Analysis of the maize (Zea mays L) genome using molecular, genetic and computational approaches

Abstract

Cloned open reading frames (ORFs) are a resource for gene discovery, peptide expression and display, and novel/hybrid peptide or enzyme discovery. Vectors have been developed to select ORFs from genomic DNA in vivo. One of the major disadvantages of these existing ORF-selecting vectors is that only ORFs that are inframe with both the 5' initiation codon and the 3' reporter protein/tag can be selected, reducing the efficiency of ORF selection nine fold. To overcome these limitations, a bacterial expression vector (pORF-Rescue) has been developed that includes two novel features: 1) three initial ATG codons (3xATGs) 5' of the multiple cloning site (MCS); and 2) a three-frame His6-tag (3xHise) 3' of the MCS. These features help initialize translation of all three reading frames of a DNA insert and tag the peptide(s) translated from any ORF(s) of that DNA insert. The ability of pORFRescue to efficiently enrich for ORFs from a complex genome was demonstrated using maize genomic and BAC DNA. Finally, the integration of the 3xATGs and 3xHis6 features of pORF-Rescue into mRNA display systems may improve the efficiency of in vitro peptide display.