Selecting open reading frames from DNA

Abstract

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.

Figure 1.

The scheme for open reading frame selection. The scheme for selecting DNA fragments encoding ORFs. Random fragments are cloned upstream of a β-lactamase gene. Those fragments that are ORFs permit readthrough into the β-lactamase gene and confer ampicillin resistance. Those that are out of frame, or contain stop codons, do not survive. After selection on ampicillin, the β-lactamase gene can be removed by passage through bacteria expressing Cre recombinase. The selected ORF can then be displayed on phage.

Figure 2.

pPAO2, plasmid map, and polylinker sequence. (A) The plasmid map of pPAO2. (B) The polylinker sequence between the HindIII and EcoRI sites of pUC119. The amino acid sequence of the in-frame construct is given above the DNA sequence. FS indicates a frameshift, which can only be overcome if an in-frame DNA fragment is cloned via the LIC approach. (C) The LIC cloning procedure in detail.

Figure 2.

pPAO2, plasmid map, and polylinker sequence. (A) The plasmid map of pPAO2. (B) The polylinker sequence between the HindIII and EcoRI sites of pUC119. The amino acid sequence of the in-frame construct is given above the DNA sequence. FS indicates a frameshift, which can only be overcome if an in-frame DNA fragment is cloned via the LIC approach. (C) The LIC cloning procedure in detail.

Figure 3.

Only clones with open reading frames survive on ampicillin. The D1.3 scFv, or an out-of-frame derivative, were cloned into pPAO2, and bacteria were plated on different concentrations of chloramphenicol or ampicillin with or without 1% glucose. Chloramphenicol resistance is encoded by the backbone of the plasmid, whereas ampicillin resistance is generated by the presence of in-frame fusions (see Fig. 2). Clone survival on ampicillin is expressed as a percentage of the number of clones growing on chloramphenicol.

Figure 4.

Ampicillin selection generates randomly sized open reading frames. (A) 20 random clones taken from a plasmid-generated fragment library, after selection on ampicillin and passage through a Cre-expressing strain to remove β-lactamase, were amplified with primers spanning the cloning site. All fragments are different sizes, indicating that different random fragments have been cloned. (B) Clones from A were digested with BstNI. All clones show different fragment sizes. (C) The supernatants from 96 random clones from the same library were spotted onto nitrocellulose after induction of protein fragment expression with IPTG. As can be seen, >80% of these clones have signals indicating that an open reading frame has been selected.

Figure 5.

Sequence analysis of ampicillin-selected open reading frame clones. The pET28b-tTG plasmid is represented linearly, with all ORFs >50 amino acids (150 bp) indicated. In addition, ORFs corresponding to kanamycin, tTG, lacI, and rop are hatched. Regions of the plasmid identified after sequencing 43 clones obtained after selection on ampicillin and removal of the β-lactamase after passage through a Cre-expressing strain are shown as black lines beneath the appropriate frame.

Figure 6.

Identification of kanamycin clones. Three kanamycin-specific primers were used in two separate PCR reactions (see Methods for details) with different numbers of estimated clones from the library indicated. Amplification is noted when 100 templates are present, indicating that the kanamycin sequence is present at an approximate level of 1/100. The 15–13 primers amplify an ORF of no biological significance that is not represented in the library. The bars on the left indicate the length of the amplification products.