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Hideaki Tojo - One of the best experts on this subject based on the ideXlab platform.
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Identification of Whey Acidic Protein (WAP) in Dog Milk
Experimental animals, 2012Co-Authors: Mami Seki, Tokuko Iwamori, Naoko Nukumi, Kiyoshi Kano, Kunihiko Naito, Keitaro Yamanouchi, Rina Matsura, Hideaki TojoAbstract:Whey Acidic Protein (WAP) has been identified as a major Whey Protein in milk of a wide range of species and reportedly plays important roles in regulating the proliferation of mammary epithelial cells. However, in some species including humans, WAP is not synthesized in the mammary gland. The presence of WAP in carnivore species has not been reported. We searched the National Center for Biotechnology Information (NCBI) database for the dog WAP gene and tried biochemically to identify WAP in dog milk. The nucleotide sequence of the examined dog genomic DNA was completely identical to that in the NCBI database and showed that the dog WAP gene, like other known functional WAP genes, has four exons. Biochemical analysis of milk Protein by reverse-phase HPLC and Western blotting demonstrated the presence of WAP in dog milk.
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Bacteriostatic activity of Whey Acidic Protein (WAP).
The Journal of veterinary medical science, 2009Co-Authors: Tokuko Iwamori, Naoko Nukumi, Kikuji Itoh, Kiyoshi Kano, Kunihiko Naito, Masamichi Kurohmaru, Keitaro Yamanouchi, Hideaki TojoAbstract:We have previously reported the action of Whey Acidic Protein (WAP) inhibiting the proliferation of mouse mammary epithelial cells in the experiments utilizing in vivo and in vitro systems. We report herein the bacteriostatic activity of WAP. Western blot analysis demonstrated successful isolation of WAP from Whey fractions of rat milk by column chromatography. The WAP fraction inhibited the growth of Staphylococcus aureus JCM2413 in a dose-dependent manner, but did not inhibit the growth of Escherichia coli. The bacteriostatic activity of WAP was highest at pH 6.6 and was not affected by the presence of 150 mM NaCl. A scanning electron micrograph of bacteria treated with WAP exhibited the disruption of the bacterial cell walls.
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Reduction of tumorigenesis and invasion of human breast cancer cells by Whey Acidic Protein (WAP).
Cancer letters, 2007Co-Authors: Naoko Nukumi, Tokuko Iwamori, Kiyoshi Kano, Kunihiko Naito, Hideaki TojoAbstract:Abstract Whey Acidic Protein (WAP) is a major component of Whey, which has two or three WAP motif domains characterized by a four-disulfide core (4-DSC) structure similar to the serine protease inhibitor. We have previously found that WAP inhibits the proliferation of mammary epithelial cells in vitro and in vivo [N. Nukumi, K. Ikeda, M. Osawa, T. Iwamori, K. Naito, H. Tojo, Regulatory function of Whey Acidic Protein in the proliferation of mouse mammary epithelial cells in vivo and in vitro , Dev. Biol. 274 (2004) 31–44]. We report herein that WAP also reduces the progression of human breast cancer cells (MCF-7 and MDA-MB-453 cells). We have demonstrated that the forced expression of WAP in MCF-7 cells reduces the proliferation in either the presence or absence of estrogen. The tumor progression of WAP-expressing MCF-7 cells in nude mice is significantly suppressed more than that of mock-MCF-7 cells following the reduced expression of angiopoietin-2 gene. We have confirmed that the invasive activity of breast cancer cells is reduced to ∼30% of that of mock cells by the forced expression of exogenous WAP through its inhibition of degradation of laminin. These data suggest that WAP has a protease-inhibitory function on the progression of breast cancer cells. It is therefore possible to utilize WAP as therapeutic Protein against tumorigenesis of breast cancer.
