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1.
We have previously characterized several genomic rearrangements of Epstein-Barr virus (EBV) DNA contained in one of the defective EBV genomes harbored by the P3HR-1 (HR-1) line (H. B. Jenson, M. S. Rabson, and G. Miller, J. Virol. 58:475-486, 1986). One recombinant clone of heterogeneous DNA (het DNA) from this defective genome is an EcoRI fragment of 16 kilobase pairs (kbp) which is a palindrome. DNA digestion fragments specific for the center of this palindrome were present in cells which contained het DNA but not in cells which lacked het DNA. Thus, the palindrome was not an artifact of DNA cloning. The organization of the center of this palindrome was studied by DNA sequencing. The comparable region of the parental HR-1 genome was also studied by DNA sequencing. The central 3,495 base pairs (bp) of the palindrome were composed of sequences derived exclusively from internal repeat 1 of EBV, represented by BamHI W fragment. At each end of the central 3,495 hp was a symmetrical recombination with sequences of BamHI-Z, located more than 50 kbp away on the standard EBV genome. The central 3,495 bp were composed of an unduplicated 341 bp flanked by two perfect palindromic repeats of 1,577 bp. The 341-bp unique region was a portion of a 387-bp region of standard HR-1 BamHI-W which was identical to the central 387 bp of the palindrome. This central 387-bp region contained numerous stretches of dyad symmetry capable of forming a large stem-and-loop structure. The palindromic rearrangement had created two novel open reading frames in het DNA derived from standard HR-1 BamHI-W sequences. These two het DNA open reading frames had different amino termini but identical carboxy termini derived from the large open reading frame in standard HR-1 BamHI-W (HR-1 BWRF1). The BamHI-W sequences found in het DNA did not include either the TATA box of standard HR-1 BamHI-W or the exons which are present in the potentially polycistronic latent mRNAs encoding EBV nuclear antigens. These marked alterations in genomic structure may relate to the unique biologic properties of virus stocks containing het DNA by creation of new polypeptides or by formation or deletion of regulatory or functional signals.  相似文献   

2.
Deletions and rearrangements in the genome of Epstein-Barr virus (EBV) strain P3HR-1 generate subgenomic infectious particles that, unlike defective interfering particles in other viral systems, enhance rather than restrict EBV replication in vitro. Reports of comparable heterogeneous (het) DNA in EBV-linked human diseases, based on detection of an abnormal juxtaposition of EBV DNA fragments BamHI W and BamHI Z that disrupts viral latency, prompted us to determine at the nucleotide level all remaining recombination joints formed by the four constituent segments of P3HR-1-derived het DNA. Guided by endonuclease restriction maps, we chose PCR primer pairs that approximated and framed junctions creating the unique BamHI M/B1 and E/S fusion fragments. Sequencing of PCR products revealed points of recombination that lacked regions of extensive homology between constituent fragments. Identical recombination junctions were detected by PCR in EBV-positive salivary samples from human immunodeficiency virus-infected donors, although the W/Z rearrangement that induces EBV reactivation was frequently found in the absence of the other two. In vitro infection of lymphoid cells similarly indicated that not all three het DNA rearrangements need to reside on a composite molecule. These results connote a precision in the recombination process that dictates both composition and regulation of gene segments altered by genomic rearrangement. Moreover, the apparent frequency of het DNA at sites of EBV replication in vivo is consistent with a likely contribution to the pathogenesis of EBV reactivation.  相似文献   

3.
Defective Epstein-Barr virus (EBV) has a deleted and rearranged genome (termed het DNA) that disrupts latency and induces standard EBV to replicate in vitro. We used the polymerase chain reaction to detect, in 2 of 10 patient samples, the junction of abnormally juxtaposed EBV DNA fragments BamHI W and Z, a genomic rearrangement responsible for the biologic activity of het DNA. By sequence analysis, the junction in wild-type defective DNA appears to be similar but not identical to the recombination in the DNA of laboratory strain P3HR-1. The presence of this marker for het DNA in the epithelial lesions of two patients suggests a role for defective EBV in a human pathologic process.  相似文献   

