scholarly journals Relative distribution of post-nuclear poly(A)-containing RNA abundance groups within the nuclear and post-nuclear polyadenylated and non-polyadenylated RNA populations of the lactating guinea-pig mammary gland

1980 ◽  
Vol 192 (2) ◽  
pp. 489-498 ◽  
Author(s):  
Ian C. Bathurst ◽  
Roger K. Craig ◽  
David G. Herries ◽  
Peter N. Campbell
Keyword(s):  
Mammary Gland ◽  
Guinea Pig ◽  
Milk Protein ◽  
Regulation Of Gene Expression ◽  
Milk Proteins ◽  
Relative Distribution ◽  
Free System ◽  
Low Salt ◽  
Nuclear Rna ◽  
System 2

1. RNA isolated from the post-nuclear supernatant of the lactating guinea-pig mammary gland was fractionated with oligo(dT)–cellulose into three populations; those that bound at ‘low salt’ [long poly(A) tracts, 78–32 nucleotides]; those that bound at ‘high salt’ [shorter poly(A) tracts, 48–21 nucleotides]; and those that did not bind [no poly(A) or short poly(A) tracts, <20 nucleotides]. Nuclear RNA was fractionated into two populations, those that bound in ‘low salt’ and those that did not bind. All the post-nuclear RNA fractions directed the synthesis of milk proteins in a Krebs II ascites cell-free system. 2. 3H-labelled DNA complementary to the post-nuclear poly-(A)-containing RNA population (low-salt fraction) was fractionated into abundant (milk-protein mRNA), moderately abundant and scarce sequences. This complementary DNA was then used to investigate the distribution of the mRNA sequences in the different RNA populations. This showed that all sequences were present in polyadenylated and non-polyadenylated fractions, but that major quantitative differences were apparent. The abundant milk-protein mRNA sequences predominated in the ‘low-salt’ post-nuclear poly(A)-containing RNA fraction, whereas the moderately abundant sequences predominated in the non-polyadenylated post-nuclear RNA fraction. In total cellular RNA, those sequences deemed initially to be moderately abundant within the ‘low-salt’ poly(A)-containing RNA population were present at a concentration very similar to those of the abundant milk-protein mRNA (approx. 6×105 copies of each sequence/cell). Similarly, analysis of the nuclear RNA populations showed that the ‘abundant’ and so-called ‘moderately abundant’ sequences were present in essentially identical concentrations (2×103 copies of each sequence/cell). The majority of these (90–95%) were non-polyadenylated. 3. The results are discussed in terms of the post-transcriptional mechanisms involved in the regulation of gene expression in the lactating guinea-pig mammary gland.

scholarly journals Uptake and metabolism of tryptophan by the isolated perfused guinea-pig mammary gland

1976 ◽  
Vol 158 (3) ◽  
pp. 659-662 ◽  
Author(s):  
B Mepham ◽  
A R Peters ◽  
S R Davis
Keyword(s):  
Amino Acids ◽  
Mammary Gland ◽  
Guinea Pig ◽  
Blood Plasma ◽  
Milk Protein ◽  
Absorption Studies ◽  
Uptake And Metabolism ◽  
Tryptophan Uptake

Tryptophan uptake by the isolated perfused lactating guinea-pig mammary gland was 46.5+/-4.6 mug/h per g. Results of absorption studies and the use of [methylene-14C]tryptophan suggest that tryptophan is one of the group of amino acids that are transferred almost quantitatively from blood plasma to milk protein.

