Eva Derman - US grants

The long range goals of our research are to understand the regulation of individual genes in multigene families, and to understand evolutionary processes that give rise to families of genes in which different family members are differentially regulated. The studies in this proposal use the mouse urinary protein (MUP) gene family as a model system. MUP genes are present in 15-25 copies in the mouse genome and are expressed in the liver and in the submandibular glands. They are under the genetic control of a cis-acting regulatory locus. The major specific aim is to elucidate the organization of MUP genes and to identify possible regulatory sequences. The research will involve a combination of electron microscope techniques, restriction mapping, and DNA sequencing techniques, all applied to MUP genomic clones isolated from BALB/c and C57BL/10 recombinant DNA libraries in the Lambda phage vector Charon 4A. DNA sequences responsible for hormonal and strain-specific regulation of MUP genes will be identified through experiments involving introduction of MUP genes and chimeric plasmids containing portions of MUP genes into heterelogous cells by DNA mediated transformation. Given suitable progress in the aforementioned studies, questions concerning the influence of chromatin structure on gene expression will be studied. In particular, it will be determined whether DNAse-I hypersensitive sites appear in the liver prior to the time that MUP genes are transcribed in this tissue. We hope, also, that it will be possible to induce, in vitro, specific chromatin changes in the vicinity of MUP genes and thereby to search for soluble factors involved in regulation.

urinary tract; gene expression;

The long range goal of this proposal is to test the hypothesis that regulatory elements derived from Mup-1.5a/5b, the submandibular gland- specific isotype of mouse major urinary protein gene (MUP), can direct the expression of biologically active peptides to the submandibular gland. There is a tremendous interest in finding methods to stimulate mucosal immunity, both to combat infections, and to develop vaccines effective at the mucosal surfaces. in all the mucosal secretions, the predominant immunoglobulin is IgA. A major goal of this proposal is to test the hypothesis that salivary gland IgA responses can be enhanced in vivo by targeting the expression of specific cytokines specifically to the epithelial cells of the submandibular gland. We identified a submandibular gland-specific enhancer/promoter in Mup- 1.5a/5b. Transgenic lines will be generated by introducing recombinant expression plasmids containing human and murine IL-6 cDNA controlled by Mud 1.5a/5b regulatory elements into mouse embryos. in the transgenic lines, the tissue- and cell type- specificity of transcription and the mode of secretion of transgene-encoded IL-6 will be determined by Northern analyses, and histochemical and immunological methods. IgA responses will be assessed in the salivary glands of mice expressing transgene-encoded IL-6, and as a control, in other mucosal tissues, e.g. the lamina propria of the small intestine and the lungs, in non-immunized mice and in mice immunized with Porhyromonas gingivalis 381 fimbriae. Histochemical methods will be used to determine whether enhanced submandibular gland levels of IL-6 bring about oral inflammation, autoimmune responses, or cell proliferation. Northern analysis will be used to detect the potential induction of acute-phase response genes in the liver. With the view of regulating the expression of transgene-encoded cytokines, minimal regulatory elements required for the submandibular gland-specific expression will be delineated using transgenic mice. Within the minimal element, DNA sequences that bind submandibular-gland enriched sequence- specific DNA-binding proteins will be identified by nuclease-protection and by electrophoretic mobility shift assays. To facilitate the identification of tissue-specific regulatory elements, comparisons to regulatory variants of the Mup-1.5a/5b gene will be performed. Following the identification of the cis-acting regulatory elements, the submandibular gland-enriched factor will be cloned in the lambda-gt11 expression vector.