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Whey Acidic Protein (WAP) regulates the proliferation of mammary epithelial cells by preventing serine protease from degrading laminin
Journal of cellular physiology, 2007Co-Authors: Naoko Nukumi, Tokuko Iwamori, Kiyoshi Kano, Kunihiko Naito, Hideaki TojoAbstract:Whey Acidic Protein (WAP) is a major Whey Protein in milk that has structural similarity to the family of serine protease inhibitors with WAP motif domains characterized by a four-disulfide core. We previously reported that enforced expression of the mouse WAP transgene in mammary epithelial cells inhibits their proliferation in vitro and in vivo by means of suppressing cyclin D1 expression (Nukumi et al., 2004, Dev Biol 274: 31-44). This study was conducted in order to clarify the molecular mechanism of the inhibitory function of WAP in HC11 cells, a mammary epithelial cell line. The assembly of laminin, a component in the extracellular matrix, was much more prominent around WAP-clonal HC11 cells that stably expressed the WAP transgene than around mock-clonal HC11 cells, and the proliferation of WAP-clonal HC11 cells was particularly inhibited in the presence of laminin. A laminin degradation assay demonstrated that WAP inhibited the activity of the pancreatic elastase-mediated cleavage of laminin B1 and the phosphorylation of ERK1/2. ERK1/2 phosphorylation was blocked by an inhibitor of the epidermal growth factor (EGF) receptor AG1478. Treatment with pancreatic elastase was found to enhance the proliferation of mock-clonal HC11 cells, but had no effect on that of WAP-clonal HC11 cells. The proliferation of WAP-clonal HC11 cells was recovered by the addition of exogenous EGF. We concluded that WAP plays some role in regulating the proliferation of mammary epithelial cells by preventing elastase-type serine protease from carrying out laminin degradation and thereby suppressing the MAP kinase signal pathway.
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Analysis of the promoter of mutated human Whey Acidic Protein (WAP) gene.
The Journal of reproduction and development, 2006Co-Authors: Naoko Nukumi, Tokuko Iwamori, Kunihiko Naito, Mami Seki, Tetsushi Yada, Hideaki TojoAbstract:Although Whey Acidic Protein (WAP) has been identified in the milk of a range of species, it has been predicted that WAP is not secreted into human milk as a result of critical point mutations within the coding region. In the present study, we first investigated computationally the promoter region of mutated human WAP genes by comparing with those of other known WAP genes. Computational database analyses showed that the human WAP promoter region was highly conserved, as in other species with milk WAP. Next, we evaluated the activity of the human WAP promoter (2.6 kb) using a reporter gene assay. MCF-7 cells were stably transfected with the hWAP/hGH (human growth hormone) fusion gene, cultured on Matrigel, and treated with lactogenic hormones. Radioimmunoassay detected hGH in the culture medium, indicating that the human WAP promoter was responsible for the lactogenic hormones. The human WAP promoter was significantly more active in MCF-7 cells than the mouse WAP promoter (2.4 kb). The present results provide us with important information on the molecular evolution of milk Protein genes.
Lothar Hennighausen - One of the best experts on this subject based on the ideXlab platform.
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Synthesis and secretion of the mouse Whey Acidic Protein in transgenic sheep
Transgenic Research, 1996Co-Authors: Robert J. Wall, Anne Powell, Robert Mcknight, Caird E. Rexroad, Avi Shamay, Lothar HennighausenAbstract:The synthesis of foreign Proteins can be targeted to the mammary gland of transgenic animals, thus permitting commercial purification of otherwise unavailable Proteins from milk. Genetic regulatory elements from the mouse Whey Acidic Protein (WAP) gene have been used successfully to direct expression of transgenes to the mammary gland of mice, goats and pigs. To extend the practical usefulness of WAP promoter-driven fusion genes and further characterize WAP expression in heterologous species, we introduced a 6.8 kb DNA fragment containing the genomic form of the mouse WAP gene into sheep zygotes. Two lines of transgenic sheep were produced. The transgene was expressed in mammary tissue of both lines and intact WAP was secreted into milk at concentrations estimated to range from 100 to 500 mg/litre. Ectopic WAP gene expression was found in salivary gland, spleen, liver, lung, heart muscle, kidney and bone marrow of one founder ewe. WAP RNA was not detected in skeletal muscle and intestine. These data suggest that unlike pigs, sheep may possess nuclear factors in a variety of tissues that interact with WAP regulatory sequences. Though the data presented are based on only two lines, these findings suggest WAP regulatory sequences may not be suitable as control elements for transgenes in sheep bioreactors.
