Construction of a new plasmid vector that can express cloned cDNA in all translational reading frames.
Construction of a bacterial expression vector, pSI4001, is described. The vector contains the lac promoter-operator and three sets of ribosome-binding sites (RBSs) tandemly arranged in all possible reading frames. cDNA can be directly cloned downstream from these translational start points in the fixed and proper orientation by using the method of Okayama and Berg [Mol. Cell. Biol. 3 (1982) 280-289]. The open reading frame of any cDNA inserted may be automatically aligned in phase with either of the three ATG start codons, thus enabling its expression with a maximum theoretical probability of unity. Fusion with the lacZ gene (coding for beta-galactosidase) has shown that at least two of the three translation initiation sites exhibit high expression capacities and the remaining one can also function at a lower but significant rate. We used the vector to construct a bovine pituitary cDNA library, from which clones coding for prolactin were detected by immunological screening with an efficiency as high as two in three clones. The construction with triple RBSs should also provide a unique experimental model to study the regulation of overlapping translations.
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002417:Cattle; D003001:Cloning, Molecular / Q000379:methods; D004247:DNA / Q000235:genetics*; D005786:Gene Expression Regulation; D005822:Genetic Vectors; D010902:Pituitary Gland / Q000502:physiology; D010957:Plasmids; D011388:Prolactin / Q000235:genetics; D014176:Protein Biosynthesis; D012270:Ribosomes / Q000378:metabolism; D001616:beta-Galactosidase / Q000235:genetics
D011388:Prolactin; D004247:DNA; D001616:beta-Galactosidase