%0 Journal Article
%T Bacterial two-hybrid screening
%A Rachel Brem
%J Genome Biology
%D 2000
%I BioMed Central
%R 10.1186/gb-2000-1-3-reports0064
%X Joung et al. test their technique by screening a library of three-finger zinc-finger proteins (derived from Zif268) which are randomized at six positions on one finger. The targets used to screen this library are sequences from the nuclear receptor element (NRE), TATA box or p53 promoters. The authors report the sequences of zinc-finger variants that bind the targets. Some are specific: they bind only one target. As results are available from a comparable phage display experiment which used the same targets, Joung et al. can compare their binders to those identified from phage display. Many of the zinc finger variants that bind the TATA and p53 sequences appear to be similar in the two cases, but those that bind the NRE are different. As a control, Joung et al. used the NRE binders identified from phage display as binders in the bacterial two-hybrid protocol. Results indicated that the phage display sequences did not bind their targets in bacteria, suggesting that the bacterial screen may be more stringent.The technique is an extension of a bacterial two-hybrid scheme previously reported by the same authors in 1997. In this implementation, targets are DNA sequences and test candidates from the library are proteins. A test candidate from the library is identified as a strong binder to the target if it can promote transcription of a reporter gene in vivo by causing interactions between a pair of mediator proteins. Joung et al. use sequences from the NRE, TATA box or p53 promoters as target DNA. The library contains variants of zinc-finger proteins and the reporter gene is the bacterial gene hisB, which encodes an enzyme of the histidine biosynthetic pathway. Their mediator proteins are Gal4 and Gal11P from yeast. It all comes together as follows. Joung et al. make one set of plasmids containing the hisB gene under the control of a promoter containing one of the targets (NRE, TATA or p53). The authors make another set of plasmids containing GAL4 fused to the gene for t
%U http://genomebiology.com/2000/1/3/reports/0064