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Severe position effects imposed on a 1 kb mouse Whey Acidic Protein gene promoter are overcome by heterologous matrix attachment regions.
Molecular reproduction and development, 1996Co-Authors: Robert A. Mcknight, Mark Spencer, Robert Wall, Lothar HennighausenAbstract:Matrix attachment regions (MARs) have been shown to participate in the insulation of transcription elements from surrounding chromatin in tissue culture cells and transgenic mice. A Whey Acidic Protein (WAP) transgene containing 1 kb promoter sequence was active in mammary tissue from 1 out of 17 lines of mice, demonstrating that the transcription elements were highly susceptible to position effects. To test whether MARs could insulate this WAP gene promoter and thereby restore transcription, we ligated MARs from the chicken lysozyme gene to the WAP transgene. Seven of the nine lines generated exhibited WAP transgene activity, expression was confined to mammary tissue, and correct regulation was observed in three of the four lines analyzed. This study provides strong additional evidence that the MAR fragments from the chicken lysozyme gene have the capacity to insulate transgenes from severe position effects.
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An Ets site in the Whey Acidic Protein gene promoter mediates transcriptional activation in the mammary gland of pregnant mice but is dispensable during lactation.
Molecular endocrinology (Baltimore Md.), 1995Co-Authors: Robert A. Mcknight, Robert J. Wall, Mark Spencer, Jiirgen Dittmer, John N. Brady, Lothar HennighausenAbstract:The Whey Acidic Protein (WAP) gene is specifically expressed in mammary tissue, and its transcription is induced several thousand-fold during pregnancy and remains high throughout lactation. A purine-rich sequence (PRS) located around -110 of the WAP gene promoter is conserved between mice, rats, and rabbits, suggesting that it features a regulatory element. This PRS contains an invariant GGAA/T core motif characteristic of the binding site for Ets transcription factors. Electromobility shift assays demonstrate that Ets1 binds specifically to the PRS. Experiments in transgenic mice further demonstrate that this PRS/Ets site plays a critical role in the activation of WAP transgenes during pregnancy, but that its presence is not required for high expression throughout lactation. Transgenes with an intact PRS/Ets site are expressed at high levels at day 13 of pregnancy, with little further increase during late pregnancy and lactation. In contrast, WAP transgenes with a mutation in the PRS/Ets site, which abr...
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Expression of the Whey Acidic Protein in transgenic pigs impairs mammary development
Transgenic Research, 1992Co-Authors: Avi Shamay, Robert J. Wall, Vernon G. Pursel, Erby Wilkinson, Lothar HennighausenAbstract:The Whey Acidic Protein has been found in milk of mice, rats, rabbits and camels, and its gene is expressed specifically in mammary tissue at late pregnancy and throughout lactation. A characteristic of Whey Acidic Protein is the ‘four-disulfide-core’ signature which is also present in Proteins involved in organ development. We have generated six lines of transgenic pigs which carry a mouse Whey Acidic Protein transgene and express it at high levels in their mammary glands. Transgenic sows from three lines could not produce sufficient quantities of milk to support normal development of healthy offspring. This phenotype appears to be similar, if not identical, to the milchlos phenotype exhibited by mice expressing Whey Acidic Protein transgenes. Mammary tissue from post-partum milchlos sows had an immature histological appearance, which was distinct from that observed during normal development or involution. Expression of the Whey Acidic Protein transgene was found in mammary tissue from sexually immature pigs from milchlos lines, but not in sows from lines that appeared to lactate normally. We suggest that precocious synthesis of Whey Acidic Protein impairs mammary development and function. Impaired mammary development due to inappropriate timing of Whey Acidic Protein expression is consistent with the notion that Proteins with the ‘four-disulfide-core’ signature participate in tissue formation.
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The Whey Acidic Protein.
Cancer treatment and research, 1992Co-Authors: Robert A. Mcknight, Avi Shamay, Vernon G. Pursel, Robert Wall, Tom Burdon, Lothar HennighausenAbstract:Although genes encoding caseins and Whey Proteins have some control mechanisms in common, namely, their mammary specificity, other aspects of their regulation are quite different. In particular, induction of gene expression during pregnancy and the dependence on steroid and peptide hormones for maxium mRNA accumulation differ between the casein and Whey Protein genes.
