A novel behavioral method to detect motoneuron disease in Wobbler mice aged three to seven days old

The severely attenuated and host range (HR) restricted modified vaccinia virus Ankara (MVA) was derived by >500 passages in chick embryo fibroblasts, during which multiple deletions and mutations occurred. To determine the basis of the HR defect, we prepared cosmids from the parental vaccinia virus Ankara genome and transfected them into nonpermissive monkey BS-C-1 cells that had been infected with MVA. Recombinant viruses that formed macroscopic plaques were detected after transfections with DNA segments that mapped near the left end of the viral genome. Plaque-forming viruses, generated by transfections with four individual cosmids and one pair of minimally overlapping cosmids, were purified, and their HRs were evaluated in BS-C-1 cells, rabbit RK-13 cells, and human HeLa, MRC-5, and A549 cells. The acquisition of the K1L and SPI-1 HR genes and the repair of large deletions were determined by polymerase chain reaction or pulse-field gel electrophoresis of NotI restriction enzyme digests of genomic DNA. The following results indicated the presence of previously unrecognized vaccinia virus HR genes: (1) the major mutations that restrict HR are within the left end of the MVA genome because the phenotypes of some recombinants approached that of the parental virus, (2) acquisition of the K1L gene correlated with the ability of recombinant viruses to propagate in RK-13 cells but did not enhance replication in human or monkey cell lines, (3) acquisition of the SPI-1 gene correlated with virus propagation in A549 cells but not with the extent of virus spread in monkey or other human cell lines, (4) there are at least two impaired HR genes because rescue occurred with nonoverlapping or minimally overlapping cosmids and recombinant viruses with intermediate HRs were isolated, and (5) at least one of the new HR genes did not map within any of the major deletions because the size of the left terminal NotI fragment was not appreciably altered in some recombinant viruses.     

A novel early antigen associated with Epstein-Barr virus productive cycle

A murine monoclonal antibody (mAb) 92A recognized a 48-kilodalton Epstein-Barr virus (EBV) early antigen (EA). The mAb stained nuclei of EBV-activated P3HR-1, B95-8 and Akata cells in a distinctive, microgranular immunofluorescence pattern. The 92A antigen was sensitive to methanol-fixation. Expression of the 92A antigen in those cells paralleled diffuse (EA-D) and restricted (EA-R) components of EA, and viral DNA (vDNA) replication. Phosphonoacetic acid did not inhibit expression of the 92A antigen. The colocalization of 92A antigen, EA-D, and vDNA was observed in viral replication compartments of B95-8 cells. On the other hand, in P3HR-1 virus-superinfected Raji cells the percentages of 92A antigen-positive cells were at much lower levels than were EA-D and -R positive cells. Immunofluorescence staining with 92A mAb was blocked by pretreatment with EBV-positive human sera, but not with EBV-negative sera. We conclude that 92A mAb recognizes a novel EA which may function in vDNA replication.     

Epstein-Barr virus nuclear antigen 2 transactivates the long terminal repeat of human immunodeficiency virus type 1

Human immunodeficiency virus type 1 (HIV-1)-infected subjects show a high incidence of Epstein-Barr virus (EBV) infection. This suggests that EBV may function as a cofactor that affects HIV-1 activation and may play a major role in the progression of AIDS. To test this hypothesis, we generated two EBV-negative human B-cell lines that stably express the EBNA2 gene of EBV. These EBNA2-positive cell lines were transiently transfected with plasmids that carry either the wild type or deletion mutants of the HIV-1 long terminal repeat (LTR) fused to the chloramphenicol acetyltransferase (CAT) gene. There was a consistently higher HIV-1 LTR activation in EBNA2-expressing cells than in control cells, which suggested that EBNA2 proteins could activate the HIV-1 promoter, possibly by inducing nuclear factors binding to HIV-1 cis-regulatory sequences. To test this possibility, we used CAT-based plasmids carrying deletions of the NF-kappa B (pNFA-CAT), Sp1 (pSpA-CAT), or TAR (pTAR-CAT) region of the HIV-1 LTR and retardation assays in which nuclear proteins from EBNA2-expressing cells were challenged with oligonucleotides encompassing the NF-kappa B or Sp1 region of the HIV-1 LTR. We found that both the NF-kappa B and the Sp1 sites of the HIV-1 LTR are necessary for EBNA2 transactivation and that increased expression resulted from the induction of NF-kappa B-like factors. Moreover, experiments with the TAR-deleted pTAR-CAT and with the tat-expressing pAR-TAT plasmids indicated that endogenous Tat-like proteins could participate in EBNA2-mediated activation of the HIV-1 LTR and that EBNA2 proteins can synergize with the viral tat transactivator. Transfection experiments with plasmids expressing the EBNA1, EBNA3, and EBNALP genes did not cause a significant HIV-1 LTR activation. Thus, it appears that among the latent EBV genes tested, EBNA2 was the only EBV gene active on the HIV-1 LTR. The transactivation function of EBNA2 was also observed in the HeLa epithelial cell line, which suggests that EBV and HIV-1 infection of non-B cells may result in HIV-1 promoter activation. Therefore, a specific gene product of EBV, EBNA2, can transactivate HIV-1 and possibly contribute to the clinical progression of AIDS.     

