Theoretical prediction of the binding free energy for mutants of replication protein A

The replication protein A (RPA) is a heterotrimeric (70, 32, and 14 kDa subunits), single stranded DNA (ssDNA) binding protein required for pivotal functions in the cell metabolism, such as chromosomal replication, prevention of hairpin formation, DNA repair and recombination, and signaling after DNA damage. Studies based on deletions and mutations have identified the high affinity ssDNA binding domains in the 70 kDa subunit of RPA, regions A and B. Individually, the domain A and B have a low affinity for ssDNA, while tandems composed of AA, AB, BB, and BA sequences bind the ssDNA with moderate to high affinity. Single and double point mutations on polar residues in the binding domains leads to a reduction in affinity of RPA for ssDNA, in particular when two hydrophilic residues are involved. In view of these results, we performed a study based on molecular dynamics simulation aimed to reproduce the experimental change in binding free energy, ΔΔG, of RPA70 mutants to further elucidate the nature of the protein-ssDNA interaction. The MM-PB(GB)SA methods implemented in Amber10 and the code FoldX were used to estimate the binding free energy. The theoretical and experimental ΔΔG values correlate better when the results are obtained by MM-PBSA calculated on individual trajectories for each mutant. In these conditions, the correlation coefficient between experimental and theoretical ΔΔG reaches a value of 0.95 despite the overestimation of the energy change by one order of magnitude. The decomposition of the MM-GBSA energy per residue allows us to correlate the change of the affinity with the residue polarity and energy contribution to the binding. The method revealed reliable predictions of the change in the affinity in function of mutations, and can be used to identify new mutants with distinct binding properties.     

The RBP-Jκ Binding Sites within the RTA Promoter Regulate KSHV Latent Infection and Cell Proliferation

Kaposi''s sarcoma-associated herpesvirus (KSHV) is tightly linked to at least two lymphoproliferative disorders, primary effusion lymphoma (PEL) and multicentric Castleman''s disease (MCD). However, the development of KSHV-mediated lymphoproliferative disease is not fully understood. Here, we generated two recombinant KSHV viruses deleted for the first RBP-Jκ binding site (RTA_1st) and all three RBP-Jκ binding sites (RTA_all) within the RTA promoter. Our results showed that RTA_1st and RTA_all recombinant viruses possess increased viral latency and a decreased capability for lytic replication in HEK 293 cells, enhancing colony formation and proliferation of infected cells. Furthermore, recombinant RTA_1st and RTA_all viruses showed greater infectivity in human peripheral blood mononuclear cells (PBMCs) relative to wt KSHV. Interestingly, KSHV BAC36 wt, RTA_1st and RTA_all recombinant viruses infected both T and B cells and all three viruses efficiently infected T and B cells in a time-dependent manner early after infection. Also, the capability of both RTA_1st and RTA_all recombinant viruses to infect CD19+ B cells was significantly enhanced. Surprisingly, RTA_1st and RTA_all recombinant viruses showed greater infectivity for CD3+ T cells up to 7 days. Furthermore, studies in Telomerase-immortalized human umbilical vein endothelial (TIVE) cells infected with KSHV corroborated our data that RTA_1st and RTA_all recombinant viruses have enhanced ability to persist in latently infected cells with increased proliferation. These recombinant viruses now provide a model to explore early stages of primary infection in human PBMCs and development of KSHV-associated lymphoproliferative diseases.     

Synonymous codon usage in forty staphylococcal phages identifies the factors controlling codon usage variation and the phages suitable for phage therapy

The immergence and dissemination of multidrug-resistant strains of Staphylococcus aureus in recent years have expedited the research on the discovery of novel anti-staphylococcal agents promptly. Bacteriophages have long been showing tremendous potentialities in curing the infections caused by various pathogenic bacteria including S. aureus. Thus far, only a few virulent bacteriophages, which do not carry any toxin-encoding gene but are capable of eradicating staphylococcal infections, were reported. Based on the codon usage analysis of sixteen S. aureus phages, previously three phages were suggested to be useful as the anti-staphylococcal agents. To search for additional S. aureus phages suitable for phage therapy, relative synonymous codon usage bias has been investigated in the protein-coding genes of forty new staphylococcal phages. All phages appeared to carry A and T ending codons. Several factors such as mutational pressure, translational selection and gene length seemed to be responsible for the codon usage variation in the phages. Codon usage indeed varied phage to phage. Of the phages, phages G1, Twort, 66 and Sap-2 may be extremely lytic in nature as majority of their genes possess high translational efficiency, indicating that these phages may be employed in curing staphylococcal infections.     