Louis Marie Houdebine - One of the best experts on this subject based on the ideXlab platform.
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Distal control of the pig Whey Acidic Protein (WAP) locus in transgenic mice.
Gene, 2007Co-Authors: Soraya Saidi, Louis Marie Houdebine, Sylvie Rival-gervier, Nathalie Daniel-carlier, Dominique Thépot, Caroline Morgenthaler, Céline Viglietta, Sonia Prince, Bruno Passet, Geneviève JolivetAbstract:Distal control of the Whey Acidic Protein (WAP) locus was studied using a transgenic approach. A series of pig genomic fragments encompassing increasing DNA lengths upstream of the mammary specific Whey Acidic Protein (WAP) gene transcription start point (tsp) and 5 kb downstream were used for microinjection in mouse fertilized eggs. Our data pointed out three regions as potent regulators for WAP but not for RAMP3 gene expression (a non mammary-specific gene located 30 kb upstream of the WAP gene). WAP gene activating elements were present in the -80 kb to -30 kb and -145 kb to -130 kb regions whereas inhibitors were present in the -130 kb to -80 kb region. The stimulatory regions were characterized by peaks of histone H4 acetylation and a poor nucleosome occupancy in lactating sow mammary glands but not in liver. These data reveal for the first time the existence of several remote potent regulatory regions of the pig WAP gene.
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Ruminants genome no longer contains Whey Acidic Protein gene but only a pseudogene
Gene, 2006Co-Authors: Siham Hajjoubi, Louis Marie Houdebine, Sylvie Rival-gervier, Hélène Hayes, Sandrine Floriot, André Eggen, François Piumi, Patrick Chardon, Dominique ThépotAbstract:Whey Acidic Protein (WAP) has been identified in the milk of only a few species, including mouse, rat, rabbit, camel, pig, tammar wallaby, brushtail possum, echidna and platypus. Despite intensive studies, it has not yet been found in the milk of Ruminants. We have isolated and characterized genomic WAP clones from ewe, goat and cow, identified their chromosomal localization and examined the expression of the endogenous WAP sequence in the mammary glands of all three species. The WAP sequences were localized on chromosome 4 (4q26) as expected from comparative mapping data. The three ruminant WAP sequences reveal the same deletion of a nucleotide at the end of the first exon when compared with the pig sequence. Due to this frameshift mutation, the putative Proteins encoded by these sequences do not harbor the features of a usual WAP Protein with two four-disulfide core domains. Moreover, RT-PCR experiments have shown that these sequences are not transcribed and are, thus, pseudogenes. This loss of functionality of the gene in Ruminants raises the question of the biological role of the WAP. Some putative roles previously suggested for WAP are discussed.
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in vitro and in vivo effects of a multimerized αs1 casein enhancer on Whey Acidic Protein gene promoter activity
Molecular Reproduction and Development, 2003Co-Authors: Thais Pantano, Louis Marie Houdebine, Céline Viglietta, Sonia Prince, Celeste Menck-le Bourhis, Caroline Maeder, Sylvie Rivalgervier, Geneviève JolivetAbstract:Experimental data obtained in previous works have led to postulate that enhancers increase the frequency of action of a linked promoter in a given cell and may have some insulating effects. The multimerized rabbit αs1-casein gene enhancer, the 6i multimer, was added upstream of the rabbit Whey Acidic Protein gene (WAP) promoter (−6,300; +28 bp) fused to the firefly luciferase (luc) gene (6i WAP-luc construct). The 6i multimer increased reporter gene expression in mouse mammary HC11 cells. In transgenic mice, a very weak but significant increase was also observed. More noticeable, no silent lines were found when the 6i multimer was associated to the WAP-luc construct. This reflects the fact that the 6i multimer tends to prevent the silencing of the WAP-luc construct. After addition of the 5′HS4 insulator region from the chicken β-globin locus upstream of the 6i multimer, similar luciferase levels were measured in 6i WAP-luc and 5′HS4 WAP-luc transgenic mice. Our present data and previous ones, which show that the 6i multimer has no insulating activity on a TK gene promoter construct indicate that the insulating activity of the 6i multimer is construct-dependent and not amplified by the 5′HS4 insulator. Mol. Reprod. Dev. 65: 262–268, 2003. © 2003 Wiley-Liss, Inc.
