Grant IM et al. (2015), The Xenopus ORFeome: A resource that enables fu...

XB-ART-51300

Dev Biol 2015 Dec 15;4082:345-57. doi: 10.1016/j.ydbio.2015.09.004.

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The Xenopus ORFeome: A resource that enables functional genomics.

Grant IM, Balcha D, Hao T, Shen Y, Trivedi P, Patrushev I, Fortriede JD, Karpinka JB, Liu L, Zorn AM, Stukenberg PT, Hill DE, Gilchrist MJ.

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Functional characterisation of proteins and large-scale, systems-level studies are enabled by extensive sets of cloned open reading frames (ORFs) in an easily-accessible format that enables many different applications. Here we report the release of the first stage of the Xenopus ORFeome, which contains 8673 ORFs from the Xenopus Gene Collection (XGC) for Xenopus laevis, cloned into a Gateway® donor vector enabling rapid in-frame transfer of the ORFs to expression vectors. This resource represents an estimated 7871 unique genes, approximately 40% of the non-redundant X. laevis gene complement, and includes 2724 genes where the human ortholog has an association with disease. Transfer into the Gateway system was validated by 5' and 3' end sequencing of the entire collection and protein expression of a set of test clones. In a parallel process, the underlying ORF predictions from the original XGC collection were re-analysed to verify quality and full-length status, identifying those proteins likely to exhibit truncations when translated. These data are integrated into Xenbase, the Xenopus community database, which associates genomic, expression, function and human disease model metadata to each ORF, enabling end-users to search for ORFeome clones with links to commercial distributors of the collection. When coupled with the experimental advantages of Xenopus eggs and embryos, the ORFeome collection represents a valuable resource for functional genomics and disease modelling.

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Species referenced: Xenopus laevis
Genes referenced: ids kif2c picalml
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	Fig. 1. Schematic overview of generation of the Xenopus ORFeome, v1.0. (a) PCR primers were designed to 9805 ORFs from the XGC cDNA library. (b) The PCR products were EST sequenced at 5â€² and 3â€² ends, and these sequences were BLASTed against the XGC ORFs. (c) BP recombination cloning reaction with the donor vector, pDONR223 gives the bare ORF in a Gateway entry construct. (d) 8673 Gateway entry clones were consolidated based on confirmation of identity from sequencing to produce Xenopus ORFeome v1.0. (e) Imunoblots of 17 proteins after transfer from Xenopus ORFeome Gateway entry vectors into a C-terminal GFP-tagged destination vector after in vitro translation. The different proteins act as internal controls for each other in this experiment, success is indicated by the relative positions of bands of predicted size, compared to standards (Bio-Rad). Weights are given in kDa, and include 36 kDa for the LAP tag (GFP-TEV-S) fusion. Faint bands may be caused by ribosomes starting at incorrect start sites, or from post-translational modifications.
	Fig. 2. Testing the full-length status of the XGC ORFs (a) using protein alignment: translated protein alignments to non-Xenopus species yields a predicted position for the start codon on a zero-based scale running from 5′ to 3′ along the length of the cDNA sequence. The position of the XGC-defined start codon on the clone cDNA sequence is marked as AA. The start of conserved protein alignment on the XGC cDNA sequence is labelledCC, and PP is the predicted position of the start codon based on the length of the protein sequence, i.e. P=C−cP=C−c, where cc is the position of the start of conservation on the non-Xenopus protein sequence. The minimum possible value of CC is zero, but PP can be negative. For each XGC ORF, PP and CC were averaged over all the respective homologous protein alignments to produce single, average values for P¯ and C¯, respectively. (b) Analysis of the XGC ORFs, over all tests, predicts 85% of the Xenopus ORFeome v1.0 to be full-length (89% including conserved ORFs, see Section 2). (c) Frequency distribution of open reading frame lengths in the source XGC cDNA library and the consolidated Xenopus ORFeome collection, showing equal likelihood of cloning long and short ORFs.
	Fig. 3. Truncated but conserved ORFS. Examples of genes where the open reading frame in X. laevis is slightly longer than in other species, including X. tropicalis, suggesting that the shorter ORFeome clone, based on the XGC predicted ORF, is conserved and likely to be functional. Sections of EST clusters showing aligned nucleotide sequences (ESTs) with in-frame stop and ATG codons in red and green respectively. ORF predicted from aligned EST codons is in purple. Start of the XGC reference ORF is indicated by arrow. Protein alignments are shown above in turquoise, with the leftmost ‘P’ being the predicted relative position of the N-terminal end of these proteins. (a) The extremely highly conserved N-terminus of histamine N-methyl transferase is extended by 15 bp. (b) The moderately conserved N-terminus of platelet-activating factor receptor is extended by 6 bp compared to Xenopus tropicalis, and both are longer then the chick, human, mouse and zebrafish proteins. Note the erroneous prediction for the Xenopus laevis protein in both cases. (c) Distribution of the numbers of lost N-terminal residues between the shorter, conserved XGC reference ORFs and the longer true ORFs in Xenopus laevis.
	Fig. 4. Example alignments of XGC cDNA sequences and matching EST clusters. (a) Brain and reproductive organ-expressed protein (TNFRSF1A), illustrating divergence between the XGC sequence and the cluster consensus sequence, and adding 60 bp to an upstream ATG. Genomic alignment indicates (data not shown) this to be a non-canonical exon; we note that the XGC prediction was for the canonical start of translation. (b) CWF19-like 2, cell cycle control (cwf19l2), illustrating 5â€² truncation of the entire cDNA sequence (i.e. the ORF and 5â€² UTR). The EST cluster and conserved homologous peptide sequences contain additional sequence that is not present in the 5â€² UTR of the XGC clone. Some interposing sequence is not shown; the numbered, horizontal scale has been clipped and compressed for clarity. (c) Lymphocyte cytosolic protein 1 (L-plastin), illustrating a 5â€² truncated XGC ORF due to erroneous identification of the correct start codon. In this case, the 5â€² UTR of the XGC cDNA contains the extra matching coding sequence from the EST cluster, and is supported by matching alignment of conserved homologous peptide sequence from non-Xenopus species.

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