Purification, primary structure and molecular cloning of a rat ribosomal protein showing homology with yeast ribosomal protein YL34.
1. A new 17-kDa mammalian ribosomal protein (PR17) was purified to homogeneity from the rat exocrine pancreas. The purification procedure was based on acidic extraction of a heat-denatured homogenate, ammonium-sulfate precipitation, hydrophobic chromatography on phenyl-Sepharose and analytical reverse-phase HPLC on mu Bondapak C18. Fractions of interest were collected using an antiserum directed against the first (1-14) moiety of somatostatin (1-28). 30 micrograms pure RP17 were obtained from 1 g fresh pancreas. 2. A short 111-b cDNA encoding RP17 was amplified from rat pancreatic first-strand cDNA template by using two 64-fold degenerate heptadecamer primers in the DNA-polymerase-chain reaction. From the sequence of amplified cDNA, an unambiguous oligonucleotide probe was designed to screen a rat pancreatic cDNA library. A cDNA clone coding for RP17 was isolated, whose nucleotide sequence, with an open reading frame coding for 155 amino acids (molecular mass of 17,199 Da), confirmed the partial amino acid sequences directly obtained from the purified protein. 3. Northern-blot analysis showed that a similar 0.75-kb transcript was present in rat pancreas, in the rat pancreatic acinar cell line AR 4-2J and in the human neuroblastoma cell-line NB-OK-1, the highest level being in the latter two preparations, despite similar levels of RP17 in all three preparations, as tested with a rabbit antiserum directed against purified RP17. 4. The N-terminal sequence of both RP17 and the ribosomal protein YL43 from Saccharomyces cerevisiae (39 amino acid residues) showed a high degree of identity (77%), indicating that RP17 is a mammalian homolog of yeast ribosomal protein YL43.
Svoboda
M
M
Department of Biochemistry and Nutrition, Medical School, Université Libre de Bruxelles, Belgium.
Ciccarelli
E
E
Vandermeers-Piret
M C
MC
Nagy
A M
AM
Van de Weerdt
C
C
Bollen
A
A
Vandermeers
A
A
Christophe
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D015153:Blotting, Western; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D008297:Male; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000235:genetics; D051381:Rats; D011919:Rats, Inbred Strains; D012269:Ribosomal Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D012441:Saccharomyces cerevisiae / Q000737:chemistry*; D012689:Sequence Homology, Nucleic Acid; D014158:Transcription, Genetic
D005656:Fungal Proteins; D012333:RNA, Messenger; D012269:Ribosomal Proteins; D004247:DNA