Preliminary characterisation and partial purification of ribosomal gene promoter-binding proteins from Trypanosoma brucei.
DNA fragments including the promoter region of the major ribosomal RNA gene of Trypanosoma brucei (r-promoter) were identified and subcloned using a synthetic oligonucleotide probe corresponding to the putative core promoter. These fragments were used in mobility shift assays with proteins extracted from T. brucei nuclei, and demonstrated the presence in 0.15 M NaCl extracts of protein(s) with specific binding affinities for the r-promoter region. Binding was stable in the presence of a 100-fold excess of competitor DNA, and occurred at the relatively low salt concentrations (< 50 mM NaCl) characteristic of many enzyme activity optima in this organism. A control DNA fragment not including the r-promoter region was not retarded in the mobility shift assays, and the r-promoter-binding activity had a molecular weight of about 140,000. Nuclear extracts from T. brucei contained large amounts of DNase activity, and the promoter-binding proteins were partially purified from the crude extract using ammonium sulphate precipitation, sephacryl S-200 and Heparin-sepharose chromatography.
Mburu
P W
PW
Biochemistry Department, University of Sussex, Falmer, Brighton, UK.
Beebee
T J
TJ
D000818:Animals; D001483:Base Sequence; D015139:Blotting, Southern; D002467:Cell Nucleus / Q000378:metabolism; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D016054:DNA, Protozoan / Q000235:genetics* / Q000302:isolation & purification; D004275:DNA, Ribosomal / Q000235:genetics* / Q000302:isolation & purification; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides; D011401:Promoter Regions, Genetic; D012335:RNA, Ribosomal / Q000235:genetics*; D015183:Restriction Mapping; D014346:Trypanosoma brucei brucei / Q000235:genetics* / Q000378:metabolism
D016054:DNA, Protozoan; D004275:DNA, Ribosomal; D004268:DNA-Binding Proteins; D009838:Oligodeoxyribonucleotides; D012335:RNA, Ribosomal