ABSTRACT
The Ku heterodimeric protein (Ku80/Ku70) is an essential component of the double-strand break DNA repair pathway in mammalian cells. We have recently defined a central region within Ku80 that is required for heterodimerization with Ku70. We now identified a core region within Ku80 (amino acids 210 to 531) that is necessary for binding of Ku to DNA ends. Interaction with Ku70 and DNA end binding are important for Ku80 function in vivo, since Ku80 mutants lacking DNA end binding activity were unable to restore radiation resistance in Ku80 deficient fibroblast cell lines. However, Ku80 mutants were identified that retained DNA end binding activity but were unable to restore radiation survival, thus pointing to additional functional properties of Ku80. An N-terminal deletional mutant of Ku80 was able to suppress wild type Ku80 function for radiation survival in several cell lines, thus demonstrating dominant negative function.
Subject(s)
Antigens, Nuclear , Cell Survival , DNA Helicases , DNA Repair , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , DNA/metabolism , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Radiation, Ionizing , Animals , Cell Line , DNA-Binding Proteins/genetics , Dimerization , Embryo, Mammalian , Fibroblasts , Humans , Ku Autoantigen , Mice , Mutation , Nuclear Proteins/genetics , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Structure-Activity Relationship , TransfectionABSTRACT
The Ku protein has a critical function in the repair of double-strand DNA breaks induced for example by ionizing radiation or during VDJ recombination. Ku serves as the DNA-binding subunit of the DNA-dependent kinase and is a heterodimeric protein composed of 80- and 70-kDa subunits. We used the two-hybrid system to analyze the interaction domains of the Ku subunits and to identify possible additional partners for Ku. Screening a human cDNA library with the Ku heterodimer did not reveal any novel partners. Screening with the individual subunits, we detected only Ku70 clones interacting with Ku80 and only Ku80 clones interacting with Ku70, indicating that these are the primary partners for one another. Ku80 and Ku70 formed only heterodimers and did not homodimerize. Ku80 was restricted to interacting with just one Ku70 molecule at a time. The minimal functional interaction domain of Ku80 that interacted with Ku70 was defined. It consisted of a 28-amino acid region extending from amino acid 449 to 477. This region was crucial for interaction with Ku70, since mutation within this critical site at amino acids 453 and 454 abrogated the ability to interact with Ku70. We furthermore verified that the same region is crucial for interaction with Ku70 using in vitro co-translation of both subunits followed by an immunoprecipitation with anti-Ku70 antibodies. This interaction domain of Ku80 does not contain any motif previously recognized in protein-protein interactions.
Subject(s)
Antigens, Nuclear , DNA Helicases , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/metabolism , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , Saccharomyces cerevisiae Proteins , Binding Sites , Cloning, Organism , DNA Repair , Humans , Ku Autoantigen , Macromolecular Substances , Mutagenesis, Site-Directed , Point Mutation , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/metabolism , Saccharomyces cerevisiae , Sequence Deletion , Transfection , beta-Galactosidase/metabolismABSTRACT
Membrane lymphotoxin (LT) complex is a trimer composed of two subunits , LT-alpha and LT-beta of which the latter is a 33-kDa transmembrane protein. The LT-beta gene is expressed in lymphoid cells and organs, but little is known about its inducible regulation. Previously, the surface expression of LT-beta in Jurkat cells has been shown to increase in response to PMA. In this report, we used this model to study the transcriptional control of the human and murine LT-beta genes. PMA strongly induced the expression of LT-beta mRNA, and the level of induction was not changed markedly by cycloheximide (CHX) treatment. The LT-beta promoter region contains conserved Egr-1, nuclear factor (NF)-kappaB, and Ets binding sites, and PMA-inducible factors bound to these sites were detected by the gel-retardation technique (electrophoretic mobility shift assay (EMSA)). To identify sequences involved in transcriptional control, sets of human and mouse promoter-chloramphenicol acetyltransferase (CAT) constructs were generated and assayed by transient transfections. The PMA response was lost after deletion of the distal Ets binding site at -110. Mutations at either the Ets or NF-kappaB sites that prevented factor binding dramatically reduced PMA-inducible promoter activity, suggesting cooperative interaction between corresponding transcription factors in PMA activation. Mutation at the Egr-1 site also resulted in substantial loss of promoter activity, and the residual activity may be attributed to binding of constitutively expressed Sp-1 to the same site. We propose that the interaction between the members of NF-kappaB and Ets families of transcription factors and their cognate sites in the promoter is the major determinant of inducible expression of the LT-beta gene in Jurkat cells.