v-representability for noninteracting systems

The Epstein-Barr virus (EBV) induces unlimited growth of B lymphocytes in vitro, a phenomenon known as immortalization. The elucidation of the mechanisms by which EBV de-regulates B-cell proliferation in vitro will permit an understanding of how the virus contributes in vivo to the genesis of Burkitt's lymphoma (BL) and of lymphoproliferations in immunosuppressed patients. At present, no single EBV immortalizing gene has been identified, and the hypothesis has been made that many viral genes cooperate in establishing an autocrine loop of secretion leading to immortalization. Constitutive expression of B-cell surface molecules such as CD21 and CD23, specifically implicated in the control of B-cell proliferation, is indeed induced at the surface of immortalized B lymphocytes. The expression of the viral nuclear antigen 2 (EBNA2) has been shown to be in part responsible for CD21 and CD23 up-regulation, and EBNA2 is suspected to be a transactivator of cellular genes, although this point remains to be demonstrated. The role of EBNA2 gene, independently of other viral genes, has been investigated by transfection into B-lymphoma lines, but conflicting results have been reported. To further investigate its role in the regulation of CD21 and CD23 molecules, we have compared the effects of EBNA2 expression in 2 sets of B-lymphoma lines infected with P3HR1 EBV strain, and/or transfected with EBNA2 gene. We report here that: (i) EBNA2 expression is not a sufficient condition to induce CD21 and CD23 upregulation, EBNA2's effects are highly dependent on the cellular context, and moreover can be modified by infection with P3HR1 virus; (ii) EBNA2 induces activation of CD23 expression in a very particular way, namely, an increased quantity of CD23 steady-state RNA coding for the form A of the protein, which is not detectable at the cell surface but directly secreted.     

Analysis of Epstein-Barr virus (EBV) type and variant in spontaneous lymphoblastoid cells and Hu-SCID mouse tumours

Epstein-Barr virus (EBV) type and strain variations were examined using both lymphoblastoid cell lines (LCLs), spontaneously derived in vitro from peripheral blood mononuclear cells (PBMC) of 15 HIV-1-seropositive individuals; and SCID mouse tumours induced by inoculation of PBMC from 11 healthy human donors (Hu-SCID tumours). Polymerase chain reaction (PCR) analysis disclosed that all but one of the 26 EBV + samples harboured EBV nuclear antigen (EBNA) 2 and 3C type A virus. On the other hand, single strand conformation polymorphism (SSCP) analysis using Epstein-Barr encoded RNA (EBER) specific primers detected an AG876-like (type B) band pattern in 21 of the 26 EBV + samples. Three Hu-SCID tumours scored as B95.8-like (type A), and two showed neither a type A nor a type B SSCP migration pattern. Sequence analysis of the amplified EBER fragments confirmed the PCR-SSCP findings; moreover, additional mutations were present not only in the two EBV + samples with anomalous SSCP pattern, but also in two other samples with a standard SSCP profile. Thus, EBER analysis did not correlate with EBNA typing, and appeared to be unsuitable for EBV type assessment. Latent membrane protein (LMP) analysis disclosed, on the whole, sever size variants: as expected, the differences were due to the variable numbers of a 33-bp repeat in the amplified fragment, as assessed by direct sequencing. The broader variability detected by LMP analysis should prove more useful than typing for assessing the presence of single and/or mixed variants resulting from EBV reactivation and/or reinfection.     