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In vitro and in vivo effects of a multimerized αs1‐casein enhancer on Whey Acidic Protein gene promoter activity
Molecular reproduction and development, 2003Co-Authors: Thais Pantano, Louis Marie Houdebine, Sylvie Rival-gervier, Céline Viglietta, Sonia Prince, Celeste Menck-le Bourhis, Caroline Maeder, Geneviève JolivetAbstract:Experimental data obtained in previous works have led to postulate that enhancers increase the frequency of action of a linked promoter in a given cell and may have some insulating effects. The multimerized rabbit αs1-casein gene enhancer, the 6i multimer, was added upstream of the rabbit Whey Acidic Protein gene (WAP) promoter (−6,300; +28 bp) fused to the firefly luciferase (luc) gene (6i WAP-luc construct). The 6i multimer increased reporter gene expression in mouse mammary HC11 cells. In transgenic mice, a very weak but significant increase was also observed. More noticeable, no silent lines were found when the 6i multimer was associated to the WAP-luc construct. This reflects the fact that the 6i multimer tends to prevent the silencing of the WAP-luc construct. After addition of the 5′HS4 insulator region from the chicken β-globin locus upstream of the 6i multimer, similar luciferase levels were measured in 6i WAP-luc and 5′HS4 WAP-luc transgenic mice. Our present data and previous ones, which show that the 6i multimer has no insulating activity on a TK gene promoter construct indicate that the insulating activity of the 6i multimer is construct-dependent and not amplified by the 5′HS4 insulator. Mol. Reprod. Dev. 65: 262–268, 2003. © 2003 Wiley-Liss, Inc.
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Pig Whey Acidic Protein gene is surrounded by two ubiquitously expressed genes
Biochimica et biophysica acta, 2003Co-Authors: Sylvie Rival-gervier, Dominique Thépot, Geneviève Jolivet, Louis Marie HoudebineAbstract:A 140-kb pig DNA fragment containing the Whey Acidic Protein (WAP) gene cloned in a bacterial artificial chromosome (BAC344H5) has been shown to contain all of the cis-elements necessary for position-independent, copy-dependent and tissue-specific expression in transgenic mice. The insert from this BAC was sequenced. This revealed the presence of two other genes with quite different expression patterns in pig tissues and in transfected HC11 mouse mammary cells. The RAMP3 gene is located 15 kb upstream of the WAP gene in reverse orientation. The CPR2 gene is located 5 kb downstream of the WAP gene in the same orientation. The same locus organization was found in the human genome. The region between RAMP3 and CPR2 in the human genome contains a WAP gene-like sequence with several points of mutation which may account for the absence of WAP from human milk.
Eve Devinoy - One of the best experts on this subject based on the ideXlab platform.
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Analysis of the efficiency of the rabbit Whey Acidic Protein gene 5' flanking region in controlling the expression of homologous and heterologous linked genes
Journal of Dairy Research, 2005Co-Authors: Eve Devinoy, Dominique Thépot, Lluis Montoliu, Mária Baranyi, László Hiripi, Marie Louise Fontaine, Lilla Bodrogi, Zsuzsanna BöszeAbstract:For 10 years, the regulatory regions of the mouse and rabbit Whey Acidic Protein gene have been used to express heterologous Proteins in the milk of transgenic mice, as well as to produce pharmaceutical Proteins, on a large scale, in the milk of transgenic livestock. To date, a broad range of expression levels have been detected, and elucidation of the structure-function relationship in these regulatory regions might help to achieve high levels of expression, reproducibly. An extended 5' regulatory region (17.6 kb v. 6.3 kb) of the rabbit Whey Acidic promoter resulted in an increased frequency of rabbit Whey Acidic Protein expression in transgenic mice. However, the expression levels were low compared with the high expression levels achieved in both transgenic mice and rabbits using the heterologous kappa-casein in the 6.3 kb rabbit Whey Acidic Protein 5' regulatory region. These results underline the importance of the 3' downstream regulatory regions, which still need to be better characterized in the Whey Acidic Protein gene.
