Isolation and Characterization of Subnuclear Compartments from Trypanosoma brucei IDENTIFICATION OF A MAJOR REPETITIVE NUCLEAR LAMINA COMPONENT

摘要: Abstract Protozoan parasites of the order Kinetoplastida are responsible for a significant proportion global morbidity and economic hardship. These organisms also represent extremely distal points within Eukarya, one such organism,Trypanosoma brucei, has emerged as major system study evolutionary cell biology. Significant technical challenges have hampered full exploitation this organism, but advances in genomics proteomics provide novel approach to acquiring rapid functional data. However, vast distance between trypanosomes higher eukaryotes presents problems with assignment based on sequence similarity, frequently homologues cannot be identified sufficient confidence informative. Direct identification proteins isolated organelles potential providing robust insight is powerful initial assignment. We selected nucleus T. brucei first target protozoan organellar proteomics. Our purification methodology was able reliably both nuclear subnuclear fractions. Analysis by gel electrophoresis, electron microscopy, immunoblotting against trypanosome subcellular markers indicated that preparations high yield purity, maintain native morphology, well resolved from other organelles. Minor developmental differences were observed proteome bloodstream procyclic stages, whereas morphological alterations visible. demonstrate direct sequencing NUP-1 envelope antigen coiled coil protein, containing ∼20 near-perfect copies 144-amino acid sequence. Immunoelectron microscopy localized inner face envelope, suggesting it filamentous component lamina.