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    Characterization of a new horse transferrin variant : a thesis presented in partial fulfilment of the requirements for the degree of Master of Science in Biochemistry at Massey University
    (Massey University, 1993) Paterson, Geoffrey Reayburn
    Transferrin is a glycoprotein with a molecular weight of approximately 80 kd. Its single polypeptide chain is formed into two lobes and it is able to bind two ferric (Fe III) ions per protein molecule. Horse serum transferrin, like the transferrins of most vertebrate species, exhibits extensive genetic polymorphism. Transferrin is one of several protein systems used for blood-typing horses. During routine blood typing a new band (designated*) was found. This variant originated from a thoroughbred stallion which was of considerable value as a sire and so it was of interest to characterize this new transferrin variant. Thoroughbred horses carry genes for only six of the fourteen known transferrin isoforms; D, Fl, F2, H2, 0 and R. The aim of this project was to characterize, by classical amino acid sequence analysis, the transferrin variant and the parental variants D and Fl, from one of which must have arisen. All three variant forms (D, Fl and*) were purified. Tryptic digests of the variants were analysed by HPLC and those peaks appearing to differ between the HPLC profiles were sequenced by automated protein sequencing. The sequences obtained confirmed that the protein isolated was a transferrin variant. Further sequencing allowed deduction of the parent transferrin variant. Two clear sequence differences between the D and Fl variant have been identified. The Fl variant was found to contain an arginine residue at amino acid position 553, whereas the D variant contains a cysteine residue at this position. At position 418 of the Fl variant a serine residue was found and at the same position in the D variant a proline residue was found. Sequence determination of peptides from the * tryptic digest revealed that a proline residue and a cysteine residue were found at positions 418 and 553 respectively, clearly indicating that the new * phenotype has arisen from the D allele and not the Fl allele.
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    The cDNA sequence and polymorphism of horse transferrin : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Veterinary Clinical Science at Massey University, Palmerston North, New Zealand
    (Massey University, 1992) Carpenter, Margaret Ann
    Transferrin, the serum protein responsible for iron transport, is highly polymorphic in many species, including the horse. In this study, the cDNA sequence of horse transferrin was determined and used to identify sequence polymorphisms which distinguish some of the many variants of horse transferrin. A horse liver cDNA library was constructed, and was screened using the human transferrin cDNA as a probe. The clones isolated gave 1800 bp of sequence. The remainder of the cDNA was obtained using PCR. The 2305 bp horse transferrin cDNA sequence included part of the 5' untranslated region and extended to the poly(A) tail. It had 80% sequence identity with the human transferrin cDNA, and encoded a protein of 706 residues, including a signal sequence of 19 amino acids. The amino acid sequence was compared to those of related proteins, i.e. the serum transferrins and lactoferrins of several species and human melanotransferrin. The horse transferrin sequence had the duplicated structure and conserved iron binding and cysteine residues which are characteristic of the transferrin family. This is consistent with the structural and functional similarities within the family. Horse transferrin has 73% amino acid sequence identity to human transferrin, 62% identity with human lactoferrin, and 53% identity with chicken transferrin. Sequence polymorphisms distinguishing the variants of horse transferrin which are found in thoroughbreds (D, F1, F2, H2, O, R and ) were identified. First, comparison of the horse transferrin cDNA and protein sequences with two reported amino acid substitutions distinguishing the D and R variants indicated that exons 12 and 15 were likely to be polymorphic. Therefore, these two regions were analysed by Southern blotting and by sequencing PCR products. The D and R variants differed by 10 nucleotide substitutions which encoded 6 amino acid substitutions. The F1, F2, H2 and variants were identical to D, and the O variant was very similar to R, in the regions studied. The data indicated that the horse transferrin variants comprise two distinct groups. Secondly, the positions of differences between the D and F1 alleles were established by producing single stranded conformation polymorphisms. Sequencing then revealed 3 nucleotide substitutions that encode 2 amino acid substitutions. All 8 of the amino acid substitutions which were observed occurred at positions which are variable between members of the transferrin family. Location of the polymorphic residues on the 3-dimensional structure of human lactoferrin revealed that all were clustered at one end of the C-lobe.