ABSTRACT
The mdm2 gene encodes a zinc finger protein that negatively regulates p53 function by binding and masking the p53 transcriptional activation domain. Two different promoters control expression of mdm2, one of which is also transactivated by p53. We cloned and characterized the mdm2 gene from a murine 129 library. It contained at least 12 exons and spanned approximately 25 kb of DNA. Sequencing of the mdm2 gene revealed three nucleotide differences that resulted in amino acid substitutions in the previously published mdm2 sequence. Sequencing of normal BalbC/J DNA and the original cosmid clone isolated from the 3T3DM cell line revealed that they are identical, suggesting that the published sequence is in error at these three positions. In addition, we analyzed the expression pattern of mdm2 and found ubiquitous low-level expression throughout embryo development and in adult tissues. Analysis of mRNA from numerous tissues for several mdm2 spliced variants that had been identified in the transformed 3T3DM cell line revealed that these variants could not be detected in the developing embryo or in adult tissues.
Subject(s)
Nuclear Proteins , Proto-Oncogene Proteins/biosynthesis , Proto-Oncogene Proteins/genetics , 3T3 Cells , Animals , Antisense Elements (Genetics) , Base Sequence , Brain/embryology , Brain/metabolism , Cloning, Molecular , Cosmids , DNA Primers , Embryo, Mammalian , Exons , Ganglia, Spinal/embryology , Ganglia, Spinal/metabolism , Gene Expression , Genomic Library , In Situ Hybridization , Introns , Mice , Mice, Inbred BALB C , Molecular Sequence Data , Neoplasm Proteins/biosynthesis , Neoplasm Proteins/genetics , Polymerase Chain Reaction , Proto-Oncogene Proteins c-mdm2 , Restriction Mapping , Zinc FingersABSTRACT
Charcot-Marie-tooth disease type 1A (CMT1A) was localized by genetic mapping to a 3 cM interval on human chromosome 17p. DNA markers within this interval revealed a duplication that is completely linked and associated with CMT1A. The duplication was demonstrated in affected individuals by the presence of three alleles at a highly polymorphic locus, by dosage differences at RFLP alleles, and by two-color fluorescence in situ hybridization. Pulsed-field gel electrophoresis of genomic DNA from patients of different ethnic origins showed a novel SacII fragment of 500 kb associated with CMT1A. A severely affected CMT1A offspring from a mating between two affected individuals was demonstrated to have this duplication present on each chromosome 17. We have demonstrated that failure to recognize the molecular duplication can lead to misinterpretation of marker genotypes for affected individuals, identification of false recombinants, and incorrect localization of the disease locus.