Subject(s)
Lymphotoxin-alpha/genetics , Membrane Proteins/genetics , Promoter Regions, Genetic/drug effects , Tetradecanoylphorbol Acetate/pharmacology , Animals , Base Sequence , Binding Sites/genetics , Cell Line , Cycloheximide/pharmacology , DNA/genetics , DNA/metabolism , DNA Probes/genetics , Gene Expression Regulation/drug effects , Humans , Lymphotoxin-beta , Mice , Molecular Sequence Data , Protein Binding , RNA, Messenger/genetics , RNA, Messenger/metabolism , Sequence Deletion , Transcription Factors/metabolismABSTRACT
DNA helicases (Hel) play a role in a number of processes involving DNA strand separation, including replication, repair, recombination and transcription. Rearrangement of receptor genes, which occurs in immature lymphocytes, could also be mediated by Hel. We report here the cloning from murine fetal thymus tissue of a novel putative Hel containing seven conserved Hel domains and belonging to the DEGH subclass of DNA Hel. We term the encoding gene lsh (lymphoid-specific Hel), since the gene is expressed in early thymocytes, but not in heart, liver, lung, muscle, brain or kidney, as judged by Northern analysis. Spleen cells expressed lsh following activation. T- and B-cell lines, at both the immature and mature stage, expressed lsh. To examine the earliest stages of lymphopoiesis, mouse embryonic tissues were examined; lsh was not detected in the yolk sac of day 12 of gestation, but was expressed in fetal liver and at high levels in fetal thymus at day 15 of gestation.
Subject(s)
DNA Helicases/genetics , Gene Expression/genetics , Lymphocytes/physiology , Thymus Gland/physiology , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , DNA Helicases/chemistry , DNA Primers , DNA, Complementary , Fetus , Mice , Mice, Inbred C57BL , Molecular Sequence Data , Polymerase Chain Reaction , Sequence Analysis , Sequence Homology, Nucleic AcidABSTRACT
In the present study we have analyzed superinduction of TNF-alpha mRNA and enhancement of TNF-alpha gene transcription by cycloheximide (Chx) in human blood monocytes isolated by continuous Percoll gradient and activated in vitro. In the same monocyte cultures, we have compared the rate of gene transcription of TNF-alpha, IL-1 beta, IL-8, and the P53-antioncogene under the influence of plastic adherence, Staphylococcus aureus Cowan 1 (SAC), and Chx added at different times of monocyte culture. It was shown that the cytokine genes have low or negligible transcriptional activity in freshly isolated monocytes, whereas P53 gene transcription was constant in freshly isolated and in vitro-stimulated cells. Transcription of the IL-1 beta and IL-8 genes was induced by adherence and was not more enhanced by SAC. Transcription of the TNF-alpha gene was not induced by adherence. Chx added at the beginning of the monocyte culture did not block TNF-alpha or IL-1 beta gene transcription. IL-8 gene transcription, however, was abrogated by Chx. Addition of SAC to monocyte culture containing Chx caused significant enhancement of TNF-alpha gene transcription. Addition of Chx after 2.5 or 4 h of SAC activation caused "superinduction" of TNF-alpha mRNA and enhancement of TNF-alpha gene transcription. The data imply that TNF-alpha gene transcription in activated human monocytes might be regulated by both positive and negative regulatory factors that differ in their stability and protein synthesis dependence. In addition, results demonstrate that TNF-alpha, IL-1 beta, IL-8, and p53 genes in human monocytes are differently regulated.
