The long term goal of this proposal is to examine the molecular basis for the defective transport of Cobalamin (Cbl; Vitamin B12) out of the enterocytes and into various peripheral tissues in patients who exhibit deficiency of transcobalamin II (TC II), a Cbl transport protein. Specific questions that will be addressed include an analysis of the factors that regulate the expression of TC II gene at the mRNA level in the absorptive enterocytes and the effect on Cbl transcytosis following hybrid arrest translation of TC II mRNA. Many of these studies, will use spontaneously differentiating human colon adenocarcinoma (Caco-2) cells, and rats (fetal and adult). Ribonuclease protection assay using [32P]- cRNA (350 bp) transcribed from Nde I digest of TC II cDNA will be used to measure TC II mRNA levels during epithelial cell differentiation, intestinal development, and along the horizontal (duodenum-ileum) and vertical (crypt to villus) axis. Furthermore, the effect of hybrid arrest translation of TC II mRNA by using anti-sense oligonucleotides generated to different regions of TC II cDNA on the intrinsic factor mediated transcytosis of [57Co]Cbl will be examined using polarized Caco- 2 cells grown on culture inserts. These studies should provide important clues on the regulation of expression and role of TC II in the normal transcytosis of Cbl across the enterocytes, which in turn may help in understanding the pathophysiology of intestinal malabsorption of Cbl noted in patients with TC II deficiency. Using in vitro mutagenesis of TC II cDNA the structural elements that contribute to or are important in the binding reactions of TC II and its secretion will be examined using the cRNA transcribed from mutagenized TC II expressed in either reticulocyte lysate or Xenopus laevis oocyte expression systems. The structural basis for TC II polymorphism, including TC II deficiency in patients will be examined at the RNA/DNA levels using skin fibroblasts obtained from these patients. Following amplification of the DNA by PCR the nature and locus of the molecular defect in TC II will be examined at the DNA level using restriction fragment length polymorphism and/or denaturing gradient gel electrophoresis. In order to begin studies on the human tissue and regional variation in the intestine of regulation of the TC II gene expression, we will first isolate a full length gene for TC II and map out the introns and exons. Analysis of structure of TC II will provide important insights into the role of TC II in maintaining flux of Cbl across cellular membranes. Such studies should help us to better understand the pathophysiology of Cbl malabsorption and transport of Cbl in patients with congenital lack of TC II.
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