Selecting Open Reading Frames From DNA

Paola Zacchi

Daniele Sblattero

Fiorella Florian

Roberto Marzari

Andrew Bradbury

^{SISSA, Trieste, Italy;}^{Dipartimento di Biologia, Università di Trieste, Trieste, Italy;}^{Bioscience Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545, USA}

^{These authors contributed equally to this work.}

^{Corresponding author.}

Received 2002 Oct 1; Accepted 2003 Mar 4.

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 β-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 β-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.

Abstract

Only ∼1.5% of the human genome comprises functional ORFs encoded by genes (Lander et al. 2001; Venter et al. 2001). The remaining 98.5% comprises RNA genes, control elements, structural elements, repeat regions, and what has been termed junk DNA. One goal of the human genome project is the identification of all human genes, and consequently the polypeptides encoded by these genes. Attempts to carry this out in silico, using EST and whole-genome sequence information, analyzed with appropriate programs (e.g., Xu and Uberbacher 1997), are having some success; however, true functional analysis of the activities of the products encoded by these genes will always require access to the physical pieces of DNA containing these genes. This has been tackled for Caenorhabditis elegans by a systematic amplification of the open reading frames (ORFs) of all predicted genes (Reboul et al. 2001), with evidence for at least 17,300 genes in this organism, of which a high proportion have structures different to those predicted in silico. The cloning of these ORFs using a recombinatorial system (Hartley et al. 2000) allows easy transfer to different vectors, and a similar strategy has been proposed for the human genome (Brizuela et al. 2001). This provides the potential means to generate complete collections of gene products, if high-throughput methods to consistently produce proteins could be found. Such a complete collection could potentially represent all the polypeptides expressed by an organism, and the interrogation of such a collection could be carried out in a protein chip format (Zhu et al. 2001). However, this approach, apart from the considerable investment required, suffers from the problem that not all proteins can be easily expressed and purified. An alternative method would be to randomly fragment DNA enriched in coding sequences, and to rely on the variable expression of different polypeptides to provide overlapping fragmented representation of individual genes.

Such an approach could be particularly useful in phage display, a technology originally developed to select peptide epitopes recognized by antibodies (Parmley and Smith 1988, 1989; Cwirla et al. 1990; Balass et al. 1993; Smith and Scott 1993; Yayon et al. 1993), but subsequently expanded to include the display of antibodies (Marks et al. 1991; Griffiths and Duncan 1998; Hoogenboom et al. 1998) and many other proteins (for reviews, see Co et al. 1991; Rada et al. 1991; Saggio and Laufer 1993; Clackson and Wells 1994; Soumillion et al. 1994; Bradbury and Cattaneo 1995; Choo and Klug 1995; Perham et al. 1995; Burritt et al. 1996; Cortese et al. 1996; Iba and Kurosawa 1997; Lowman 1997). Although traditional phage display has been successfully applied to gene rich bacterial genomes (Jacobsson and Frykberg 1995, 1996, 1998; Jacobsson et al. 1997) and individual genes (Parmley and Smith 1989; Du Plessis et al. 1995; Petersen et al. 1995; Wang et al. 1995; Bluthner et al. 1996, 1999), to identify antibody epitopes or binding partners, it suffers from the problem that only one clone in 18, if starting with DNA encoding an ORF, will be correctly in frame (one clone in three will start correctly, one clone in three will end correctly, and one clone in two will have the correct orientation), although experiments with synthetic peptide libraries have indicated that stop codons do not necessarily prevent display (Carcamo et al. 1998). Although this high rate of nonfunctional inserts may be tolerable when starting with DNA from a single gene or even a small gene rich genome, in which complete functional representation can be obtained with relatively small libraries, it may become impractical if using more complex DNA sources.

In general, attempts to display random ORFs on filamentous phage, such as those encoded by cDNA fragments, have not been very successful, notwithstanding the development of vectors in which random fragments are displayed at the C terminus of a Jun peptide that interacts with Fos displayed at the N terminus of p3 (Crameri et al. 1994; Crameri and Blaser 1996), at the C terminus of p3 (Fuh and Sidhu 2000), p8 (Fuh et al. 2000), p6 (Jespers et al. 1995), or at the C terminus of an artificial protein that is able to replace p8 in filamentous phage (Weiss and Sidhu 2000), although successful display of a cDNA library was recently reported (Butteroni et al. 2000). Greater success appears to have been achieved with λ-based vectors for cDNA display (Santini et al. 1998; Beghetto et al. 2001). However, even though such C-terminal intracellular vectors increase the likelihood that ORFs will be displayed, they do not per se provide any selective pressure for ORFs.

This indicates the need for a selective step to filter DNA fragments encoding ORFs away from those that do not. Conceptually, the easiest way to do this would be to integrate an antibiotic selection step, in which DNA encoding an ORF permitted read-through into an antibiotic resistance gene, but DNA containing stop codons or frameshifts did not. Seehaus et al. (1992) have described a vector in which antibody genes were cloned upstream of a β-lactamase gene, with the rationale that only those antibody genes that were in frame would be capable of conferring ampicillin resistance by the creation of an antibody–lactamase fusion protein, whereas those that contained deletions or frameshifts would not. The extension of such a concept to the selection of ORFs generally, rather than just antibody genes, would have wide utility.

In this paper, we describe a vector that selects for ORFs directly within a vector with a subsequent functional context (phage display; see Fig. Fig.1).1). This is carried out by cloning random fragments upstream of a β-lactamase gene flanked by two homologous lox sites in frame with gene 3. Only those phage carrying fragments in frame with the β-lactamase are able to confer ampicillin resistance. Once selection for ORFs has occurred, the lactamase gene can be removed by Cre-recombinase-induced recombination, allowing full display of selected fragments (see Fig. Fig.1).1). We demonstrate the utility of this vector by showing that 100% of fragments randomly cloned into this vector contain ORFs, allowing us to identify epitopes from human tissue transglutaminase, an enzyme involved in protein cross-linking, recognized by a specific monoclonal antibody.

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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.

ELISA signals generated for phage containing the D1.3 scFv cloned into a standard phage display vector (pDAN5), pPAO2 with β-lactamase still present (before cre), and after β-lactamase has been removed.

The number of clones before (chloramphenicol) and after ampicillin preselection is given.

Detailed analysis of the 43 different sequenced clones, giving their lengths, the open reading frames from which they are derived, and the number of times they were isolated.

Details of clones recognized by CUB, an anti-tTG monoclonal recognizing an epitope encoded by DNA 860–768 (reverse and complemented), are given.

Acknowledgments

This work was funded by a DOE grant, DE-FG02–98ER62647 awarded to A.R.M.B. We would like to thank Brian Sauer for the gift of BS1365.

The publication costs of this article were defrayed in part by payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC section 1734 solely to indicate this fact.

Acknowledgments

Notes

E-MAIL vog.lnal@bma; FAX (505) 667-2891.

Article and publication are at http://www.genome.org/cgi/doi/10.1101/gr.861503.

Notes

E-MAIL vog.lnal@bma; FAX (505) 667-2891.

Article and publication are at http://www.genome.org/cgi/doi/10.1101/gr.861503.

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