Molecular Cloning and Characterization of Novel Morus alba Germin-Like Protein Gene Which Encodes for a Silkworm Gut Digestion-Resistant Antimicrobial Protein

Background

Silkworm fecal matter is considered one of the richest sources of antimicrobial and antiviral protein (substances) and such economically feasible and eco-friendly proteins acting as secondary metabolites from the insect system can be explored for their practical utility in conferring broad spectrum disease resistance against pathogenic microbial specimens.

Methodology/Principal Findings

Silkworm fecal matter extracts prepared in 0.02 M phosphate buffer saline (pH 7.4), at a temperature of 60°C was subjected to 40% saturated ammonium sulphate precipitation and purified by gel-filtration chromatography (GFC). SDS-PAGE under denaturing conditions showed a single band at about 21.5 kDa. The peak fraction, thus obtained by GFC wastested for homogeneityusing C18reverse-phase high performance liquid chromatography (HPLC). The activity of the purified protein was tested against selected Gram +/− bacteria and phytopathogenic Fusarium species with concentration-dependent inhibitionrelationship. The purified bioactive protein was subjected to matrix-assisted laser desorption and ionization-time of flight mass spectrometry (MALDI-TOF-MS) and N-terminal sequencing by Edman degradation towards its identification. The N-terminal first 18 amino acid sequence following the predicted signal peptide showed homology to plant germin-like proteins (Glp). In order to characterize the full-length gene sequence in detail, the partial cDNA was cloned and sequenced using degenerate primers, followed by 5′- and 3′-rapid amplification of cDNA ends (RACE-PCR). The full-length cDNA sequence composed of 630 bp encoding 209 amino acids and corresponded to germin-like proteins (Glps) involved in plant development and defense.

Conclusions/Significance

The study reports, characterization of novel Glpbelonging to subfamily 3 from M. alba by the purification of mature active protein from silkworm fecal matter. The N-terminal amino acid sequence of the purified protein was found similar to the deduced amino acid sequence (without the transit peptide sequence) of the full length cDNA from M. alba.     

Identification and characterization of the Arabidopsis gene encoding the tetrapyrrole biosynthesis enzyme uroporphyrinogen III synthase

UROS (uroporphyrinogen III synthase; EC 4.2.1.75) is the enzyme responsible for the formation of uroporphyrinogen III, the precursor of all cellular tetrapyrroles including haem, chlorophyll and bilins. Although UROS genes have been cloned from many organisms, the level of sequence conservation between them is low, making sequence similarity searches difficult. As an alternative approach to identify the UROS gene from plants, we used functional complementation, since this does not require conservation of primary sequence. A mutant of Saccharomyces cerevisiae was constructed in which the HEM4 gene encoding UROS was deleted. This mutant was transformed with an Arabidopsis thaliana cDNA library in a yeast expression vector and two colonies were obtained that could grow in the absence of haem. The rescuing plasmids encoded an ORF (open reading frame) of 321 amino acids which, when subcloned into an Escherichia coli expression vector, was able to complement an E. coli hemD mutant defective in UROS. Final proof that the ORF encoded UROS came from the fact that the recombinant protein expressed with an N-terminal histidine-tag was found to have UROS activity. Comparison of the sequence of AtUROS (A. thaliana UROS) with the human enzyme found that the seven invariant residues previously identified were conserved, including three shown to be important for enzyme activity. Furthermore, a structure-based homology search of the protein database with AtUROS identified the human crystal structure. AtUROS has an N-terminal extension compared with orthologues from other organisms, suggesting that this might act as a targeting sequence. The precursor protein of 34 kDa translated in vitro was imported into isolated chloroplasts and processed to the mature size of 29 kDa. Confocal microscopy of plant cells transiently expressing a fusion protein of AtUROS with GFP (green fluorescent protein) confirmed that AtUROS was targeted exclusively to chloroplasts in vivo.     

