ABSTRACT
The POU transcription factor Oct-4 is expressed specifically in the germ line, pluripotent cells of the pregastrulation embryo and stem cell lines derived from the early embryo. Osteopontin (OPN) is a protein secreted by cells of the preimplantation embryo and contains a GRGDS motif that can bind to specific integrin subtypes and modulate cell adhesion/migration. We show that Oct-4 and OPN are coexpressed in the preimplantation mouse embryo and during differentiation of embryonal cell lines. Immunoprecipitation of the first intron of OPN (i-opn) from covalently fixed chromatin of embryonal stem cells by Oct-4-specific antibodies indicates that Oct-4 binds to this fragment in vivo. The i-opn fragment functions as an enhancer in cell lines that resemble cells of the preimplantation embryo. Furthermore, it contains a novel palindromic Oct factor recognition element (PORE) that is composed of an inverted pair of homeodomain-binding sites separated by exactly 5 bp (ATTTG +5 CAAAT). POU proteins can homo- and heterodimerize on the PORE in a configuration that has not been described previously. Strong transcriptional activation of the OPN element requires an intact PORE. In contrast, the canonical octamer overlapping with the downstream half of the PORE is not essential. Sox-2 is a transcription factor that contains an HMG box and is coexpressed with Oct-4 in the early mouse embryo. Sox-2 represses Oct-4 mediated activation of i-opn by way of a canonical Sox element that is located close to the PORE. Repression depends on a carboxy-terminal region of Sox-2 that is outside of the HMG box. Expression, DNA binding, and transactivation data are consistent with the hypothesis that OPN expression is regulated by Oct-4 and Sox-2 in preimplantation development.
Subject(s)
DNA-Binding Proteins/chemistry , DNA-Binding Proteins/physiology , Embryonic Development/genetics , Enhancer Elements, Genetic , Nuclear Proteins/physiology , Sialoglycoproteins/antagonists & inhibitors , Sialoglycoproteins/chemistry , Transcription Factors/chemistry , Amino Acid Sequence , Animals , Base Composition , Base Sequence , Binding Sites/genetics , Blastocyst/metabolism , Blastocyst/physiology , Carcinoma, Embryonal , Cell Differentiation/genetics , Chromatin/isolation & purification , Chromatin/metabolism , DNA-Binding Proteins/isolation & purification , DNA-Binding Proteins/metabolism , Dimerization , Embryo, Mammalian , Female , HMGB Proteins , Introns , Mice , Mice, Inbred Strains , Molecular Sequence Data , Mutagenesis, Site-Directed , Octamer Transcription Factor-3 , Osteopontin , POU Domain Factors , Pregnancy , Protein Conformation , SOXB1 Transcription Factors , Sialoglycoproteins/biosynthesis , Transcription Factors/physiology , Tumor Cells, CulturedABSTRACT
SUMMARY: Metallothionein genes can be induced in vivo by heavy metals, glucocorticoids, and toxins. In all transgenic mice carrying the MT-I promoter, that have been reported so far, induction by glucocorticoids failed. This study reports two mouse lines, transgenic for the murine MT-I-HBV (hepatitis B virus; map position site: 30-1986) construct, which secrete the viral surface antigen (HBsAg) in their serum. In both lines, males produce more HBsAg than females, and in all cases the MT-I promoter can be induced by dexamethasone, lipopolysaccharide (LPS), and heavy metals. A glucocorticoid-responsive element, which is situated in the HBV fragment used, can explain the dexamethasone induction of the MT-I promoter. ZUSAMMENFASSUNG: Expression des Hepatitis B oberflächen Antigens (HBsAg) unter Kontrolle des mMT-I Promoters kann in transgenen Mäusen durch Zink Sulfat, Dexamethason und Lipopolysacchariden induziert werden Metallothioneingene werden in vivo durch Schwermetalle, Glucocorticoide und Toxine induziert. Soweit bisher bekannt, konnte jedoch in transgenen Mäusen mit dem MT-I-Promotor keine Expression durch Glucocorticioide beobachtet werden. Wir berichten hier von zwei transgenen Mäuselinien mit dem murinen MT-I-Promotor, der das Oberflächenantigen des Hepatitis B Virus (HbsAg, map position site 30-1986) exprimierte. In beiden Linien produzierten die männlichen Tiere mehr HBsAg im Blutserum als die weiblichen. Ohne Ausnahme reagierte der MT-I-Promotor bei Applikation von Dexamethason, Lipopolysaccharid (LPS) und Schwermetall. Ein Glucocorticoid-responsives Element in den HBV-Sequenzen kann die Induktion des MT-I-Promotors erklären.
ABSTRACT
Bovine microsatellites were used to amplify DNA of red deer (Cervus elaphus). Fourteen of 27 bovine systems (52%) displayed polymorphism, while no (CA)n-repeat was detected in seven systems and six systems gave no amplificates in red deer. The allele number ranged from 2 to 7, the polymorphism information content between 0.24 and 0.76. The results demonstrate that transfer of microsatellite systems between families of the same order (artiodactyla) is possible. Molecular genetic research will help to clarify the differentiation and ecology of wild animals and will contribute to define criteria needed for the preservation of endangered species.
