ABSTRACT
Two regions of the EBNA-3A protein of Epstein-Barr virus were shown to be capable of binding to the cell protein RBP-Jk (also known as CBF-1), a component of the Notch signaling pathway. Consistent with this binding, EBNA-3A inhibited reporter gene expression from plasmids containing RBP-Jk DNA binding sites within their promoters, including the Cp promoter. When EBNA-3A was linked to a GAL4 DNA binding domain, it repressed the activity of a promoter containing GAL4 binding sites at all plasmid concentrations tested. However, a deletion mutant of EBNA-3A lacking amino acids 100 to 364 showed a biphasic response in the GAL4 assay: it inhibited transcription at low DNA concentrations but activated it at high DNA concentrations. There appears to be a gene activation function within EBNA-3A that is masked in the full-length protein in this assay. Current models for EBNA-3 function have stressed transcription repression through binding to RBP-Jk, but we consider an alternative scheme in which the role of the binding of EBNA-3A, -3B, and -3C to RBP-Jk is to buffer the levels of active EBNA-3 protein. We have also found that the behavior of EBNA-3A in a cell fractionation procedure that distinguishes insoluble matrix from soluble cell fractions is modified by EBNA-LP, indicating a further novel level of interplay between the EBNA proteins.
Subject(s)
DNA-Binding Proteins/metabolism , Epstein-Barr Virus Nuclear Antigens/metabolism , Herpesvirus 4, Human/metabolism , Nuclear Proteins , Binding Sites , Cell Line, Transformed , Epstein-Barr Virus Nuclear Antigens/genetics , HeLa Cells , Herpesvirus 4, Human/genetics , Humans , Immunoglobulin J Recombination Signal Sequence-Binding Protein , Promoter Regions, Genetic , Repressor Proteins/genetics , Repressor Proteins/metabolism , Sequence Deletion , Transcription, Genetic , Tumor Cells, CulturedABSTRACT
Human B lymphocytes infected with Epstein-Barr virus (EBV) express 11 viral genes, of which six are essential for efficient transformation. The protein products of these genes appear to cause cell growth by modifying cell signal transduction pathways. For example, EBNA-2 mimics the Notch 1 pathway and LMP-1 interacts with the signalling from CD40/CD40-L, which promotes growth in normal B cells. In the human cancers linked to EBV, most of the viral transforming genes are not expressed. It is likely that growth of these cells is controlled by a combination of the EBV genes whose expression continues with altered cell proto-oncogenes and tumour suppressor genes, but other explanations of the role of EBV in cancer cells are also possible. The presence of the virus in the tumour cells of EBV-associated cancers constitutes a potentially useful tumour specific marker that might be used to direct therapy to the tumour cells.
Subject(s)
Genes, Viral , Herpesvirus 4, Human/genetics , Neoplasms/virology , Burkitt Lymphoma/virology , Gene Expression , Hodgkin Disease/virology , Humans , Lymphoma, T-Cell/virology , Nasopharyngeal Neoplasms/virologyABSTRACT
Bovine leukaemia virus (BLV) is the aetiologic agent of bovine leucosis. The virus induces malignancies of the B-cell lineage (leukaemia/lymphoma). The role played by interleukin 6 (IL-6) in the BLV-induced leukemogenesis process was evaluated. Six cell lines derived from BLV-induced tumours were tested for the expression of IL-6 receptors. Two cell lines (LB155 and YR2) display 250-300 receptor per cell (kd = 1.7 10(-10) M and 1.4 10(-10) M, respectively) whereas the other four (LB159, LB167, YR1 and M51) do not display detectable amounts of receptors. Very low (if any) expression of IL-6 receptors has been found in the case of the B lymphocytes of animals in persistent lymphocytosis (PL). Despite the presence of IL-6 receptors on the surface of LB155 and YR2 cells, no influence of exogenous IL-6 on their growth has been observed. Northern analyses indicated the presence of IL-6 transcripts only in the case of mRNA isolated from LB155 cells. Since this cell line also expresses receptors for the cytokine, an autocrine loop may exist in these cells. Experiments in which bovine and bovine epithelial cell lines were transfected with a plasmid containing the bovine IL-6 promoter controlling the expression of the reporter cat gene failed to indicate any influence of the viral transactivator p34tax on the activity of this promoter. We conclude that IL-6 receptors and IL-6 mRNA can be found in some BLV-induced tumours, but this does not correlate with viral expression in BLV-induced leukaemia/lymphoma.