15N enrichment of casein amino acids in the milk from goats given a single intravenous dose of l-[15N]leucine

1999 ◽  
Vol 66 (2) ◽  
pp. 283-288 ◽  
Author(s):  
JOAQUIN RUBERT-ALEMÁN ◽  
GUIDO RYCHEN ◽  
FLORENCE CASSERON ◽  
FRANCOIS LAURENT ◽  
GERARD JEAN MARTIN
Keyword(s):  
Amino Acids ◽  
Mammary Gland ◽  
Milk Protein ◽  
Carbon Sources ◽  
Amino Nitrogen ◽  
Milk Proteins ◽  
15N Enrichment ◽  
N Enrichment ◽  
Branched Chain ◽  
Plasma Leucine

Branched-chain amino acids (AA) are mostly metabolized in the splanchnic area, but some are metabolized within the mammary gland and thus could contribute to the synthesis of non-essential AA (Wohlt et al. 1977). The extraction rate of leucine from plasma by the mammary gland is particularly high (64% in the goat; Roets et al. 1983), in excess of that used for the synthesis of milk proteins (Davis & Mepham, 1976; Wohlt et al. 1977; Roets et al. 1983). Thus, although mammary leucine is mainly used for milk protein, it also provides amino nitrogen and carbon sources for the synthesis of non-essential AA.To our knowledge, no information is available on the transfer and distribution of plasma leucine amino nitrogen to milk protein AA. Using the technique for chromatographic fractionation of AA recently developed by Casseron et al. (1997), we studied the specific 15N enrichment of casein (CN) AA in the goat given a single intravenous injection of [15N]leucine.

scholarly journals Identification and subsequent phosphorylation of sequestered partially processed caseins in the lactating guinea-pig mammary gland

1984 ◽  
Vol 222 (2) ◽  
pp. 501-510 ◽  
Author(s):  
A P Boulton ◽  
J C Pascall ◽  
R K Craig
Keyword(s):  
Endoplasmic Reticulum ◽  
Mammary Gland ◽  
Guinea Pig ◽  
Milk Protein ◽  
Secretory Pathway ◽  
Short Pulse ◽  
Early Event ◽  
Post Translational Modification ◽  
Electrophoretic Mobilities ◽  
Membrane Bound

Golgi and endoplasmic-reticulum fractions were prepared from the lactating guinea-pig mammary gland. The endoplasmic-reticulum fraction was highly active in the processing and sequestration of milk-protein primary translation products. Explants from the lactating gland in organ culture were used to identify milk-protein intermediates present in the secretory pathway, and the timing of the events leading to their post-translational modification. With [35S]methionine, the milk proteins labelled after a short pulse (3 min) were represented by the partially processed (but not phosphorylated) caseins and alpha-lactalbumin sequestered within membrane-bound vesicles. After a 30 min labelling period, higher-Mr caseins with electrophoretic mobilities identical with those of the phosphorylated caseins isolated from milk were identified in the incubation medium, and sequestered within membrane-bound vesicles. Pulse-chase experiments established a precursor-product relationship between these forms. Secretion is apparent approx. 30 min after sequestration. Caseins are highly phosphorylated; removal of the phosphate residues with acid phosphatase results in proteins with increased electrophoretic mobility, similar to those of the partially processed early casein intermediates found sequestered in explants after a 3 min pulse with [35S]methionine, and those sequestered within microsomal membranes after mRNA-directed cell-free protein synthesis. A comparison of the proteins labelled during both short (5 min) and long (30 min) pulses with [35S]methionine and [32P]Pi shows that, in contrast with the 35S-labelled caseins, those labelled with [32P]Pi exhibit only electrophoretic mobilities identical with those of the mature caseins isolated from milk and those identified after long labelling periods with [35S]methionine. No phosphorylated early intermediate forms of caseins were identified. We conclude that the synthesis and post-translational modification of guinea-pig caseins occurs in two stages, (i) an early event involving synthesis and sequestration within the endoplasmic reticulum, an event that involves signal-peptide removal, followed (ii) 10-20 min later by phosphorylation at a different point in the secretory pathway, probably in the Golgi complex. Secretion of the phosphorylated caseins occurs 10-20 min later.

scholarly journals Secretory proteins compete for production in the mammary gland of transgenic mice