A cosmid and yeast artificial chromosome contig containing the complete ryanodine receptor (RYR1) gene

The ryanodine receptor (RYR1) gene is responsible for some forms of malignant hyperthermia and has been localized to 19q13.1. Central core disease is a genetic myopathy that is genetically linked to RYR1. We have identified an overlapping set of cosmid and YAC clones that spans more than 800 kb and includes the RYR1 gene (approximately 205 kb). Cosmids from this region were identified by screening three chromosome 19 cosmid libraries (11-fold coverage) with six subclones representing the entire RYR1 cDNA. Genomic sequences from positive cosmids were then used as probes to identify additional cosmids. A minimally overlapping set of 23 cosmids was assembled into two contigs on the basis of restriction fragment analysis and hybridization data. Three YAC clones were isolated by screening a human YAC library with selected cosmid inserts. Overlaps among these YACs and the cosmid contigs were determined by hybridizing YAC Alu-PCR products to cosmid DNAs. The YACs bridged the gap between the cosmid contigs and extended the contig on both sides. Fluorescence in situ hybridization experiments positioned the RYR1 contig between GPI, MAG, and D19S191 on the proximal side and D19S190, CYP2A, CYP2F, SNRPA, BCKDHA, and other markers on the distal side. The 800-kb contig of cloned reagents will facilitate the detailed characterization of the RYR1 gene and other loci that may be closely related to central core disease.     

Genetic analysis of type 5 capsular polysaccharide expression by Staphylococcus aureus

Capsules are produced by over 90% of Staphylococcus aureus strains, and approximately 25% of clinical isolates express type 5 capsular polysaccharide (CP5). We mutagenized the type 5 strain Reynolds with Tn918 to target genes involved in CP5 expression. From a capsule-deficient mutant, we cloned into a cosmid vector an approximately 26-kb EcoRI fragment containing the transposon insertion. In the absence of tetracycline selection, Tn918 was spontaneously excised, thereby resulting in a plasmid containing 9.4 kb of S. aureus DNA flanking the Tn918 insertion site. The 9.4-kb DNA fragment was used to screen a cosmid library prepared from the wild-type strain. Positive colonies were identified by colony hybridization, and a restriction map of one clone (pJCL19 with an approximately 34-kb insert) carrying the putative capsule gene region was constructed. Fragments of pJCL19 were used to probe genomic DNA digests from S. aureus strains of different capsular serotypes. Fragments on the ends of the cloned DNA hybridized to fragments of similar sizes in most of the strains examined. Blots hybridized to two fragments flanking the central region of the cloned DNA showed restriction fragment length polymorphism. A centrally located DNA fragment hybridized only to DNA from capsular types 2, 4, and 5. DNA from pJCL19 was subcloned to a shuttle vector for complementation studies. A 6.2-kb EcoRI-ClaI fragment complemented CP5 expression in a capsule-negative mutant derived by mutagenesis with ethyl methanesulfonate. These experiments provide the necessary groundwork for identifying genes involved in CP5 expression by S. aureus.     

Fibromuscular dysplasia of the internal carotid artery: a clinicopathological study

A combination of Southern blot analysis on a panel of tumor-derived somatic cell hybrids and fluorescence in situ hybridization techniques was used to map YACs, cosmids and DNA markers from the Xp11.2 region relative to the X chromosome breakpoint of the renal cell carcinoma-associated t(X;1)(p11;q21). The position of the breakpoint could be determined as follows: Xcen-OATL2-DXS146-DXS255-SYP-t(X;1)-TFE 3-OATL1-Xpter. Fluorescence in situ hybridization experiments using TFE3-containing YACs and cosmids revealed split signals indicating that the corresponding DNA inserts span the breakpoint region. Subsequent Southern blot analysis showed that a 2.3-kb EcoRI fragment which is present in all TFE3 cosmids identified, hybridizes to aberrant restriction fragments in three independent t(X;1)-positive renal cell carcinoma DNAs. The breakpoints in these tumors are not the same, but map within a region of approximately 6.5 kb. Through preparative gel electrophoresis an (X;1) chimaeric 4.4-kb EcoRI fragment could be isolated which encompasses the breakpoint region present on der(X). Preliminary characterization of this fragment revealed the presence of a 150-bp region with a strong homology to the 5' end of the mouse TFE3 cDNA in the X-chromosome part, and a 48-bp segment in the chromosome 1-derived part identical to the 5' end of a known EST (accession number R93849). These observations suggest that a fusion gene is formed between the two corresponding genes in t(X;1)(p11;q21)-positive papillary renal cell carcinomas.     

