Raji revisited: cytogenetics of the original Burkitt's lymphoma cell line.
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Burkitt Lymphoma↗
Biomedical subjects
I Ernberg
Publications and source records attributed to I Ernberg.
Functional interaction of Oct transcription factors with the family of repeats in Epstein-Barr virus oriP.
The family of repeats (FR) is a major upstream enhancer of the Epstein-Barr virus (EBV) latent C promoter (Cp) that controls transcription of six different latent nuclear proteins following interaction with the EBV nuclear protein EBNA1. Here, it was shown that Cp could also be activated by octamer-binding factor (Oct) proteins. Physical binding to the FR by the cellular transcription factors Oct-1 and Oct-2 was demonstrated by using an electrophoretic mobility-shift assay. Furthermore, Oct-1 in combination with co-regulator Bob.1, or Oct-2 alone, could drive transcription of a heterologous thymidine kinase promoter linked to the FR in both B cells and epithelial cells. Cp controlled by the FR was also activated by binding of Oct-2 to the FR. This may have direct implications for B cell-specific regulation of Cp.
B-Lymphocytes↗
Capturing whole-genome characteristics in short sequences using a naïve Bayesian classifier.
Bacterial genomes have diverged during evolution, resulting in clearcut differences in their nucleotide composition, such as their GC content. The analysis of complete sequences of bacterial genomes also reveals the presence of nonrandom sequence variation, manifest in the frequency profile of specific short oligonucleotides. These frequency profiles constitute highly specific genomic signatures. Based on these differences in oligonucleotide frequency between bacterial genomes, we investigated the possibility of predicting the genome of origin for a specific genomic sequence. To this end, we developed a naïve Bayesian classifier and systematically analyzed 28 eubacterial and archaeal genomes. We found that sequences as short as 400 bases could be correctly classified with an accuracy of 85%. We then applied the classifier to the identification of horizontal gene transfer events in whole-genome sequences and demonstrated the validity of our approach by correctly predicting the transfer of both the superoxide dismutase (sodC) and the bioC gene from Haemophilus influenzae to Neisseria meningitis, correctly identifying both the donor and recipient species. We believe that this classification methodology could be a valuable tool in biodiversity studies.
Archaea↗
C-terminal domain of the Epstein-Barr virus LMP2A membrane protein contains a clustering signal.
The latency-regulated transmembrane protein LMP2A interferes with signaling from the B-cell antigen receptor by recruiting the tyrosine kinases Lyn and Syk and by targeting them for degradation by binding the cellular E3 ubiquitin ligase AIP4. It has been hypothesized that this constitutive activity of LMP2A requires clustering in the membrane, but molecular evidence for this has been lacking. In the present study we show that LMP2A coclusters with chimeric rat CD2 transmembrane molecules carrying the 27-amino-acid (aa) intracellular C terminus of LMP2A and that this C-terminal domain fused to the glutathione-S-transferase protein associates with LMP2A in cell lysates. This molecular association requires neither the cysteine-rich region between aa 471 and 480 nor the terminal three aa 495 to 497. We also show that the juxtamembrane cysteine repeats in the LMP2A C terminus are the major targets for palmitoylation but that this acylation is not required for targeting of LMP2A to detergent-insoluble glycolipid-enriched membrane microdomains.
Amino Acid Sequence↗
Differences in the immunogenicity of latent membrane protein 1 (LMP1) encoded by Epstein-Barr virus genomes derived from LMP1-positive and -negative nasopharyngeal carcinoma.
