Isolation of dominant negative mutants and inhibitory antisense RNA sequences by expression selection of random DNA fragments.
Selective inhibition of specific genes can be accomplished using genetic suppressor elements (GSEs) that encode antisense RNA, dominant negative mutant proteins, or other regulatory products. GSEs may correspond to partial sequences of target genes, usually identified by trial and error. We have used bacteriophage lambda as a model system to test a concept that biologically active GSEs may be generated by random DNA fragmentation and identified by expression selection. Fragments from eleven different regions of lambda genome, encoding specific peptides or antisense RNA sequences, rendered E. coli resistant to the phage. Analysis of these GSEs revealed some previously unknown functions of phage lambda, including suppression of the cellular lambda receptor by an 'accessory' gene of the phage. The random fragment selection strategy provides a general approach to the generation of efficient GSEs and elucidation of novel gene functions.
Holzmayer
T A
TA
Department of Genetics, University of Illinois, Chicago 60612.
Pestov
D G
DG
Roninson
I B
IB
D001425:Bacterial Outer Membrane Proteins; D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D004279:DNA, Viral / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D005799:Genes, Dominant / Q000235:genetics*; D016153:Genes, Suppressor / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D018272:Porins; D016372:RNA, Antisense / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics; D011991:Receptors, Virus / Q000235:genetics; D013489:Suppression, Genetic / Q000235:genetics*; D014764:Viral Proteins / Q000235:genetics
D001425:Bacterial Outer Membrane Proteins; D004279:DNA, Viral; D018272:Porins; D016372:RNA, Antisense; D012367:RNA, Viral; D011991:Receptors, Virus; D014764:Viral Proteins; C016532:maltoporins