Rep*: a viral element that can partially replace the origin of plasmid DNA synthesis of Epstein-Barr virus

Replication of the Epstein-Barr viral (EBV) genome occurs once per cell cycle during latent infection. Similarly, plasmids containing EBV's plasmid origin of replication, oriP, are replicated once per cell cycle. Replication from oriP requires EBV nuclear antigen 1 (EBNA-1) in trans; however, its contributions to this replication are unknown. oriP contains 24 EBNA-1 binding sites; 20 are located within the family of repeats, and 4 are found within the dyad symmetry element. The site of initiation of DNA replication within oriP is at or near the dyad symmetry element. We have identified a plasmid that contains the family of repeats but lacks the dyad symmetry element whose replication can be detected for a limited number of cell cycles. The detection of short-term replication of this plasmid requires EBNA-1 and can be inhibited by a dominant-negative inhibitor of EBNA-1. We have identified two regions within this plasmid which can independently contribute to this replication in the absence of the dyad symmetry element of oriP. One region contains native EBV sequences within the BamHI C fragment of the B95-8 genome of EBV; the other contains sequences within the simian virus 40 genome. We have mapped the region contributing to replication within the EBV sequences to a 298-bp fragment, Rep*. Plasmids which contain three copies of Rep* plus the family of repeats support replication more efficiently than those with one copy, consistent with a stochastic model for the initiation of DNA synthesis. Plasmids with three copies of Rep* also support long-term replication in the presence of EBNA-1. These observations together indicate that the latent origin of replication of EBV is more complex than formerly appreciated; it is a multicomponent origin of which the dyad symmetry element is one efficient component. The experimental approach described here could be used to identify eukaryotic sequences which mediate DNA synthesis, albeit inefficiently.     

Molecular effects of 2',2'-difluorodeoxycytidine (Gemcitabine) on DNA replication in intact HL-60 cells

The ability of pH-step alkaline elution to isolate different size species of nascent DNA (nDNA) from intact cells was utilized to study the effects of 2',2'-difluorodeoxycytidine (dFdC) on DNA replication in HL-60 cells. Preincubation with dFdC caused a concentration-dependent decrease in overall [3H]thymidine incorporation into DNA, accompanied by an increase in the proportion of radiolabel accumulated in small nDNA fragments. Twenty-four hours following removal of dFdC, radiolabel progressed from smaller to larger fragments and into genomic-length DNA. At initial concentrations of exposures to dFdC or cytosine arabinoside (ara-C) that caused 50% lethality (LC50) to HL-60 cells (40 and 50 nM, respectively), slower and less complete transit of nDNA from small subreplicon-length fragments through larger intermediates to genomic-length DNA was observed for nDNA fragments containing incorporated [3H]dFdC than for fragments containing [3H]ara-C. This was accomplished with less [3H]dFdC incorporated into DNA than [3H]ara-C at these extracellular concentrations of drug. Pulse-chase studies, using higher concentrations of radiolabeled drug, similarly revealed that nDNA fragments containing incorporated dFdC, like those containing ara-C, progressed with respect to time into larger nDNA intermediates and ultimately into genomic-length DNA; however, such progression for nDNA fragments containing dFdC was less complete than for fragments containing ara-C. The radioactivity incorporated into DNA represented authentic dFdC, as determined by DNA degradation studies, and was stable in DNA for at least 48 hr after removal of extracellular [3H]dFdC. Some of the effects of dFdC on ribonucleotide reduction in HL-60 cells were assessed by measurement of the intracellular pools of dCTP and dGTP. The drug had a greater effect on pools of dGTP than of dCTP, with transient reductions in dGTP observed at concentrations that encompass the LC50 for dFdC. These studies suggest that the interaction with DNA synthesis is an important component of the cytotoxicity of dFdC in HL-60 cells. Because it is incorporated progressively through nDNA compartments and ultimately into genomic-length DNA, dFdC should be categorized as an agent that slows DNA elongation in the intact cell, and not as a chain terminator in the absolute sense.     