We have previously shown that an EBV-encoded latent membrane protein 1 (LMP1) gene derived from a nude mouse-propagated nasopharyngeal carcinoma (NPC) tumor and expressed in nonimmunogenic murine mammary carcinoma S6C cells failed to convey immunogenicity (rejectability) in syngeneic mice, whereas the corresponding B-cell derived LMP1 gene made the mice highly immunogenic. This raised the question of whether LMPL-expressing NPCs have been selected for low immunogenicity at the viral gene expression level. If so, LMP1-negative tumors that carry highly methylated LMP1 regulatory sequences may not have been exposed to a similar immunoselection. In the present study, we have compared LMP1 genes derived from two LMP1-positive NPCs and two LMP1-negative NPCs. All four genes were expressed in S6C cells in parallel with the previously tested isolates from a B-cell (B95-8)-derived and a nude mouse-propagated NPC (Cao)-derived gene. As in the previous study, we have found that the B-cell-derived LMP1 isolate was highly immunogenic. LMP1-positive tumor-derived isolates were poorly immunogenic, whereas the isolates from the LMP1-negative NPC tumor had intermediate immunogenicity. Sequence data revealed that LMP1 genes from LMP1-expressing NPC had 16 amino acid substitutions, whereas LMP1 from non-LMP1-expressing NPC had only 9 amino acid changes in the coding region. Three of the changes were at shared sites, but with different modifications. The fact that the gene from non-LMP1-expressing NPC mutated at a low frequency but was more immunogenic than the LMP1 gene derived from LMP1-expressing NPC, which was highly mutated but less immunogenic, favors the idea that LMP1-positive tumors escape immunosurveillance in immunocompetent hosts by either a selective down-regulation of LMP1 expression, methylation in the LMP1 promoter sequence, or mutation of LMP1 in LMP1-expressing samples.
Animals↗
NotI clones in the analysis of the human genome.
Not I linking clones contain sequences flanking Not I recognition sites and were previously shown to be tightly associated with CpG islands and genes. To directly assess the value of Not I clones in genome research, high density grids with 50 000 Not I linking clones originating from six representative Not I linking libraries were constructed. Altogether, these libraries contained nearly 100 times the total number of Not I sites in the human genome. A total of 3437 sequences flanking Not I sites were generated. Analysis of 3265 unique sequences demonstrated that 51% of the clones displayed significant protein similarity to SWISSPROT and TREMBL database proteins based on MSPcrunch filtering with stringent parameters. Of the 3265 sequences, 1868 (57.2%) were new sequences, not present in the EMBL and EST databases (similarity < or =90%). Among these new sequences, 795 (24.3%) showed similarity to known proteins and 712 (21.8%) displayed an identity of >75% at the nucleotide level to sequences from EMBL or EST databases. The remaining 361 (11.1%) sequences were completely new, i.e. <75% identical. The work also showed tight, specific association of Not I sites with the first exon and suggest that the so-called 3' ESTs can actually be generated from 5'-ends of genes that contain Not I sites in their first exon.
Base Sequence↗
Epstein-Barr virus (EBV) load in bone marrow transplant recipients at risk to develop posttransplant lymphoproliferative disease: prophylactic infusion of EBV-specific cytotoxic T cells.
A semiquantitative polymerase chain reaction assay was used to monitor the blood levels of Epstein-Barr virus (EBV)-DNA in 9 patients receiving allogeneic bone marrow transplants (BMT). Four of 5 recipients of HLA-mismatched T-cell-depleted grafts showed a 4- to 5-log increase of EBV-DNA within 1 to 3 months after BMT. Administration of 2 to 4 infusions of 10(7) EBV-specific cytotoxic T-lymphocytes (CTLs)/m(2) starting from the time of maximal virus load resulted in a 2- to 3-log decrease of virus titers in 3 patients. One patient, who received a T-cell culture lacking a major EBV-specific component, progressed to fatal EBV-positive lymphoma. Administration of EBV-CTLs before the onset of the EBV-DNA peak resulted in stabilization of the virus titers within 2 to 3 logs above the normal levels in the fifth patient. A moderate increase of virus titers was also detected in 3 of 4 patients receiving unmanipulated HLA-matched grafts, whereas 1 patient with Wiskott-Aldrich syndrome reached a 5-log increase of EBV-DNA load within 70 days after BMT. Our results suggest that a rapid increase of circulating EBV-DNA occurs in the absence of EBV-specific T-cell precursors or in the presence of congenital immune defects that prevent the reestablishment of virus-specific immunity. Prophylactic administration of EBV-CTLs early after BMT appears to provide the most effective protection against the development of EBV-associated lymphoproliferative disease.