Subject(s)
Cycloheximide/pharmacology , Genes, p53/immunology , Interleukin-1/genetics , Interleukin-8/genetics , Monocytes/immunology , Transcription, Genetic/drug effects , Tumor Necrosis Factor-alpha/genetics , Cells, Cultured , Genes, p53/drug effects , Humans , Interleukin-1/immunology , Interleukin-8/immunology , Macrophage Activation/genetics , Monocytes/drug effects , Monocytes/metabolism , RNA, Messenger/drug effects , Staphylococcus aureus/immunology , Tumor Necrosis Factor-alpha/drug effects , Tumor Necrosis Factor-alpha/immunologyABSTRACT
The p53 gene has been associated with malignant transformation as well as "anti-oncogene" activity. In the present report expression of p53 in resting and activated human blood monocytes and lymphocytes is analyzed. It is found that human monocytes freshly isolated by continuous percoll gradient centrifugation contained detectable level of p53 mRNA. Stimulation of monocytes by potent activation inducer Staphylococcus Aureus Cowan I for 3-5 hr caused disappearance of r53 mRNA. In contrast, induction of high level of TNF-alpha mRNA was detected. Addition of cycloheximide had no effect on p53 mRNA content in stimulated monocytes, and caused disappearance of mRNA in resting cells. In lymphocytes cultures p53 mRNA was absent in freshly isolated cells and in resting lymphocytes cultured for 20 hr. Activation of lymphocytes by lectin caused accumulation of p53 mRNA. We suggest that r53 gene regulation and functions might be different in human monocytes and lymphocytes.
Subject(s)
Gene Expression , Genes, p53 , Lymphocytes/ultrastructure , Monocytes/ultrastructure , Blotting, Northern , Cells, Cultured , Culture Media , Humans , Lymphocyte Activation , RNA, Messenger/genetics , Tumor Necrosis Factor-alpha/pharmacologyABSTRACT
The p53 gene is associated with malignant transformation as well as 'antioncogene' activity. In this report expression of p53 in resting and activated human blood monocytes and lymphocytes was studied. It is shown that human monocytes freshly isolated by continuous Percoll-gradient centrifugation contain detectable levels of p53 mRNA. Stimulation of monocytes by the potent activation inducer Staphylococcus aureus Cowan I (SAC) for 3-5 h caused the disappearance of p53 mRNA. In contrast, induction of a high level of tumor necrosis factor alpha mRNA was detected. The addition of cycloheximide did not increase the p53 mRNA content in stimulated monocytes, and decreased the mRNA level in resting cells. p53 mRNA was absent in freshly isolated lymphocytes and in resting cells cultured for 20 h. Activation of lymphocytes by phytohemagglutinin caused accumulation of p53 mRNA. We suggest that p53 gene regulation and functions might be different in human monocytes and lymphocytes.
Subject(s)
Genes, Tumor Suppressor , Genes, p53 , Lymphocytes/physiology , Monocytes/physiology , Tumor Suppressor Protein p53/genetics , Blotting, Northern , Cycloheximide/pharmacology , Gene Expression/drug effects , Humans , RNA, Messenger/genetics , Transcription, Genetic , Tumor Necrosis Factor-alpha/geneticsABSTRACT
The presence of TNF-alpha mRNA and protein in circulating human blood monocytes isolated by continuous Percoll gradient fractionation was studied. The technique of RNA isolation from the blood samples was used to study TNF-alpha mRNA expression. It was shown that human blood monocytes of healthy donors contained no presynthesized pool of TNF-alpha mRNA as well as no TNF-alpha protein.
Subject(s)
Leukocytes, Mononuclear/metabolism , Tumor Necrosis Factor-alpha/biosynthesis , Blotting, Northern , Cells, Cultured , Chemical Fractionation , Humans , RNA, Messenger/isolation & purificationABSTRACT
The present study was undertaken to assess the presence of tumor necrosis factor (TNF)-alpha mRNA and protein in circulating human blood monocytes and to study the TNF-alpha gene expression in human monocytes isolated by continuous Percoll gradient fractionation. The technique of RNA isolation directly from the blood samples was used to study TNF-alpha mRNA expression in circulating human blood leukocytes. It was shown that human blood leukocytes of healthy donors contained no presynthesized pool of TNF-alpha mRNA as well as no TNF-alpha protein. It was found that early pretreatment with cycloheximide interferes with TNF-alpha mRNA induction by Staphylococcus aureus.
