Sodium Dependent Vitamin C Transporters in the Sheep Corpus Luteum: Sequence Analysis
Advisor:Wick, Macdonald P.
Ottobre, Joseph S.
Keywords:Sodium Dependent Vitamin C Transporter
Polymerase Chain Reaction
MetadataShow full item record
Publisher:The Ohio State University
Series/Report no.:The Ohio State University. Department of Animal Sciences Honors Theses;2005
Vitamin C is a multifunctional antioxidant that is sequestered within the corpus luteum (CL). The concentrations of vitamin C found in the functional CL are 50-100 fold greater than concentrations in systemic plasma. Vitamin C may play a protective antioxidant role in the CL and has been shown to prevent apoptosis. The loss of vitamin C from the CL has been associated with luteolysis. Two sodium dependent vitamin C transporters (SVCT1 and SVCT2) have been described in various species, and the primary sequences of these proteins are known (e.g., human, pig, guinea pig, rats, mice). The primary sequences for SVCT1 and SVCT2 have not yet been reported in the sheep. Vitamin C transporters are highly conserved, and it is likely that the sheep CL expresses similar transport proteins. Since these transport proteins would represent critical elements in the regulation of vitamin C concentrations in the CL, the objective of the current work was to determine the primary protein sequences for SVCT1 and SVCT2 in the sheep. CL were surgically collected from regularly cycling sheep on day 3 of the estrous cycle. Luteal tissue was immediately snap frozen in liquid nitrogen and stored at -80° C. RNA was extracted from CL and transcribed into cDNA using MMLV reverse transcriptase. PCR was then run using primers that were designed from known SVCT sequences. PCR-amplified cDNA fragments of the predicted lengths were resolved by agarose electrophoresis, excised, and cloned into plasmids. Plasmids were transformed into Escherichia coli for amplification prior to DNA sequence analyses. We have sequenced a 296 base pair portion of the message for ovine SVCT1 and an 1860 base pair portion of the message for ovine SVCT2. This encodes for 98 amino acids for oSVCT1 and 618 amino acids plus the stop codon for oSVCT2. The length of the oSVCT1 amino acid sequence corresponds to 14% of the presumptive sequence based upon that of the human. We have found the sheep message to have high homology with that of the human (93%), the pig (92%), the rat (90%), and mouse (90%). The protein sequence was also found to have high homology with that of the human (100%), the pig (98%), the rat (100%), and mouse (98%). The length of the oSVCT2 amino acid sequence corresponds to 95% of the presumptive sequence based upon that of the human. We have found the sheep message to have high homology with that of the human (90%), the pig (93%), the rat (87%), and the mouse (87%). The protein sequence was also found to have high homology with that of the human (89%), the pig (88%), the rat (86%), and mouse (86%). These data are important in furthering studies of the regulation of SVCT proteins and vitamin C concentrations in the CL of the sheep.
Ohio Agricultural Research and Development Center
Items in Knowledge Bank are protected by copyright, with all rights reserved, unless otherwise indicated.