Adolescent↗
CIS--cloning of identical sequences between two complex genomes.
Development of the methods permitting cloning of identical sequences between two sources of DNA can be very useful for many purposes, including isolation of disease genes. Here we describe a new method called CIS (cloning of identical sequences). A combination of digestion with MvnI, treatment with mung bean nuclease, UDG (uracil-DNA glycosylase) and PCR with 5'-methyl-dCTP and dUTP was used to isolate identical sequences between two micro-cell hybrid lines (MCH). In a control experiment, mouse MCH903.1 and MCH939.2 containing human chromosome 3 from different individuals, were compared using the CIS procedure. Only background fluorescence in-situ hybridization (FISH) was achieved. In another experiment, mouse MCH903.1, containing complete human chromosome 3, and rat MCH429.11, containing a part of human 3q from the same chromosome were compared. The experiment showed that the original MCH429.11 and the DNA purified using the CIS procedure had identical FISH patterns to human metaphase chromosomes, thus demonstrating the efficiency of CIS.
Animals↗
A repetitive sequence of Epstein-Barr virus nuclear antigen 6 comprises overlapping T cell epitopes which induce HLA-DR-restricted CD4(+) T lymphocytes.
Most human adults carry the Epstein-Barr virus (EBV) and develop immunological memory against the structural and the virus-encoded cellular proteins. The EBV nuclear antigen 6 (EBNA6) elicits cytotoxic T cell responses and it also maintains a persistent antibody response. The majority of sera from EBV-seropositive individuals reacts with a synthetic peptide, p63, comprising 21 amino acids of a repetitive region of EBNA6. CD4(+) T lymphocytes, with specificity for p63, could be recalled from the T cell repertoire of EBV carriers that expressed certain HLA-DR allotypes which were identified as good binders of p63 by an in vitro flow cytometric assay. Analysis of the HLA-DR/p63 interaction by molecular mechanics calculations indicated the presence of multiple overlapping epitopes which were predicted to bind in a HLA-DRB1 allo- and subtype-specific manner. Specific activation of p63-selected long-term CD4(+) T cell cultures resulted in a proliferative response, in the production of IL-2 and in the secretion of high levels of tumor necrosis factor as measured by bioassays. Proliferation and cytokine production of p63-specific T cells could be induced by p63-loaded HLA-DR-matched antigen-presenting cells and by B cells co-expressing relevant HLA-DR molecules and EBNA6. Our results show that peptides of an EBNA6 repeat region induce CD4(+) T cells which can react with EBNA6-carrying cells in many individuals. We suggest that these T(h) cells may be important in conditioning dendritic cells for initiation potent virus-specific immune responses, provide help for EBV-specific B cells, drive IgG isotype switch and support the sustained effector function of memory cytotoxic T lymphocytes.
Adult↗
Latent membrane protein 2A of Epstein-Barr virus binds WW domain E3 protein-ubiquitin ligases that ubiquitinate B-cell tyrosine kinases.
