alpha S-and-beta casein gene frequencies in ewe and cow genome.
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Animals↗
Biomedical subjects
L M Houdebine
Publications and source records attributed to L M Houdebine.
Absence of poly (A) in a large part of newly synthesized casein mRNAs.
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Animals↗
Purification of the mRNAs for Ewe alphaS-casein and beta-casein by immunoprecipitation of polysomes.
Specific polysomes synthesizing alphas-casein and beta-casein were immunoprecipitated from total polysomes of the lactating ewe mammary gland. The polysome - anti-casein complex was immunoprecipitated by anti-immunoglobulins. 22%, 32% and 10% of polysomes were immunoprecipitated with saturating amounts of anti-alphas-casein, anti-beta-casein and anti-chi-casein respectively. Poly(U)-Sepharose chromatography of the immunoprecipitated RNAs permitted the isolation of the corresponding poly(A)-containing RNA which migrated as one major band in polyacrylamide gel electrophoresis. As judged by the contamination with the messenger activity for one of the other caseins, the purity of the mRNA for alphas-casein as well as for beta-casein was estimated to be between 75% and 80%.
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Isolation and characteristics of mRNPs associated with bound polyribosomes from the ewe lactating mammary gland.
The dissociation of bound polyribosomes from ewe lactating mammary gland, by EDTA or Puromycin-KC1 treatment releases Messenger Ribonucleoprotein particles (mRNP'S) which sediment in sucrose gradients as a single component having a sedimentation coefficient of about 22S. RNAs derived from mRNPs sediment between 9S and 15S and direct the synthesis of milk proteins in reticulocyte cell free systems. The mRNP protein moiety contains two major polypeptides with molecular weights of 52,000 and 72,000 daltons and two minor polypeptides with apparent molecular weights of 58,000 and 60,000 daltons.
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Synthesis of DNA complementary to the mRNAs for milk proteins by E. coli DNA polymerase I.
E.Coli DNA polymerase I (Klenow subfragment) was used for the synthesis of complementary DNA with the mRNAs for rabbit milk proteins as templates. The cDNA formed, contained 200 nucleotides and represented about 20% of the mRNA template. The cDNA was hybridized specifically to the mRNA templates. The Klenow subfragment of the E.Coli DNA polymerase I was as efficient as the avian myeloblastosis virus reverse transcriptase in the synthesis of cDNA. The mean size of the cDNA fragments obtained with the Klenow enzyme proved to be 70% of the value obtained with the AMV reverse transcriptase and at least twice the value generally obtained with the complete E.Coli DNA polymerase I. The cDNA was used for the detection and the quantification of the mRNA template in various RNA fractions.
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Effects of prolactin and progesterone on expression of casein genes. Titration of casein mRNA by hybridization with complementary DNA.
Prolactin injected into pseudopregnant rabbits induced lactogenesis. Progesterone injected simultaneously considerably reduced this effect. The content of mRNA for casein in total cellular RNA and in total polysomal RNA was estimated during these two hormonal actions using a hybridization technique. The synthesis of DNA complementary to casein mRNA was carried out with mRNA obtained from polysomes immunoprecipitated with anti-casein antibody. The content of casein mRNA was determined by hybridization of this cDNA with the RNA fractions. This technique revealed that prolactin induced the synthesis of mRNA for casein whereas progresterone greatly limited this effect of prolactin. These variations of casein mRNA content are in good agreement with values previously determined by translation and with the ability of mammary gland to synthesize casein.
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Studies on the synthesis of casein messenger RNA during pregnancy in the rabbit.
The quantity of casein mRNA in the rabbit mammary gland was assayed during the course of pregnancy, by translation of casein mRNA in a reticulocyte lysate and by hybridization to DNA complementary to purified casein mRNA. Both methods indicated that the major increases in the concentration of casein mRNA in both polysomal and total cellular RNA occurred between the 18th and 25th day of pregnancy. The change in casein mRNA concentration during this period coincided with a sharp rise in casein synthesis in mammary gland explants suggesting that the levels of casein mRNA determine the rate of casein synthesis in the mammary gland. The sensitivity of the hybridization assay made it possible to detect the presence of casein mRNA in virgin animals and during the very early stages of pregnancy. At day 5, casein mRNA was found associated with polysomes indication that there was probably some casein synthesis at this early stage of gestation. These results suggest that the hormones controlling lactogenesis in the rabbit may function by augmenting the rate of casein mRNA synthesis rather that initiating transcription of previously inactive genes.
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Regulation of casein synthesis in the rabbit mammary gland. Titration of mRNA activity for casein under prolactin and progesterone treatments.
