Cloning 된 효모의 RNAI 유전자의 특성에 관하여 = Characterization of the cloned RNA1 gene of Saccharomyces cerevisiae

다국어 초록 (Multilingual Abstract)

The RNAI mutation of Saccharomyces cerevisia is a recessive and temperature sensitive lethal mutation which interferes with the production of mRNA, rRNA, and tRNA. However, the precise role of RNAI gene have not been revealed until yet. We have cloned rnal-1 mutant gene from rnal-1 mutant yeast strain(R49 ; trpl, ura3-52, rnal-1). The 3.4kb BglII fragment of wild type RNAI clone(81-2-6) contains whole RNAI gene. The genomic southern blotting with BglII digested R49 genomic DNA as a probe shows the unique and identical band with wild type 3.4kb BglII fragment. Therefore, We prepared partial BglII genomic library(3∼4kb BglII fragments) into BamH I site of pUC19. The rna 1-1 mutant clone was screened with Digoxigenin(DIG)-lableled probe by high density colony hybridization. The 5`-flanking region of rnal-1 gene was sequenced by dideoxy chain termination method. The 5`-flanking sequence of RNAI gene contains three TATA-like sequence ; TAATA, TATA and TTTTAA at position of -67, -45, and -36 from first ATG codon respectively. The 5`-flanking region of wild type RNAI gene from ATG codon to -103nt was deleted with Bal31 exonuclease digestion, generating pUC△/RNAI. After constructing pYEP△RNA I (consists of -103nt deleting RNAI gene, URA3 gene, 2㎛ rep. origin), pYEPrnal-1(consists of Xba I fragment of pUCrnal-1. URA3 gene, 2㎛ rep. origin), and pYEPRNAI. each plasmid was transformed into host strain(trpl, ura3-52, rnal-1) by electroporation, respectively. Yeast transformant carrying pYEP△RNAI did not complement the thermal sensitivity of rnal-1 gene. It means that TATA-like sequences in 5`-flanking region is not TATA sequence for transcribing RNAI gene and there may be other essential sequence in upstream region for the transcription of RNAI gene.