PURIFICATION AND IDENTIFICATION OF 2 MAJOR SINGLE-STRANDED BINDING-PROTEINS OF YEAST SACCHAROMYCES-CEREVISIAE AS RIBOSOMAL-PROTEIN L4 AND HISTONE H2B

Affinity chromatography on single-stranded DNA cellulose (ssDC) is use ful for purification of single-stranded RNA and single-stranded DNA bi nding proteins. Most of the proteins purified off of this resin have p roven to be ribonucleoproteins, with various roles in RNA processing. A homogenate of the yeast Saccharomyces cerevisiae shows perhaps a doz en major protein species, and many more minor protein species, upon el ution from ssDC. A major protein species of 30-31 kDa that elutes from ssDC between 0.35 and 0.45 M NaCl was purified to homogeneity. V8 pro tease was used to fragment this protein, and the peptides so generated were purified by HPLC and sequenced. From the sequence so derived six synthetic oligonucleotides were made. These oligonucleotides were use d to pull out the corresponding gene from a yeast genomic library. The entire gene was eventually found on a 4.4 kb BamHI fragment. This ent ire fragment was sequenced. The sequence showed three open reading fra mes (ORFs). ORF1 was the p30 gene, for all six V8 peptide fragment seq uences were found in it. Published here is the sequence for ORF1. Sequ ence comparisons of this sequence to the protein sequence databases sh owed that it is ribosomal protein L4. Another major ssb of yeast, whic h migrates as a doublet of 15-16 kDa, was also purified. N-terminal pe ptide sequencing of this protein produced a sequence identical to that for histone H2B.