The latent membrane protein (LMP) 2A of Epstein-Barr virus (EBV) is implicated in the maintenance of viral latency and appears to function in part by inhibiting B-cell receptor (BCR) signaling. The N-terminal cytoplasmic region of LMP2A has multiple tyrosine residues that upon phosphorylation bind the SH2 domains of the Syk tyrosine kinase and the Src family kinase Lyn. The LMP2A N-terminal region also has two conserved PPPPY motifs. Here we show that the PPPPY motifs of LMP2A bind multiple WW domains of E3 protein-ubiquitin ligases of the Nedd4 family, including AIP4 and KIAA0439, and demonstrate that AIP4 and KIAA0439 form physiological complexes with LMP2A in EBV-positive B cells. In addition to a C2 domain and four WW domains, these proteins have a C-terminal Hect catalytic domain implicated in the ubiquitination of target proteins. LMP2A enhances Lyn and Syk ubiquitination in vivo in a fashion that depends on the activity of Nedd4 family members and correlates with destabilization of the Lyn tyrosine kinase. These results suggest that LMP2A serves as a molecular scaffold to recruit both B-cell tyrosine kinases and C2/WW/Hect domain E3 protein-ubiquitin ligases. This may promote Lyn and Syk ubiquitination in a fashion that contributes to a block in B-cell signaling. LMP2A may potentiate a normal mechanism by which Nedd4 family E3 enzymes regulate B-cell signaling.
Amino Acid Motifs↗
Genome-wide allelotype analysis of sporadic primary nasopharyngeal carcinoma from southern China.
Nasopharyngeal carcinoma (NPC) is one of the most common malignant tumors in Southern China, especially in the Guangdong area. To demonstrate a comprehensive profile of loss of heterozygosity (LOH) in NPC, we applied a large panel of 382 microsatellite polymorphism markers covering all the 22 autosomes in 98 cases of sporadic primary NPC. Of the 335 informative markers, 83 loci showed high level of LOH (presence in equal to or more than 30% cases) and most of the high frequent loci were clustered to chromosome 1p36 and 1p34, 3p14-p21, 3p24-p26, 3q25-q26 and 3q27, 4q31 and 4q35, 5q15-21 and 5q32-q33, 8p22-p23, 9p21-p23 and 9q33-q34, 11p12-p14, 13q14-q13 and 13q31-q32, 14q13-q11, 14q24-q23 and 14q32. High frequency of LOH was found in chromosomes 3, 5, 9 and 11 (>/=50%), while medium frequency of LOH was found in chromosomes 1, 4, 6, 14, 17 and 19 (40-49%). Several new regions showing high frequency of LOH were found in chromosome 1p36, 3q25-q26, 3q27, 5q15-q21, 8p22-p23 and 11p12-14. The relationship between LOH and TNM stage of NPC was evaluated. Regions 6p23 (D6S289), 8p23.1 (D8S549) and 9q34.2 (D9S1826) showed higher frequency of LOH in later stages (III and IV) than in earlier stages (I and II) (P<0.05). Thus, our study provides a global view on allelic loss in the development of NPC and should shed light on the way for localization of putative tumor suppressor genes associated with the pathogenesis of NPC.
Adult↗
Cell phenotype-dependent splicing reflecting differential promoter usage for EBNA transcripts in EBV-carrying cells.
Three types of virus-host cell interactions have been described in cells latently infected with EBV: EBNA 1 expression in type I Burkitt's lymphoma cell lines (BL), EBNA 1, LMP1 and 2 expression in most nasopharyngeal carcinomas (NPC) and EBNA 1-6 with LMP 1 and 2 expression in group III BL-lines as well as lymphoblastoid cell lines (LCL). Two group I BL lines that express only EBNA 1 were found to initiate their EBNA 1 mRNA transcription from a promoter in the Bam HI Q-fragment. They use a sequence at +210 bp relative to the Fp transcription initiation site in group I BL cell lines. The Fp promoter-region seems to be activated in the lytic cycle. LCLs initiate their transcription from one of several upstream sites, usually the Cp promoter or, less frequently, one of several Wp-promoters. Using RNA-reverse transcription polymerase chain reaction (RT-PCR), we have now shown that EBV carrying cells that do not express EBNA 2-6 always splice their EBNA mRNA at the Q-exon, while EBNA 2-6 positive cells use either the Cp or one of the Wp promotors. When EBNA 2-6 are downregulated by somatic cell hybridisation between EBNA 1-6 positive B-cell lines and non B-cells of hematopoetic, epithelial or fibroblastic origin that express the phenotype of the non-B cell parent, the parental usage of Cp/Wp is switched off, and the Q-exon is activated. NPC cells show the same pattern of promoter usage as the hybrids with non-B phenotype. Group III BL cells use both promoter regions. Thus, the virus can use two alternative programs, depending on the cell phenotype. The "EBNA-1 only" program is activated from the Q-promoter. In cells with an immunoblastic (LCL or BL group III) phenotype, the upstream Cp/Wp promoters generate a 100 kb. long pre-mRNA, from which all the EBNAs are spliced. As a rule, only one of the two programs is used for each phenotype, except for the BL group III cells that began as group I but subsequently developed into a more LCL-like cell. Such cells used both promoter regions, with or without activation of the lytic cycle.
