Robert P. Erickson - US grants

We seek to understand molecular events involved in the differentiation of the mammalian male embryonic gonad. Such knowledge should be of benefit for improved diagnosis, and possibly prenatal diagnosis, of human intersex conditions as well as increasing our understanding of normal human development. Our studies will use both mouse models and human material. Studies in mice will focus on genetic loci in the proximal arm of chromosome 17 which interact with the mouse Y chromosome (which also has variants) to try and elucidate how these autosomal loci interact with the Y chromosome and other autosomal loci in contributing to normal testicular development. Among the loci on chromosome 17 may be those containing Banded krait minor satellite (Bkm) sex specific sequences. Since certain large deletions (T-Or1 and T-Hp) in this area of chromosome 17 can cause hermaphroditism, we will determine whether or not these deletions alter the amounts of Bkm-sequences detected on chromosome 17 by in situ hybridization. We will use the Bkm probe to look for mRNAs containing Bkm sequences in mouse and human fetal gonads and appropriate other tissues. Cosmid clones from the proximal region of mouse chromosome 17 and lambda clones from the human X chromosome will be screened with a Bkm probe. Subportions of positive clones will be used to screen fetal gonad RNA. The human Y chromosomal clones previously obtained, and new ones obtained by PERT and chromosome "hop" techniques, will be used to study XX males and their parents in an attempt to determine the source of the Y chromosomal DNA usually found in such XX males. The work on human material will also use human Y chromosome clones to look for expressed Y sequences in the male fetal gonad. Genomic clones corresponding to mouse or human male fetal gonad specific expressed sequences will be sought by screening cDNA libraries with the identified probe and using the cDNA clone to screen genomic clones (when the probe is not unique sequence).

Neurofibromatosis (NF) is a common human autosomal dominant condition characterized by cafe-au-lait spots, neurofibromas, and a broad range of other manifestations. Its high spontaneous mutation rate (among the highest described in man) and preliminary linkage analysis using protein polymorphisms has suggested that the responsible mutation may be at a different locus in different families. The power of linkage analysis to identify the location of a mutant gene has recently been greatly extended by the introduction of DNA polymorphisms as genetic markers, which should allow any disease gene to be mapped if appropriate families are available for study, and if a sufficient number of informative markers can be generated. NF is a highly appropriate target disorder for this approach. We propose to ascertain three or four three-generation families with NF and sufficient available family members to allow linkage analysi on single families, so that potential locus heterogeneity will not destroy any associations. Using EB virus transformed lymphocytes, we will look for evidence of linkage to GC blood group (chr. 4) and secretor (chr. 19) both of which have been suggested to show linkage to NF in some but not all families. We will also develop and study markers on chromosome 8 (where circumstantial evidence suggests the NF locus might be, and for which DNA probes are needed anyway). We will maximize the rapidity of obtaining linkage information on other autosomal chromosomes by using probes for multigene families, as well as the "minisatellite" probes recently described which detect a large family of polymorphic markers on a single Southern blot.

We propose to create a deletion in the mouse genome for the mouse analogue of the human met oncogene. Mice homozygous for the deletion would be a possible model for the human disorder cystic fibrosis (CF). It has recently been found that met, which maps to human chromosome 7, might even be the CF gene or is very closely linked to cystic fibrosis (less than 0.7 cM). Since there is a high degree of conservation of linkage between mouse and man, a radiation-induced deletion of the met locus in mice may well also remove the mouse analogue of the CF gene. We will search for such a deletion by screening DNA of F1 mice where one of the parents has been irradiated as part of an ongoing mutation project at Research Triangle Institute. This is a high risk project because (1) the screening of progeny from irradiated animals will not be efficient unless we are able to identify a restriction fragment length polymorphism between the parental strains whose F1 progeny we will be screening, (2) the number of progeny mice which will need to be screened is large, (3) the met-"CF" portion of the mouse genome may include genes necessary for embryonic development such that the homozygotes for the deletion would not survive long enough to be useful, and (4) the linkage may not be maintained. Nonetheless, we believe that the immense value of a murine model for treatment efforts, including gene therapy, warrants taking the risk.

RNA; antisense nucleic acid; gene expression; genetic manipulation; genetic promoter element; mammalian embryology; growth factor receptors; pregnancy loss; morphology; developmental genetics; oncogenes; genetically modified animals; biotechnology; congenital disorders; spermatogenesis; growth factor; model design /development; nucleic acid inhibitor; radionuclides; complementary DNA; molecular cloning; polymerase chain reaction; laboratory mouse; injection /infusion; genetic mapping;