rolC gene homologue from Lettuce was cloned and its expression was confirmed in plant tissues. Firstly, the homologous region in several plant chromosomal DNA was detected by DNA slot blot hybridization. Lettuce total RNA was analyzed by northern blot analysis and revealed that the rolC gene was being expressed. $Poly(A)^+$ RNA was selected from total RNA, analyzed by RNA slot blot analysis, and revealed that the rolC gene does not be expressed after about 6 week. Then, Lettuce genomic DNA was digested with HindIII and analyzed by southern blot hybridization. DNA fragments of 4-6 kb range were subcloned to pUC19 and screened by colony hybridization. 4 clones were mapped with several restriction enzyme. After localization of pKC01, Avaii fragment was subcloned to pSELECT-1.
1. Slot blot hybridization 으로 여러 가지 쌍떡잎 식물을 screening 하여 rolC genomic homologue 가 존재함을 확인 하였다.
2. Lettuce 의 total RNA 로 Nothern blot hybridization 하여 뿌리에서 RNA transcript 의 존재를 확인하였다.
3. 발생 단계별로 얻은 poly(A)'RNA로 slot blot 한 결과 6주 이전의 모든 sample 에서 transcript의 존재를 확인 하였다.
4. Lettuce의 subgenomic library를 colony hybridization 으로 screening하여 6개의 clone을 얻었고, 이 중 4 clone을 여러개의 제한효소로 절단하여 mapping하였다.
5. pKC01을 localization하여 pSELECT-1으로 subcloning 하였다.
