title: p53 splice acceptor site mutation and increased HsRAD51 protein expression in Bloom's syndrome GM1492 fibroblasts.
abstract: GM1492 human diploid skin fibroblasts derived from a patient with Bloom's syndrome (BS), lack detectable p53 mRNA and protein as shown by Northern and Western blotting, and express an increased RecA-like activity. Here we demonstrate that the p53 gene is grossly intact in GM1492 cells according to Southern blotting. DNA sequencing did not reveal any mutations in the promoter region of p53. A highly sensitive RT-PCR produced a p53 cDNA fragment that was shorter than expected. DNA sequence analysis of p53 cDNA showed that exon 6 was missing, explaining the shorter PCR product. Furthermore, sequencing of genomic DNA revealed a base substitution at the nucleotide preceding the AG splice acceptor site of intron 5. The omission of exon 6 creates a frameshift at the junction of exons 5 and 7, and a premature stop codon in exon 7. The aberrant transcript is predicted to encode a truncated p53 protein containing 189 amino acid residues. Moreover, Western blotting demonstrated elevated HsRAD51 protein levels in GM1492 cells. The lack of sufficient levels of wild-type p53 and increased levels of HsRad51 protein may contribute to the elevated RecA-like activity in the GM1492 fibroblasts.
authors.affiliation: National Institute of Environmental Medicine, Karolinska Institutet, Box 210, S-171 77, Stockholm, Sweden.
authors.forename: K P
authors.initials: KP
authors.lastname: Magnusson
authors.forename: M
authors.initials: M
authors.lastname: Sandström
authors.forename: M
authors.initials: M
authors.lastname: Ståhlberg
authors.forename: M
authors.initials: M
authors.lastname: Larsson
authors.forename: J
authors.initials: J
authors.lastname: Flygare
authors.forename: D
authors.initials: D
authors.lastname: Hellgren
authors.forename: K G
authors.initials: KG
authors.lastname: Wiman
authors.forename: S
authors.initials: S
authors.lastname: Ljungquist
mesh_terms: D017398:Alternative Splicing / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D001816:Bloom Syndrome / Q000235:genetics* / Q000473:pathology; D015153:Blotting, Western; D002460:Cell Line; D002478:Cells, Cultured; D004247:DNA / Q000737:chemistry / Q000235:genetics; D004252:DNA Mutational Analysis; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D004268:DNA-Binding Proteins / Q000235:genetics*; D005347:Fibroblasts / Q000166:cytology / Q000378:metabolism*; D016368:Frameshift Mutation; D005786:Gene Expression Regulation; D006367:HeLa Cells; D006801:Humans; D009154:Mutation; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics; D051135:Rad51 Recombinase; D016159:Tumor Suppressor Protein p53 / Q000235:genetics* / Q000378:metabolism
chemical_list: D018076:DNA, Complementary; D004268:DNA-Binding Proteins; D012333:RNA, Messenger; D016159:Tumor Suppressor Protein p53; D004247:DNA; C495153:RAD51 protein, human; D051135:Rad51 Recombinase