1995 ◽  
Vol 310 (2) ◽  
pp. 637-641 ◽  
Author(s):  
M McClenaghan ◽  
A Springbett ◽  
R M Wallace ◽  
C J Wilde ◽  
A J Clark
Keyword(s):  
Mammary Gland ◽  
Transgenic Mice ◽  
Milk Protein ◽  
Milk Proteins ◽  
Whey Acidic Protein ◽  
Acidic Protein ◽  
Host Protein ◽  
Secretory Proteins ◽  
Mrna Levels ◽  
Endogenous Proteins

To explore the possibility that genes might compete for expression, we have studied transgenic mice producing high levels of the sheep milk protein, beta-lactoglobulin (BLG), in the mammary gland. Mice carrying one or more transgene loci expressed BLG in milk at levels ranging from 7 to 33 mg/ml. The effects of BLG synthesis on the levels of endogenous milk gene expression were examined. No significant increase in total milk protein concentration was recorded even in mice expressing the largest amounts of BLG. Measurement of individual milk proteins showed that transgene protein was manufactured at the expense of host protein synthesized in the gland. Whey acidic protein production was more suppressed than casein production. Suppression of endogenous proteins was matched by a reduction in the corresponding steady-state mRNA levels; in double-transgenic mice, which expressed the largest amounts of BLG, beta-casein and whey acidic protein mRNA populations were reduced to 75 and 56% of control levels respectively. We demonstrate that an exogenous gene competes effectively for expression with endogenous genes. Possible mechanisms of competition are discussed.

scholarly journals Guinea-pig milk-protein synthesis. Isolation and characterization of messenger ribonucleic acids from lactating mammary gland and identification of caseins and pre-α-lactalbumin as translation products in heterologous cell-free systems

1976 ◽  
Vol 160 (1) ◽  
pp. 57-74 ◽  
Author(s):  
R K Craig ◽  
P A Brown ◽  
O S Harrison ◽  
D McIlreavy ◽  
P N Campbell
Keyword(s):  
Molecular Weight ◽  
Protein Synthesis ◽  
Mammary Gland ◽  
Guinea Pig ◽  
Wheat Germ ◽  
Milk Proteins ◽  
N Terminus ◽  
Lactating Mammary Gland ◽  
Heterologous Cell ◽  
Alpha Lactalbumin

1. The major milk proteins synthesized by the lactating mammary gland of the guinea pig were identified and designated as caseins A, B and C and alpha-lactalbumin, with estimated mol.wts. of 28000, 25500, 20500 and 14500 respectively. 2. Antisera to the total casein fraction and to alpha-lactalbumin were prepared from rabbits. The milk proteins were also iodinated with either 131I or 125I. 3. A poly(A)-rich RNA fraction was isolated from lactating guinea-pig mammary glands. Isolation was by affinity chromatography on oligo(dT)-cellulose. 4. Examination of this RNA fraction by electrophoresis on polyacrylamide gels containing formamide indicated three major species 1, 2 and 3, with estimated wol.wts. of 5.4 × 10(5) and 3.3 × 10(5), and the apparent absence of rRNA species. 5. The poly(A)-rich RNA stimulated protein synthesis in heterologous cell-free systems based on wheat germ, Krebs II ascites-tumour cells, and the latter supplemented with an initiation factor-3 fraction from rabbit reticulocyte ribosomes. 6. Between 80 and 90% of the protein synthesis directed by the mRNA was for milk proteins. 7. Analysis of the proteins immunoprecipitated by the alpha-lactalbumin antiserum showed in the wheat-germ system that the product was a protein with a molecular weight greater than that of alpha-lactalbumin, whereas in the ascites-tumour-cell systems both this protein and alpha-lactalbumin were found. When the larger protein was treated with CNBr and the resulting peptides were examined, it was shown that the extra peptide was at the N-terminus. This and other evidence is adduced for the initial translation product of alpha-lactalbumin being a precursor with an addition of about ten amino acids at the N-terminus. 8. Similar analysis of the casein immlnospecific proteins produced under the direction of mRNA indicated that the products had a molecular weight that was apparently a littel smaller than that of the caseins synthesized in vivo. This was not consistent with higher-molecular weight casein precursors. 9. Possible explanations for the results obtained are discussed, especially in terms of the physiological significance of the pre-alpha-lactalbumin as a secretory protein.

