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Transposon tools for recombination DNA manipulation: characterization of transcriptional regulators from yeast, Xenopus, and mouse

Author:
Morgan, B.A., Conlon, F.L., Manzanares, M., Millar, J.B.A., Kanuga, N., Sharpe, J., Krumlauf, R., Smith, J.C., Sedgwick, S.G.
Source:
Proceedings of the National Academy of Sciences of the United States of America 1996 v.93 no.7 pp. 2801-2806
ISSN:
0027-8424
Subject:
Saccharomycetales, Saccharomyces cerevisiae, Xenopus laevis, mice, gene expression, DNA-binding proteins, plasmid vectors, reporter genes, beta-galactosidase, recombinant DNA, genetic transformation, genetic markers, genes, transgenic animals, transcription (genetics), promoter regions, mutation, Schizosaccharomyces pombe, transcription factors
Abstract:
Transposon Tn1000 has been adapted to deliver novel DNA sequences for manipulating recombinant DNA. The transposition procedure for these "tagged" Tn1000s is simple and applicable to most plasmids in current use. For yeast molecular biology, tagged Tn1000s introduce a variety of yeast selective markers and replication origins into plasmids and cosmids. In addition, the beta-globin minimal promoter and lacZ gene of Tn beta lac serve as a mobile reporter of eukaryotic enhancer activity. In this paper, Tn beta lac was used to localize a mouse HoxB-complex enhancer in transgenic mice. Other tagged transposons create Ga14 DNA-binding-domain fusions, in either Escherichia coli or yeast plasmids, for use in one- and two-hybrid tests of transcriptional activation and protein-protein interaction, respectively. With such fusions, the Saccharomyces cerevisiae Swi6 G1/S-phase transcription factor and the Xenopus laevis Pintallavis developmental regulator are shown to activate transcription. Furthermore, the same transposon insertions also facilitated mapping of, the Swi6 and Pintallavis domains responsible for transcriptional activation. Thus, as well as introducing novel sequences, tagged transposons share the numerous other applications of transposition such as producing insertional mutations, creating deletion series, or serving as mobile primer sites for DNA sequencing.
Agid:
2355963