Selecting karyophilic DNA cis elements in Xenopus laevis oocytes; a new approach.
The intracellular localisation and mobility of exogenous DNA introduced into Xenopus laevis oocytes is largely unknown. In this paper, we report a new technique to investigate the cytoplasmic/nuclear transport of a random pool of linear, double-stranded, oligomeric DNA of 147 bp in length. We chose a combinatorial approach which made use of repetitive rounds of selection and amplification to search for new cis elements mediating nuclear import or retention. A new PCR-based methodology was established to reliably detect exogenous DNA in subcellular and total extracts prepared from Xenopus laevis oocytes. Studies in vivo and with cellular extracts indicate the presence of a highly efficient nuclease activity in the nuclear compartment. The described combinatorial approach constitutes a promising tool for the isolation of novel DNA cis elements which may play an important role in the nuclear internalisation and retention of exogenous DNA in Xenopus laevis oocytes.
Marcello
Marco
M
Deutsches Krebsforschungszentrum, Heidelberg, Germany.
Fischer
Roger
R
Tröster
Helmut
H
Trendelenburg
Michael F
MF
Sczakiel
Georg
G
D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D002467:Cell Nucleus / Q000235:genetics; D003593:Cytoplasm / Q000235:genetics; D004247:DNA / Q000235:genetics*; D017931:DNA Primers; D005260:Female; D009865:Oocytes / Q000502:physiology*; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods; D014982:Xenopus laevis / Q000235:genetics*
D017931:DNA Primers; D004247:DNA