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Anthrax is a highly fatal disease caused by the gram-positive, endospore-forming, rod-shaped bacterium Bacillus anthracis. Spores, rather than vegetative bacterial cells, are the source of anthrax infections. Spores of B. anthracis are enclosed by a prominent loose-fitting structure called the exosporium. The exosporium is composed of a basal layer and an external hair-like nap. Filaments of the hair-like nap are made up largely of a single collagen-like glycoprotein called BclA. A second glycoprotein, BclB, has been identified in the exosporium layer. The specific location of this glycoprotein within the exosporium layer and its role in the biology of the spore are unknown. We created a mutant strain of B. anthracis [DELTA] Sterne that carries a deletion of the bclB gene. The mutant was found to possess structural defects in the exosporium layer of the spore (visualized by electron microscopy, immunofluorescence, and flow cytometry) resulting in an exosporium that is more fragile than that of a wild-type spore and is easily lost. Immunofluorescence studies also indicated that the mutant strain produced spores with increased levels of the BclA glycoprotein accessible to the antibodies on the surface. The resistance properties of the mutant spores were unchanged from those of the wild-type spores. A bclB mutation did not affect spore germination or kinetics of spore survival within macrophages. BcLB plays a key role in the formation and maintenance of the exosporium structure in B. anthracis.

Certain carbohydrates (rhamnose, 3-O-methyl rhamnose, and galactosamine) have been demonstrated to be present in Bacillus anthracis spores but absent in vegetative cells. Others have demonstrated that these spore-specific sugars are constituents of the glycoprotein BclA. In the current work, spore extracts were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A second collagen-like glycoprotein, BclB, was identified in B. anthracis. The protein moiety of this glycoprotein was identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MS) and the carbohydrate components by gas chromatography-mass spectrometry and tandem mass spectrometry. Spore-specific sugars were also demonstrated to be components of BclB.

ESI (electrospray ionization) MS and tandem mass spectrometry (MS/MS) were used for the analysis of single nucleotide polymorphisms (SNPs) and more complex genetic variations. Double-stranded (ds) PCR products were studied. PCR products of the proline [5'-(mathematical expression not reproducible in ASCII) (G(sub 17))- (mathematical expression not reproducible in ASCII) (C(sub 38)) (mathematical expression not reproducible in ASCII)-3'] and arginine variants [(5'-(mathematical expression not reproducible in ASCII)(G(sub 17))- (mathematical expression not reproducible in ASCII) (G(sub 38)) (mathematical expression not reproducible in ASCII)-3'] of the p53 gene are distinguished by an SNP (cytosine or guanine) and were discriminated using both quadrupole and quadrupole ion trap MS analysis. A 69 bp arginine mutant PCR product [5'-(mathematical expression not reproducible in ASCII)(C(sub 17))- (mathematical expression not reproducible in ASCII)(G(sub 38)) (mathematical expression not reproducible in ASCII)-3'] with a negating switch has the same mass as the proline variant but was readily distinguishable on ion trap MS/MS analysis; fragments containing the mutation site, but not the polymorphism, were identified. The 69 bp PCR products were restriction-enzyme-digested, to create 43 bp fragments. ESI quadrupole ion trap MS/MS analysis of the 43 bp product-ion spectra readily demonstrated both polymorphism and negating switch sites. MS and MS/MS are powerful and complemenmor techniques for analysis of DNA. MS can readily distinguish SNPs but MS/MS is required to differentiate isomeric PCR products (same nucleotide composition but different sequence).

ESI FTICR mass spectrometry is the only technique currently used for accurate molecular weight analysis of PCR products above 100 bp in size. This is important in demonstrating the potential for MS in making major contributions in the molecular biology and genomics areas. In the near future, it is more likely that less expensive, more user friendly MS techniques will be used for high-throughput analyses (including MALDI TOF and ESI quadrupole). There have been numerous reports on the use of MALDI TOF. The current report is to the first to evaluate the use of ESI-quadrupole analysis of PCR products. Synthetic oligonucleotides (30 and 89 mers) and polymerase chain reaction products of varying molecular weight (62, 88, 89, and 114 bp) were analyzed by ESI using a quadrupole MS. The mass accuracy for nucleic acids in the 30-62 bp range was shown to allow determination of nucleotide substitutions and additions/deletions. For higher molecular weight PCR products (88-114 bp), the mass accuracy of ESI-MS distinguishes single or multiple nucleotide insertions/deletions. In addition, ESI quadrupole MS allows determination of molecular weight of both strands of higher molecular weight ds PCR products and can distinguish nucleotide modifications (e.g., with biotin). In conclusion, it is demonstrated that ESI-MS occupies an intermediate position (as compared to MALDI TOF and ESI FTICR) with regard to mass accuracy and resolution in analysis of nucleic acids.

A segment of the 16S/23S rDNA interspace region of a Bacillus cereus strain examined through polymerase chain reaction (PCR) amplification created several products. Upon examination of the PCR products using the electrospray ionization-Fourier transform ion cyclotron resonance technique, it was observed that a T and an A has disappeared from the coding and noncoding strand respectively. In addition, the existence of unreported sequence variations between rRNA operons in the organism is observed.

A procedure for rapid purification of polymerase chain reaction (PCR) products allowing precise molecular weight determination using electrospray ionization-Fourier transform ion cyclotron resonance (ESI-FTICR) mass spectrometry is described. PCR amplification utilized the DNA polymerase from Pyrococcus furiosus (Pfu) which, unlike Taq, does not incorporate a nontemplated terminal deoxyadenosine phosphate. An 89-base pair nucleotide portion of the spacer region between the 16S and 23S ribosomal rRNA genes was amplified from the genome of three members of Bacillus cereus group and a 114 nucleotide region from the Bacillus subtilis. PCR involves polymerization of nucleotide precursors using two oligonucleotide primers and an amplification enzyme, as well as the presence of metal ions. Mass spectrometric analysis greatly benefits from removal of the oligonucleotide primers (15- and 17-mers in this instance) and nucleotide precursors since they adversely affect sensitivity and metal ion adduction results in an inaccurate molecular weight determination. In the presence of guanidinium hydrochloride, the PCR products bind preferentially to a silica resin, allowing removal of other components (i.e., dNTP's, primers, and salts). Further removal of metal ions was accomplished using a microdialysis device, allowing samples to be pumped through a hollow cellulose fiber with an external countercurrent flow of 2.5 mM ammonium acetate. Prior to injection into the mass spectrometer, the sample buffer was adjusted to 50 vol % acetonitrile, 25 mM piperidine, and 25 mM imidazole, which enhanced signal intensity. The molecular weights of the PCR products determined by nucleotide sequence and MS analysis were in excellent agreement, and several PCR products were analyzed where mass differences corresponding to single base substitutions could be accurately assigned. These assignments were possible due to the high mass precision, accuracy, and resolution FTICR inherently affords. This constitutes the first report demonstrating the ionization and detection of PCR products by mass spectrometry with mass precision and accuracy for assignment of such modifications or substitutions.

Values and benefits derived from the study of American history have changed over the years. Historians must more adequately describe the principal benefits to be obtained from the study of American history and consider ways in which history instruction can be revitalized to help those benefits to be realized. (CJ)

The application of marketing to the promotion of social causes was proposed a decade ago. The authors position social marketing as an approach to social change, describe its evolution, and review social marketing applications and assess their impact. [ABSTRACT FROM AUTHOR]

Discusses issues and implication underlying the graduation competency testing movement in the social studies. Presents advantages and disadvantages of commercially and locally developed tests and outlines steps in selecting competencies and preparing tests. (KC)