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Query: UNIPROT:P06889 (Mol)
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We designed synthetic oligonucleotide primers and hybridization probe for use in polymerase chain reaction (PCR) amplification and hybridization detection of Epstein-Barr virus (EBV) nucleic acid sequences. Primer sequences were chosen from the coding region for the Epstein-Barr virus nuclear antigen-1 (EBNA-1). PCR amplification and hybridization with these oligonucleotides was carried out on standard laboratory cell lines including African Burkitt's lymphoma and infectious mononucleosis derived cell lines, as well as cell lines recently established from clinical EBV isolates from bone marrow transplant recipients. All EBV cell lines tested were positive. No false-positives were detected with uninfected cell lines, human placental DNA or with other viruses. The sensitivity of the detection procedure was such that four copies of the EBV genome could consistently be detected in a background of 1 microgram of placental DNA. EBV was detected in DNA extracts from the peripheral blood mononuclear cells of two patients with infectious mononucleosis and one patient with viral-associated hemophagocytic syndrome. Three of 18 EBV seropositive patients without known ongoing EBV-associated illness undergoing ambulatory surgery also had EBV detected in DNA extracts from their peripheral blood mononuclear cells. EBV was detected in DNA extracts from lymphoma tissue from two patients with post-transplant lymphomas and two AIDS patients with primary CNS lymphomas. EBV was not detected in 12 B-cell lymphoma specimens from patients without history of immunocompromise. DNA extracts from formalin-fixed paraffin-embedded Hodgkin's tissues previously shown to be EBV positive by Southern blot were also demonstrated to be EBV positive by PCR. Thus, with the oligonucleotides described, PCR is applicable to the detection of EBV in a spectrum of clinical isolates. The broad specificity of these oligonucleotides for all strains of EBV tested is probably a function of the highly conserved sequence of the EBNA-1 DNA binding domain.
Mol Cell Probes 1990 Oct
PMID:Oligonucleotides for polymerase chain reaction amplification and hybridization detection of Epstein-Barr virus DNA in clinical specimens. 217 46

The human MAbs, anti-D, -c, -E and -G of the Rh blood group system, produced by Epstein-Barr virus transformed B-cell lines, were purified by protein A-Sepharose chromatography and used to characterize the Rh antigens of human red cells. Scatchard plot analyses performed with the radiolabelled MABs indicated that each R2R2 red cell carries 0.43, 0.32 and 0.38 x 10(5) D, c and E binding sites, respectively. About half this number of antigen sites are present on erythrocytes from heterozygote individuals using the appropriate antibody. We found, however, that only 0.18 x 10(5)G antigenic sites were present on each R1R1 red cell. The affinity constants of the anti-D, -E and -G were similar varying from 0.6 to 1.5 x 10(8) M-1 whereas that of the anti-c was much lower (0.035 x 10(8) M-1). The blood group specificity and binding properties indicate that the MAbs behave like the polyclonal anti-Rh reagents. Immunoprecipitation experiments carried out with membranes from R2R2 red cells show that a 30-32 kDa component can be identified whatever the antibody used. The immune complexes involving anti-c, -E or -G antibodies could be formed with the detergent lysates from red cell membranes. In contrast, membrane integrity was a prerequisite for the binding of the anti-D antibodies. Finally, from extraction studies of immunocomplexes with non-ionic detergents it was concluded that all the Rh-active components are bound to the membrane skeleton, suggesting that these molecules may have important function for maintaining red cell shape and viability.
Mol Immunol 1988 Sep
PMID:Characterization of the D, c, E and G antigens of the Rh blood group system with human monoclonal antibodies. 246 84

