Hydrolysis of filter-paper cellulose to glucose by two recombinant endogenous glycosyl hydrolases of Coptotermes formosanus

Abstract Genes encoding for glycosyl hydrolases (GH) in multiple families were recovered from an expression sequence tag library of Coptotermes formosanus, a xylophagous lower termite species. Functional analyses of these genes not only shed light on the mechanisms the insect employs to successfully use cellulosic materials as energy sources, which may serve as strategic targets for designing molecular-based bio-pesticides, but also enrich discoveries of new cellulolytic enzymes for conversion of biomass into biofuel. Our study demonstrated that cellulose could be converted to glucose by two recombinant endogenous glycosyl hydrolases (endo-β-1,4 glucanase in GH9 and β-glucosidase in GH1). While the former cleaved cellulose to cellobiose and cellotriose, the resulting simple cellodextrins were digested to glucose. Both of the Escherichia coli-expressed recombinant proteins showed properties that could be incorporated in a glucose-based ethanol production program.     

Thymineless death is inhibited by CsrA in Escherichia coli lacking the SOS response

Thymineless death (TLD) is the rapid loss of colony-forming ability in bacterial, yeast and human cells starved for thymine, and is the mechanism of action of common chemotherapeutic drugs. In Escherichia coli, significant loss of viability during TLD requires the SOS replication-stress/DNA-damage response, specifically its role in inducing the inhibitor of cell division, SulA. An independent RecQ- and RecJ-dependent TLD pathway accounts for a similarly large additional component of TLD, and a third SOS- and RecQ/J-independent TLD pathway has also been observed. Although two groups have implicated the SOS-response in TLD, an SOS-deficient mutant strain from an earlier study was found to be sensitive to thymine deprivation. We performed whole-genome resequencing on that SOS-deficient strain and find that, compared with the SOS-proficient control strain, it contains five mutations in addition to the SOS-blocking lexA(Ind⁻) mutation. One of the additional mutations, csrA, confers TLD sensitivity specifically in SOS-defective strains. We find that CsrA, a carbon storage regulator, reduces TLD in SOS- or SulA-defective cells, and that the increased TLD that occurs in csrA⁻ SOS-defective cells is dependent on RecQ. We consider a hypothesis in which the modulation of nucleotide pools by CsrA might inhibit TLD specifically in SOS-deficient (SulA-deficient) cells.     

Method for hull‐less barley transformation and manipulation of grain mixed‐linkage beta‐glucan

Hull‐less barley is increasingly offering scope for breeding grains with improved characteristics for human nutrition; however, recalcitrance of hull‐less cultivars to transformation has limited the use of these varieties. To overcome this limitation, we sought to develop an effective transformation system for hull‐less barley using the cultivar Torrens. Torrens yielded a transformation efficiency of 1.8%, using a modified Agrobacterium transformation method. This method was used to over‐express genes encoding synthases for the important dietary fiber component, (1,3;1,4)‐β‐glucan (mixed‐linkage glucan), primarily present in starchy endosperm cell walls. Over‐expression of the HvCslF6 gene, driven by an endosperm‐specific promoter, produced lines where mixed‐linkage glucan content increased on average by 45%, peaking at 70% in some lines, with smaller increases in transgenic HvCslH1 grain. Transgenic HvCslF6 lines displayed alterations where grain had a darker color, were more easily crushed than wild type and were smaller. This was associated with an enlarged cavity in the central endosperm and changes in cell morphology, including aleurone and sub‐aleurone cells. This work provides proof‐of‐concept evidence that mixed‐linkage glucan content in hull‐less barley grain can be increased by over‐expression of the HvCslF6 gene, but also indicates that hull‐less cultivars may be more sensitive to attempts to modify cell wall composition.     

p120 catenin is required for normal renal tubulogenesis and glomerulogenesis

Defects in the development or maintenance of tubule diameter correlate with polycystic kidney disease. Here, we report that absence of the cadherin regulator p120 catenin (p120ctn) from the renal mesenchyme prior to tubule formation leads to decreased cadherin levels with abnormal morphologies of early tubule structures and developing glomeruli. In addition, mutant mice develop cystic kidney disease, with markedly increased tubule diameter and cellular proliferation, and detached luminal cells only in proximal tubules. The p120ctn homolog Arvcf is specifically absent from embryonic proximal tubules, consistent with the specificity of the proximal tubular phenotype. p120ctn knockdown in renal epithelial cells in 3D culture results in a similar cystic phenotype with reduced levels of E-cadherin and active RhoA. We find that E-cadherin knockdown, but not RhoA inhibition, phenocopies p120ctn knockdown. Taken together, our data show that p120ctn is required for early tubule and glomerular morphogenesis, as well as control of luminal diameter, probably through regulation of cadherins.     

