Zdenka Hradecna
University of Wisconsin-Madison
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Featured researches published by Zdenka Hradecna.
Gene | 1996
Jadwiga Wild; Zdenka Hradecna; György Pósfai; Waclaw Szybalski
A prerequisite for sequencing large genomes is to obtain 30- to 150-kb genomic DNA fragments in adequate quantity. Previously, we developed a system which enables one to excise and amplify in vivo such segments directly from the Escherichia coli genome. This system, which employed the yeast Flp/FRT elements for excision and the plasmid R6K-based replication machinery for DNA amplification, permits one to bypass conventional cloning [Pósfai et al. (1994) Nucleic Acids Res. 22, 2392-2398]. To extend the applicability of such a system to many species, we describe here a broad-host-range (bhr) system in which the amplification of the excised DNA fragment depends on the oriV element and the Rep (TrfA) protein from the promiscuous RK2/RP4 plasmid. We have constructed insertion plasmids which carry the FRT and oriV sites. To introduce such plasmids into the appropriate position in the host genome, a short genomic sequence homologous to this position was cloned into the multiple cloning site (MCS) of the FRT/oriV insertion plasmid and then recombined into this position in the genome by RecA-mediated recombination. In such a manner, many strains with single FRT/oriV insertions at various positions could be generated. Subsequent genetic crosses or phage transduction allow two neighboring FRT/oriV sites (less than 150 kb apart) to be brought into a single genome. In the present report, the lacZ and phoB sites, which are 51 kb apart in the E. coli genome, were used for the introduction of the FRT/oriV sites. To deliver the Flp (excision) and Rep (amplification) functions in trans, the yeast FLP and RK2 plasmid trfA genes were placed under the control of the Ptet promoter/operator which is tightly regulated by the TetR repressor. The addition of heated chlortetracycline (cTc) inactivates TetR, turning on the synthesis of Flp and TrfA, which respectively, execute (i) excision of the 51-kb genomic segment between the two FRT sites (in lacZ and in phoB), and (ii) its amplification.
Genome Research | 2002
Jadwiga Wild; Zdenka Hradecna; Waclaw Szybalski
Virology | 1967
Zdenka Hradecna; Waclaw Szybalski
Nucleic Acids Research | 1994
György Pósfai; Michael Koob; Zdenka Hradecna; Noaman Hasan; Marcin Filutowicz; Waclaw Szybalski
Journal of Cellular Physiology | 1969
Waclaw Szybalski; Kjell Bøvre; M. Fiandt; Arabinda Guha; Zdenka Hradecna; S. Kumar; Homer A. Lozeron; R. V. M. Maher; H. J. J. Nijkamp; William C. Summers; K. Taylor
Nature | 1969
Sushil Kumar; Kjell Bøvre; Arabinda Guha; Zdenka Hradecna; Veronica Mary Maher; Waclaw Szybalski
Virology | 1969
Zdenka Hradecna; Waclaw Szybalski
Gene | 1998
Jadwiga Wild; Marian Sektas; Zdenka Hradecna; Waclaw Szybalski
Archive | 2001
Waclaw Szybalski; Jadwiga Wild; Zdenka Hradecna
Archive | 2002
Waclaw Szybalski; Jadwiga Wild; Zdenka Hradecna