Total polysomal RNA or poly(A)-containing RNA isolated from membrane-bound polysomes of normal lactating rabbits directed the synthesis of casein in a reticulocyte lysate. Casein was identified by immunoprecipitation followed by SDS-polyacrylamide gel electrophoresis of the immunoprecipitate. The poly(A)-containing RNA was heterogeneous with one major peak corresponding to a 12-S sedimentation coefficient as determined by polyacrylamide gel electrophoresis. Using the same procedure, mRNAs isolated from the non-secreting tissue of pseudopregnant rabbits were found not to contain the 12-S peak and were unable to direct the synthesis of casein in vitro. Prolactin injected into pseudopregnant rabbits induced the synthesis of proteins immunoprecipitable by anti-casein anti-serum and induced the simultaneous appearance of the 12-S mRNA. Progesterone injected with prolactin prevented the induction of casein synthesis and the appearance of mRNA for casein. A close relationship was established between the ability of the tissue to synthesize immunoprecipitable casein and the corresponding mRNA content of polysomes.
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Absence of mRNA for casein in free polysomes of lactating ewe mammary gland.
Total polysomal RNA or poly A- containing RNA extracted from free polysomes of lactating ewe mammary gland was unable to direct a significant synthesis of alpha-s- and beta-casein. Polyacrylamide gel electrophoresis of poly A- containing RNA revealed that the two major peaks of mRNA isolated from membrane-bound polysomes were not observed in mRNA isolated from free polysomes. Purified 125-I anti-alpha-s-casein could bind significantly to membrane-bound polysomes only. These data suggest that casein synthesis occurs almost exclusively on membrane-bound polysomes and that this specificity may be attributed to the presence of casein mRNAs on these polysomes and their absence from free polysomes.
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Isolation and characterization of casein mRNAs from lactating ewe mammary glands.
RNA from bound polysomers of lactating ewe's mammary gland directs the synthesis of the three major milk proteins (alphas, beta and kappa-caseins) in a cell-free system derived from rabbit reticuyocytes. The "in vitro" product was identified by immunoprecipitation with specific antibodies and by electrophoresis in SDS polyacrylamide gel. Each of these messengers was purified from 20 to 25 fold from total membrane-bound polysomal RNA using poly U-Sepharose chromatography. This purified fraction assayed in a reticulocyte cell-free system is able to direct also the synthesis of 2 minor secretory proteins (beta-lactoglobulin and alpha-lactalbumin). The messenger RNAs purified by hybridization to poly U-Sepharose have a sedimentation coefficient of about 12 S and an apparent molecular weight of approximatively 3.5 s 10-5 daltons was observed by polyacrylamide gel electrophoresis under denaturing contitions. This value which correspond to about 900 nucleotides is significantly greater than the number expected for coding milk proteins.
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Inhibition by progesterone of the lactogenic effect of prolactin in the pseudopregnant rabbit.
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Lack of poly(A) sequence in half of the messenger RNA coding for ewe alpha S casein.
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Animals↗
Expression of heat shock-regulated human growth hormone genes containing or lacking introns by NIH-3T3 and Wish cell lines.
A plasmid containing the complete genomic DNA of the human growth hormone (ghGH) comprising four introns and driven by the human promoter of the human gene of the 70 kDa heat shock protein (hsp70) has been used to transfect mouse NIH-3T3 and human Wish cells. Selected cell lines were characterized for stable hGH secretion. Similarly in the same NIH-3T3 cells, the stable expression of the same plasmid construct, but containing the complementary DNA of the hGH gene (chGH), was compared in terms of the effect of introns on heterologous protein synthesis. Genomic hGH recombined cells synthetized, in a heat regulated fashion, matured hsp70/hGH hybrid mRNA able to drive the secretion of a 22 kDa polypeptide. Like the natural hGH, this polypeptide expressed the functional hormonal activity of prolactin on casein secretion by mammary cells. The time course of hGH secretion was prolonged in ghGH transcripts, while that of mRNA degradation appeared delayed, especially in Wish cells, as compared to chGH expression. In the human Wish cells the decay of endogenous hsp mRNA has been compared to that of recombinant hsp mRNA, demonstrating that this human hsp70/hGH hybrid mRNA was present in the cytoplasm during a longer period than the human endogenous hsp70 mRNA. In conclusion, similar levels of expression and resulting gene products were expressed from the chGH or the ghGH gene in an inducible manner.
3T3 Cells↗
The effect of matrix attached regions (MAR) and specialized chromatin structure (SCS) on the expression of gene constructs in cultured cells and in transgenic mice.