scholarly journals The ribonucleic acids of the mammary gland of the guinea pig

1975 ◽  
Vol 146 (3) ◽  
pp. 575-583
Author(s):  
P Ashby ◽  
P N Campbell
Keyword(s):  
Mammary Gland ◽  
Guinea Pig ◽  
Gel Electrophoresis ◽  
Milk Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Mammary Glands ◽  
Ribosomal Subunits ◽  
Ribonucleic Acids ◽  
Lactating Mammary Gland ◽  
Zone Velocity

1. 32-P-labelled polyribosome preparations were made from the mammary glands of lactating and late-pregnant guinea pigs after injection of (32-P)i into the animals. 2. The RNA of polyribosomes, ribosomal subunits and that released from polyribosomes by EDTA were analysed by zone velocity centrifugation and by polyacrylamide-gel electrophoresis. 3. RNA species which have the physical properties expected for the milk protein mRNA were detected. RNA species of a size which could code for the caseins were present in lactating but not in pre-lactating mammary-gland polyribosomes.

scholarly journals Esterification of glycerol 3-phosphate in lactating guinea-pig mammary gland

1967 ◽  
Vol 105 (1) ◽  
pp. 213-223 ◽  
Author(s):  
N. J. Kuhn
Keyword(s):  
Mammary Gland ◽  
Guinea Pig ◽  
Phosphatidic Acid ◽  
Guinea Pigs ◽  
Particulate Fraction ◽  
Mammary Tissue ◽  
Acyl Donor ◽  
Free System ◽  
Kennedy Pathway ◽  
A Cell

1. The presence of palmitoyl-CoA–l-glycerol 3-phosphate palmitoyltransferase (EC 2.3.1.15) has been demonstrated in a particulate fraction of mammary tissue from lactating guinea pigs. 2. Cell-free preparations also catalysed the activation of palmitate and oleate, and the conversion of enzymically formed phosphatidic acid into glycerides, in accord with the Kennedy pathway of glyceride formation. 3. The properties of the system that esterifies l-glycerol 3-phosphate were studied with respect to substrates and cofactors, and the reaction product was shown to be phosphatidic acid (1,2-diacyl glycerol 3-phosphate). 4. The extent to which newly formed phosphatidic acid was converted into glyceride in a cell-free system was dependent on the nature of the acyl donor, the concentration of subcellular particles, the time of incubation and the concentration of Mg2+.

Evidence of Hormone Induction of Milk Proteins in Normal and Malignant Mammary Gland Cultures

1973 ◽  
Vol 31 ◽  
pp. 566-567
Author(s):  
Kenneth S. McCarty ◽  
Richard F. Jones ◽  
Jon C. Nixon ◽  
Kenneth S. McCarty
Keyword(s):  
Mammary Gland ◽  
Dna Synthesis ◽  
Protein Production ◽  
Milk Protein ◽  
Milk Proteins ◽  
Mammary Glands ◽  
Secretory Protein ◽  
Mammary Adenocarcinoma ◽  
Mammary Tissue ◽  
Tissue Cultures

Normal epithelial cells from mammary glands of mature virgin mice synthesize increasing levels of casein and α-lactalbumin when cultured in the presence of insulin, hydrocortisone, and prolactin. in the absence of DNA synthesis or when DNA synthesis is blocked, the augmentation of milk protein production does not occur. It would appear that a necessary coupling of secretory protein production to DNA synthesis or mitosis or both is essential.Mammary tissue from 10-12 day pregnant nulliparous C3H mice, BA type mammary adenocarcinoma, and H2712 type mammary adenocarcinoma have been compared in their response to insulin, dexamethasone and prolactin in organ and tissue cultures.

