The rolC gene in Agrobacterium rhizogenes Ri plasmid behaves as oncogene in plant. As compared with the viral oncogenes, host genomic counterpart of the rolC gene was searched for in Nicotiana tabacum. The rolC gene contained in HindIII-EcoRI fragment of pPCV002-C in Agrobacterium tumefaciens (GV3101) was transferred into the corresponding site of pUC19 and constructed a new plasmid pYTS-C. The 1kb HpaI-EcoRI fragment excised from pYTS-C included the rolC gene and was used as a probe in hybridization analysis and in screening libraries. Before screening libraries, the existence of homologous region in several plant chromosomal DNAs with the rolC gene was demonstrated by slot blot hybridization. One of samples shown positive signals, Nicotiana tabacum's lambada genomic library was obtained. Screening of recombinant bacteriophage was then carried out by in situ plaque hybridization with $^{32}P$-labeled rolC-containing fragment. As a result of first screening, 15 positive recombinant plaques were obtained. Ten positive plaques were randomly selected and were performed up to tertiary screeing. Two of them were restriction endonuclease mapped.
토양 박테리아 Agrobacterium rhizogenes의 Ri plasmid TL-DNA의 rolC 유전자는 식물체 내에서 oncogene으로 작용한다. 세포내 염색체상의 counterpart를 가지고 있는 viral oncogenes과 비교하여, rolC 유전자의 host genomic counterpart를 Nicotiana tabacum에서 찾아 보았다.
먼저 pPCV002-C의 HindIII-EcoRI 단편 내에 존재하는 rolC 유전자를 분리한 후 pUC19의 동일한 효소부위로 subcloning하여 새로운 plasmid pYTS-C를 만들었다. pYTS-C에서 절제된 1kb Hpal-EcoRI 단편에 rolC 유전자가 포함되어 있었고, 뒤의 여러 hybridizations과 libraries screening에 probe 로 사용되었다. Libraries screening에 앞서, 여러 식물체의 염색체 DNA들에 대한 slot blot hybridization을 행함으로써, rolC 유전자 상동부위가 염색체 내에 존재함을 증명하였다. 그리고 나서 positive signals을 보인 samples중 하나인 Nicotiana tabacum의 genomic library를 얻었고 그 library에 대한 screening을 실시하였다. 1차 screening 결과, 15개 가량이 rolC 유전자 상동부위를 가진 것으로 나타났는데, 10개를 임의로 선택해서 3차까지 screening했다. 이중에서 2개 만의 restriction endonuclease mapping이 이루어졌다.
