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POLY(A)+ RNA FROM SHEEP OMASAL EPITHELIUM INDUCES EXPRESSION OF A PEPTIDE TRANSPORT PROTEIN(S) IN XENOPUS LAEVIS OOCYTES

by

YuanXiang Pan

Thesis submitted to the Faculty of the Virginia Tech in partial fulfillment of the requirements for the degree of

Master of Science

in

Animal and Poultry Sciences

Approved

K. E. Webb, Jr., Co-Chair
E. A. Wong, Co-Chair
J. R. Bloomquist
J. H. Herbein

August 9, 1996
Blacksburg, Virginia


Abstract

In order to verify the research from this laboratory that sheep omasal epithelium contains mRNA encoding for a peptide transporter (s) and to determine di- to octapeptide transport capability, poly(A)+ RNA isolated from sheep omasal epithelium was injected into Xenopus laevis oocytes. Poly(A)+ RNA was functionally expressed in Xenopus oocytes 4 to 7 d post-injection. Peptide (5 di-, 10 tri-, 6 tetra-, 2 penta-, 1 hepta-, 1 septa-, 1 octapeptide) transport capability was measured by impaling oocytes with a microelectrode to monitor membrane potential (Vm). Oocytes were maintained in pH 5.5 buffer. Peptide transport was identified as being expressed when, in the presence of a buffered peptide substrate (1 mM), the oocyte membrane showed persistent depolarization (a more positive Vm). In the absence of peptide transport, the membrane became depolarized with the addition of buffered substrate, but rapidly repolarized to the resting potential. Peptide transport was expressed for some di-, tri-, and tetrapeptides. Measured depolarization ranged from 9.6 mV to 42.1 mV. Larger peptides were not transported by the oocytes. When transport expression was measured with the substrates in a pH 7.5 buffer, no transport occurred indicating that transport was dependent on a proton gradient. The data indicate that sheep omasal epithelium contains mRNA that code for a protein(s) capable of proton-dependent di-, tri-, and tetrapeptide transport. This provides further evidence that absorption of peptides from the ruminant stomach is possible.

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