Milk protein gene expression in the rat mammary gland

Qasba, Pradman K.; Dandekar, Abhaya M.; Horn, Toby M.; Losonczy, Ilona; Siegel, Mary; Sobiech, Krzysztof A.; Nakhasi, Hira L.; Devinoy, Ève

doi:10.1080/10408398209527332

Cited by 13 publications

(11 citation statements)

References 53 publications

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“…The in vitro-synthesized protein was immunoprecipitated by a-LA antiserum and immunocompeted with pure rat a-LA. Total poly(A)+RNA from the lactating gland synthesizes several [3S]cysteine-labeled lactoproteins (22) (Fig. 1, track 1), but only one of these was immunoprecipitable with a-LA antiserum and immunocompeted by pure rat a-LA ( Fig.…”

Section: Resultsmentioning

confidence: 99%

“…It has been proposed (9,22) that the higher molecular weight form of a-LA could be larger than the 123-amino acid-long rat a-LA (10). Our present data, based on DNA sequence analyses oftwo cDNA clones (p17 and p18), now confirm the previously proposed higher molecular weight form of rat a-LA (9,22) and show it to be 17 residues longer than 123 amino acids (10,27). DNA sequence analysis of the 630-nucleotide insert ofthe recombinant plasmid p18, containing gene sequences complementary to rat a-LA mRNA, showed that the insert encodes residues 25-140 ofthe a-LA protein.…”

Section: Resultsmentioning

confidence: 99%

“…These changes increased the efficiency of translation (22). Aliquots of the resulting labeled products (2 x 105 cpm) were immunoprecipitated with rat a-LA antibody (9) in the presence-and absence of 1 ug ofunlabeled rat a-LA.…”

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

Dandekar¹,

Qasba²

1981

Proc. Natl. Acad. Sci. U.S.A.

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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...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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

Dandekar¹,

Qasba²

1981

Proc. Natl. Acad. Sci. U.S.A.

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“…This group ofwhey phosphoproteins (Wp-protein) constitutes 1 to 2 mg/ml in whole rat milk. These Wp-proteins initially copurify with rat a-LA in the whey fraction, but they can be separated from a-LA on DEAE-cellulose into three charged forms (1,5,6). These proteins are rich in aspartic and glutamic acids (or their amides) and in serine and half-cystine but lack tyrosine (5).…”

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confidence: 99%

“…From rat milk, a-lactalbumin (a-LA) (1, 2) and a number of caseins (42, 29, and 25 kilodaltons) (3,4) have been purified and characterized. A unique group of phosphoproteins, electrophoretically distinct but having similar amino acid compositions, has been isolated from rat milk whey (1,5,6). This group ofwhey phosphoproteins (Wp-protein) constitutes 1 to 2 mg/ml in whole rat milk.…”

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confidence: 99%

Complete sequence analysis of cDNA clones encoding rat whey phosphoprotein: homology to a protease inhibitor.

Dandekar¹,

Robinson²,

Appella³

et al. 1982

Proc. Natl. Acad. Sci. U.S.A.

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Lactoprotein clones have been isolated from a rat mammary gland recombinant library of cDNA plasmids. Clones p-Wp 52 and p-Wp 47 were shown by hybrid selection, in vitro translation, and immunoprecipitation to represent a cloned DNA sequence encoding rat whey phosphoprotein. We report here the nucleotide sequence of the cDNA insert ofp-Wp 52 and show that it encodes the complete whey phosphoprotein sequence. The encoded sequence shows a high content ofhalf-cystine, glutamic acid, aspartic acid, and serine but an absence of tyrosine. The half-cystines appear in unique arrangements and are repeated in two domains of the protein. The second domain has striking similarities with the second domain of the red sea turtle protease inhibitor. Clone p-Wp 52 has allowed the study of expression of whey phosphoprotein mRNA during functional differentiation of rat mammary gland and in mammary tumors. The whey phosphoprotein mRNA is detected during midpregnancy and lactation in the rat mammary gland but is barely detected in mammary tumors in which other milk protein mRNAs are expressed. The whey phosphoprotein gene in these tumors is hypermethylated, correlating with the reduced expression of this gene.Hormonal regulation of milk protein synthesis is being studied in a number of laboratories. From rat milk, a-lactalbumin (a-LA) (1, 2) and a number of caseins (42, 29, and 25 kilodaltons) (3, 4) have been purified and characterized. A unique group of phosphoproteins, electrophoretically distinct but having similar amino acid compositions, has been isolated from rat milk whey (1,5,6). This group ofwhey phosphoproteins (Wp-protein) constitutes 1 to 2 mg/ml in whole rat milk. These Wp-proteins initially copurify with rat a-LA in the whey fraction, but they can be separated from a-LA on DEAE-cellulose into three charged forms (1, 5, 6). These proteins are rich in aspartic and glutamic acids (or their amides) and in serine and half-cystine but lack tyrosine (5). The role ofthese proteins in the rat mammary gland is at present unknown, but their expression shows variability during functional differentiation of the gland.cDNA clones corresponding to some of the rat milk proteins have been isolated (7,8). For studies on the regulation of individual milk proteins, which are under different hormonal controls (9), it is essential that the individual cDNA clone be correctly identified with respect to its corresponding protein.Previously, we reported the isolation of a rat a-LA cDNA clone and established its identity by comparing the DNA sequence with the a-LA protein sequence (7).From taining a-LA also contained Wp-proteins. These fractions were then loaded on a DEAE-cellulose column and the proteins were eluted as described (1, 5, 6). The three charged forms of the phosphoproteins that did not have a-LA activity eluted after the a-LA protein peaks and were designated as Wpl, Wp2, and

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Prolactin

Vonderhaar¹

1987

The Mammary Gland

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Milk protein gene expression in the rat mammary gland

Cited by 13 publications

References 53 publications

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

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

Complete sequence analysis of cDNA clones encoding rat whey phosphoprotein: homology to a protease inhibitor.

Prolactin

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