[The structure, function and mutation of Rb gene promoter in normal individuals and retinoblastoma patients]

To investigate the DNA sequence of the 5' untranslated region of the Rb gene in normal individuals and retinoblastoma patients and identify the elements involved in transcription regulation of Rb gene.

SSCP analysis and direct genomic DNA sequencing were used to identify variations or alterations in DNA of normal adult WBC and tumor DNA of retinoblastoma patients. The different DNA fragments located in the 5' untranslated region of Rb gene were amplified and inserted into an expression plasmid with a CAT reporter gene. The capacity of transcription regulation of isolated DNA fragments was determined by CAT assay.

A 240 bp DNA fragment located between -327(-)-87 bp upstream of the first starting codon was shown to be the sequence with essential promoter function. The Sp1/RBF-1, ATF and E2F binding sites were present in this region and positive and negative elements were found further upstream and downstream. Four of 5 naturally occurring mutations were identified in either Sp1/RBF-1 or ATF binding sites from 302 patients with retinoblastoma. All these mutants had reduced CAT activity. However, 100 cases of normal adults did not show any variant DNA sequence in the same region.

The promoter DNA sequence of Rb gene is quite stable. Its variation usually leads to reduction in promoter activity which may be associated with genetic susceptibility to retinoblastoma.