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Abstract

Objective

To use canine glyceraldehyde-3-phosphate dehydrogenase complementary DNA (GAPDH cDNA) as a template in ribonuclease (RNase) protection assays to measure canine GAPDH mRNA expression.

Design and Procedure

Primers designed from the human GAPDH gene were used to amplify a 191 -base pair canine GAPDH cDNA by reverse-transcription polymerase chain reaction. The cDNA was sequenced, and used as a template for RNase protection assay.

Sample Population

Total RNA was isolated from a canine squamous carcinoma cell line.

Results

Canine GAPDH cDNA had a high degree of homology to human, rat, and mouse GAPDH. In vitro transcription of canine GAPDH cDNA was used to produce complementary RNA that detected canine GAPDH mRNA by RNase protection assay.

Conclusion

Canine GAPDH cDNA is a useful loading control to be used in RNase protection assays measuring mRNA expression in canine cells or tissues.

Free access
in American Journal of Veterinary Research

Abstract

Objective—To clone and sequence the cDNA for feline preproparathyroid hormone (preproPTH) and to compare that sequence with other known parathyroid hormone (PTH) sequences.

Sample Population—Parathyroid glands from 1 healthy cat.

Procedure—A cDNA library was constructed in λ phage from feline parathyroid gland mRNA and screened with a radiolabeled canine PTH probe. Positive clones were sequenced, and nucleic acid and deduced amino acid sequences were analyzed and compared with known preproPTH and PTH sequences.

Result—Screening of approximately 2 X 105 recombinant plaques revealed 3 that hybridized with the canine PTH probe; 2 clones comprised the complete sequence for feline preproPTH. Feline preproPTH cDNA consisted of a 63-base pair (bp) 5'-untranslated region (UTR), a 348-bp coding region, and a 326-bp 3'-UTR. The coding region encoded a 115-amino acid peptide. Mature feline PTH consisted of 84 amino acids. Amino acid sequence analysis revealed that feline PTH was > 83% identical to canine, bovine, swine, equine, human, and macaque PTH and 69, 71, and 44% identical to mouse, rat, and chicken PTH, respectively. Within the region responsible for hormonal activity (amino acids 1 to 34), feline PTH was > 79% identical to other mammalian PTH sequences and 64% identical to the chicken sequence.

Conclusions and Clinical Relevance—The amino acid sequence of PTH is conserved among mammalian species. Knowledge of the cDNA sequence for feline PTH may be useful to investigate disturbances of calcium metabolism and alterations in PTH expression in cats. (Am J Vet Res 2002;63:194–197)

Full access
in American Journal of Veterinary Research