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Titolo:
EPSTEIN-BARR-VIRUS RECOMBINANTS FROM OVERLAPPING COSMID FRAGMENTS
Autore:
TOMKINSON B; ROBERTSON E; YALAMANCHILI R; LONGNECKER R; KIEFF E;
Indirizzi:
HARVARD UNIV,SCH MED,DEPT MED,75 FRANCIS ST BOSTON MA 02115 HARVARD UNIV,SCH MED,DEPT MED,75 FRANCIS ST BOSTON MA 02115 HARVARD UNIV,SCH MED,DEPT MICROBIOL & MOLEC GENET BOSTON MA 02115
Titolo Testata:
Journal of virology
fascicolo: 12, volume: 67, anno: 1993,
pagine: 7298 - 7306
SICI:
0022-538X(1993)67:12<7298:ERFOCF>2.0.ZU;2-C
Fonte:
ISI
Lingua:
ENG
Soggetto:
2ND-SITE HOMOLOGOUS RECOMBINATION; GROWTH TRANSFORMATION INVITRO; LYMPHOCYTE-B TRANSFORMATION; MARMOSET LEUKOCYTES; NUCLEAR PROTEIN-2; EPITHELIAL-CELLS; EBV RECOMBINANTS; P3HR-1 DELETION; GENES; DNA;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Science Citation Index Expanded
Citazioni:
53
Recensione:
Indirizzi per estratti:
Citazione:
B. Tomkinson et al., "EPSTEIN-BARR-VIRUS RECOMBINANTS FROM OVERLAPPING COSMID FRAGMENTS", Journal of virology, 67(12), 1993, pp. 7298-7306

Abstract

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, afrequency 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 fragmentand 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 3Cgene, 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%. Other than through incorporation into recombinants arising from among the five cosmids, this marker was rarely incorporated into recombinants which had any marker from the P3HR-1 genome. Thus, the five-cosmid transfection strategy is particularly useful for incorporation of a nonselected marker mapping near the end of a transfected cosmid or near the site of a large deletion.

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Documento generato il 12/07/20 alle ore 01:41:02