Feeding and milk protein production

1984 ◽  
Vol 9 ◽  
pp. 53-67 ◽  
Author(s):  
P. C. Thomas
Keyword(s):  
Amino Acids ◽  
Mammary Gland ◽  
Protein Production ◽  
Milk Protein ◽  
Milk Proteins ◽  
Protein Yield ◽  
Dietary Energy ◽  
Milk Protein Content ◽  
Protein Supply ◽  
The Impact

This paper reviews the effects of feeding on milk protein production. It deals, first, with the chemical composition of the milk proteins and the extent to which the composition is influenced by diet, the synthesis of proteins in the mammary gland and the effects of variations in the supply of amino acids and of the energy-yielding nutrients that are absorbed from the gastrointestinal tract. There is then an examination of the impact of changes in dietary energy and protein supply on the content and yield of protein in milk and specific consideration of particular features of ration formulation, including supplementary energy concentrate foods, supplementary lipids and amount and type of supplementary protein foods. Finally, it is argued that the effects of diet on milk protein production are evaluated best simply in terms of milk protein yield; some of the pitfalls of interpreting information on milk protein content in practice are pointed out.It is concluded that the yield of milk protein is determined by the dietary supply of energy-yielding constituents and protein but that presently employed systems for ration formulation do not provide a satisfactory means of interrelating milk protein yield and the intake of nutrients in the cow's diet.

scholarly journals Studies on the intracellular segregation of polyribosome-associated messenger ribonucleic acid species in the lactating guinea-pig mammary gland

1979 ◽  
Vol 181 (3) ◽  
pp. 737-756 ◽  
Author(s):  
R K Craig ◽  
A P Boulton ◽  
O S Harrison ◽  
D Parker ◽  
P N Campbell
Keyword(s):  
Protein Synthesis ◽  
Mammary Gland ◽  
Guinea Pig ◽  
Milk Protein ◽  
Complexity Analysis ◽  
Free Protein ◽  
Mrna Species ◽  
Run Off ◽  
Membrane Bound ◽  
Cell Free Protein Synthesis

1. Free and membrane-bound polyribosomes were isolated and the associated mRNA species characterized by cell-free protein synthesis, RNA-complexity analysis and polyribosome run-off in vitro. 2. Of the recovered polyribosomal RNA 85% was associated with membrane-bound polyribosomes and contained 87–93% of the total milk-protein mRNA species as assessed by cell-free protein synthesis or RNA-complexity analysis. 3. RNA-complexity analysis showed that the abundant (milk-protein mRNA assumed) species constituted 55% of the post-nuclear poly(A)-containing RNA population, the remainder consisting of a moderately abundant population (18%) and a low abundance population (27%). Calculations suggest that each population contained up to 2, 48 and 5000 different species respectively. 4. RNA-complexity analysis of the free polyribosomal poly(A)-containing RNA demonstrated that all the species in the post-nuclear fraction were present, though in different proportions, the abundant, moderately abundant and low-abundance groups representing 38, 30 and 32% of this population. 5. RNA-complexity analysis of the membrane-bound polyribosomal poly(A)-containing RNA revealed a more limited population, 72% consisting of the abundant (milk-protein mRNA) species, and 28% a population of up to 900 RNA species. 6. Polyribosome run-off confirmed that milk-protein mRNA was associated with the membrane-bound and free polyribosomes, but represented only a small fraction of the total protein synthesized by the latter. 7. Comparative analysis of milk proteins synthesized in mRNA-directed cell-free systems, or by run-off of free and of membrane-bound polyribosomes, is consistent with the interpretation that in vivo the initiation of protein synthesis occurs on free polyribosomes, followed by the attachment of a limited population to the endoplasmic reticulum. After attachment, but before completion of peptide synthesis, the detachable N-terminal peptide sequence of one of these(pre-alpha-lactalbumin) is removed. 8. The results are discussed in terms of the mechanisms involved in the intracellular segregation of mRNA species in the lactating guinea-pig mammary gland.

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