The complete amino acid sequence of human serum transferrin has been determined by aligning the structures of the 10 CNBr fragments. The order of these fragments in the polypeptide chain is deduced from the structures ofpeptides overlapping methionine residues and other evidence. Human transferrin contains 678 amino acid residues and-including the two asparagine-linked glycans-has an overall molecular weight of 79,550. The polypeptide chain contains two homologous domains consisting of residues 1-336 and 337-678, in which 40% of the residues are identical when aligned by inserting gaps at appropriate positions. Disulfide bond arrangements indicate that there are seven residues between the last half-cystine in the first domain and the first half-cystine in the second domain and therefore, a maximum of seven residues in the region of polypeptide between the two domains. Transferrin-which contains two Fe-binding siteshas clearly evolved by the contiguous duplication of the structural gene for an ancestral protein that had a single Fe-binding site and contained -340 amino acid residues. The two domains show some interesting differences including the presence of both N-linked glycan moieties in the COOH-terminal domain at positions 413 and 610 and the presence of more disulfide bonds in the COOH-terminal domain (11 compared to 8). The locations of residues that may function in Fe-binding are discussed.The iron-transportprotein ofserum, transferrin, is a monomeric glycoprotein with Mr =80,000. The properties and functions of serum transferrins have been recently reviewed in detail (1). Briefly, the transferrin molecule possesses two independent metal binding sites, each of which can bind a ferric ion with a Ka of -1022 M-1 together with a bicarbonate anion. The protein ligands for Fe3" at each site include two or three tyrosine residues, one or two histidine residues, and-the concomitantly bound bicarbonate anion (1). The view that transferrin consists of two homologous domains-each associated with one metal binding site-is supported by the demonstration of internal homology in a partial sequence for human transferrin (2) and by the production offragments ofvarious transferrins by partial proteolysis that have approximately half the molecular weight ofthe native protein and single sites for Fe3" binding (3-6). Low resolution x-ray crystallographic studies also support a two-domain structure for rabbit serum transferrin (7).The delivery of iron from transferrin to cells is mediated by the binding of transferrin-Fe3+ complexes to specific cellular receptors (e.g., see refs. 8-10). Transferrin molecules therefore possess a specific receptor-recognition site in addition to the two metal binding sites. We report here the complete amino acid sequence ofhuman serum transferrin, which confirms the presence of extensive internal homology within the polypeptide chain and permits the identification of the locations of the substitutions in some previously reported genetic variants. Together with structural information fr...

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Galactosyltransferases constitute a family of enzymes, each member of which transfers galactose from UDPgalactose to a specific acceptor molecule, generating a specific galactose-acceptor linkage. Two synthetic oligonucleotides, 27mer and 21mer, were synthesized, based on the amino acid sequences of two peptides derived from bovine milk N-acetylglucosaminide (,Bl-4)galactosyltransferase (EC 2.4.1.90), and used as hybridization probes to isolate cDNA clones for galactosyltransferase from a bovine mammary gland cDNA library. One of the plasmids, designated pLbGT-1, contains an insert of about 3.7 kilobases that hybridizes to both of the probes and encodes the amino acid sequences of five peptides obtained from bovine milk (,Bl-4)galactosyltransferase. A second plasmid, designated pLbGT-2, contains an insert of about 4.1 kilobases that hybridizes to only the 27mer and that encodes a polypeptide containing the sequence of the carboxyl-terminal 120 residues identical to the peptide encoded by pLbGT-1; the rest of the protein sequence, however, does not contain known sequences from bovine galactosyltransferase. The two cDNAs contain a 3'.untranslated region of about 2.7 kilobases that includes two copies of the Alu-equivalent sequences. pLbGT-1 and pLbGT-2 hybridize to mRNAs of various sizes obtained from the bovine and rat mammary gland and the human mammary tumor cell line MCF-7, with the longest mRNA from each species being around 4.5 kilobases. The results show that pLbGT-1 is a cDNA clone for bovine (,Bl-4)galactosyltransferase, and pLbGT-2 encodes a protein that is structurally and may be functionally related to transferases.Glycosyltransferases are a family of enzymes that are involved in the synthesis and extension of the oligosaccharide chains of glycoproteins and glycolipids (for review, see refs.

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Human forensic casework requires sensitive quantitation of human nuclear (nDNA), mitochondrial (mtDNA), and male Y-chromosome DNA from complex biomaterials. Although many such systems are commercially available, no system is capable of simultaneously quantifying all three targets in a single reaction. Most available methods either are not multiplex compatible or lack human specificity. Here, we report the development of a comprehensive set of human-specific, target-specific multiplex polymerase chain reaction (PCR) assays for DNA quantitation. Using TaqMan-MGB probes, our duplex qPCR for nDNA/mtDNA had a linear quantitation range of 100 ng to 1 pg, and our triplex qPCR assay for nDNA/mtDNA/male Y DNA had a linear range of 100-0.1 ng. Human specificity was demonstrated by the accurate detection of 0.05 and 5% human DNA from a complex source of starting templates. Target specificity was confirmed by the lack of cross-amplification among targets. A high-throughput alternative for human gender determination was also developed by multiplexing the male Y primer/probe set with an X-chromosome-based system. Background cross-amplification with DNA templates derived from 14 other species was negligible aside from the male Y assay which produced spurious amplifications from other nonhuman primate templates. Mainstream application of these assays will undoubtedly benefit forensic genomics.

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