4.
Antigens associated with the Epstein-Barr virus (EBV) replicative cycle were found in the nucleus and cytoplasm of human placental, Vero, BSC-1, and owl monkey kidney cells transfected with EBV DNA prepared from several different strains of virus. The number of antigen-positive nuclei increased when transfection was followed by cell fusion induced by inactivated Sendai virus. About 1,200 antigen-positive foci were induced per micrograms of EBV DNA. On the basis of their reactivity with various well-characterized human sera, it appears that the antigens are part of the early antigen complex. None of the four restriction endonucleases, EcoRI, HindIII, SalI, and BamHI, destroyed the ability of EBV DNA to induce early antigen. However, only SalI seemed to leave intact the full spectrum of antigen expression by the HR-1 and FF41 strains of EBV DNA. By means of transfection with recombinant DNA plasmids containing different EBV (FF41) DNA fragments regenerated by EcoRI, we showed that the coding region for early antigen was at least partially contained on the 17.2-megadalton EcoRI B fragment.  相似文献   

5.
The P3J-HR-1 strain of Epstein-Barr virus (EBV) fails to immortalize human lymphocytes. We wished to understand the nature of the genomic alterations which correlated with the loss of this ability. As a first step, the heterogeneity of DNA molecules in the P3J-HR-1 line was eliminated by cell cloning. Then a physical map was prepared of virion DNA from one cell clone, designated FF452-3. By comparison with the genomes of two EBVs, B95-8 and FF41, which are competent to immortalize lymphocytes, we identified a total of eight modifications of BamHI and EcoRI restriction endonuclease fragments of EBV (FF452-3) DNA consisting of insertions, deletions, or loss of a restriction endonuclease recognition site. To determine which of these alterations might be responsible for the loss of transforming phenotype, we examined homologous DNA fragments of the Jijoye strain of EBV, the progenitor of the HR-1 strain which still retains the ability to immortalize lymphocytes. We also studied viral DNA in lymphocytes transformed in vitro by Jijoye virus. Six of the eight alterations were found both in Jijoye and in clonal HR-1 DNA and were presumably genomic traits characteristic of this lineage of EBV. A small deletion in the BamHI-K fragment of HR-1 DNA was not found in Jijoye virion DNA, but this deletion was present in intracellular Jijoye DNA. Thus only one major genomic lesion in HR-1 DNA, a deletion of at least 2.4 x 10(6) molecular weight of DNA from a fused BamHI-H-Y fragment, consistently distinguished Jijoye DNA from its non-immortalizing P3J-HR-1 derivative. This deletion is likely to affect EBV genes which are directly or indirectly involved in immortalizing lymphocytes.  相似文献   

