A new promoter, designated as $\underline{YEP1}$ (Yeast and $\underline{E}$. $\underline{coil}$ dual Promoter), was isolated from yeast genomic DNA and characterized.
A strategy was developed for isolating yeast genomic DNA fragments containing promoter activities of both $\underline{E}$. $\underline{coli}$ and yeast. The bacterial $Cm^R$ gene mediating the drug resistance to chloramphenicol by encoding an enzyme chloramphenicol acetyl transferase (CAT) was used as an indicator gene after removing its own promoter region. A promoter-probe plasmid vector, pK15 (7.6 kb), was constructed by the insertion of promoterless $Cm^R$ gene into a yeast 2 um-based shuttle plasmid between $\underline{E}$. $\underline{coil}$ and yeast. Another vector, pKW4 (5.9 kb), was constructed by removing a nonessential 1.7 kb-long region from pK15. The pK15 or pKW4 vector contains an unique BamHI restriction site immediately upstream of the ribosome-binding sequences of the promoterless $Cm^R$ gene.
The constructed vectors, pK15 and pKW4, were proven to be useful for the promoter-probing in either organism of $\underline{E}$. $\underline{coil}$ and yeast. A well-known promoter of $\underline{E}$. $\underline{coli}$, $P_{tac}$, or yeast, $P_{ADHI}$, was inserted into the BamHI site upstream of the $Cm^R$ gene in pK15 or in pKW4. The $\underline{E}$. $\underline{coli}$ cells carrying the $P_{tac}$ -CAT-fusion plasmids or yeast cells carrying the $P_{ADHI}$ -CAT-fusion plasmids were able to grow in the presence of the drug, while the control $\underline{E}$. $\underline{coli}$ or yeast cells carrying the parental plasmids, pK15 or pKW4, were not.
A yeast-$\underline{E}$. $\underline{coli}$ dual promoter ($\underline{YBP1}$), 2.2 kb in length, was isolated from Alul-Haelll-generated fragments of yeast genomic DNA by its ability of conferring the drug resistance in $\underline{E}$. $\underline{coli}$ and in yeast. The phenotypic selection for chloramphenicol resistance was sequentially performed, initially in $\underline{E}$. $\underline{coli}$ and then in yeast, to isolate the promoter-active DNA fragments.
The nucleotide sequence of about 840 bp, 3'-region of $\underline{YEP1}$ was determined. Several 5'-regions of the $\underline{YBP1}$ were deleted and the CAT activities promoted from the deletion derivatives were subsequently assayed. The 840 bp sequence of $\underline{YBP1}$ was found to be essential for the dual promoter activity.
The start points of $Cm^R$ transcripts from $\underline{YBP1}$ in $\underline{E}$. $\underline{coli}$ or in yeast were determined by analyzing the size of a DNA primer which had been annealed to the $Cm^R$ transcripts in the cellular extracted RNA and extended to the 5'-end of the RNA by the enzyme reverse transcriptase.
The nucleotide sequencing and primer extension analysis showed that the $\underline{YBP1}$ sequence contains sets of consensus sequences pertaining to prokaryotic- and yeast-type promoter elements. TATTTT was found from -12 to -7, and TTGTCC from -35 to -30, from the single, mRNA start point determined in $\underline{E}$. $\underline{coli}$, and the spacing between the two sequences was 17 bp. These sequences are presumed to be the promoter elements typically found in most of the known E. coli promoters.
The $\underline{YEP1}$ caused multiple transcription initiation in yeast at more than 20 different points that were spaced over a 100-bp region with variable efficiencies. The DNA region is composed of A-T-rich sequences. Putative TATA-like sequences are found at several places upstream from the transcription start points.
A 276-bp DNA sequence of $\underline{YRPI}$, between -872 and -596 from the initiating ATG codon, was required for the maximal promoter activity in yeast but not in $\underline{E}. $\underline{coli}. The sequence is presumed to be an upstream activation site (UAS) frequently found in yeast promoters.
Northern blot analysis of $Cm^R$ transcripts from $\underline{YEP1}$ in polyadenylated RNA prepared from yeast cells showed that the transcription termination and polyadenylation occurred using the corresponding yeast signals present at several places located within the yeast 2 um plasmid-portion of pK15.
It has been found that the dual promoter region as has been characterized in this thesis is conserved in natural Saccharomyces cerevisiae genome. Southern blot analysis of the YEPI in yeast genome suggested that more than one copies of the dual promoter sequence do exist in the natural yeast genome.
