Murine polypyrimidine tract binding protein

Purification, cloning, and mapping of the RNA binding domain

Alfred L M Bothwell, Dean W. Ballard, William M. Philbrick, Glen Lindwall, Stephen E. Maher, Margot M. Bridgett, Sharon F. Jamison, Mariano Garcia-Blanco

Research output: Contribution to journalArticle

38 Citations (Scopus)

Abstract

A complex of nucleic acid binding proteins (100, 35, and 25 kDa) was purified to apparent homogeneity from nuclear extracts of the murine plasmacytoma J558L. Amino-terminal sequence analysis of the 25-kDa subunit enabled the isolation of a cDNA that encodes a 528-amino acid protein that is highly homologous to the human 62-kDa human polypyrimidine tract binding protein (PTB) (Garcia-Blanco, M. A., Jamison, S. F., and Sharp, P. A. (1989) Genes & Dev. 3, 1874-1886; Gil, A., Sharp, P. A., Jamison, S. F., and Garcia-Blanco, M. A. (1991) Genes & Dev. 5, 1224-1236; Patton, J. G., Mayer, S. A., Tempst, P., and Nadal-Ginard, B. (1991) Genes & Dev. 5, 1237-1251). Sequence comparison programs suggested the presence of domains related to the RNA recognition motif found in other RNA-binding proteins, and deletion analysis revealed that the carboxyl-terminal 195 amino acids of the recombinant PTB was sufficient for specific binding to pre-mRNAs. Cross-linking experiments identified a 25-kDa protein in crude nuclear extracts of J558L cells that possessed the RNA binding properties of PTB, while a ∼60-kDa protein is detected in other murine cell lines tested. Thus, the 25-kDa protein found in J558L is likely a proteolytic product of the murine polypyrimidine tract binding protein. A probe derived from the PTB cDNA detected a ubiquitous 3.3-kb mRNA in murine cell lines and a 3.6-kb mRNA in human lines. Southern blot analysis revealed three strongly hybridizing DNA fragments and several more weakly hybridizing bands in mouse, human, and yeast DNA. The role of PTB in pre-mRNA splicing is discussed.

Original languageEnglish (US)
Pages (from-to)24657-24663
Number of pages7
JournalJournal of Biological Chemistry
Volume266
Issue number36
StatePublished - Dec 25 1991
Externally publishedYes

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Polypyrimidine Tract-Binding Protein
Cloning
Purification
Organism Cloning
RNA
Genes
RNA Precursors
Proteins
Complementary DNA
Cells
Amino Acids
Cell Line
Messenger RNA
Plasmacytoma
RNA-Binding Proteins
DNA
Southern Blotting
Complex Mixtures
Recombinant Proteins
Yeast

ASJC Scopus subject areas

  • Biochemistry

Cite this

Bothwell, A. L. M., Ballard, D. W., Philbrick, W. M., Lindwall, G., Maher, S. E., Bridgett, M. M., ... Garcia-Blanco, M. (1991). Murine polypyrimidine tract binding protein: Purification, cloning, and mapping of the RNA binding domain. Journal of Biological Chemistry, 266(36), 24657-24663.

Murine polypyrimidine tract binding protein : Purification, cloning, and mapping of the RNA binding domain. / Bothwell, Alfred L M; Ballard, Dean W.; Philbrick, William M.; Lindwall, Glen; Maher, Stephen E.; Bridgett, Margot M.; Jamison, Sharon F.; Garcia-Blanco, Mariano.

In: Journal of Biological Chemistry, Vol. 266, No. 36, 25.12.1991, p. 24657-24663.

Research output: Contribution to journalArticle

Bothwell, ALM, Ballard, DW, Philbrick, WM, Lindwall, G, Maher, SE, Bridgett, MM, Jamison, SF & Garcia-Blanco, M 1991, 'Murine polypyrimidine tract binding protein: Purification, cloning, and mapping of the RNA binding domain', Journal of Biological Chemistry, vol. 266, no. 36, pp. 24657-24663.
Bothwell ALM, Ballard DW, Philbrick WM, Lindwall G, Maher SE, Bridgett MM et al. Murine polypyrimidine tract binding protein: Purification, cloning, and mapping of the RNA binding domain. Journal of Biological Chemistry. 1991 Dec 25;266(36):24657-24663.
Bothwell, Alfred L M ; Ballard, Dean W. ; Philbrick, William M. ; Lindwall, Glen ; Maher, Stephen E. ; Bridgett, Margot M. ; Jamison, Sharon F. ; Garcia-Blanco, Mariano. / Murine polypyrimidine tract binding protein : Purification, cloning, and mapping of the RNA binding domain. In: Journal of Biological Chemistry. 1991 ; Vol. 266, No. 36. pp. 24657-24663.
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abstract = "A complex of nucleic acid binding proteins (100, 35, and 25 kDa) was purified to apparent homogeneity from nuclear extracts of the murine plasmacytoma J558L. Amino-terminal sequence analysis of the 25-kDa subunit enabled the isolation of a cDNA that encodes a 528-amino acid protein that is highly homologous to the human 62-kDa human polypyrimidine tract binding protein (PTB) (Garcia-Blanco, M. A., Jamison, S. F., and Sharp, P. A. (1989) Genes & Dev. 3, 1874-1886; Gil, A., Sharp, P. A., Jamison, S. F., and Garcia-Blanco, M. A. (1991) Genes & Dev. 5, 1224-1236; Patton, J. G., Mayer, S. A., Tempst, P., and Nadal-Ginard, B. (1991) Genes & Dev. 5, 1237-1251). Sequence comparison programs suggested the presence of domains related to the RNA recognition motif found in other RNA-binding proteins, and deletion analysis revealed that the carboxyl-terminal 195 amino acids of the recombinant PTB was sufficient for specific binding to pre-mRNAs. Cross-linking experiments identified a 25-kDa protein in crude nuclear extracts of J558L cells that possessed the RNA binding properties of PTB, while a ∼60-kDa protein is detected in other murine cell lines tested. Thus, the 25-kDa protein found in J558L is likely a proteolytic product of the murine polypyrimidine tract binding protein. A probe derived from the PTB cDNA detected a ubiquitous 3.3-kb mRNA in murine cell lines and a 3.6-kb mRNA in human lines. Southern blot analysis revealed three strongly hybridizing DNA fragments and several more weakly hybridizing bands in mouse, human, and yeast DNA. The role of PTB in pre-mRNA splicing is discussed.",
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