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Hormone-induced modifications of the chromatin structure surrounding upstream regulatory regions conserved between the mouse and rabbit Whey Acidic Protein genes.
Biochemical Journal, 2003Co-Authors: Benjamin Millot, Marie Louise Fontaine, Lluis Montoliu, Teresa Mata, Eve DevinoyAbstract:The upstream regulatory regions of the mouse and rabbit Whey Acidic Protein (WAP) genes have been used extensively to target the efficient expression of foreign genes into the mammary gland of transgenic animals. Therefore both regions have been studied to elucidate fully the mechanisms controlling WAP gene expression. Three DNase I-hypersensitive sites (HSS0, HSS1 and HSS2) have been described upstream of the rabbit WAP gene in the lactating mammary gland and correspond to important regulatory regions. These sites are surrounded by variable chromatin structures during mammary-gland development. In the present study, we describe the upstream sequence of the mouse WAP gene. Analysis of genomic sequences shows that the mouse WAP gene is situated between two widely expressed genes (Cpr2 and Ramp3). We show that the hypersensitive sites found upstream of the rabbit WAP gene are also detected in the mouse WAP gene. Further, they encompass functional signal transducer and activator of transcription 5-binding sites, as has been observed in the rabbit. A new hypersensitive site (HSS3), not specific to the mammary gland, was mapped 8 kb upstream of the rabbit WAP gene. Unlike the three HSSs described above, HSS3 is also detected in the liver, but similar to HSS1, it does not depend on lactogenic hormone treatments during cell culture. The region surrounding HSS3 encompasses a potential matrix attachment region, which is also conserved upstream of the mouse WAP gene and contains a functional transcription factor Ets-1 (E26 transformation-specific-1)-binding site. Finally, we demonstrate for the first time that variations in the chromatin structure are dependent on prolactin alone.
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A distal region, hypersensitive to DNase I, plays a key role in regulating rabbit Whey Acidic Protein gene expression
Biochemical Journal, 2001Co-Authors: Benjamin Millot, Dominique Thépot, Marie Louise Fontaine, Eve DevinoyAbstract:The aim of the present study was to identify the functional domains of the upstream region of the rabbit Whey Acidic Protein (WAP) gene, which has been used with considerable efficacy to target the expression of several foreign genes to the mammary gland. We have shown that this region exhibits three sites hypersensitive to DNase I digestion in the lactating mammary gland, and that all three sites harbour elements which can bind to Stat5 in vitro in bandshift assays. However, not all hypersensitive regions are detected at all stages from pregnancy to weaning, and the level of activated Stat5 detected in the rabbit mammary gland is low except during lactation. We have studied the role of the distal site, which is only detected during lactation, in further detail. It is located within a 849bp region that is required to induce a strong expression of the chloramphenicol acetyltransferase reporter gene in transfected mammary cells. Taken together, these results suggest that this region, centred around a Stat5-binding site and surrounded by a variable chromatin structure during the pregnancy–lactation cycle, may play a key role in regulating the expression of this gene in vivo. Furthermore, this distal region exhibits sequence similarity with a region located around 3kb upstream of the mouse WAP gene. The existence of such a distal region in the mouse WAP gene may explain the differences in expression between 4.1 and 2.1kb mouse WAP constructs.
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Rabbit Whey Acidic Protein gene upstream region controls high-level expression of bovine growth hormone in the mammary gland of transgenic mice.