The flanking sequences of several genes have been shown to direct a position independent expression of transgenes. Attempts to completely identify the insulating sequences have failed so far. Some of these sequences contain a matrix attached region (MAR) located in the flanking part of the genes. This article will show that the MARs in cultured cells located in the 3' OH region of the human apolipoprotein B100 (Apo B100) and within the SV40 genome were unable to stimulate and insultate transgene expression directed by the promoters from a rabbit whey acidic protein (WAP) gene or from human cytomegalovirus (hCMV) early genes. In transgenic mice, the MAR from the Apo B100 and SV40 genes did not enhance the expression of a transgene containing the rabbit whey acid protein (WAP) promotor, the late gene SV40 intron (VP1 intron), the bovine growth hormone (bGH) cDNA and the SV40 late gene terminator. This construct was even toxic for embryos. Similarly, the specialized chromatin structure (SCS) from the Drosophila 87A7 HSP70 gene reduced chloramphenicol acetyl transferase (CAT) activity when added between a cytomegalovirus (CMV) enhancer and a Herpes simplex thymidine kinase (TK) gene promoter. This inhibitory action was almost complete when a second SCS sequence was added before the CMV enhancer. Sequences from the firefly luciferase and from the human gene cathepsin D cDNA used as control unexpectedly showed a similar inhibitory effect when added to the CMVTKCAT construct instead of SCS. When added before the CMV enhancer and after the transcription terminator in the CMVTKCAT construct, the SCS sequence was unable to insulate the integrated gene as seen by the fact that the level of CAT in cell extracts were by no means correlated with the number of copies in individual clones. From these data, it is concluded that i) a MAR containing the canonical AT rich sequences does not amplify the expression of all gene constructs ii) At rich MAR sequences do not have per se an insulating effect iii) Drosophila SCS from the 87A7 HSP70 gene has no insulating effect in all gene constructs (at least in mammalian cells) iv) and the addition of a DNA fragment between an enhancer and a promoter in a gene construct cannot be used as a reliable test to evaluate its insulating property.
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[Recent data on the mechanism of action of prolactin].
Prolactin is a hormone involved in the control of many functions, from osmoregulation in fishes to lactation in mammals. For that reason, the control of its secretion by hypophysis is particularly complex. Multiple factors of hypothalamic origin (dopamine, GABA, VIP, etc . . .) and hormones (oestrogens, TRH, thyroxine, . . .) are involved in this control. Prolactin molecule contains about 200 aminoacids. It has three disulfide bridges, of which one, at the center of the molecule, is required for the lactogenic activity. The expression of prolactin gene is dependent upon oestrogens, TRH, Ca++ ion and cAMP which favour its transcription. In contrast, dopaminergics such as CB 154 lower the expression rate of this gene. Prolactin receptor is located essentially on the plasma and intracellular membranes of target cells. Its essential binding part has a molecular weight of about 40,000. In mammary gland and liver prolactin receptor is up-regulated following a slow process. It is also down-regulated following a rapid and reversible process. In mammary gland, prolactin controls the expression of milk protein genes by enhancing their transcription rate and also by increasing the stability and the translation rate of the mRNAs. The transfer of the prolactin information to genes takes place through a relay which is released from plasma membrane when the receptor is occupied by the hormone or by anti-prolactin receptor antibodies. This relay which seems to be a small peptide (less than 1000 daltons and inactivated by trypsin) acts directly and specifically on isolated mammary nuclei via a dephosphorylation of nuclear proteins.
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[Control of the expression of milk protein genes by prolactin, glucocorticoids and progesterone].
The expression of casein genes is under the control of several hormones of which the most important are prolactin, glucocorticoids and progesterone. In pseudopregnant or mid-pregnant rabbit having partially developped but inactive mammary gland, prolactin induces casein synthesis. The phenomenon is accompanied by an accumulation of casein mRNAs and by a stimulation of their translation. The accumulation of casein mRNAs results from an acceleration of the transcription of the corresponding genes and from a stabilization of the mRNAs. These prolactin effects are amplified by glucocorticoids which are not per se inducers and they are inhibited by progesterone. The essential action of prolactin and glucocorticoids can be obtained in cultured mammary explants and epithelial cells. This induction is accompanied by a transformation of the mammary cell in which are accumulated ribosomes and membranes involved in milk synthesis and exportation. This transformation is favoured by prolactin and inhibited by progesterone. Hence, the abundant milk secretion is triggered only after parturition when the predominence of progesterone is reversed in favour of prolactin. Prolactin incubated with mammary membranes promotes the formation of a factor capable of accelerating beta-casein gene transcription when added to isolated mammary nuclei. This factor is formed only by lactogen hormones and from prolactin receptor containing membranes. The information contained in the factor seems to be understood only by prolactin target genes. The generation of the factor can be provoked by anti-prolactin receptor antibodies and it is inhibited by tubulin binding drugs such as colchicine. The molecule exhibiting prolactin-like activity has a small molecular weight, it is thermostable and inactivated by trypsin. The stimulation of beta-casein gene transcription is abolished when the factor is incubated with nuclei in the presence of phosphatase inhibitors. These facts suggest that prolactin after its binding to its peripheral receptors triggers the release of a small peptide which migrates to nuclei where it activates the transcription of the prolactin target genes through a dephosphorylation of nuclear proteins. This small peptide is a good candidate to the prolactin intracellular relay.
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Generating biological models through gene transfer to domestic animals.
Transgenic domestic animals remain infrequently used as models for biochemical, biomedical or pharmaceutical studies. The difficulty in obtaining these animals and the cost of their maintenance explains this situation. This review briefly summarizes the different techniques of gene transfer, targeted or not, and also the techniques for constructing vectors for transgene expression. A few examples of domestic animal models are also reported.
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