Epstein-Barr virus (EBV)-positive NK cell line YT, and ALL/LBL of NK-lineage

The YT cells, already known as natural killer (NK) line, were tested for Epstein-Barr virus (EBV). The simply maintained YT cells (YT-O) and the two different subclones showed and identical length of junctional DNA of terminal repeats in Southern blot with LMP-1 probe, indicating that the 3 had already been positive for EBV before subcloning. YT-O expressed limited amount of EBNA2 or LMP-1 mRNA, whereas the 2 subclones expressed abundant EBNA2 or LMP-1 mRNA in the Northern blot analysis with EBNA2 or LMP-1 probe. Thus, the ex vivo cells were positive for EBV, since the BL (Burkitt lymphoma) type EBV gene expression in the YT-O does not generally occur in in vitro infection. The remaining clinical record indicated that the lymphoma was extranodal (angiocentric lymphoma), involving mediastinum and liver, but not nodal or lymphoblastic lymphoma (LBL). Acute lymphoblastic lymphoma (ALL)/LBL of the NK-lineage has not been defined, although such neoplasms should exist. Since T- and NK-lineages are so close in immature stages of differentiation that ALL/LBL of NK may have been sorted into T-lineage. The phenotypic records of the T-ALL/LBL in our laboratory indicated that CD7+CD5+CD2- is much higher in incidence than CD7+CD5-CD2+. This may reflect the size difference of physiological populations of T- and NK-lineage cells. Furthermore, the latter is of CD45RO type in contrast to the rest of the early-thymic (pro-thymic) T-ALL/LBL groups of CD45RA type. A CD56+ case of 4 CD7+CD5-CD2+ cases have been published as a case of LBL of NK-lineage. It is necessary to scrutinize CD7+CD5-CD2+ cells in order to clarify the phenotype of neoplastic and physiological NK cells in immature stages.     

An enhanced EBNA1 variant with reduced IR3 domain for long-term episomal maintenance and transgene expression of oriP-based plasmids in human cells

The latent replication of oriP-based plasmids in human cells depends on the viral oriP-binding transactivator EBNA1. In this report, the effect of the internal repeat 3 (IR3 or GlyAla repeat) domain of EBNA1 on long-term maintenance and transgene expression of OriP-based plasmids was examined in dividing human cells. To assess the potential contribution of different isoforms of EBNA1 specifically, the long-term stability of oriP-based plasmids was determined after stable transfection of various CMV-driven EBNA1 genes in EBV-negative human B cells. Episome copy number was quantified using a novel sensitive assay based on human mitochondrial DNA as an internal extrachromosomal control. Using this assay, the standard B95.8-derived EBNA1 was compared with its truncated IR3-deleted, form, as well as a new EBNA1 isoform cloned from Raji. The results of a 6-month study indicate that the isoforms of EBNA1 differ with respect to their efficiency of plasmid maintenance. While the EBNA-1 Raji encoding plasmid was the most stable, the oriP-based vector expressing the truncated EBNA1 (IR3del) gene was lost at a much higher rate than those transducing full size EBNA1s. In parallel, long-term reporter gene expression in various human B cell lines was shown to persist at the highest level with the oriP-based Raji EBNA-1 construct. These results show that the GlyAla domain can positively influence long-term plasmid stability and episomal transgene expression.     

Human histone gene organization: nonregular arrangement within a large cluster

We have previously located the genes of the five human main type H1 genes and the gene encoding the testicular subtype H1t to the region 21.1 to 22.2 on the short arm of chromosome 6. To investigate the organization of the histone genes in this region, we isolated two YACs from a human YAC library by PCR screening with primers specific for histone H1.1. This screen revealed two YAC clones, YAC Y23 (corresponding to ICRFy901D1223) contains an insert of about 480 kb, whereas the smaller YAC 4A (corresponding to ICRFy900C104) spans about 340 kb and is completely covered by YAC Y23. We have subcloned the YAC inserts in cosmids, determined the linear orientation of the cosmids by cosmid walking, and constructed a restriction map of the entire region by mapping the individual cosmids using partial digests and hybridization with labeled oligonucleotides complementary to the cos site of the vector. Hybridization analysis, subcloning, restriction mapping, and sequencing revealed that most of the previously isolated phage and cosmid clones containing histone genes are part of this YAC including the clones containing the four human main type H1 histone genes H1.1 to H1.4, the H1t gene, and core histone genes. Thirty-five histone genes map within 260 kb of the YAC Y23 insert. All newly identified histone genes were sequenced, and the sequences were deposited with the EMBL nucleotide sequence database. The histone H1.5 gene is not part of this region, and we therefore conclude that the H1.5 gene and the associated core histone genes form a separate subcluster within this chromosomal region.