NAST: a multiple sequence alignment server for comparative analysis of 16S rRNA genes.

Microbiologists conducting surveys of bacterial and archaeal diversity often require comparative alignments of thousands of 16S rRNA genes collected from a sample. The computational resources and bioinformatics expertise required to construct such an alignment has inhibited high-throughput analysis. It was hypothesized that an online tool could be developed to efficiently align thousands of 16S rRNA genes via the NAST (Nearest Alignment Space Termination) algorithm for creating multiple sequence alignments (MSA). The tool was implemented with a web-interface at http://greengenes.lbl.gov/NAST. Each user-submitted sequence is compared with Greengenes' 'Core Set', comprising approximately 10,000 aligned non-chimeric sequences representative of the currently recognized diversity among bacteria and archaea. User sequences are oriented and paired with their closest match in the Core Set to serve as a template for inserting gap characters. Non-16S data (sequence from vector or surrounding genomic regions) are conveniently removed in the returned alignment. From the resulting MSA, distance matrices can be calculated for diversity estimates and organisms can be classified by taxonomy. The ability to align and categorize large sequence sets using a simple interface has enabled researchers with various experience levels to obtain bacterial and archaeal community profiles.

Synthesis of (S)-isoprenoid thiodiphosphates as substrates and inhibitors.

Thiolo thiophosphate analogues of isopentenyl diphosphate (IPP), dimethylallyl diphosphate (DMAPP), geranyl diphosphate (GPP), farnesyl diphosphate (FPP), and geranylgeranyl diphosphate (GGPP) were synthesized. Inorganic thiopyrophosphate (SPP(i)) was prepared from trimethyl phosphate in four steps. The tris(tetra-n-butylammonium) salt was then used to convert isopentenyl tosylate to (S)-isopentenyl thiodiphosphate (ISPP). (S)-Dimethylallyl (DMASPP), (S)-geranyl (GSPP), (S)-farnesyl (FSPP), and (S)-geranylgeranyl thiodiphosphate (GGSPP) were prepared from the corresponding bromides in a similar manner. ISPP and GSPP were substrates for avian farnesyl diphosphate synthase (FPPase). Incubation of the enzyme with ISPP and GPP gave FSPP, whereas incubation with IPP and GSPP gave FPP. GSPP was a substantially less reactive than GPP in the chain elongation reaction and was an excellent competitive inhibitor, K(I)(GSPP) = 24.8 microM, of the enzyme. Thus, when ISPP and DMAPP were incubated with FPPase, GSPP accumulated and was only slowly converted to FSPP.

Synthesis of geranyl S-thiolodiphosphate. A new alternative substrate/inhibitor for prenyltransferases.

The tris(tetra-n-butylammonium) salt of thiopyrophosphate 5 was prepared from trimethyl phosphate in four steps. Treatment of geranyl bromide with 5 gave an 80% yield of geranyl S-thiolodiphosphate (6). Thiolodiphosphate 6 is substantially less reactive than geranyl diphosphate (7) in the prenyl transfer reaction catalyzed by farnesyl diphosphate synthase and is a good inhibitor of the enzyme.

Differentiation of Mycoplasma hyopneumoniae, M flocculare, and M hyorhinis on the basis of amplification of a 16S rRNA gene sequence.

To differentiate Mycoplasma hyopneumoniae, the cause of mycoplasmal pneumonia in pigs, from M flocculare and M hyorhinis, an assay, using the polymerase chain reaction to amplify a segment of the 16S rRNA gene sequence, was developed. The assay was found to be useful for identification of field isolates, as well as for identification of laboratory-adapted strains. Amplification of DNA from M hyopneumoniae and M flocculare resulted in products of 200 and 400 base pairs, respectively. The DNA from M hyorhinis was not amplified. The assay was sensitive enough to detect as little as 1,000 genome equivalents of M hyopneumoniae and M flocculare DNA. Sensitivity was increased 100-fold by increasing the concentration of magnesium ion in the reaction buffer from 2 to 4 mM; however, DNA from M hyorhinis was also amplified under these conditions. The DNA from several walled bacteria and from other mycoplasmas was also tested, but none of these DNA samples was amplified, suggesting that the assay was specific for porcine mycoplasmas.

[The Limulus test in studies of pyrogenicity. Applications in radiopharmacy]. / Test Limule dans les études de pyrogénicité. Applications en radiopharmacie.

Preparation, standardization and use of the Limulus test, in relation to the official Rabbit test. Applications of the Limulus test to radiopharmaceuticals.