The long-term goal of this proposal is to understand the molecular basis for the genetic control of mammalian development. The albino-deletion complex represents one of few regions of the mouse genome where a set of specific developmental defects are associated with a series of overlapping chromosomal deletions. A total of 37 deletions exist, all of which remove the region of mouse chromosome 7 that surrounds and includes the albino coat color locus. The complementation patterns among these deletion chromosomes indicate hat at least 9 distinct genetic units exist in this region. Four of these units are needed for normal embryonic development during the preimplantation or early postimplantation stages. The work proposed here will concentrate on a molecular genetic analysis of two of these units, each of which is known to contain a gene(s) whose expression is required during the time that the basic body plan is being established in the early postimplantation mouse embryo. The homozygous (null) lethal phenotype associated with deletions that remove this areal has been determined and molecular markers for this region have been generated by chromosome microdissection and microcloning. Chromosome walking and pulsed-field gel electrophoresis will be used to isolate and physically map additional markers. Transcription units will be identified by screening cDNA libraries made from early postimplantation embryos and also by NOrthern blot analysis using RNA from embryo-derived stem cells. Once a transcription unit has been identified, its tissue- and stage-specific pattern of expression will be determined either by Northern blot analysis or by in situ hybridization to tissue sections. A molecular characterization of cDNAs and corresponding genomic sequences will be done with the purpose of identifying putative protein coding regions, direction of transcription, 5' and 3' ends, and intron/exon boundaries. Antibodies will be made to synthetic peptides with the purpose of locating the protein product in the embryo. Transgenic mice will be produced to determine if any of the identified transcription units can complement the homozygous defect. Analysis of the lethal phenotype associated with the e=homozygous state of the five deletions of interest will be extended by examining specific gene expression in mutant embryos. Homeobox gene expression in wild-type mouse embryos has been extensively studied, and it is known that some of these genes begin to be expressed at a time which is coincident with the appearance of abnormalities in the homozygous individuals. Therefore, by determining the pattern of homeobox gene expression in the mutant embryos, a more accurate assessment of their developmental potential can be made.
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