Rat alpha-lactalbumin has a 17-residue-long COOH-terminal hydrophobic extension as judged by sequence analysis of the cDNA clones.

Abstract

cDNA for rat a-lactalbumin has been cloned in bacterial plasmid, and its sequence has been analyzed. The DNA sequence analysis shows that rat a-lactalbumin has 17 extra residues beyond the COOH terminus of the a-lactalbumin isolated and sequenced to date from other species. The predicted COOHterminal sequence is hydrophobic and proline rich and bears some resemblance to #-casein sequences. a-Lactalbumin (a-LA) is a mammary gland-specific protein involved in lactose synthesis. It is a modifier protein that changes the substrate specificity of galactosyl transferase, an enzyme normally involved in the biosynthesis of glycoproteins. Specifically, it promotes the binding ofglucose, otherwise a poor substrate, to galactosyl transferase, thereby permitting synthesis of lactose (for review, see refs. 1 and'2).The complete amino acid sequences of human (3), bovine (4), goat (5), guinea pig (6), and rabbit (7) and the partial sequence ofkangaroo (8) a-LA are known. These a-LAs are single polypeptides of 123 amino acid (except rabbit a-LA, which has 122) residues that have sequences somewhat similar to that of lysozyme (1, 2). a-LA from rat has been shown to be a glycoprotein (9, 10) and was separated on a DEAE-cellulose column into two molecular species (9). One of these has been shown by sedimentation analysis to have a higher molecular weight than any other known a-LA (9).For studies of hormonal regulation of milk protein genes in normal and neoplastic tissue, we have fractionated individual RNA species from the total poly(A)+RNA of the lactating rat mammary gland and have identified and characterized rat a-LA mRNA (11,12). It is difficult to obtain pure RNA absolutely free of other RNA species by conventional fractionation techniques. However, this problem can be resolved if a cDNA clone containing sequences complementary to the corresponding mRNA can be obtained. We now report the isolation of cDNA clone of rat a-LA gene sequences and present its nucleotide sequence.This sequence encodes part of the known partial sequence for rat a-LA protein. From the DNA sequence, the remainder of the rat a-LA polypeptide sequence can be deduced; it is homologous with the other known a-LA sequences but contains a COOH-terminal 17-residue extension that has a sequence resembling that of a casein.MATERIALS AND METHODS Synthesis of Double-Stranded cDNA. Rat mammary gland poly(A)+RNA was prepared from 5-day lactating Sprague-Dawley rats as described (12). cDNA was synthesized from 100 ,g of poly(A)+RNA by using 600 units of reverse transcriptase (RNAdependent DNA nucleotidyltransferase, avian myeloblastosis virus, a gift of J. W. Beard, Life Sciences, St. Petersburg, FL) in a 1.2-ml reaction mixture of 50 mM Tris HCl, pH 8.3/6 mM MgCl2/1.5 mM dithiothreitol/40 mM KCI/400 ,uM each dATP, dCTP, dGTP, and dTTP containing oligo(dT)12_18 (12.5 ,Ag/ml), actinomycin D (300 ,ug/ml), and 0.4 mCi of [3H]dATP (15 Ci/mmol; 1 Ci = 3.7 x 1010 becquerels) (12). The yield of single-stranded cDNA was =18 ,Ag. The DNA was fractionated on the b...

Programmed cell death in bovine mammary tissue during lactation and involution

“…Northern blot analysis was performed by hybridization of immobilized RNA with 32P-labelled complementary DNA (cDNA) or oligonucleotide probes. An a-lactalbumin cDNA was prepared by polymerase chain reaction (PCR) from a lactating bovine mammary cDNA library using oligonucleotide primers encoding two 30-mer sequences of a-lactalbumin mRNA (5'-GTCAGTTTGCCTGAA-TGGGTCTGTACCACG-3' and 5'-GACTCGAGTCGACATCGA(Tl7)-3' (Dandekar & Qasba, 1981) as described by Hurley & Schuler (1987). The reaction product was purified for Klenow polymerase labelling or insertion into the pGEM-T vector (Promega) for confirmation of sequence.…”

Sulphated proteins secreted by rat mammary epithelial cells

“…Unlike other α-lactalbumin, rat α-lactalbumin has 17 extra amino acid residues which form an extension at the carboxyl terminus of the protein [19,20]. The C-terminal sequence extension is hydrophobic and proline rich, two features which might explain the anomalous electrophoretic mobility of the protein, which migrated with an Mr of at least 23 000 in our conditions, compared with an Mr of approximately 14 000−15 000 in other species.…”

Cloning and molecular characterization of a yak α-lactalbumin cDNA from mammary tissue

“…Several investigations have been carried out on α-lactalbumin cDNA in some mammals, such as cattle (Hurley and Schuler, 1987), goat (Kumagai et al, 1987), sheep (Gaye et al, 1987), pig (Das Gupta et al, 1992), rat (Dandekar and Qasba, 1981), guinea pig (Hall et al, 1982), mouse (Vilotte et al, 1992), and canine (Watanabe et al, 2000). However, the yak α-lactalbumin cDNA has been less extensively investigated.…”