Molecular reproduction and development, 1995Co-Authors: Dominique Thépot, Eve Devinoy, Marie Louise Fontaine, M. Massoud, Marie-georges Stinnakre, Guy Kann, Louis Marie HoudebineAbstract:Transgenic mice were produced which secreted high levels of bGH into milk. The 6.3-kb upstream region of the rabbit Whey Acidic Protein (rWAP) gene was linked to the structural part of the bovine growth hormone (bGH) gene, and the chimeric gene was introduced into mouse oocytes. bGH was detected by radioimmunoassay in the milk of all resulting transgenic mice. bGH concentrations in milk varied from line to line, from 1.0–16 mg/ml. This expression was not correlated to the number of transgene copies. In all lines studied, the mammary gland was the major organ expressing bGH mRNA during lactation. bGH mRNA concentrations were barely detectable in the mammary gland of cyclic females; they increased during pregnancy. These results show that the upstream region of the rWAP gene harbors powerful regulatory elements which target high levels of bGH transgene expression to the mammary gland of lactating transgenic mice. © 1995 wiley-Liss, Inc.
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Hormone responsive elements within the upstream sequences of the rabbit Whey Acidic Protein (WAP) gene direct chloramphenicol acetyl transferase (CAT) reporter gene expression in transfected rabbit mammary cells.
Molecular and cellular endocrinology, 1991Co-Authors: Eve Devinoy, Rachel Malienou-ngassa, Claudine Puissant, Dominique Thépot, L.m. HoudebineAbstract:Abstract Whey Acidic Protein gene transcription is induced in the mammary gland under the influence of lactogenic hormones: prolactin, insulin and cortisol. The rabbit WAP gene has already been isolated and sequenced in a previous work. In the present study, we have evaluated the role of the 5' flanking region of the rabbit WAP gene in the transcriptional regulation of the WAP gene by using a reporter CAT gene. Chimeric genes containing the upstream region of the WAP gene have been linked to the bacterial CAT gene and transfected into rabbit primary mammary cells. The results reported here show that two regions carrying important regulatory elements of the rabbit WAP gene are located between -6300 and -3000 bp, and between -3000 and -1800 bp upstream from the WAP transcription start point, respectively. They contribute to the high level of expression of the rabbit WAP gene in the mammary cell.
Dominique Thépot - One of the best experts on this subject based on the ideXlab platform.
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Distal control of the pig Whey Acidic Protein (WAP) locus in transgenic mice.
Gene, 2007Co-Authors: Soraya Saidi, Louis Marie Houdebine, Sylvie Rival-gervier, Nathalie Daniel-carlier, Dominique Thépot, Caroline Morgenthaler, Céline Viglietta, Sonia Prince, Bruno Passet, Geneviève JolivetAbstract:Distal control of the Whey Acidic Protein (WAP) locus was studied using a transgenic approach. A series of pig genomic fragments encompassing increasing DNA lengths upstream of the mammary specific Whey Acidic Protein (WAP) gene transcription start point (tsp) and 5 kb downstream were used for microinjection in mouse fertilized eggs. Our data pointed out three regions as potent regulators for WAP but not for RAMP3 gene expression (a non mammary-specific gene located 30 kb upstream of the WAP gene). WAP gene activating elements were present in the -80 kb to -30 kb and -145 kb to -130 kb regions whereas inhibitors were present in the -130 kb to -80 kb region. The stimulatory regions were characterized by peaks of histone H4 acetylation and a poor nucleosome occupancy in lactating sow mammary glands but not in liver. These data reveal for the first time the existence of several remote potent regulatory regions of the pig WAP gene.
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Ruminants genome no longer contains Whey Acidic Protein gene but only a pseudogene
Gene, 2006Co-Authors: Siham Hajjoubi, Louis Marie Houdebine, Sylvie Rival-gervier, Hélène Hayes, Sandrine Floriot, André Eggen, François Piumi, Patrick Chardon, Dominique ThépotAbstract:Whey Acidic Protein (WAP) has been identified in the milk of only a few species, including mouse, rat, rabbit, camel, pig, tammar wallaby, brushtail possum, echidna and platypus. Despite intensive studies, it has not yet been found in the milk of Ruminants. We have isolated and characterized genomic WAP clones from ewe, goat and cow, identified their chromosomal localization and examined the expression of the endogenous WAP sequence in the mammary glands of all three species. The WAP sequences were localized on chromosome 4 (4q26) as expected from comparative mapping data. The three ruminant WAP sequences reveal the same deletion of a nucleotide at the end of the first exon when compared with the pig sequence. Due to this frameshift mutation, the putative Proteins encoded by these sequences do not harbor the features of a usual WAP Protein with two four-disulfide core domains. Moreover, RT-PCR experiments have shown that these sequences are not transcribed and are, thus, pseudogenes. This loss of functionality of the gene in Ruminants raises the question of the biological role of the WAP. Some putative roles previously suggested for WAP are discussed.