Proteolytic processing of specific antigen was studied using Epstein Barr virus transformed B-lymphoblastoid cells expressing membrane IgG against tetanus toxin. As previously reported (Watts, C., and H.W. Davidson. 1988. EMBO (Eur. Mol. Biol. Organ.) J. 7:1937-1945), receptor-mediated endocytosis of monovalent antigen bound at 0 degrees C began immediately upon shifting the cells to 37 degrees C. In contrast, degradation of antigen, assessed either by the release of acid-soluble radiolabel into the incubation medium, or by SDS-PAGE analysis of total cell-associated antigen, proceeded after a lag of 10-20 min. Degradation was abolished by exposure of the cells to metabolic inhibitors, or by incubation at 20 degrees C, and inhibited in a dose-dependent fashion by chloroquine and by the lysosomal protease inhibitors leupeptin, E-64, and pepstatin A. Analysis of the cell-associated radiolabel by SDS-PAGE and autoradiography after incubations at 37 degrees C revealed the time-dependent generation of distinct antigen fragments. Virtually quantitative immunoprecipitation of these fragments was obtained using a monoclonal anti-human IgG antibody, indicating that the antigen/mIg complex is the initial substrate for processing. We show that the pattern of fragmentation observed varies from one B cell line to another (a) depending on the epitope through which the antigen is bound and endocytosed and (b) depending on whether additional epitopes in the antigen are complexed with anti-tetanus Fabs. The implications of these results for the presentation of major histocompatibility complex restricted antigen fragments, and for intracellular trafficking of ligand/receptor complexes are discussed.
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PMID:Epitope-directed processing of specific antigen by B lymphocytes. 247 83

Transcription from the Moloney murine leukemia virus (Mo-MuLV) long terminal repeat (LTR) is inhibited in murine stem cells and induced during maturation of these cells. We have investigated whether alterations in the activity of this viral regulatory element also occur during differentiation of human myeloid leukemia cells. The Mo-MuLV LTR and the simian virus 40 (SV40) early promoter were introduced into HL-60 promyelocytes on Epstein-Barr virus-derived chloramphenicol acetyltransferase expression vectors. When these cells were induced to terminally differentiate, transcription from the Mo-MuLV LTR was induced approximately 10-fold. Expression from the SV40 promoter remained constant during differentiation of these cells. Replacing the SV40 transcriptional enhancer with the Mo-MuLV LTR transcriptional enhancer rendered the SV40 promoter inducible during differentiation. We conclude that sequences within the transcriptional enhancer of the Mo-MuLV LTR contain cis-acting elements responsible for induction of gene expression during differentiation of human myeloid cells.
Mol Cell Biol 1989 Aug
PMID:Induced expression from the Moloney murine leukemia virus long terminal repeat during differentiation of human myeloid cells is mediated through its transcriptional enhancer. 247 90

Charge interactions are of great importance for protein function and structure, and for a variety of cellular and biochemical processes. We present a systematic approach to the detection of distinctive clusters, runs and periodic patterns of charged residues in a protein sequence. Criteria and formulae are set forth to assess statistical significance of these charge configurations. For the 80-odd proteins potentially encoded by the Epstein-Barr virus, only the major nuclear antigens of the latent state and the transactivator of the lytic cycle contain separated charge clusters of opposite sign as well as periodic charge patterns. From our studies of the polypeptides of the human herpesviruses and of a broad collection of human and other viral protein sequences, distinctive charge configurations appear to be associated with viral capsid and core proteins (positive clusters or runs, mostly at the carboxyl terminus), with many viral glycoproteins and membrane-associated proteins (negative charge clusters), and with transactivators and transforming proteins (multiple charge structures). The statistics developed in this paper apply more generally to other than charge properties of a protein and should aid in the evaluation of a large variety of sequence features.
J Mol Biol 1989 Jan 05
PMID:A method to identify distinctive charge configurations in protein sequences, with application to human herpesvirus polypeptides. 253 22

We have isolated a heterogeneous collection of human genomic sequences which replicate autonomously when introduced into human cells. The novel strategy for the isolation of these sequences involved cloning random human DNA fragments into a defective Epstein-Barr virus vector. This vector alone was unable to replicate in human cells, but appeared to provide for the nuclear retention of linked DNA. The human sequences persist in a long-term replication assay (greater than 2 months) in the presence of the viral nuclear retention sequences. Using a short-term (4-day) assay, we showed that the human sequences are able to replicate in the absence of all viral sequences. The plasmids bearing human sequences were shown to replicate based on the persistence of MboI-sensitive plasmid DNA in the long-term assay and the appearance of DpnI-resistant DNA in the short-term assay. The human sequences were shown to be responsible for the replication activity and may represent authentic human origins of replication.
Mol Cell Biol 1989 Mar
PMID:Isolation of human sequences that replicate autonomously in human cells. 254 63

