Regulation of gene transcription requires an interaction between specific segments of nuclear DNA and specific proteins. We describe a method to localize the specific DNAbinding proteins using haptenized double-stranded (ds) DNAs. To demonstrate this method, an oligodeoxynucleotide (oligo-DNA) with a consensus base sequence of cyclic adenosine monophasphate-"& element (CRE) (TGACGTCA) with three 'MA repeats at the 5' end was synthesized. Since the CRE sequence is palindromic, the oligo-DNA was allowed to self-anneal and form ds DNA with three 'MA repeats at both ends. The CRE ds-oligo-DNA was irradiated with w light to form haptenic thymine-thymine (T-T) dimers. The haptenized CRE ds-oligo-DNA reacted by Southwestern analysis with a distinct set of proteins, previously identified as CRE-binding proteins, ranging from

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It is well known that sake yeast has a high tolerance for ethanol, as compared to baker's yeast. To investigate the relationship between the ethanol tolerance of sake yeast and the palmitoyl-CoA pool for protein modification, the growth of yeast cells with depletion of the palmitoyl-CoA pool was monitored in the presence of ethanol. The overexpression of SNC1 was used to achieve the depletion of the palmitoyl-CoA pool, because the loss of Snc palmitoylation does not affect the general growth characteristics of yeast and does not interfere with the secretory processes (Couve, A. et al., Proc. Natl. Acad. Sci. USA, 92, 5987-5991 (1995)). Although the sake yeast UT-1 exhibited much better growth in the presence of ethanol than laboratory strains, the overexpression of Snc1 was accompanied by sparse growth with increasing ethanol concentration. Exogenous palmitic acid rescued the poor growth caused by Snc1 overexpression, and the overexpression of Snc1(ser95) (which could not palmitoylated) had no effect on the growth characteristics of strain UT-1, suggesting that the poor growth with Snc1 overexpression was due to an overall increase in proteins in the unpalmitoylated form. To ascertain that fatty acid activation has a distinct role in the growth of yeast in the presence of ethanol, FAA genes encoding long chain acyl-CoA synthetases were overexpressed in combination with snc1 overexpression. Interestingly, the growth defect caused by snc1 overexpression was rescued by the overexpression of FAA4, but not of FAA1, which plays a predominant role in laboratory strains. On the contrary, disruption of faa1 led to faster growth in the presence of ethanol. These results suggest that Faa1p and Faa4p play reciprocal roles in regulating protein modification during growth in the presence of ethanol, since Faa1p and Faa4p both function to incorporate palmitic acid into phospholipids and neutral lipids. Moreover, Northern hybridization analysis revealed that faa1 mRNA was expressed strongly in a laboratory strain, and weakly in the sake yeast strain K-7 which exhibited good growth in the presence of ethanol. The combination of the disruption of faa1 and exogenously supplied palmitic acid was highly effective for growth in the presence of ethanol even under the normal snc1 expression level, implying that activation of exogenous palmitic acid by Faa4p is of particular importance in growth in ethanol.

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