ABSTRACT
The retinoblastoma-related p130 protein is a member of a conserved family, consisting of Rb, p107 and p130, which are believed to play important roles in cell-cycle control and cellular differentiation. We have generated a null mutation in p130 by gene targeting and crossed the null allele into Balb/cJ and C57BL/6J strains of mice. In an enriched Balb/cJ genetic background, p130(-/-) embryos displayed arrested growth and died between embryonic days 11 and 13. Histological analysis revealed varying degrees of disorganization in neural and dermamyotomal structures. Immunohistochemistry with antibody reactive with Islet-1 indicated markedly reduced numbers of neurons in the spinal cord and dorsal root ganglia. Immunohistochemistry with antibody reactive with desmin indicated a similar reduction in the number of differentiated myocytes in the myotome. The myocardium of mutant embryos was abnormally thin and resembled an earlier staged two-chambered heart consisting of the bulbus cordis and the ventricular chamber. TUNEL analysis indicated the presence of extensive apoptosis in various tissues including the neural tube, the brain, the dermomyotome, but not the heart. Immunohistochemistry with antibody reactive with PCNA revealed increased cellular proliferation in the neural tube and the brain, and decreased proliferation in the heart. The placentas of p130(-/-) embryos did not display elevated apoptosis and were indistinguishable from wild type suggesting that the phenotype was not due to placental failure. Following a single cross with the C57BL/6 mice, p130(-/-) animals were derived that were viable and fertile. These results indicate that p130 in a Balb/cJ genetic background plays an essential role that is required for normal development. Moreover, our experiments establish that second-site modifier genes exist that have an epistatic relationship with p130.
Subject(s)
Embryonic and Fetal Development/genetics , Fetal Death/genetics , Phosphoproteins/physiology , Proteins , Retinoblastoma Protein/physiology , Animals , Apoptosis , Crosses, Genetic , Embryo, Mammalian/cytology , Embryo, Mammalian/pathology , Embryo, Mammalian/physiology , Female , Gestational Age , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Phenotype , Phosphoproteins/deficiency , Phosphoproteins/genetics , Pregnancy , Retinoblastoma Protein/deficiency , Retinoblastoma Protein/genetics , Retinoblastoma-Like Protein p130 , Species SpecificityABSTRACT
The mouse p130 cDNA was cloned from a thymus-derived library using the human p130 cDNA as a probe. The 4515-bp mouse cDNA encodes a putative 1092 aa protein with predicted molecular mass of 123 kD. Comparison of mouse and human sequences reveals the mouse protein lacks 43 aa in a conserved domain, relative to the human sequence, located amino-terminal to the pocket region. Northern analysis of P19 embryonal carcinoma (EC) and RA-induced P19 cultures indicates p130 mRNA is not expressed at detectable levels in undifferentiated stem cells and is strongly upregulated after post-mitotic neurons begin to accumulate. Northern analysis of adult mouse tissues indicated that the 4.8 kb p130 mRNA is expressed ubiquitously, however, a putative 5'-truncated 1.7 kb isoform is detected solely in testis. Forced expression of mouse p130 induced growth suppression of P19 EC cells and Western analysis of transfected P19 cells suggested the cloned cDNA encodes the full-length p130 protein.
Subject(s)
Phosphoproteins/genetics , Proteins , RNA, Messenger/genetics , Amino Acid Sequence , Animals , Base Sequence , Blotting, Northern , Carcinoma, Embryonal/genetics , Carcinoma, Embryonal/pathology , Cell Differentiation , Cloning, Molecular , DNA, Complementary , Humans , Mice , Mice, Inbred CBA , Molecular Sequence Data , Retinoblastoma-Like Protein p130 , Sequence Homology, Amino AcidABSTRACT
Murine cytomegalovirus infection of spleen cultures induced the production of a small (less than 10,000 molecular weight) immunosuppressive factor (VISF), which suppressed concanavalin-A mitogenesis in fresh mouse spleen cells, and in fresh human peripheral blood leukocytes. The factor did not affect the growth of two murine T-cell lines or of mouse fibroblasts. A similar factor was also found in the serum of infected mice, at the time of maximum immune suppression. The properties of VISF indicate that the mechanism of MCMV immune suppression is different from that caused by several other viruses which are important in human and veterinary medicine.
Subject(s)
Cytomegalovirus/immunology , Immunosuppressive Agents/immunology , Lymphocytes/immunology , Spleen/immunology , Viral Proteins/immunology , Animals , Cell Division , Cell Line , Cells, Cultured , Chromatography , Concanavalin A/pharmacology , Cytomegalovirus Infections/immunology , DNA/biosynthesis , Humans , Immunosuppressive Agents/blood , Lymphocyte Activation , Mice , Spleen/microbiology , Thymidine/metabolism , Viral Proteins/bloodABSTRACT
Murine cytomegalovirus infection in spleen cultures resulted in the production of a soluble factor, VISF (virus-induced suppressive factor), which inhibited concanavalin A mitogenesis in fresh spleen cells. Its production was specific for MCMV, since infection of spleen cultures by Sindbis virus, or bacteriophages PM 2 and T 4, and the phagocytosis of latex beads, all failed to elicit VISF. Maximum appearance of the factor occurred within 24 hours p.i. in spleen cultures, and its source was identified as the population of spleen cells which adhered to a plastic culture dish within two hours at 37 degrees C. Non-adherent cells did not produce the factor. Its production was not inhibited by indomethacin. VISF could be concentrated by ultrafiltration on a YM 2 membrane filter, and it was readily fractionated by chromatography on sephadex G-25. In relation to peptides of known molecular weight it appeared to be smaller than 1,400 daltons. Its ability to suppress concanavalin A mitogenesis was largely removed by digestion with proteinase K. Thus VISF appears to be a relatively small peptide or peptide-containing substance. It was purified further by HPLC.