6.
7.
Epstein-Barr virus recombinants from overlapping cosmid fragments.   总被引:14,自引:12,他引:2       下载免费PDF全文
Five overlapping type 1 Epstein-Barr virus (EBV) DNA fragments constituting a complete replication- and transformation-competent genome were cloned into cosmids and transfected together into P3HR-1 cells, along with a plasmid encoding the Z immediate-early activator of EBV replication. P3HR-1 cells harbor a type 2 EBV which is unable to transform primary B lymphocytes because of a deletion of DNA encoding EBNA LP and EBNA 2, but the P3HR-1 EBV can provide replication functions in trans and can recombine with the transfected cosmids. EBV recombinants which have the type 1 EBNA LP and 2 genes from the transfected EcoRI-A cosmid DNA were selectively and clonally recovered by exploiting the unique ability of the recombinants to transform primary B lymphocytes into lymphoblastoid cell lines. PCR and immunoblot analyses for seven distinguishing markers of the type 1 transfected DNAs identified cell lines infected with EBV recombinants which had incorporated EBV DNA fragments beyond the transformation marker-rescuing EcoRI-A fragment. Approximately 10% of the transforming virus recombinants had markers mapping at 7, 46 to 52, 93 to 100, 108 to 110, 122, and 152 kbp from the 172-kbp transfected genome. These recombinants probably result from recombination among the transfected cosmid-cloned EBV DNA fragments. The one recombinant virus examined in detail by Southern blot analysis has all the polymorphisms characteristic of the transfected type 1 cosmid DNA and none characteristic of the type 2 P3HR-1 EBV DNA. This recombinant was wild type in primary B-lymphocyte infection, growth transformation, and lytic replication. Overall, the type 1 EBNA 3A gene was incorporated into 26% of the transformation marker-rescued recombinants, a frequency which was considerably higher than that observed in previous experiments with two-cosmid EBV DNA cotransfections into P3HR-1 cells (B. Tomkinson and E. Kieff, J. Virol. 66:780-789, 1992). Of the recombinants which had incorporated the marker-rescuing cosmid DNA fragment and the fragment encoding the type 1 EBNA 3A gene, most had incorporated markers from at least two other transfected cosmid DNA fragments, indicating a propensity for multiple homologous recombinations. The frequency of incorporation of the nonselected transfected type 1 EBNA 3C gene, which is near the end of two of the transfected cosmids, was 26% overall, versus 3% in previous experiments using transfections with two EBV DNA cosmids. In contrast, the frequency of incorporation of a 12-kb EBV DNA deletion which was near the end of two of the transfected cosmids was only 13%.(ABSTRACT TRUNCATED AT 400 WORDS)  相似文献   

8.
We present results of biological experiments which indicate that the subpopulation of Epstein-Barr virus strain P3HR-1 with heterogeneous (het) DNA consists of self-contained replicons which multiply alongside, but independently of, Epstein-Barr virus strain HR-1 containing standard DNA. When a population of HR-1 virions containing het DNA was introduced into X50-7 cells, the input heterogeneous DNA increased in abundance, as did the DNA of the endogenous virus of X50-7 cells. The input standard HR-1 viral DNA, however, was not amplified. When parental HR-1 cells or a cellular subclone containing het DNA were grown for several weeks in the presence of human serum with neutralizing antibody, the het DNA was lost from the culture; standard HR-1 DNA, however, was not affected by antiserum. Furthermore, virions containing het DNA could be serially propagated through cellular subclones of HR-1 cells which lack het DNA. After each serial passage, cells which acquired het DNA released virions with the ability to induce early antigens in Raji cells. These experiments define a novel in vitro life cycle of an Epstein-Barr virus variant which is maintained, not vertically by partitioning to daughter cells in cell division, but horizontally by cell-to-cell spread.  相似文献   

9.
Incubation of the DNA of the B95-8 strain of Epstein-Barr virus [EBV (B95-8) DNA] with EcoRI, Hsu I, Sal I, or Kpn I restriction endonuclease yielded 8 to 15 fragments separable on 0.4% agarose gels and ranging in molecular weight from less than 1 to more than 30 x 10(6). Bam I and Bgl II yielded fragments smaller than 11 x 10(6). Preincubation of EBV (B95-8) DNA with lambda exonuclease resulted in a decrease in the Hsu I A and Sal I A and D fragments, indicating that these fragments are positioned near termini. The electrophoretic profiles of the fragments produced by cleavage of the DNA of the B95-8, HR-1, and Jijoye strains of EBV were each distinctive. The molecular weights of some EcoRI, Hsu I, and Sal I fragments from the DNA of the HR-1 strain of EBV [EBV (HR-1) DNA] and of EcoRI fragments of the DNA of the Jijoye strain of EBV were identical to that of fragments produced by cleavage of EBV (B95-8) DNA with the same enzyme, whereas others were unique to each strain. Some Hsu I, EcoRI, and Sal I fragments of EBV (HR-1) DNA and Kpn I fragments of EBV (B95-8) DNA were present in half-molar abundance relative to the majority of the fragments. In these instances, the sum of the molecular weights of the fragments was in excess of 10(8), the known molecular weight of EBV (HR-1) and (B95-8) DNA. The simplest interpretation of this finding is that each EBV (HR-1), and possibly also (B95-8), DNA preparation contains two populations of DNA molecules that differ in the arrangement of DNA sequences about a single point, such as has been described for herpes simplex virus DNA. Minor fragments could also be observed if there were more than one difference in primary structure of the DNAs. The data do not exclude more extensive heterogeneity in primary structure of the DNA of the HR-1 strain. However, the observation that the relative molar abundance of major and minor fragments of EBV (HR-1) DNA did not vary between preparations from cultures that had been maintained separately for several years favors the former hypothesis over the latter.  相似文献   

