An episomally replicating vector binds to the nuclear matrix protein SAF-A in vivo.
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.
Jenke
Bok Hee C
BH
Institute of Cell Biology, Stockumer Strasse 10, University of Witten/Herdecke, D-58448 Witten, 1Heinrich Pette-Institute, University of Hamburg, Hamburg, Germany.
Fetzer
Christian P
CP
Stehle
Isa M
IM
Jönsson
Franziska
F
Fackelmayer
Frank O
FO
Conradt
Harald
H
Bode
Jürgen
J
Lipps
Hans J
HJ
D000818:Animals; D026501:Blotting, Southwestern; D015153:Blotting, Western; D016466:CHO Cells; D002945:Cisplatin / Q000378:metabolism; D006224:Cricetinae; D005822:Genetic Vectors / Q000378:metabolism*; D034722:Heterogeneous-Nuclear Ribonucleoprotein U; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D012261:Ribonucleoproteins / Q000378:metabolism*
D034722:Heterogeneous-Nuclear Ribonucleoprotein U; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D012261:Ribonucleoproteins; D002945:Cisplatin