Selecting open reading frames from DNA.
We describe a method to select DNA encoding functional open reading frames (ORFs) from noncoding DNA within the context of a specific vector. Phage display has been used as an example, but any system requiring DNA encoding protein fragments, for example, the yeast two-hybrid system, could be used. By cloning DNA fragments upstream of a fusion gene, consisting of the beta-lactamase gene flanked by lox recombination sites, which is, in turn, upstream of gene 3 from fd phage, only those clones containing DNA fragments encoding ORFs confer ampicillin resistance and survive. After selection, the beta-lactamase gene can be removed by Cre recombinase, leaving a standard phage display vector with ORFs fused to gene 3. This vector has been tested on a plasmid containing tissue transglutaminase. All surviving clones analyzed by sequencing were found to contain ORFs, of which 83% were localized to known genes, and at least 80% produced immunologically detectable polypeptides. Use of a specific anti-tTG monoclonal antibody allowed the identification of clones containing the correct epitope. This approach could be applicable to the efficient selection of random ORFs representing the coding potential of whole organisms, and their subsequent downstream use in a number of different systems.
Zacchi
Paola
P
SISSA, Trieste, Italy.
Sblattero
Daniele
D
Florian
Fiorella
F
Marzari
Roberto
R
Bradbury
Andrew R M
AR
D000911:Antibodies, Monoclonal; D001483:Base Sequence / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods; D004247:DNA / Q000032:analysis* / Q000235:genetics; D000939:Epitopes / Q000235:genetics / Q000276:immunology; D015723:Gene Library; D005822:Genetic Vectors / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D016366:Open Reading Frames / Q000235:genetics*; D016364:Reading Frames / Q000235:genetics; D011995:Recombination, Genetic / Q000235:genetics; D011503:Transglutaminases / Q000235:genetics / Q000276:immunology
D000911:Antibodies, Monoclonal; D000939:Epitopes; D004247:DNA; D011503:Transglutaminases