10.
Mutations in five phenotypically distinct mutants derived from herpes simplex virus type 1 strain KOS which lie in or near the herpes simplex virus DNA polymerase (pol) locus have been fine mapped with the aid of cloned fragments of mutant and wild-type viral DNAs to distinct restriction fragments of 1.1 kilobase pairs (kbp) or less. DNA sequences containing a mutation or mutations conferring resistance to the antiviral drugs phosphonoacetic acid, acyclovir, and arabinosyladenine of pol mutant PAAr5 have been cloned as a 27-kbp Bg+II fragment in Escherichia coli. These drug resistance markers have been mapped more finely in marker transfer experiments to a 1.1-kbp fragment (coordinates 0.427 to 0.434). In intratypic marker rescue experiments, temperature-sensitive (ts), phosphonoacetic acid resistance, and acyclovir resistance markers of pol mutant tsD9 were mapped to a 0.8-kbp fragment at the left end of the EcoRI M fragment (coordinates 0.422 to 0.427). The ts mutation of pol mutant tsC4 maps within a 0.3-kbp sequence (coordinates 0.420 to 0.422), whereas that of tsC7 lies within the 1.1-kbp fragment immediately to the left (coordinates 0.413 to 0.420). tsC4 displays the novel phenotype of hypersensitivity to phosphonoacetic acid; however, the phosphonoacetic acid hypersensitivity phenotype is almost certainly not due to the mutation(s) conferring temperature sensitivity. The ts mutation of mutant tsN20--which does not affect DNA polymerase activity--maps to a 0.5-kbp fragment at the right-hand end of the EcoRI M fragment (coordinates 0.445 to 0.448). The mapping of the mutations in these five mutants further defines the limits of the pol locus and separates mutations differentially affecting catalytic functions of the polymerase.  相似文献   

11.
Summary A PstI 7.7 kbp fragment from chloroplast (ct) DNA of spinach shows homology to an EcoRI 8.3 kbp fragment of mitochondrial (mt) DNA and in turn, both are homologous to a number of common regions of nuclear (n) DNA. The common area of homology between the chloroplast and mitochondrial fragments is between a KpnI 1.8 segment internal to the PstI sites in the ctDNA and an EcoRI/BamHI 2.9 kbp fragment at one end of the mitochondrial 8.3 kbp fragment. The KpnI 1.8 kbp ctDNA fragment is within a structural gene for the P700 chlorophyll a apoprotein. Further analysis of this KpnI 1.8 kbp fragment confined the homologous region in mtDNA to a ct 0.8 kbp HpaII fragment. These smaller pieces of the organellar genomes share homologies with nuclear DNA as well as displaying unique hybridization sites. The observations reported here demonstrate that there is a common or closely related sequence in all three genetic compartments of the cell.  相似文献   

