Fragile X linked mental retardation or Martin-Bell syndrome (MBS) is the single most common inherited form of mental retardation. Clinical diagnosis and cytogenetic detection of the fragile site at Xq27 are fraught with difficulties, particularly carrier detection and prenatal diagnosis. Somatic cell hybrid analysis offers unique advantages in dissecting the genetic and biochemical factors which influence fragile X expression, including: 1) produces higher expression rates than lymphocytes in low expressing and normal X chromosomes, 2) allows isolation of different mutations on a homogeneous, xenogeneic background, away from human autosomes, 3) allows isolation of the fragile X from the normal X homolog in carrier females in an active or inactive state, and 4) produces spontaneous expression by thymidine (Td) deprivation without the use of antimetabolites in a thymidylate synthase deficient (TS-) hybrid system. These unique properties of hybrid analysis will be further exploited with the following aims: 1) Test the hypothesis: The fragile site at Xq27 is a common fragile site present in all humans which is amplified by uniqual crossover in female meiosis, disrupting a neighboring gene and producing MBS. 3 of 3 normal X chromosomes are fragile X negative in lymphocytes but positive in hybrids; more normal humans and other primates will be tested. Degree of amplification and expression should be correlated, forming a continuum: lowest in normal Xs, higher in clinically normal transmitting males, highest in affected males. This explains many non-Mendelian aspects of MBS inheritance. 2) determine why lymphocyte expression in heterozygous females is absent or lower than that in hemizygous males. Females differ from males in two obvious ways: presence of a normal X homolog and X inactivation. Using hybrids, the fragile X can be isolated from the normal X in an active or inactive state. 3) Develop methods to enhance fragile X expression to improve diagnostic and prenatal diagnostic capabilities. In standard or TS- hybrids, fragile X expression is induced by inhibitors of DNA replication such as FUdR, Td deprivation, excess Td, or aphidicolin and significantly enhanced by caffein post-treatment. Using a TS- hybrid system which can be synchronized and spontaneous expression obtained without use of replication inhibitors, we will further investigate caffeine's enhancement of fragile X expression with these and other inhibitors of DNA replication. 4) Isolate and identify the DNA sequence responsible for fragile site expression. A cosmid library has been constructed from Xq24-qter which contains the fragile site. Individual cosmids will be assayed for their ability to """"""""create"""""""" a fragile site in the permissive Chinese hamster cell line after DNA transfection.
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