Animals↗
Novel mode of ligand binding by the SH2 domain of the human XLP disease gene product SAP/SH2D1A.
BACKGROUND: The Src homology 2 (SH2) domains of cytoplasmic signaling proteins generally bind phosphotyrosine (pTyr) sites in the context of carboxy-terminal residues. SAP (also known as SH2D1A or DSHP), the product of the gene that is mutated in human X-linked lymphoproliferative (XLP) disease, comprises almost exclusively a single SH2 domain, which may modulate T-cell signaling by engaging T-cell co-activators such as SLAM, thereby blocking binding of other signaling proteins that contain SH2 domains. The SAP-SLAM interaction can occur in a phosphorylation-independent manner. RESULTS: To characterize the interaction between SAP and SLAM, we synthesized peptides corresponding to the SAP-binding site at residue Y281 in SLAM. Both phosphorylated and non-phosphorylated versions of an 11-residue SLAM peptide bound SAP, with dissociation constants of 150 nM and 330 nM, respectively. SLAM phosphopeptides that were truncated either at the amino or carboxyl terminus bound with high affinity to SAP, suggesting that the SAP SH2 domain recognizes both amino-terminal and carboxy-terminal sequences relative to the pTyr residue. These results were confirmed by nuclear magnetic resonance (NMR) studies on (15)N- and (13)C-labeled SAP complexed with three SLAM peptides: an amino-terminally truncated phosphopeptide, a carboxy-terminally truncated phosphopeptide and a non-phosphorylated Tyr-containing full-length peptide. CONCLUSIONS: The SAP SH2 domain has a unique specificity. Not only does it bind peptides in a phosphorylation-independent manner, it also recognizes a pTyr residue either preceded by amino-terminal residues or followed by carboxy-terminal residues. We propose that the three 'prongs' of a peptide ligand (the amino and carboxyl termini and the pTyr) can engage the SAP SH2 domain, accounting for its unusual properties. These data point to the flexibility of modular protein-interaction domains.
Adaptor Proteins, Signal Transducing↗
Epstein-Barr virus genomes are found predominantly in IgA-positive B cells in the blood of healthy carriers.
B lymphocytes have been identified as the main reservoir of latent Epstein-Barr virus (EBV) in healthy virus carriers. We have established a semi-quantitative PCR method to estimate the EBV genome load in the blood B-cell subpopulation in healthy individuals. EBV DNA was detected in subfractionated IgM-, IgG- and IgA-positive B cells. Between 80% and 90% of the viral DNA was found in the IgA-positive compared with the IgA-negative fraction.
B-Lymphocytes↗
Horizontal transfer of DNA by the uptake of apoptotic bodies.
In this study we have raised the question of whether DNA can be transferred from one cell to another by phagocytosis of apoptotic bodies. We have used integrated copies of the Epstein-Barr virus (EBV) as a marker to follow the fate and expression pattern of apoptotic DNA in the phagocytotic host. Apoptosis was induced in EBV-carrying cell lines by irradiation before cultivation with either human fibroblasts, macrophages, or bovine aortic endothelial cells. Analysis of the expression pattern of EBV-encoded genes was performed by immunofluorescent staining as well as in situ hybridization. Cocultivation of apoptotic bodies from lymphoid cell lines containing integrated but not episomal copies of EBV resulted in expression of the EBV-encoded genes EBER and EBNA1 in the recipient cells at a high frequency. Fluorescence in situ hybridization analysis showed uptake of human chromatin as well as integrated EBV-DNA into the nuclei of bovine aortic endothelial cells. These data show that DNA may be rescued and reused from apoptotic bodies by somatic cells. In addition, our findings suggest that apoptotic bodies derived from EBV-carrying B lymphocytes may serve as the source of viral transfer to cells that lack receptors for the EBV virus in vivo.