12.
13.
The cleavage of the DNAs of the B95-8 and P3HR-1 virus strains of Epstein-Barr virus by the restriction endonucleases EcoRI, HindIII and BamI was investigated using a new technique for quantitative evaluation of the fluorescence of ethidium stained DNA fragments separated on agarose gels. The results obtained with B95-8 DNA showed that in addition to the limited repetitions of nucleotide sequences observed in the EcoRI and HindIII cleavage patterns, the molecule contained a BamI fragment with a molecular mass of 2.0 megadaltons which was present in a total of about 11 copies and localized to a limited part of the DNA molecule. The same sequences were also present in the P3HR-1 DNA albeit in a lower molar ratio. P3HR-1 DNA yielded restriction enzyme cleavage patterns suggesting DNA sequence heterogeneity of P3HR-1 virus. No fragment was present in more than about 4 copies per molecule of P3HR-1 DNA. Comparison of the restriction enzyme cleavage patterns of P3HR-1 and B95-8 DNA revealed a high degree of structural homology emphasized by nucleic acid hybridization experiments with EBV complementary RNA synthesized in vitro.  相似文献   

14.
Isolation of a repeated DNA sequence from Bordetella pertussis   总被引:8,自引:0,他引:8  
A repeated DNA sequence in the genome of Bordetella pertussis has been demonstrated. At least 20 copies of this sequence could be observed in either BamHI or EcoRI restriction enzyme digests of chromosomal DNA; fragments carrying the repeated DNA sequence ranged in size from about 1.5 to 20 kbp. The repeated DNA sequence was cloned from two separate regions of the B. pertussis genome, as shown by restriction enzyme site maps of the two clones and by hybridization studies. A small number of differences in the pattern of hybridization of the repeated DNA sequence to chromosomal DNA from several strains of B. pertussis was observed. No repeated DNA sequences were observed in one strain each of B. parapertussis and B. bronchiseptica, and there was no hybridization of B. pertussis DNA to Escherichia coli chromosomal DNA. The repeated DNA sequence was subcloned on a 2.54 kbp BamHI fragment from one of the two original clones. Restriction enzyme digests and hybridization studies showed that the repeated DNA sequence was about 1 kbp in size and had a single, internal ClaI site.  相似文献   

15.
The highly complex G + C-rich satellite DNA of the Bermuda land crab Gecarcinus lateralis has been studied by denaturation mapping. Following digestion of the satellite with EndoR.Eco RI, the major 2.07-kilobase pair (kbp) basic repeating unit and a minor 4.14-kbp fragment were exposed to 254 nm light in the presence of silver ions, conditions which resulted in essentially irreversible denaturation of regions rich in adjacent pyrimidines by the formation of pyrimidine dimers. The positions and sizes of the denatured regions were determined in electron micrographs of partially denatured 2.07-kbp and 4.14-kbp fragments spread in the presence of formamide. After 15 min exposure to UV, 90% of the 2.07-kbp fragments had a denaturation bubble averaging 0.17 kbp centered around one-third (0.64 kbp) the total length; 20% exhibited another in the region from 1.8 kbp to 2.07 kbp. Similarly, about 90% of the 4.14-kbp fragments had denatured regions centered at 0.64 kbp and 2.75 kbp and 20% of the fragments had denaturation bubbles in regions centered at 1.92 kbp and 3.9 kbp. The positions of the denaturation bubbles in the 4.14-kbp fragments support restriction enzyme mapping evidence that it is a dimer of the 2.07-kbp fragment arranged head to tail. Sequencing data show that the predominant sequence of a 0.29-kbp region centered around 0.64 kbp in the basic repeat unit is 49% A + T and that 42% of the bases are adjacent TTs and CTs capable of dimerization under the conditions used.  相似文献   

