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The Preferred Substrate for RecA-Mediated Cleavage of Bacteriophage 434 Repressor Is the DNA-Bound Dimer. David R. Pawlowski, 2004.Induction of a lysogen of a lambdoid bacteriophage usually involves RecA-stimulated autoproteolysis of the bacteriophage repressor protein . Previous work on the phage repressors showed that the monomeric form of the protein is the target of RecA . Our previous work indicated that in the case of bacteriophage 434, virtually none of the repressor is present as a monomer in vivo . Hence, if the repressor in a lysogen is present as a dimer, how can RecA-stimulated autoproteolysis play a role in bacteriophage induction? We examined this question by determining the rate of RecA-stimulated 434 repressor cleavage as a function of repressor concentration and added DNA . Our results show that binding of 434 repressor to a specific DNA binding site dramatically increases the velocity of repressor autocleavage compared to the velocity of cleavage of the monomer and concentration-induced dimer . DNA binding-deficient hemidimers formed between the intact repressor and its C-terminal domain fragment have a lower rate of cleavage than DNA-bound dimers . These results show that the DNA-bound 434 repressor dimer, which is the form of the repressor that is required for its transcriptional regulatory functions, is the preferred form for RecA-stimulated autocleavage . We also show that the rate of repressor autocleavage is influenced by the sequence of the bound DNA . Kinetic analysis of the autocleavage reaction indicated that the DNA sequence influences the velocity of 434 repressor autocleavage by affecting the affinity of the repressor-DNA complex for RecA, not the chemical cleavage step . Regardless of the mechanism, the finding that the presence and precise sequence of DNA modulate the autocleavage reaction shows that DNA allosterically affects the function of 434 repressor . Virus-Binding Proteins Recovered from Bacterial Culture Derived from Activated Sludge by Affinity Chromatography Assay Using a Viral Capsid Peptide. Daisuke Sano, 2004.The contamination of water environments by pathogenic viruses has raised concerns about outbreaks of viral infectious diseases in our society . Because conventional water and wastewater treatment systems are not effective enough to inactivate or remove pathogenic viruses, a new technology for virus removal needs to be developed . In this study, the virus-binding proteins (VBPs) in a bacterial culture derived from activated sludge were successfully recovered . The recovery of VBPs was achieved by applying extracted crude proteins from a bacterial culture to an affinity column in which a custom-made peptide of capsid protein from the poliovirus type 1 (PV1) Mahoney strain (H2N-DNPASTTNKDKL-COOH) was immobilized as a ligand . VBPs exhibited the ability to adsorb infectious particles of PV1 Sabin 1 as determined by enzyme-linked immunosorbent assay . The evaluation of surface charges of VBPs with ion-exchange chromatography found that a majority of VBP molecules had a net negative charge under the conditions of affinity chromatography . On the other hand, a calculated isoelectric point implied that the viral peptide in the affinity column was also charged negatively . As a result, the adsorption of the VBPs to the viral peptide in the affinity column occurred with a strong attractive force that was able to overcome the electrostatic repulsive force . Two-dimensional electrophoresis revealed that the isolated VBPs include a number of proteins, and their molecular masses were widely distributed but smaller than 100 kDa . Amino acid sequences of N termini of five VBPs were determined . Homology searches for the N termini against all protein sequences in the National Center for Biotechnology Information (NCBI) database showed that the isolated VBPs in this study were newly discovered proteins . These VBPs that originated with bacteria in activated sludge might be stable, because they are existing in the environment of wastewater treatments . Therefore, a virus removal technology utilizing VBPs as viral adsorbents can be developed, since it is possible to replicate VBPs by protein cloning techniques . Karyotype Rearrangements in a Wine Yeast Strain by rad52-Dependent and rad52-Independent Mechanisms. David Carro, 2003.Yeast strains isolated from the wild may undergo karyotype changes during vegetative growth, a characteristic that compromises their utility in genetic improvement projects for industrial purposes . Karyotype instability is a dominant trait, segregating among meiotic derivatives as if it depended upon only a few genetic elements . We show that disrupting the RAD52 gene in a hypervariable strain partially stabilizes its karyotype . Specifically, RAD52 disruption eliminated recombination at telomeric and subtelomeric sequences, had no influence on ribosomal DNA rearrangement rates, and reduced to 30% the rate of changes in chromosomal size . Thus, there are at least three mechanisms related to karyotype instability in wild yeast strains, two of them not requiring RAD52-mediated homologous recombination . When utilized for a standard sparkling-wine second fermentation,  rad52 strains retained the enological properties of the parental strain, specifically its vigorous fermentation capability . These data increase our understanding of the mechanisms of karyotype instability in yeast strains isolated from the wild and illustrate the feasibility and limitations of genetic remediation to increase the suitability of natural strains for industrial processes .
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