Report from the second cytomegalovirus and immunosenescence workshop

The Second International Workshop on CMV & Immunosenescence was held in Cambridge, UK, 2-4th December, 2010. The presentations covered four separate sessions: cytomegalovirus and T cell phenotypes; T cell memory frequency, inflation and immunosenescence; cytomegalovirus in aging, mortality and disease states; and the immunobiology of cytomegalovirus-specific T cells and effects of the virus on vaccination. This commentary summarizes the major findings of these presentations and references subsequently published work from the presenter laboratory where appropriate and draws together major themes that were subsequently discussed along with new areas of interest that were highlighted by this discussion.     

Proteogenomics to discover the full coding content of genomes: A computational perspective

Proteogenomics has emerged as a field at the junction of genomics and proteomics. It is a loose collection of technologies that allow the search of tandem mass spectra against genomic databases to identify and characterize protein-coding genes. Proteogenomic peptides provide invaluable information for gene annotation, which is difficult or impossible to ascertain using standard annotation methods. Examples include confirmation of translation, reading-frame determination, identification of gene and exon boundaries, evidence for post-translational processing, identification of splice-forms including alternative splicing, and also, prediction of completely novel genes. For proteogenomics to deliver on its promise, however, it must overcome a number of technological hurdles, including speed and accuracy of peptide identification, construction and search of specialized databases, correction of sampling bias, and others. This article reviews the state of the art of the field, focusing on the current successes, and the role of computation in overcoming these challenges. We describe how technological and algorithmic advances have already enabled large-scale proteogenomic studies in many model organisms, including arabidopsis, yeast, fly, and human. We also provide a preview of the field going forward, describing early efforts in tackling the problems of complex gene structures, searching against genomes of related species, and immunoglobulin gene reconstruction.     

Epitope-specific human influenza antibody repertoires diversify by B cell intraclonal sequence divergence and interclonal convergence

We generated from a single blood sample five independent human mAbs that recognized the Sa antigenic site on the head of influenza hemagglutinin and exhibited inhibitory activity against a broad panel of H1N1 strains. All five Abs used the V(H)3-7 and J(H)6 gene segments, but at least four independent clones were identified by junctional analysis. High-throughput sequence analysis of circulating B cells revealed that each of the independent clones were members of complex phylogenetic lineages that had diversified widely using a pattern of progressive diversification through somatic mutation. Unexpectedly, B cells encoding multiple diverging lineages of these clones, including many containing very few mutations in the Ab genes, persisted in the circulation. Conversely, we noted frequent instances of amino acid sequence convergence in the Ag combining sites exhibited by members of independent clones, suggesting a strong selection for optimal binding sites. We suggest that maintenance in circulation of a wide diversity of somatic variants of dominant clones may facilitate recognition of drift variant virus epitopes that occur in rapidly mutating virus Ags, such as influenza hemagglutinin. In fact, these Ab clones recognize an epitope that acquired three glycosylation sites mediating escape from previously isolated human Abs.     

Over‐expression of specific HvCslF cellulose synthase‐like genes in transgenic barley increases the levels of cell wall (1,3;1,4)‐β‐d‐glucans and alters their fine structure

Cell walls in commercially important cereals and grasses are characterized by the presence of (1,3;1,4)‐β‐d ‐glucans. These polysaccharides are beneficial constituents of human diets, where they can reduce the risk of hypercholesterolemia, type II diabetes, obesity and colorectal cancer. The biosynthesis of cell wall (1,3;1,4)‐β‐d ‐glucans in the Poaceae is mediated, in part at least, by the cellulose synthase‐like CslF family of genes. Over‐expression of the barley CslF6 gene under the control of an endosperm‐specific oat globulin promoter results in increases of more than 80% in (1,3;1,4)‐β‐d ‐glucan content in grain of transgenic barley. Analyses of (1,3;1,4)‐β‐d ‐glucan fine structure indicate that individual CslF enzymes might direct the synthesis of (1,3;1,4)‐β‐d ‐glucans with different structures. When expression of the CslF6 transgene is driven by the Pro35S promoter, the transgenic lines have up to sixfold higher levels of (1,3;1,4)‐β‐d ‐glucan in leaves, but similar levels as controls in the grain. Some transgenic lines of Pro35S:CslF4 also show increased levels of (1,3;1,4)‐β‐d ‐glucans in grain, but not in leaves. Thus, the effects of CslF genes on (1,3;1,4)‐β‐d ‐glucan levels are dependent not only on the promoter used, but also on the specific member of the CslF gene family that is inserted into the transgenic barley lines. Altering (1,3;1,4)‐β‐d ‐glucan levels in grain and vegetative tissues will have potential applications in human health, where (1,3;1,4)‐β‐d ‐glucans contribute to dietary fibre, and in tailoring the composition of biomass cell walls for the production of bioethanol from cereal crop residues and grasses.