tailieunhanh - Báo cáo khoa học: Identification of three proteins that associate in vitro with the Leishmania (Leishmania) amazonensis G-rich telomeric strand

The chromosomal ends ofLeishmania (Leishmania) ama-zonensis contain conserved 5¢-TTAGGG-3¢ telomeric repeats. Protein complexes that associatein vitro with these DNA sequences,Leishmania amazonensisG-strand telo-meric protein (LaGT1-3), were identified and characterized byelectrophoreticmobilityshift assays andUVcross-linking using protein fractions purified from S100 and nuclear extracts. The three complexes did not form (a) with double-stranded DNA and the C-rich telomeric strand, (b) in competition assays using specific telomeric DNA oligo-nucleotides, or (c) after pretreatment with protein-ase K. . | Eur. J. Biochem. 271 3050-3063 2004 FEBS 2004 doi Identification of three proteins that associate in vitro with the Leishmania Leishmania amazonensis G-rich telomeric strand Maribel F. Fernandez1. Rafael R. Castellari2. Fabio F. Conte1. Fabio C Gozzo3. Adao A Sabino3 . . . . . Hildete Pinheiro4 Jose C. Novello2 Marcos N. Eberlin3 and Maria I. N. Cano1 1 Departamento de Patologia Clinica Faculdade de Ciencias Medicas 2Laboratorio de Quimica de Proteinas LAQUIP Departamento de Bioquimica Instituto de Biologia 3Instituto de Quimica 4Departamento de Estatistica Instituto de Matematica Estatistica e Computapão Cientfica Universidade Estadual de Campinas UNICAMP Brazil The chromosomal ends of Leishmania Leishmania ama-zonensis contain conserved 5 -TTAGGG-3 telomeric repeats. Protein complexes that associate in vitro with these DNA sequences Leishmania amazonensis G-strand telomeric protein LaGT1-3 were identified and characterized by electrophoretic mobility shift assays and UV cross-linking using protein fractions purified from S100 and nuclear extracts. The three complexes did not form a with doublestranded DNA and the C-rich telomeric strand b in competition assays using specific telomeric DNA oligonucleotides or c after pretreatment with proteinase K. LaGT1 was the most specific and did not bind a Tetrahymena telomeric sequence. All three LaGTs associated with an RNA sequence cognate to the telomeric G-rich strand and a complex similar to LaGT1 is formed with a double-stranded DNA bearing a 3 G-overhang tail. The protein components of LaGT2 and LaGT3 were purified by affinity chromatography and identified after renaturation as w 35 and w 52 kDa bands respectively. The 15 kDa protein component of LaGT1 was gel-purified as a UV cross-linked complex of w 18-20 kDa. Peptides generated from trypsin digestion of the affinity and gel-purified protein bands were analysed by matrix-assisted laser desorption ionization-time of flight and .