The DNA-binding domain of Epstein-Barr virus nuclear antigen 1 was found by hydroxyl radical footprinting to protect backbone positions on one side of its DNA-binding site. The guanines contacted in the major groove by the DNA-binding domain of Epstein-Barr virus nuclear antigen 1 were identified by methylation protection. No difference was found in the interaction of the DNA-binding domain of Epstein-Barr virus nuclear antigen 1 with tandemly repeated and overlapping binding sites.
Mol Cell Biol 1989 Jun
PMID:High-resolution footprints of the DNA-binding domain of Epstein-Barr virus nuclear antigen 1. 254 88

Previous work has demonstrated that ultraviolet (UV) irradiation of SV40-based plasmids can strikingly enhance the frequency of stable transformation of human cells. In this study we compared the effect of UV-induced DNA damage on transformation mediated by integrative versus autonomously replicating plasmids derived from human Epstein-Barr virus (EBV). We report that transfection of human fibroblasts with UV-irradiated integrative EBV-based plasmid results in enhanced transformation. However, transfection of UV-damaged episomal EBV-based constructs into the same human cell line does not enhance transformation; in fact, the extrachromosomal status of the plasmid is maintained irrespective of the UV dose to the plasmid. We conclude that enhanced transformation of human cells by damaged DNA requires its chromosomal integration.
Mol Carcinog 1989
PMID:DNA damage stimulates human cell transformation by integrative but not episomal Epstein-Barr virus-derived plasmid. 255 55

A long-term culture Epstein-Barr virus (EBV)-negative malignant lymphoid cell line (NAK) was established from a lymph node biopsy of a chronic lymphocytic leukemia patient. This cell line is of particular interest because it grows as an adherent cell line and depends on the presence of autologous conditioned medium for growth. After 6 months of growth in vitro, doubling time and cell cycle parameters were derived. Doubling time was 48 hours with over 45% cycling cells. Cell viability was over 90%. Expression of B-cell markers (CD19 and CD20) and surface immunoglobulin of the original tumor cell biopsy were roughly the same as in passage 14 (3 months in culture), including the expression of the original patient idiotype and IgM-lambda. Furthermore, binding of antiidiotypic antibodies was only slightly decreased at passage 14. Cytogenetic studies of chromosomal abnormalities in the primary tumor tissue and in later passages indicated similar abnormalities, with no translocations t(8;14), t(14;22), or t(2;8). However, frequent trisomies, deletions, and t(1;4) translocations were observed. Negative results for EBV nuclear antigen indicate that this cell line is an EBV-negative cell line.
Mol Biother 1989
PMID:Establishment and characterization of a malignant lymphoid cell line from a chronic lymphocytic leukemia patient. 261 Sep 52

Two previously reported insulin receptor cDNA sequences differ by 36 base pairs (bp) in the distal alpha-subunit, suggesting that alternative mRNA splicing within the coding region may occur (two insulin receptor isoforms). We developed a quantitative modification of the polymerase chain reaction technique in order to detect and characterize differential mRNA splicing at this site within the distal alpha-subunit. Using RNA derived from a variety of human cell types, we detected two polymerase chain reaction-amplified cDNA species reflecting the presence or absence of the above 36 nucleotides. Identity of the two cDNA species was confirmed by Southern blots, the use of a BANI restriction site present only in the 36 base pair segment and dideoxy sequencing. The relative expression of the two mRNA forms varied markedly in a tissue-specific manner. Buffy coat leukocytes and Epstein-Barr virus-transformed lymphocytes express only the shorter mRNA. Placenta expresses both species equally; muscle, isolated adipocytes and cultured fibroblasts express somewhat more of the longer mRNA (relative ratios of mRNA abundance of 1.51, 3.18, and 2.77, respectively); liver expresses mostly the longer mRNA (relative ratio of 9.8). In RNA derived from cultured and fresh cells from patients with several states of insulin resistance, the relative expression of the two mRNA species was similar to results obtained with comparable normal tissues. Although the functional significance of alternative splicing of the insulin receptor mRNA is unknown, differential expression of these two receptor mRNAs may provide a structural basis for previously observed tissue-specific differences in insulin binding and action.
Mol Endocrinol 1989 Aug
PMID:Tissue-specific expression of two alternatively spliced insulin receptor mRNAs in man. 277 82


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