Signal-exon trap: a novel method for the identification of signal sequences from genomic DNA.
We describe a genomic DNA-based signal sequence trap method, signal-exon trap (SET), for the identification of genes encoding secreted and membrane-bound proteins. SET is based on the coupling of an exon trap to the translation of captured exons, which allows screening of the exon-encoded polypeptides for signal peptide function. Since most signal sequences are expected to be located in the 5'-terminal exons of genes, we first demonstrate that trapping of these exons is feasible. To test the applicability of SET for the screening of complex genomic DNA, we evaluated two critical features of the method. Specificity was assessed by the analysis of random genomic DNA and efficiency was demonstrated by screening a 425 kb YAC known to contain the genes of four secretory or membrane-bound proteins. All trapped clones contained a translation initiation signal followed by a hydrophobic stretch of amino acids representing either a known signal peptide, transmembrane domain or novel sequence. Our results suggest that SET is a potentially useful method for the isolation of signal sequence-containing genes and may find application in the discovery of novel members of known secretory gene clusters, as well as in other positional cloning approaches.
Péterfy
M
M
Department of Biomedical Science, Amgen Inc., Thousand Oaks, CA 91320, USA.
Gyuris
T
T
Takács
L
L
D000818:Animals; D019556:COS Cells; D003001:Cloning, Molecular / Q000379:methods*; D003176:Complement C3 / Q000235:genetics; D004247:DNA; D018819:Dipeptidyl Peptidase 4 / Q000235:genetics; D005091:Exons; D017930:Genes, Reporter; D005821:Genetic Techniques; D005822:Genetic Vectors; D016678:Genome; D006801:Humans; D007378:Interleukins / Q000235:genetics; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D021382:Protein Sorting Signals / Q000235:genetics* / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D014158:Transcription, Genetic
D003176:Complement C3; D007378:Interleukins; D008565:Membrane Proteins; D021382:Protein Sorting Signals; D011506:Proteins; D011993:Recombinant Fusion Proteins; D004247:DNA; D018819:Dipeptidyl Peptidase 4