16.
The arrangement of EcoRI, Hsu I, and Sal I restriction enzyme sites in the DNA of the B95-8 and W91 isolates of Epstein-Barr virus (EBV) has been determined from the size of the single-enzyme-cleaved fragments and from blot hybridizations that identify which fragments cut from the DNA with one enzyme contain nucleotide sequences in common with fragments cut from the DNA with a second enzyme. The DNA of the B95-8 isolate was the prototype for this study. The data indicate that (i) approximately 95 X 10(6) to 100 X 10(6) daltons of EBV (B95-8) DNA is in a consistent and unique sequence arrangement. (ii) Both termini are variable in length. One end of the molecule after Hsu I endonuclease cleavage consists of approximately 3,000 base pairs, with as many as 10 additional 500-base pair segments. The opposite end of the molecule after Sal I endonuclease cleavage consists of approximately 1,500 base pairs, with as many as 10 additional 500-base pair segments. (iii) The opposite ends of the molecule contain homologous sequences. The high degree of homology between the opposite ends of the molecule and the similarity in size of the "additional" 500-base pair segments suggests that there are identical repeating units at both ends of the DNA. The arrangement of restriction endonuclease fragments of the DNA of the W91 isolate of EBV is similar to that of the B95-8 isolate and differs from the latter in the presence of approximately 7 X 10(6) daltons of "extra" DNA at a single site. Thus, the size of almost all EcoRI, Hsu I, and Sal I fragments of EBV (W91) DNA is identical to that of fragments of EBV (B95-8) DNA. A single EcoRI fragment, C, of EBV (W91) DNA is approximately 7 X 10(6) daltons larger than the corresponding EcoRI fragment of EBV (B95-8) DNA. Digestion of EBV (W91) DNA with Hsu I or Sal I restriction endonucleases produces two fragments (Hsu I D1 and D2 or Sal I G2 and G3) which differ in total size by approximately 7 X 10(6) daltons from the fragments of EBV (B95-8) DNA. Furthermore, the EcoRI, Hsu I, and Sal I fragments of EBV (W91) and (B95-8) DNAs, which are of similar molecular weight, have homologous nucleotide sequences. Moreover, the W91 fragments contain only sequences from a single region of the B95-8 genome. Two lines of evidence indicate that the "extra" sequences present in W91 EcoRI fragment C are viral DNA and not cellular. (i) The molecular weight of the "enlarged" EcoRI C fragment of EBV (W91) DNA is identical to that of the EcoRI C fragment of another isolate of EBV (Jijoye), (ii) The HR-1 clone of Jijoye has previously been shown to contain DNA which is not present in the B95-8 strain but is present in the EcoRI C and Hsu I D2 and D1 fragments of EBV (W91) DNA (N. Raab-Traub, R. Pritchett, and E. Kieff, J. Virol. 27:388-398, 1978).  相似文献   

17.
We have cloned and characterized a novel endogenous murine mammary tumor virus proviral unit of the C3H/He strain of mice. The cloned proviral unit is 16 kilobase pairs (kbp) in size and is composed of a 5.6-kbp 5' EcoRI segment of an endogenous provirus with 10.4-kbp flanking cellular sequences. A comparison of the restriction map of the cloned proviral DNA with that of an endogenous provirus of the GR strain of mice has revealed minor differences in restriction sites on the two proviruses. The restriction enzyme SstI, which does not cleave the 5' EcoRI fragment of GR DNA, cleaves the C3H/He proviral sequences once; MspI has an additional site in the C3H/He proviral sequences. By using a subcloned fragment containing unique cellular sequences as a hybridization probe, we (i) mapped the C3H/He proviral unit to chromosome 14 by using mouse-hamster somatic cell hybrids, and (ii) demonstrated that this proviral unit is also present in the genome of DBA/2 mice. From these results we conclude that the C3H/He strain of mice acquired this proviral unit from DBA stock by genetic transmission. Our data also indicate that the murine mammary tumor virus sequences present in the gag-specific proviral unit of C3H/He mice extend at least 2.45 kbp downstream of the EcoRI site in the genomic DNA. Since the structural organization and chromosomal location of this proviral unit are distinct from those of previously reported proviral units represented by similar-sized (16.7-kbp) EcoRI fragments, we tentatively propose to designate this proviral unit Mtv-7a.  相似文献   

