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Albusin B, a Bacteriocin from the Ruminal Bacterium Ruminococcus albus 7 That Inhibits Growth of Ruminococcus flavefaciens. Junqin Chen, 2004.An  32-kDa protein (albusin B) that inhibited growth of Ruminococcus flavefaciens FD-1 was isolated from culture supernatants of Ruminococcus albus 7 . Traditional cloning and gene-walking PCR techniques revealed an open reading frame (albB) encoding a protein with a predicted molecular mass of 32,168 Da . A BLAST search revealed two homologs of AlbB from the unfinished genome of R . albus 8 and moderate similarity to LlpA, a recently described 30-kDa bacteriocin from Pseudomonas sp . strain BW11M1 .
Proteome and Transcriptome Analysis of the Virulence Genes Regulated by the VirR/VirS System in Clostridium perfringens. Takeshi Shimizu, 2002.The proteins under the control of the two-component system VirR/VirS in Clostridium perfringens were analyzed by using two-dimensional gel electrophoresis of the culture supernatant from the wild type and the virR mutant . Based on matrix-assisted laser desorption ionization-time of flight/mass spectrometry, seven positively regulated proteins and eight negatively regulated proteins were identified . Transcriptome analysis confirmed that 7 of the 15 proteins were regulated by the VirR/VirS system at the transcriptional level, but the remaining proteins were modified with a VirR/VirS-directed protease at the posttranslation and secretion levels . We purified and characterized the VirR/VirS-directed protease from the culture supernatant and identified it as a kind of clostripain . Because this proteolytic activity was strongly inhibited by leupeptin and antipain, it was concluded that this protease was a member of the family of cysteine proteases of C . perfringens . Synthesis of Triacylglycerols by the Acyl-Coenzyme A:Diacyl-Glycerol Acyltransferase Dga1p in Lipid Particles of the Yeast Saccharomyces cerevisiae . Daniel Sorger, 2002.The terminal step of triacylglycerol (TAG) formation in the yeast Saccharomyces cerevisiae is catalyzed by the enzyme acyl-CoA:diacylglycerol acyltransferase (DAGAT) . In this study we demonstrate that the gene product of YOR245c, Dga1p, catalyzes a major yeast DAGAT activity which is localized to lipid particles . Enzyme measurements employing a newly established assay containing radioactively labeled diacylglycerol (DAG) as a substrate and unlabeled palmitoyl-CoA as a cosubstrate revealed a 70- to 90-fold enrichment of DAGAT in lipid particles over the homogenate but also a 2- to 3-fold enrichment in endoplasmic reticulum fractions . In a dga1 deletion strain, the DAGAT activity in lipid particles is dramatically reduced, whereas the activity in microsomes is affected only to a minor extent . Thus, we propose the existence of DAGAT isoenzymes in the microsomal fraction . Furthermore, we unveiled an acyl-CoA-independent TAG synthase activity in lipid particles which is distinct from Dga1p and the phosphatidylcholine:DAGAT Lro1p . This acyl-CoA-independent TAG synthase utilizes DAG as an acceptor and free fatty acids as cosubstrates and occurs independently of the acyl-CoA synthases Faa1p to Faa4p . Based on lipid analysis of the respective deletion strains, Lro1p and Dga1p are the major contributors to total cellular TAG synthesis, whereas other TAG synthesizing systems appear to be of minor importance . In conclusion, at least three different pathways are involved in the formation of storage TAG in the yeast . Subunit Oligomerization and Substrate Recognition of the Escherichia coli ClpYQ (HslUV) Protease Implicated by In Vivo Protein-Protein Interactions in the Yeast Two-Hybrid System. Yi-Ying Lee, 2003.The Escherichia coli ClpYQ (HslUV) is an ATP-dependent protease that consists of an ATPase large subunit with homology to other Clp family ATPases and a peptidase small subunit related to the proteasomal ß-subunits of eukaryotes . Six identical subunits of both ClpY and ClpQ self-assemble into an oligomeric ring, and two rings of each subunit, two ClpQ rings surrounded by single ClpY rings, form a dumbbell shape complex . The ClpYQ protease degrades the cell division inhibitor, SulA, and a positive regulator of capsule transcription, RcsA, as well as RpoH, a heat shock sigma transcription factor . Using the yeast-two hybrid system, we explored the in vivo protein-protein interactions of the individual subunits of the ClpYQ protease involved in self-oligomerization, as well as in recognition of specific substrates . Interactions were detected with ClpQ/ClpQ, ClpQ/ClpY, and ClpY/SulA . No interactions were observed in experiments with ClpY/ClpY, ClpQ/RcsA, and ClpQ/SulA . However, ClpY, lacking domain I (ClpY  I) was able to interact with itself and with intact ClpY . The C-terminal region of ClpY is important for interaction with other ClpY subunits . The previously defined PDZ-like domains at the C terminus of ClpY, including both D1 and D2, were determined to be indispensable for substrate binding . Various deletion and random point mutants of SulA were also made to verify significant interactions with ClpY . Thus, we demonstrated in vivo hetero- and homointeractions of ClpQ and ClpY molecules, as well as a direct association between ClpY and substrate SulA, thereby supporting previous in vitro biochemical findings .
Intraspecies Polymorphism of Cryptosporidium parvum Revealed by PCR-Restriction Fragment Length Polymorphism (RFLP) and RFLP-Single-Strand Conformational Polymorphism Analyses. Zhiliang Wu, 2003.A glycoprotein (Cpgp40/15)-encoding gene of Cryptosporidium parvum was analyzed to reveal intraspecies polymorphism within C . parvum isolates . Forty-one isolates were collected from different geographical origins (Japan, Italy, and Nepal) and hosts (humans, calves, and a goat) . These isolates were characterized by means of DNA sequencing, PCR-restriction fragment length polymorphism (PCR-RFLP), and RFLP-single-strand conformational polymorphism (RFLP-SSCP) analyses of the gene for Cpgp40/15 . The sequence analysis indicated that there was DNA polymorphism between genotype I and II, as well as within genotype I, isolates . The DNA and amino acid sequence identities between genotypes I and II differed, depending on the isolates, ranging from 73.3 to 82.9% and 62.4 to 80.1%, respectively . Those among genotype I isolates differed, depending on the isolates, ranging from 69.0 to 85.4% and 54.8 to 79.2%, respectively . Because of the high resolution generated by PCR-RFLP and RFLP-SSCP, the isolates of genotype I could be subtyped as genotypes Ia1, Ia2, Ib, and Ie . The isolates of genotype II could be subtyped as genotypes IIa, IIb, and IIc . The isolates from calves, a goat, and one Japanese human were identified as genotype II . Within genotype II, the isolates from Japan were identified as genotype IIa, those from calves in Italy were identified as genotype IIb, and the goat isolate was identified as genotype IIc . All of the genotype I isolates were from humans . The Japanese isolate (code no . HJ3) and all of the Nepalese isolates were identified as genotypes Ia1 and Ia2, respectively . The Italian isolates were identified as genotype Ib, and the Japanese isolate (code no . HJ2) was identified as genotype Ie . Thus, the PCR-RFLP-SSCP analysis of this glycoprotein Cpgp40/15 gene generated a high resolution that has not been achieved by previous methods of genotypic differentiation of C . parvum .
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