Cloning and expression of the toxin B gene ofClostridium difficile

Results from our cloning studies on toxin A indicated that the gene for toxin B resided approximately 1 kb upstream of the toxin A gene. Clone pCD19, which contains the 5-end of the toxin A gene and a small open reading frame, was found to contain 1.2 kb of DNA which, when subcloned, expressed a nontoxic peptide that reacted with toxin B antibodies. The rest of the toxin B gene was located on the 6.8 kb cloned fragment of plasmid pCD19L. The two fragments overlapped 0.8 kb. Lysates containing protein expressed by the 6.8 fragment were cytotoxic and lethal, and were neutralized by toxin B antibody. The two fragments were ligated to give the complete toxin B gene. The protein expressed by the complete gene was cytotoxic and lethal, and showed complete immunological identity with toxin B. Further analysis of the expressed protein and the toxin B gene confirmed our earlier findings showing that toxin B has a molecular weight of 240,000 or greater.     

Multistep mass tagging coupled with 2D LC-MS--an approach to increasing the number of identified proteins.

Identification of large numbers of proteins from complex biological samples is a continuing challenge in the area of quantitative proteomics. We introduce here a simple and reliable multistep mass tagging technique using our recently developed solid phase mass tagging reagents. When coupled with two-dimensional liquid chromatography/nano-electrospray ionization ion trap mass spectrometry (2D-LC/nano-ESI-MS), this method allows enhanced protein identification when tested on samples from prokaryotic and eukaryotic sources. The proteome of Escherichia coli D21 grown to either mid-exponential or stationary phase, and the membrane proteome from established breast cancer cell lines BT474 and MCF7 were used as model systems in these experiments. In both experiments, the numbers of total identified proteins are at least twice the numbers identified from a single tagging cycle. The sample complexity can be effectively reduced with corresponding increases in protein identification using the multistep method. The strategy described here represents a potentially powerful technique for large-scale qualitative and quantitative proteome research.     

Thomitrems A and E,two indole-alkaloid isoprenoids from Penicillium crustosum Thom

Two indole-alkaloid isoprenoids were isolated from extracts of Penicillium crustosum Thom grown on rice. Their structures were elucidated on the basis of various NMR experiments and by comparison to the structurally related penitrems. The two compounds, designated thomitrem A and thomitrem E, contain a 18(19)-double bond and lack the characteristic penitrem 17(18)-ether linkage.     

Transparent Plastic Incubator for the Anaerobic Glove Box

An incubator designed for use inside an anaerobic glove box is described. The incubator is made of transparent plastic material, has sliding plastic doors, and can be made in various sizes from readily available materials.     

The BSP30 salivary proteins from cattle,LUNX/PLUNC and von Ebner's minor salivary gland protein are members of the PSP/LBP superfamily of proteins

Saliva influences rumen function in cattle, yet the biochemical role for most of the bovine salivary proteins (BSPs) has yet to be established. Two cDNAs (BSP30a and BSP30b) from bovine parotid salivary gland were cloned and sequenced, each coding for alternate forms of a prominent protein in bovine saliva. The BSP30 cDNAs share 96% sequence identity with each other at the DNA level and 83% at the amino acid level, and appear to arise from separate genes. The predicted BSP30a and BSP30b proteins share 26-36% amino acid identity with parotid secretory protein (PSP) from mouse, rat and human. BSP30 and PSP are in turn more distantly related to a wider group of proteins that includes lung-specific X protein, also known as palate, lung, and nasal epithelium clone (LUNX/PLUNC), von Ebner's minor salivary gland protein (VEMSGP), bactericidal permeability increasing protein (BPI), lipopolysaccharide binding protein (LBP), cholesteryl ester transfer protein (CETP), and the putative olfactory ligand-binding proteins RYA3 and RY2G5. Bovine cDNAs encoding homologs of LUNX/PLUNC and VEMSGP were isolated and sequenced. Northern blot analysis showed that LUNX/PLUNC, BSP30 and VEMSGP are expressed in bovine salivary tissue and airways, and that they have non-identical patterns of expression in these tissues. The expression of both BSP30a and BSP30b is restricted to salivary tissue, but within this tissue they have distinct patterns of expression. The proximity of the human genes coding for the PSP/LBP superfamily on HSA20q11.2, their similar amino acid sequence, and common exon segmentation strongly suggest that these genes evolved from a common ancestral gene. Furthermore, they imply that the BSP30a and BSP30b proteins may have a function in common with other members of this gene family.     

Real-time measurements of DNA hybridization on microparticles with fluorescence resonance energy transfer

When capture oligonucleotides are tethered on planar surfaces, mass transport limitations influence the kinetics of solid-phase nucleic acid hybridizations. By diffusion theory, however, hybridization of oligonucleotides on microparticles should be reaction-rate limited. In an initial effort to understand the kinetics of microparticle hybridization reactions, we developed a fluorescence resonance energy transfer method for monitoring oligonucleotide hybridization on microparticles. Microparticles were coated with a fluoresceinated oligomer at surface densities of 20, 40, and 80% saturation, hybridized to a complementary oligonucleotide labeled with tetramethylrhodamine, and monitored over time for quenching of the fluorescein signal as hybridization occurred on the particle surface. Association rate constants were compared for microparticle-based hybridization and solution-phase hybridization. Rate constants for hybridizations on the particle surface were about an order of magnitude less than those for hybridization in solution, but decreasing the surface density of the capture oligonucleotide to 20% saturation improved particle hybridization rates. Although a bimolecular reaction model adequately described solution-phase hybridization kinetics, oligonucleotide hybridization on microparticles did not fit this model but exhibited biphasic reaction kinetics. Based on two different lines of reasoning, we argue that microparticle-based oligonucleotide hybridization was indeed reaction-rate limited in our system and not diffusion-rate limited.