Molecular Cloning of Chitinase Genes Family from Serratia marcescens

  • Song, Young-Hwan (Department of Microbiology, National Fisheries University Pusan) ;
  • Kweon, Oh-Gun (Department of Microbiology, National Fisheries University Pusan)
  • Published : 1993.12.30

Abstract

Total genomic DNA library of Serratia marcescens was prepared by inserting Sau3AI partial digesting fragments(above 5 kb) into the dephosphorylated BamHl site of pUC19. In primary screening, two colonies were selected by observing the halo around E. coli transformants grown on the swollen colloidal chitin media. Secondary screening was performed by soaking two colonies with a few drops of 4-methylumbelleliferryl N-acetyl-$\beta$-D-glucocosaminide(4-MuNGlcNAc). As 4-MuNGlcNAc is a specific, fluorogenic substrate for chitinase, the positive clones produce light fluorescence by the exposure under the long wave U.V. light(360 nm). From genomic DNA library derived from pUC19, we have isolated two different chitinase clones, pCH1(11.0Kb) and pCH2(7.5Kb), which show completely different restriction map to each other. The cross-hybridization of pCH1EA and pCH2 have not revealed any hybridization signals to each other.

Sau3AI으로 부분절단한 Serratia marcescens genomic DNA(5Kb 이상)을 pUC19의 BamHI site에 삽입하여 total genomic library를 준비하였다. Swollen colloidal chitin media에서 halo를 형성하는 2개의 E.coli 형질전환주를 선별하였다. 이들 colony가 chitinase 유전자를 갖음을 재확인하기 위하여 4-methylumbelliferyl N-acetyl-$\beta$-D-glucosaminide(4-MuFGlcNAc)를 이용하였다. 4-MuFGlcNAc는 chitinase에 대한 기질특이성을 나타내며 형광을 나타내는 기질로서 positive clone들은 360nm의 자외선을 조사하였을 경우 밝은 형광을 나타낸다. pUC19으로 부터 유래된 2 종류의 다른 chitinase clone, pCH1(11.0Kb) 및 pCH2(7.5Kb)를 genomic DNA library로 부터 분리하였으며, 이들의 제한효소지도를 작성한 결과 서로 다른 제한효소지도를 나타내었다. pCH1EA 및 pCH2로 부터 각각의 EcoRI-Xbal fragment를 subcloning함으로써 두개의 다른 chitinase 유전자의 위치를 결정하였다. pCH1EA 및 pCH2를 cross hybridization 한 결과 hybridization signal을 나타내지 않아 서로 유사성이 없는 것으로 사료된다.

Keywords