Identification of Chromobacterium violaceum genes with potential biotechnological application in environmental detoxification

Chromobacterium violaceum is a Gram-negative bacterium found in a wide variety of tropical and subtropical ecosystems. The complete genome sequence of C. violaceum ATCC 12472 is now available, and it has considerable biotechnological potential for various applications, such as environmental detoxification, as well as medical and agricultural use. We examined the biotechnological potential of C. violaceum for environmental detoxification. Three operons, comprising the ars operon, involved in arsenic resistance, the cyn operon, involved in cyanate detoxification, and the hcn operon, encoding a cyanase, responsible for biogenic production of cyanide, as well as an open reading frame, encoding an acid dehalogenase, were analyzed in detail. Probable catalytic mechanisms for the enzymes were determined, based on amino acid sequence comparisons and on published structural information for these types of proteins.     

Sequence-specific oligonucleotide purification using peptide nucleic acid amphiphiles in hydrophobic interaction chromatography

We present a methodology to perform sequence-specific separations of oligonucleotides using peptide nucleic acids covalently linked to alkane chains, or "PNA amphiphiles (PNAAs)". The PNAA/DNA duplexes are discriminated from unbound DNA using hydrophobic interaction chromatography on a phenyl-substituted Sepharose column. Nearly quantitative recovery is achieved at concentrations of 50 microM after incubation of oligomers with a stoichiometric amount of PNAA for 1 min or so. The method exhibits high sequence specificity, selectivity, and resolution when applied to mixtures of various oligomers up to 60 base pairs in length.     

In vivo bioluminescence imaging for integrated studies of infection

Understanding biological processes in the context of intact organ systems with fine temporal resolution has required the development of imaging strategies that reveal cellular and molecular changes in the living body. Reporter genes that confer optical signatures on a given biological process have been used widely in cell biology and have been used more recently to interrogate biological processes in living animal models of human biology and disease. The use of internal biological sources of light, luciferases, to tag cells, pathogens, and genes has proved to be a versatile tool to provide in vivo indicators that can be detected externally. The application of this technology to the study of animal models of infectious disease has not only provided insights into disease processes, but has also revealed new mechanisms by which pathogens may avoid host defences during infection.     

Structural genomics of membrane proteins

Improvements in the fields of membrane-protein molecular biology and biochemistry, technical advances in structural data collection and processing, and the availability of numerous sequenced genomes have paved the way for membrane-protein structural genomics efforts. There has been significant recent progress, but various issues essential for high-throughput membrane-protein structure determination remain to be resolved.     

POSaM: a fast,flexible, open-source,inkjet oligonucleotide synthesizer and microarrayer

DNA arrays are valuable tools in molecular biology laboratories. Their rapid acceptance was aided by the release of plans for a pin-spotting microarrayer by researchers at Stanford. Inkjet microarraying is a flexible, complementary technique that allows the synthesis of arrays of any oligonucleotide sequences de novo. We describe here an open-source inkjet arrayer capable of rapidly producing sets of unique 9,800-feature arrays.     

Natively unfolded domains in endocytosis: hooks, lines and linkers

It is commonly assumed that a protein must adopt a tertiary structure to achieve its active native state and that regions of a protein that are devoid of alpha-helix or beta-sheet structures are functionally inert. Although extended proline-rich regions are recognized as presenting binding motifs to, for example, Src homology 2 (SH2) and SH3 domains, the idea persists that natively unfolded regions in functional proteins are simply 'spacers' between the folded domains. Such a view has been challenged in recent years and the importance of natively unfolded proteins in biology is now being recognized. In this review, we highlight the role of natively unfolded domains in the field of endocytosis, and show that some important endocytic proteins lack a traditionally folded structure and harbour important binding motifs in their unstructured linker regions.     

Endocytic recycling

After endocytosis, most membrane proteins and lipids return to the cell surface, but some membrane components are delivered to late endosomes or the Golgi. We now understand that the pathways taken by internalized molecules that eventually recycle to the cell surface can be surprisingly complex and can involve a series of sorting events that occur in several organelles. The molecular basis for many of these sorting processes is only partly understood.     

Identification of cardiac malformations in mice lacking <Emphasis Type="Italic">Ptdsr</Emphasis>using a novel high-throughput magnetic resonance imaging technique

Background  

Congenital heart defects are the leading non-infectious cause of death in children. Genetic studies in the mouse have been crucial to uncover new genes and signaling pathways associated with heart development and congenital heart disease. The identification of murine models of congenital cardiac malformations in high-throughput mutagenesis screens and in gene-targeted models is hindered by the opacity of the mouse embryo.     

Isotope-differentiated binding energy shift tags (IDBEST) for improved targeted biomarker discovery and validation

Mass spectrometry has proved to be an important tool for protein biomarker discovery, identification and characterization. However, global proteomic profiling strategies often fail to identify known low-abundance biomarkers as a result of the limited dynamic range of mass spectrometry (two to three orders of magnitude) compared with the large dynamic range of protein concentrations in biologic fluids (11 to 12 orders of magnitude for serum). In addition, the number of peptides generated in such methods vastly overwhelms the resolution capacity of mass spectrometers, requiring extensive sample clean-up (e.g., affinity tag, retentate chromatography and/or high-performance liquid chromatography) before mass spectrometry analysis. Baiting and affinity pre-enrichment strategies, which overcome the dynamic range and sample complexity issues of global proteomic strategies, are very difficult to couple to mass spectrometry. This is due to the fact that it is nearly impossible to sort target peptides from those of the bait since there will be many cases of isobaric peptides. IDBEST (Target Discovery, Inc.) is a new tagging strategy that enables such pre-enrichment of specific proteins or protein classes as the resulting tagged peptides are distinguishable from those of the bait by a mass defect shift of approximately 0.1 atomic mass units. The special characteristics of these tags allow: resolution of tagged peptides from untagged peptides through incorporation of a mass defect element; high-precision quantitation of up- and downregulation by using stable isotope versions of the same tag; and potential analysis of protein isoforms through more complete peptide coverage from the proteins of interest.