Animals↗
Demethylation of the Epstein-barr virus origin of lytic replication and of the immediate early gene BZLF1 is DNA replication independent. Brief report.
Epstein-Barr virus (EBV) episomal DNA is extensively methylated in Burkitt lymphoma derived cell lines. In this study we examined whether lytic viral cycle reactivation is dependent on demethylation of critical viral genes. Viral replication was induced in the Burkitt's lymphoma cell line Daudi by the combination of 12-O-tetradecanoylphorbol-13-acetate (TPA) and sodium-butyrate. Two regions necessary for EBV replication, the BZLF1 immediate early region and the origin of lytic cycle replication (ori Lyt) were demethylated during the early phase of the lytic virus cycle. Demethylation was observed while production of new (unmethylated) viral DNA was blocked by phosphonoformic acid (PFA). This suggests that demethylation, which may be instrumental for the onset of the lytic cycle, is an active process independent of viral DNA replication
Antigens, Viral↗
Specific methylation patterns in two control regions of Epstein-Barr virus latency: the LMP-1-coding upstream regulatory region and an origin of DNA replication (oriP).
The Epstein-Barr virus (EBV) can establish at least four different forms of latent infection. Previously, we have shown that the level of methylation of the EBV genome varies, depending on the form of latency. The methylation status of CpGs was analyzed by the bisulfite genomic sequencing technique in four different cell types representing different forms of latency. The dyad symmetry element of the origin of replication (oriP) region and the latent membrane protein 1 (LMP-1) regulatory sequence (LRS) were studied. The dyad symmetry element has four binding sites for EBNA-1. In a cell with type I latency, a region upstream of the dyad symmetry element was highly methylated, whereas the dyad symmetry element was unmethylated in the EBNA-1-binding region. The LRS was extensively methylated in the LMP-1-negative cell line Rael, in contrast to a LMP-1-expressing nasopharyngeal carcinoma tumor (NPC C15), which was almost completely unmethylated. The methylation pattern of LRS in type I and type III Burkitt lymphoma cells of similar parental origins confirmed that demethylation of some regions takes place upon phenotypic drift.
DNA Methylation↗
Direct identification by PCR of EBV types and variants in clinical samples.
Both Epstein-Barr virus (EBV) type A and type B, and variants of type A, were identified simultaneously by polymerase chain reaction (PCR) amplification of a DNA region coding for a 13 amino acid repeat in the Epstein-Barr virus nuclear antigen (EBNA) 6. Whereas this region varies extensively in type A isolates, no variation was seen in type B isolates. When a repetitive region in the LMP1-coding region was amplified by PCR, it was possible to distinguish individual variants of type B isolates from each other. Forty-two saliva samples from HIV-1-carrying individuals were examined for the presence of type A and type B virus. Both types and multiple variants of each type were found with a much higher frequency than in the saliva samples from healthy individuals. Type A EBV alone was detected in mouthwash samples from 6 infectious mononucleosis (IM) patients. Both type A and B were detected in the peripheral blood B-lymphocytes (PBL) from 1 healthy individual. The same type A variant was demonstrated both in PBL and in the mouthwash sample from another healthy individual. In this study it was shown that a combination of the EBNA 6- and LMP 1-specific PCRs followed by Southern hybridisation can be used to identify both type A and type B virus, as well as to distinguish between multiple variants of the same strain, in saliva and B-cells from both healthy and immunosuppressed individuals.
Cell Line↗