18.
Identification of the gene encoding Marek''s disease herpesvirus A antigen.   总被引:5,自引:5,他引:0  
The gene encoding the glycoprotein Marek's disease herpesvirus A antigen (MDHV-A) precursor polypeptide pr47 was delineated by using Northern blot (RNA blot) analysis and hybrid selection of its mRNA with cloned MDHV DNA, cell-free translation of the mRNA, and immunoprecipitation of the polypeptide. The resulting piece of DNA with strongly positive hybrid selection results was a 2.2-kilobase-pair (kbp) PvuII-EcoRI restriction fragment localized to the center of the 18.3-kbp MDHV BamHI B fragment of the total virus genome. The localization was specific since no other small restriction subfragment of the larger BamHI B fragment was able to hybrid select significant MDHV-A mRNA and the gene mapped only in the BamHI B fragment of the total virus genome. Northern blot analysis confirmed the localization of the MDHV-A gene on the 2.2-kbp fragment and detected its mRNA as a 1.8-kilobase species, a size consistent with encoding a 47-kilodalton polypeptide. This is the first report of an MDHV gene being mapped to the MDHV viral genome. This opens the way for the use of recombinant DNA technology to study the nature of the gene encoding a secreted virus-specific glycoprotein that could possibly be involved in immunoprevention, immunosuppression, or immunoevasion, immune phenomena known or speculated to be involved in this oncogenic herpesvirus system.  相似文献   

19.
Deleted, rearranged, heterogeneous (het) Epstein-Barr virus (EBV) DNA with the distinctive capability of disrupting EBV latency has been reported in biopsy samples of EBV-associated tumors whose onset in immunocompetent hosts is characteristically preceded by an antibody response indicative of EBV reactivation. Using the EBV P3HR-1 strain, we have reproduced in long-term culture of SVK epithelial cells an unusual pattern of infection previously observed in a subset of tumor biopsy samples: the persistence of het DNA in the absence of the parental helper virus. Fluorescence in situ hybridization (FISH) of infected cell subclones indicated the retention of het DNA in an integrated form. Incorporation of an intact het DNA molecule was confirmed by PCR, using primers that framed junctions of the four rearranged EBV DNA segments comprising P3HR-1-derived het DNA. Structural analysis of EBV terminal repeats revealed a banding pattern consistent with the integration of het DNA as a concatemer. Linkage of concatemeric monomers was defined at a nucleotide level, and that junctional sequence was detected in cell-free P3HR-1 virion DNA, confirming that subgenomic het DNA was packaged into infectious particles in a concatemeric configuration. Stable integration into cells having lost the standard viral genome allowed the unambiguous designation of het DNA as the source for viral gene products potentially encoded by both. Continuous expression of the latency-to-lytic switch protein Zta and detection of the BALF4 gene product gB, known to expand the target cell range of standard virus when incorporated at augmented levels into infectious progeny, add to a presumption of het DNA-enhanced pathogenesis in diseases of EBV reactivation.  相似文献   

20.
We used cloned BamHI fragments from Epstein-Barr virus strain B95-8 [EBV(B95-8)]DNA to obtain detailed restriction maps of the region of the genome adjacent to the large internal repeat cluster. These maps together with the results of hybridization experiments using a 3.1-kilobase repeat probe defined more precisely the location of the injection between the internal repeat cluster and the flanking unique-sequence DNA. On one side (UL), the repeat sequences extended 600 +/- 80 base pairs (bp) into BamHI-Y; on the other side (US), they extended 1,300 +/- 200 bp into BamHI-C. Therefore, EBV(B95-8) DNA contained a nonintegral number of 3.1-kilobase repeat units, namely, 12.6 copies. The mapping studies also revealed a second series of internal tandem repetitions in EBV(B95-8) DNA located within the BamHI-H fragment. This cluster comprised 11 copies of a 135-bp repeat unit which contained a single site for the NotI restriction endonuclease. Hybridization to these cloned EBV(B95-8) fragments using total EBV(HR-1) DNA as probe indicated that the deletion in EBV(HR-1) removed all 3,000 bp of unique-sequence DNA which lay between the large 3.1-kilobase and the small 135-bp repeat clusters. Thus, the deletion which destroyed the transforming ability in the EBV(HR-1) virus was bounded on either side by tandem repetitions.  相似文献   

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