(14) with eight pools of a YCp50-based genomic library (kindly supplied by M. Rose). Because the sin3-1 mutation suppresses the requirement of the HO:lacZ promoter for SWI5, the parent strain will have high levels of ,-galactosidase activity relative to the activity of a similar strain bearing a functional copy of SIN3. A total of 104 Ura+ transformants were selected on SD-uracil plates and screened for 3-galactosidase production on SD-uracil plates containing 50 mM KPi (pH 7.0) and 600 ,ug of 5-bromo-4-chloro-3-indolyl-,-D-galactoside (X-Gal) per ml. White colonies were selected and counterscreened for a-factor production by a halo assay (10). Plasmid DNA was prepared from transformants that could produce a-factor, and the transformants were tested for the ability to complement sin3-1 when reintroduced into strain S596-3D. One

A DNA-binding protein has been identified from extracts of the budding yeast Saccharomyces cerevisiae which binds to sites present in the promoter regions of a number of yeast genes transcribed by RNA polymerase II, including SIN3 (also known as SDI]), SWIS, CDC9, and TOP]. This protein also binds to a site present in the enhancer for the 35S rRNA gene, which is transcribed by RNA polymerase I, and appears to be identical to the previously described REB1 protein (B. E. Morrow, S. P. Johnson, and J. R. Warner, J. Biol. Chem. 264:9061-9068, 1989 Along with bypassing the SWI5 requirement, the sin3 mutation permits HO expression in daughters (27,33). Additionally, it was observed that a DNA-binding protein which recognizes a sequence in the HO promoter was deficient in sin3 extracts (27)

The SIN3 gene (also known as SDI1) is a negative regulator of the yeast HO gene. Mutations in SIN3 suppress the requirement for the SWI5 activator for expression of the yeast HO gene and change the normal asymmetric pattern of HO expression in mother and daughter cells. Furthermore, the in vitro DNA-binding activity of several DNA-binding proteins is reduced in extracts prepared from sin3 mutants. We have cloned the SIN3 gene and determined that a haploid strain with a SIN3 gene disruption is viable. We determined the sequence of the SIN3 gene, which is predicted to encode a 175-kDa polypeptide with four paired amphipathic helix motifs. These motifs have been identified in the myc family of helix-loop-helix DNA-binding proteins and in the TPR family of regulatory proteins. The SIN3 transcript was mapped, and it was determined that the SIN3 transcript was absent in stationary-phase cells. Immunofluorescence microscopy with anti-SIN3 antibody demonstrated that SIN3 protein was present in nuclei. A comparison of restriction map and sequence data revealed that SIN3 is the same as regulatory genes UME4 and RPD1.

A DNA-binding protein has been identified from extracts of the budding yeast Saccharomyces cerevisiae which binds to sites present in the promoter regions of a number of yeast genes transcribed by RNA polymerase II, including SIN3 (also known as SDI1), SWI5, CDC9, and TOP1. This protein also binds to a site present in the enhancer for the 35S rRNA gene, which is transcribed by RNA polymerase I, and appears to be identical to the previously described REB1 protein (B. E. Morrow, S. P. Johnson, and J. R. Warner, J. Biol. Chem. 264:9061-9068, 1989). When oligonucleotides containing a REB1-binding site are placed between the CYC1 upstream activating sequence and TATA box, transcription by RNA polymerase II in vivo is substantially reduced, suggesting that REB1 acts as a repressor of RNA polymerase II transcription. The in vitro levels of the REB1 DNA-binding activity are reduced in extracts prepared from strains bearing a mutation in the SIN3 gene. A greater reduction in REB1 activity is observed if the sin3 mutant strain is grown in media containing galactose as a carbon source.