Protocol for the Generation of a Transcription Factor Open Reading Frame Collection (TFome)

The construction of a physical collection of open reading frames (ORFeomes) for genes of any model organism is a useful tool for the exploration of gene function, gene regulation, and protein-protein interaction. Here we describe in detail a protocol that has been used to develop the first collection of transcription factor (TF) and co-regulator (CR) open reading frames (TFome) in maize (Burdo et al., 2014). This TFome is being used to establish the architecture of gene regulatory networks (GRNs) responsible for the control of transcription of all genes in an organism. The protocol outlined here describes how to proceed when only an incomplete genome with partial annotation is available. TFome clones are made in a recombination-ready vector of the Gateway? system, allowing for the facile transfer of the ORFs to other Gateway?-compatible vectors, such as those suitable for expression in other host species. Although this protocol was developed for the maize TFome it can readily be applied to the generation of complete ORFeome collections in other eukaryotic species.[Protocol overview] An important aspect of successful TFome generation is the initial effort spent to establish a reliable set of gene models so that they can be subsequently amplified or synthesized. An actual TFome construction protocol for a particular species will depend on available resources such as a full-length cDNA (flcDNA) collection and a reliable reference genome (Figure 1). In the case of maize, a flcDNA collection and a draft genome was available, but the former provided only 30% of the needed clones, and the latter contained gaps and some erroneous gene models. In order to develop a near-complete set of target gene models for maize TFs, a bioinformatics pipeline was developed as described by Yilmaz et al. (2009). In brief, a two-pronged search process was developed. The first involved making a collection of protein sequences of TFs in other species and available from databases such as PlantTFDB, PlnTFDB and DBDTF. These sequences were then used to search gene models from the draft maize genome using BLASTP. The second process involved developing a collection of domains that define TF families and that are mostly annotated in the PFAM database (Finn et al., 2014). These domains were then used to search the draft maize genome using BLASTX. The number of TF families that exist and their naming is subject to change as new members are discovered and studied. Table 1 provides a list of kn