staphylococcus aureus, cross-reacts with that fungal protein. By immunoblot analysis we show that MAb 1.H12 recognizes gp43. This interaction is able to inhibit the laminin-mediated adhesion to epithelial cells as well as the P. brasiliensis infection in vivo. Moreover, through immunoenzymatic assays, we show that MAb 1.H12 recognizes gp43 in solid phase and that this interaction is partially inhibited by the addition of anti-gp43 MAbs. These results show that MAb 1.H12 recognizes the gp43, suggesting the presence of an epitope similar to those found in the other laminin-binding proteins from phylogenetically very distant cells. These findings reinforce the possibility of evolutionary conservation of such epitopes.

Comparative analysis of noncoding sequences of orthologous bovine and human gene pairs

Genomic sequence comparison across species has enabled the elucidation of important coding and regulatory sequences encoded within DNA. Of particular interest are the noncoding regulatory sequences, which influence gene transcriptional and posttranscriptional processes. A phylogenetic footprinting strategy was employed to identify noncoding conservation patterns of 39 human and bovine orthologous genes. Seventy-three conserved noncoding sequences were identified that shared greater than 70% identity over at least 100 bp. Thirteen of these conserved sequences were also identified in the mouse genome. Evolutionary conservation of noncoding sequences across diverse species may have functional significance, and these conserved sequences may be good candidates for regulatory elements.

Characterization of esterase patterns in hepatopancreas of three species of Macrobrachium (Palaemonidae)

The genus Macrobrachium (Bate, 1868) belongs to the Palaemonidae family. These species are commonly found in lakes, floodplains and rivers in tropical and subtropical regions of South America. The Macrobrachium genus encompasses nearly 210 species of ecological and economic importance. In this study, three species of Macrobrachium (M acrobrachium jelskii, M acrobrachium amazonicum and M acrobrachium brasiliense) were studied in order to characterize the esterase patterns in the hepatopancreas, which were still unknown. Esterases are enzymes which catalyze the hydrolysis of esters. In the hepatopancreas, these enzymes play important roles in several metabolic processes involved in some functions of this organ, such as detoxification and digestion. Twelve esterase bands (EST1 to EST12) were detected in these species, and a comparison among them showed no qualitative differences in interspecific bands, or between males and females. Inhibitors were used to classify the esterase bands. The results indicated seven acetylesterases, two carboxylesterases, one arylesterase, and one cholinesterase. The EST11 band was not detected in these procedures because of its lower frequency. Statistical analyses showed no variability among the species, in either interspecific or intraspecific assays. These results support the hypothesis of a high evolutionary conservation of esterases in the hepatopancreas of these crustaceans. The data enabled us to assess the genetic structure of these species through the use of esterasic enzymes. It also contributes to our knowledge about the biology of these poorly studied species. Knowledge on the genetic structure of populations and species are essential when defining priorities for their management and conservation. © 2012 Elsevier Ltd.