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An episomally replicating vector binds to the nuclear matrix protein SAF‐A in vivo
Author(s) -
Jenke Bok Hee C,
Fetzer Christian P,
Stehle Isa M,
Jönsson Franziska,
Fackelmayer Frank O,
Conradt Harald,
Bode Jürgen,
Lipps Hans J
Publication year - 2002
Publication title -
embo reports
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 4.584
H-Index - 184
eISSN - 1469-3178
pISSN - 1469-221X
DOI - 10.1093/embo-reports/kvf070
Subject(s) - nuclear matrix , biology , immunoprecipitation , microbiology and biotechnology , nuclear protein , mitosis , vector (molecular biology) , matrix (chemical analysis) , dna replication , viral matrix protein , scaffold/matrix attachment region , in vivo , dna binding protein , dna , genetics , virus , cell culture , gene , chemistry , transcription factor , recombinant dna , chromatography , chromatin remodeling , chromatin
pEPI‐1, a vector in which a chromosomal scaffold/matrix‐attached region (S/MAR) is linked to the simian virus 40 origin of replication, is propagated episomally in CHO cells in the absence of the virally encoded large T‐antigen and is stably maintained in the absence of selection pressure. It has been suggested that mitotic stability is provided by a specific interaction of this vector with components of the nuclear matrix. We studied the interactions of pEPI‐1 by crosslinking with cis ‐diamminedichloroplatinum II, after which it is found to copurify with the nuclear matrix. In a south‐western analysis, the vector shows exclusive binding to hnRNP‐U/SAF‐A, a multifunctional scaffold/matrix specific factor. Immunoprecipitation of the crosslinked DNA–protein complex demonstrates that pEPI‐1 is bound to this protein in vivo . These data provide the first experimental evidence for the binding of an artificial episome to a nuclear matrix protein in vivo and the basis for understanding the mitotic stability of this novel vector class.