Expression patterns of glycine transporters (xGlyT1, xGlyT2, and xVIAAT) in Xenopus laevis during early development

Glycine, a major inhibitory neurotransmitter in the vertebrate nervous system, not only functions in synaptic signaling, but has also been implicated in regulating neuronal differentiation, neuronal proliferation, synaptic modeling, and neural network stability. Elements of the glycinergic phenotype include the membrane-bound glycine transporters (GLYT1 and GLYT2), which remove glycine from the synaptic cleft, and the vesicular inhibitory amino acid transporter (VIAAT or VGAT), which sequesters both glycine and GABA into synaptic vesicles. Here, we describe the spatial and temporal expression patterns of xGlyT1, xGlyT2, and xVIAAT during early developmental stages of Xenopus laevis. In situ hybridization reveals that xGlyT1 is first expressed in early tailbud stages in the midbrain, hindbrain, and anterior spinal cord; it extends posteriorly through the spinal cord and appears in the forebrain, retina, between the somites, and in the blood islands by swimming tadpole stages. xGlyT2 and xVIAAT initially appear in late neurula stages in the anterior spinal cord. By swimming tadpole stages, the expression of these genes appears in the forebrain, midbrain, and hindbrain and extends posteriorly through the spinal cord; xVIAAT is also expressed in the retina. Confocal analysis of multiplex fluorescent in situ hybridization signal in the spinal cord reveals that xGlyT1 and xGlyT2 share little cellular colocalization. While there is significant coexpression between xVIAAT and xGlyT2, xVIAAT and the GABAergic marker glutamic acid decarboxylase (xGAD67), and xGlyT2 and xGAD67, each gene also appears to have discrete, non-colocalized areas of expression.     

Computational Approaches and Tools Used in Identification of Dispersed Repetitive DNA Sequences

It has become clear that dispersed repeat sequences have played multiple roles in eukaryotic genome evolution including increasing genetic diversity through mutation, inducing changes in gene expression, and facilitating generation of novel genes. Growing recognition of the importance of dispersed repeats has fueled development of computational tools designed to expedite discovery and classification of repeats. Here we review major existing repeat exploration tools and discuss the algorithms utilized by these tools. Special attention is devoted to ab initio programs, i.e., those tools that do not rely upon previously identified repeats to find new repeat elements. We conclude by discussing the strengths and weaknesses of current tools and highlighting additional approaches that may advance repeat discovery/characterization.     

Identification and characterization of rabbit small intestinal villus cell brush border membrane Na-glutamine cotransporter

Glutamine, the primary metabolic fuel for the mammalian small intestinal enterocytes, is primarily assimilated by Na-amino acid cotransporters. Although Na-solute cotransport has been shown to exist in the brush border membrane (BBM) of the absorptive villus cells, the identity of Na-glutamine cotransport in rabbit small intestinal villus cells was unknown. Na-dependent glutamine uptake is present in villus BBM vesicles. An intravesicular proton gradient did not stimulate this Na-dependent glutamine uptake, whereas Li+ did not significantly suppress this uptake. These observations in concert with amino acid substitution studies suggested that Na-glutamine cotransporter in the villus cell BBM was the newly identified cotransporter B0AT1 (SLC6A19). Quantitative real-time PCR identified the message for this cotransporter in villus cells. Thus a full-length cDNA of B0AT1 was cloned and expressed in MDA-MB-231 cells. This expressed cotransporter exhibited characteristics similar to those observed in villus cells from the rabbit small intestine. Antibody was generated for B0AT1 that demonstrated the presence of this cotransporter protein in the villus cell BBM. Kinetic studies defined the kinetic parameters of this cotransporter. Thus this study describes the identification, cloning, and characterization of the Na-amino acid cotransporter responsible for the assimilation of a critical amino acid by the absorptive villus cells in the mammalian small intestine.