Purification and identification of two 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 useful for purification of single-stranded RNA and single-stranded DNA binding proteins. Most of the proteins purified off of this resin have proven to be ribonucleoproteins, with various roles in RNA processing. A homogenate of the yeast Saccharomyces cerevisiae shows perhaps a dozen major protein species, and many more minor protein species, upon elution 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 protease 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 used 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 entire fragment was sequenced. The sequence showed three open reading frames (ORFs). ORF1 was the p30 gene, for all six V8 peptide fragment sequences were found in it. Published here is the sequence for ORF1. Sequence comparisons of this sequence to the protein sequence databases showed that it is ribosomal protein L4. Another major ssb of yeast, which migrates as a doublet of 15-16 kDa, was also purified. N-terminal peptide sequencing of this protein produced a sequence identical to that for histone H2B.
Cusick
M E
ME
Department of Medical Biochemistry and Genetics, Texas A&M College of Medicine, College Station 77843-1114.
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004277:DNA, Single-Stranded; D010450:Endopeptidases; D005800:Genes, Fungal; D006657:Histones / Q000235:genetics / Q000302:isolation & purification*; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D012269:Ribosomal Proteins / Q000235:genetics / Q000302:isolation & purification*; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000235:genetics
D004277:DNA, Single-Stranded; D006657:Histones; D010446:Peptide Fragments; D012269:Ribosomal Proteins; C056368:ribosomal protein L4; D010450:Endopeptidases