The crypts and villi of the intestine are lined by a complex continuous single cell layer epithelium. Multipotential anchored stem cells located near the base of the crypts give rise to four terminally differentiated cell types, the majority of which migrate to the tips of the villi and are extruded within three to four days. The intestinal crypt cells represent one of the most rapidly proliferating cell populations in adult animals, with each crypt producing about 300 cells per day. The mechanisms controlling the rapid proliferation and cell differentiation in the intestine are not well understood. The family of tyrosine kinases includes a number of growth factor receptors and oncogenes, and these function to regulate growth and differentiation in a variety of systems. We hypothesize that tyrosine kinases also play an important regulatory role in the intestinal crypts. To identify the tyrosine kinase genes expressed in the crypts, we used the polymerase chain reaction primed by degenerate oligonucleotide probes corresponding to two invariant amino acid sequence motifs found in all tyrosine kinases, to amplify tyrosine kinase catalytic domains using crypt cell cDNA as a template. The amplified tyrosine kinase fragments were cloned and 285 clones have been characterized. Sequencing has identified several tyrosine kinase genes expressed in the crypts, including 14 novel genes. By examining their expression, we are determining which of these tyrosine kinase genes is preferentially transcribed in the intestinal crypts. Full length cDNA clones encoding these tyrosine kinases will be isolated and characterized. We will express these cDNAs in cell lines and in transgenic mice, to determine if inappropriate expression results in a detectable phenotype. Antibodies will be generated against fusion proteins encoded by these clones, and these will be used to begin our characterization of the tyrosine kinase proteins. Identification of the tyrosine kinases expressed in the intestine, and isolation of novel crypt cell tyrosine kinase cDNA clones will provide the molecular tools for the genetic and biochemical dissection of differentiation programs taking place in the intestine. Our long-term goal is to understand the function and regulation of crypt cell tyrosine kinases both in vivo and in vitro.
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