Isolation of a series of single missense mutants of a dna gene of phage φ29, gene 1, utilizing their inhibitory effect on E. coli growth

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概要

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• Gene 1 product (gp1) of Bacillus subtilis phage φ29 is known to promote DNA replication of the phage. Although its role in the DNA replication is not clear, gp1 is reported to exhibit multiple characteristics, including RNA binding, cell membrane localization, and self-association. To investigate these characteristics, we undertook the isolation of a series of missense mutants of gene 1 bearing substitutions at various regions. During cloning of gene 1, we found that its expression severely inhibited the growth of its host Escherichia coli cells. In this study, we utilized this growth-inhibition phenomenon to screen a random library muta-genized by error-prone PCR, expecting that mutants which could not inhibit cell growth would be affected in the authentic functions of gp1. Using this approach, we obtained 31 different mutants bearing single amino acid substitutions at 26 positions along the entire length of gp1. As a preliminary analysis of these mutants, we compared the deduced amino acid sequences of gp1s from φ29 and its related phages PZA, B103 and M2. Alignment of these sequences revealed three conserved regions, i.e. a hydrophobic region near the carboxyl terminus (assumed to be involved in the membrane localization and self-association of gp1), coiled-coil motif (essential for self-association), and a region of unknown function near the amino terminus. Interestingly, many of the substitutions in the isolated mutants occurred at strongly conserved residues in these regions and affected characteristic features of the regions (e.g. hydrophobicity of the hydrophobic region). These substitutions are expected to affect authentic functions of gp1, and the mutants will be useful for studies of the structure and functions of gp1.

• 日本遺伝学会の論文
• 2005-10-25

著者

• Makino Osamu

  Department Of Genetics Life Science Institute Sophia University

• TAKEUCHI Ari

  Department of Genetics, Life Science Institute, Sophia University

• HASHIYAMA Kazuya

  Department of Genetics, Life Science Institute, Sophia University

• ARAI Rewa

  Department of Genetics, Life Science Institute, Sophia University

• Arai Rewa

  Department Of Genetics Life Science Institute Sophia University

• Hashiyama Kazuya

  Department Of Genetics Life Science Institute Sophia University

• Takeuchi Ari

  Department Of Genetics Life Science Institute Sophia University

関連論文

• Isolation of a series of single missense mutants of a dna gene of phage φ29, gene 1, utilizing their inhibitory effect on E. coli growth
• A Variant Protein from φ29 Replication Gene, Gene 1, Did Not Form Homo-Polymer Due to a Single Amino Acid Substitution Near the Carboxyl Terminus
• Stereochemical Studies on the Nuclear Alkylation of Phenols with 1-Phenylethyl Chloride and the Acidic Rearrangement of the 1-Phenylethyl Ethers of Phenols in the Phenolic Solvents

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