Subject(s)
Enzootic Bovine Leukosis/immunology , Gene Expression , Interleukin-6/biosynthesis , Leukemia Virus, Bovine/immunology , Receptors, Interleukin/biosynthesis , Animals , B-Lymphocytes/immunology , Blotting, Northern , Cattle , Cell Division , Cell Line , Cell Membrane/metabolism , Chloramphenicol O-Acetyltransferase/biosynthesis , Enzootic Bovine Leukosis/virology , Humans , Kinetics , RNA, Messenger/biosynthesis , Receptors, Interleukin/metabolism , Receptors, Interleukin-6 , Recombinant Proteins/metabolism , Sheep , Transfection , Tumor Cells, CulturedABSTRACT
The screening of a bovine genomic library with a human tumour necrosis factor-alpha (TNF-alpha) cDNA probe resulted in the isolation of a 7.2 kb DNA fragment containing the entire bovine TNF-alpha gene. Analysis of this genomic clone showed that it also contains the bovine lymphotoxin (LT, TNF-beta) gene. Comparison to published sequences of human, murine, ovine and rabbit counterparts allowed us to delineate the coding sequences, the promoters and the enhancers of these two genes. Sequences involved in the regulation of translation and in the mRNA stability were found in the 3' untranslated regions.
Subject(s)
Cattle/genetics , Lymphotoxin-alpha/genetics , Tumor Necrosis Factor-alpha/genetics , Amino Acid Sequence , Animals , Base Sequence , Cloning, Molecular , Conserved Sequence , DNA/genetics , DNA Probes , Genomic Library , Hominidae/genetics , Humans , Lymphotoxin-alpha/biosynthesis , Mice/genetics , Molecular Sequence Data , Restriction Mapping , Sequence Homology, Amino Acid , Tumor Necrosis Factor-alpha/biosynthesisABSTRACT
We report the cloning of bovine interleukin-6 (IL-6) cDNA. The clone was isolated from a bovine-leukemia virus (BLV)-induced B cell-lymphosarcoma cDNA library cloned in the bacteriophage lambda gt11. The cDNA encodes a full length IL-6 protein made of 208 amino acids with 65, 53, 42 and 42% homology to published sequences of porcine, human, mouse and rat IL-6, respectively. The significance of IL-6 expression in a BLV-induced tumor is briefly discussed.
Subject(s)
Interleukin-6/genetics , Amino Acid Sequence , Animals , Base Sequence , Cattle , Cloning, Molecular , DNA , Genomic Library , Humans , Molecular Sequence Data , Sequence Homology, Amino AcidABSTRACT
We report the cloning and sequencing of a 1252 base pairs (bp) DNA fragment containing the bovine interleukin-6 (IL-6) gene promoter. This fragment was isolated from a bovine genomic library constructed in the lambda GEM11 vector. Comparison with human, murine and rat IL-6 gene promoters reveals a high degree of conservation of the 200 bp immediately upstream of the RNA CAP site. This region contains nucleotide stretches matching with consensus sequences recognized by transcription factors, including NF-KB, CREB and NF-IL6. A potential AP-1 binding site is found 284 nucleotides upstream of the RNA CAP site. The bovine IL-6 gene promoter cloned upstream of the bacterial chloramphenicol acetyl transferase (CAT) gene was shown to be active in bovine and ovine cells.
Subject(s)
Interleukin-6/genetics , Promoter Regions, Genetic , Animals , Base Sequence , Cattle , Cloning, Molecular , DNA , Humans , Molecular Sequence Data , Sequence Homology, Nucleic AcidABSTRACT
We report the cloning and the sequencing of a cDNA coding for the mature bovine interleukin 7 (IL-7). The clone was isolated from a bovine leukemia virus (BLV)-induced B cell-lymphosarcoma cDNA library. The 5' non-coding sequence and the sequence of the signal peptide were obtained from a clone isolated from a bovine genomic library. The entire bovine IL-7 protein is 176 amino acids long and shows 75 and 65% homology to published sequences of human and murine IL-7, respectively.
Subject(s)
Interleukin-7/genetics , Amino Acid Sequence , Animals , Base Sequence , Cattle , Cloning, Molecular , DNA , Enzootic Bovine Leukosis/genetics , Humans , Leukemia Virus, Bovine/metabolism , Molecular Sequence Data , Protein Sorting Signals/genetics , Sequence Homology, Amino Acid , Sequence Homology, Nucleic AcidABSTRACT
Neuroleukin (NLK) is a protein of relative molecular mass (Mr) 56,000 (56K) secreted by denervated rat muscle and found in large amounts in muscle, brain, heart and kidneys. The protein is a neurotrophic factor for spinal and sensory neurons and a lymphokine product of lectin-stimulated T-cells. It also induces immunoglobulin secretion by human mononuclear cells. Molecular clones of NLK have been expressed in monkey COS cells and the product was shown to have the same biological and biochemical properties as the extracted protein. NLK is abundant in muscle, brain and kidney, but is active at concentrations of 10(-9) to 10(-11) M, similar to those for other polypeptide factors. We have cloned the gene for pig muscle phosphohexose isomerase (PHI) (EC 5.3.1.9) which catalyses the conversion of glucose-6-phosphate to fructose-6-phosphate, an obligatory step in glycolysis, and determined its amino-acid sequence. Surprisingly, it is 90% homologous to the sequence of mouse neuroleukin.