• BMB Reports
• /
• v.29 no.1
• /
• pp.17-21
• /
• 1996

Ile91 of restriction endonuclease EcoRV, which has not been known to take part directly in catalytic activity, was substituted with Leu by site-directed mutagenesis. The Ile91Leu mutant shows over 1000-fold less activity than the wild type EcoRV under standard reaction condition. The metal ion dependency of the reaction was altered. In contrast to the wild type EcoRV, the mutant prefers $Mn^{2+}$ to $Mn^{2+}$ as the cofactor. In $Mn^{2+}$ buffer the mutant is as active as the wild type enzyme in $Mn^{2+}$ buffer. Like the wild type enzyme, the mutant shows an unspecific binding of DNA in gel shift experiments. In contrast to the wild type enzyme, the mutant did not cleave at noncognate sites of DNA under star condition.

• BMB Reports
• /
• v.29 no.2
• /
• pp.99-104
• /
• 1996

Site-directed substitutions were made to change the Ile91 of restriction endonuclease EcoRV to either Val, Ala or Gly to identify the role of Ile91 in recognition and catalysis, since substitution of Ile91 with Leu afforded dramatic effects on the activity and properties of restriction endonuclease EcoRV. These changes alter the size of the hydrophobic side chain at position 91 and thus might have revealed the reason for the altered phenotype of Ile91Leu. However, the properties of Ile91Val and Ile91Ala mutants were much like wild type EcoRV, in both activity and metal ion preference. Ile91Gly had very little activity with either $Mg^{2+}$ or $Mn^{2+}$ as cofactors. To try to understand the unusual $Mn^{2+}$ profile of the Ile91Leu mutant, two double mutants, Ile91Leu;Asp90Asn and Ile91Leu;Glu45Met were created. Both double mutants were seriously disabled by the second amino acid change. Ile91Leu;Glu45Met had some residual activity in the $Mn^{2+}$ reaction buffer, whereas the Ile91Leu;Asp90Asn displayed no detectable activity.

• Bulletin of the Korean Chemical Society
• /
• v.20 no.1
• /
• pp.109-111
• /
• 1999

• Korean Journal of Veterinary Research
• /
• v.39 no.1
• /
• pp.90-95
• /
• 1999

A species-specific 760 base pair(bp) BamHI to EcoRI DNA fragment(fMG-2) of lipoprotein gene was isolated from a Mycoplasma gallisepticum(M gallisepticum) genomic library. Based on the DNA sequence data of fMG-2, a pair of 25bp primers was synthesized. When used in the polymerase chain reaction(PCR), 732bp DNA products were amplified from 6 standard strains and 10 field isolates of M gallisepticum, but not from 2 Mycoplasma synoviae and 7 other Mycoplasma species. The lower detection limit was 100fg of the genomic DNA. Identity of the PCR products was confirmed by comparison of patterns of restriction endonuclease analysis with AseI, DraI, EcoRV and SspI.