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Analysis of the efficiency of the rabbit Whey Acidic Protein gene 5' flanking region in controlling the expression of homologous and heterologous linked genes
Journal of Dairy Research, 2005Co-Authors: Eve Devinoy, Dominique Thépot, Lluis Montoliu, Mária Baranyi, László Hiripi, Marie Louise Fontaine, Lilla Bodrogi, Zsuzsanna BöszeAbstract:For 10 years, the regulatory regions of the mouse and rabbit Whey Acidic Protein gene have been used to express heterologous Proteins in the milk of transgenic mice, as well as to produce pharmaceutical Proteins, on a large scale, in the milk of transgenic livestock. To date, a broad range of expression levels have been detected, and elucidation of the structure-function relationship in these regulatory regions might help to achieve high levels of expression, reproducibly. An extended 5' regulatory region (17.6 kb v. 6.3 kb) of the rabbit Whey Acidic promoter resulted in an increased frequency of rabbit Whey Acidic Protein expression in transgenic mice. However, the expression levels were low compared with the high expression levels achieved in both transgenic mice and rabbits using the heterologous kappa-casein in the 6.3 kb rabbit Whey Acidic Protein 5' regulatory region. These results underline the importance of the 3' downstream regulatory regions, which still need to be better characterized in the Whey Acidic Protein gene.
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Pig Whey Acidic Protein gene is surrounded by two ubiquitously expressed genes
Biochimica et biophysica acta, 2003Co-Authors: Sylvie Rival-gervier, Dominique Thépot, Geneviève Jolivet, Louis Marie HoudebineAbstract:A 140-kb pig DNA fragment containing the Whey Acidic Protein (WAP) gene cloned in a bacterial artificial chromosome (BAC344H5) has been shown to contain all of the cis-elements necessary for position-independent, copy-dependent and tissue-specific expression in transgenic mice. The insert from this BAC was sequenced. This revealed the presence of two other genes with quite different expression patterns in pig tissues and in transfected HC11 mouse mammary cells. The RAMP3 gene is located 15 kb upstream of the WAP gene in reverse orientation. The CPR2 gene is located 5 kb downstream of the WAP gene in the same orientation. The same locus organization was found in the human genome. The region between RAMP3 and CPR2 in the human genome contains a WAP gene-like sequence with several points of mutation which may account for the absence of WAP from human milk.
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A distal region, hypersensitive to DNase I, plays a key role in regulating rabbit Whey Acidic Protein gene expression
Biochemical Journal, 2001Co-Authors: Benjamin Millot, Dominique Thépot, Marie Louise Fontaine, Eve DevinoyAbstract:The aim of the present study was to identify the functional domains of the upstream region of the rabbit Whey Acidic Protein (WAP) gene, which has been used with considerable efficacy to target the expression of several foreign genes to the mammary gland. We have shown that this region exhibits three sites hypersensitive to DNase I digestion in the lactating mammary gland, and that all three sites harbour elements which can bind to Stat5 in vitro in bandshift assays. However, not all hypersensitive regions are detected at all stages from pregnancy to weaning, and the level of activated Stat5 detected in the rabbit mammary gland is low except during lactation. We have studied the role of the distal site, which is only detected during lactation, in further detail. It is located within a 849bp region that is required to induce a strong expression of the chloramphenicol acetyltransferase reporter gene in transfected mammary cells. Taken together, these results suggest that this region, centred around a Stat5-binding site and surrounded by a variable chromatin structure during the pregnancy–lactation cycle, may play a key role in regulating the expression of this gene in vivo. Furthermore, this distal region exhibits sequence similarity with a region located around 3kb upstream of the mouse WAP gene. The existence of such a distal region in the mouse WAP gene may explain the differences in expression between 4.1 and 2.1kb mouse WAP constructs.
