ABSTRACT
The suggested common regulator of the eye lens crystallin genes is c-Maf. Maf responsive elements have been detected in a number of crystallin promoters including that of the rat betaB2-crystallin gene. The betaB2-crystallin gene is active in the post-natal lens and its mRNA reaches its maximal level in the rat lens 6 months after birth. Yet c-Maf has been reported to be present in the rat lens only up to 3 months of age. This discrepancy prompted an investigation into the role of the Maf responsive element (MARE) in the regulation of activity of the rat betaB2-crystallin promoter in rat lens fiber cells. Although betaB2 promoter activity is enhanced by c-Maf in both in vitro differentiating rat lens fiber cells and CHO cells, deletion of the betaB2 MARE, which was mapped to -143/-123, does not decrease betaB2 promoter activity in lens fiber cells. Furthermore, a dominant negative c-Maf construct did not inhibit activity of the betaB2 promoter in lens fiber cells. The data suggest that the betaB2 MARE does not play a major role in regulating activity of the betaB2 promoter. Rather, a putative Sox binding site at -164/-159 and a positive element at -14/-7 seem to be the prime regulatory elements.
Subject(s)
Crystallins/genetics , Genes, Regulator/physiology , Promoter Regions, Genetic/physiology , Saccharomyces cerevisiae Proteins , Transcription Factors , Animals , Cell Differentiation , Epithelial Cells/cytology , Fungal Proteins/genetics , Gene Expression , Genes, Reporter/physiology , Lens, Crystalline/metabolism , Rats , Transfection/methodsABSTRACT
Recently identified mutations affecting different domains of the RET proto-oncogene are associated with Multiple Endocrine Neoplasia type 2A (MEN 2A) and type 2B (MEN 2B), familial and sporadic Medullary Thyroid Carcinomas (MTC) and Hirschsprung disease (HSCR). In order to facilitate the screening for RET mutations, and to study possible genotype-phenotype correlations, we established exon-intron junctions and extended the intronic sequences flanking the 20 exons of this gene. This made it possible to design primers and to develop PCR conditions useful for SSCP analysis of the whole RET coding sequence. Nine conformational variants were observed which after sequencing turned out to be 8 silent mutations and a conservative amino acid substitution. Restriction analysis performed on DNA samples from unrelated controls confirmed the polymorphic nature of six of these nucleotide changes and made it possible to estimate the frequency of the corresponding alleles.
