title: Alu-DNA repeat-binding protein p68 is a part of Alu-RNA containing alpha-RNP.
abstract: An Alu-DNA repeat-binding protein with a molecular mass of 68 kDa (p68) is identified in the somatic human cell nucleoplasm. Gel mobility shift assay (GMSA), South-western blotting and affinity purification on DNA attached to the carrier were used in the identification. GMSA revealed multiple complexes with the exponential dependence of their relative mobility. A narrow binding site of the p68 was revealed using synthetic oligonucleotides. It is located between the A-box and B-box of the RNA polymerase III promoter and is identical to that reported for the Alu-binding protein from human spermatozoids. The same narrow binding site, the similarity of the isolation procedure from germ and somatic cells, and similar binding properties and molecular masses suggest homology of the two proteins. Antibodies raised against Alu-protein complexes led to hypershift of the complexes in GMSA and stained p68 in active fractions in human spermatozoids and in Alu-RNA-containing alpha-RNP particles. Immunofluorescence of a HeLa cell monolayer revealed an intranuclear dot pattern with the dots corresponding to euchromatin areas and some dots located at the cell periphery in the cytoplasm. alpha-RNP particles bound Alu-DNA in vitro and contained p68 as shown using the immunogold procedure. Alu-DNA binding activity was revealed in cytoplasm as well as in nucleoplasm. The possible nature of the main Alu-DNA binding protein and its involvement in the particle structure are discussed.
authors.affiliation: Institute of Cytology, Russian Academy of Sciences, St. Petersburg, Russia.
authors.forename: D V
authors.initials: DV
authors.lastname: Lukyanov
authors.forename: M E
authors.initials: ME
authors.lastname: Urusova
authors.forename: K M
authors.initials: KM
authors.lastname: Shcherba
authors.forename: O I
authors.initials: OI
authors.lastname: Podgornaya
mesh_terms: D020087:Alu Elements / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D005455:Fluorescent Antibody Technique; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000737:chemistry*; D009841:Oligonucleotides / Q000378:metabolism; D011401:Promoter Regions, Genetic; D012320:RNA Polymerase III / Q000235:genetics; D016601:RNA-Binding Proteins / Q000737:chemistry; D012261:Ribonucleoproteins / Q000737:chemistry*; D016415:Sequence Alignment; D013094:Spermatozoa
chemical_list: C408965:Alu-binding protein, 68 kDa, human; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D009841:Oligonucleotides; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins; C094973:sperm Alu-binding protein, human; D012320:RNA Polymerase III