Identification of three proteins that associate in vitro with the Leishmania (Leishmania) amazonensis G-rich telomeric strand.
The chromosomal ends of Leishmania (Leishmania) amazonensis 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 double-stranded 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 approximately 35 and approximately 52 kDa bands, respectively. The <or= 15 kDa protein component of LaGT1 was gel-purified as a UV cross-linked complex of approximately 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 electrospray ionization tandem mass spectrometry. The fingerprint and amino acid sequence analysis showed that the protein components of LaGT2 and of LaGT3 were, respectively, similar to the kinetoplastid Rbp38p and to the putative subunit 1 of replication protein A of Leishmania spp., whereas the <or= 15 kDa protein component of LaGT1 was probably a novel Leishmania protein.
Fernández
Maribel F
MF
Departamento de Patologia Clínica, Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Brazil.
Castellari
Rafael R
RR
Conte
Fábio F
FF
Gozzo
Fábio C
FC
Sabino
Adão A
AA
Pinheiro
Hildete
H
Novello
José C
JC
Eberlin
Marcos N
MN
Cano
Maria I N
MI
D000818:Animals; D001482:Base Composition; D001483:Base Sequence; D002458:Cell Fractionation; D016054:DNA, Protozoan / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D006801:Humans; D007891:Leishmania / Q000235:genetics* / Q000502:physiology; D046911:Macromolecular Substances; D013058:Mass Spectrometry; D010449:Peptide Mapping; D015800:Protozoan Proteins / Q000302:isolation & purification / Q000378:metabolism*; D012091:Repetitive Sequences, Nucleic Acid; D012492:Salts / Q000737:chemistry; D016615:Telomere / Q000235:genetics*; D034501:Telomere-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*
D016054:DNA, Protozoan; D046911:Macromolecular Substances; D015800:Protozoan Proteins; D012492:Salts; D034501:Telomere-Binding Proteins