Adding to the recombineering toolbox: interrogating internal transcriptional complexity in Caenorhabditis elegans operons via recombineered fosmid-based reporters
Operons are common in prokaryotic genomes but, except for Nematoda, are uncommon in eukaryotes. Nematoda operons comprise gene clusters, containing between two to eight genes, with short, approximately. 100 bp, intergenic regions. Operons are common in C. elegans with an estimated 17% of genes clustered in operons. The significance of Nematoda operons is unclear although evidence suggests they may have arisen during genome evolution and compaction - the so-called "easy come, slow go" theory. Conventionally a discrete nascent transcript, transcribed from a major promoter upstream of the first gene in the operon, undergoes frans-splicing and 5’ addition of one of two splice-leader sequences followed by ds-splicing of the resulting pre-mRNAs to form mature, discrete mRNAs. However evidence suggests operon gene transcription is likely to be more complex. For example, microarray data indicated that correlated operon gene expression is weaker with increasing intergenic distance suggesting expression is influenced by sequence elements located internally within the operon structure. Further evidence, derived from comparison of expression data generated with fluorescent protein (FP) transcriptional reporters driven by either the upstream "operon promoter" alone or by such potential intergenic "internal promoter" sequences, generated different expression patterns in different tissues. Such data indicated the presence of internal regulatory elements within a subset of operons which were subsequently termed "hybrid operons". An alternative method to further dissect the presence of such internal regulatory elements would be to utilize translational style reporters built directly from genomic clones in which an entire operon structure is contained within the insert sequence. Tagging each operon gene with a different FP would permit operon gene expression analysis within the genomic context of that operon. To facilitate construction of fosmid-based translational reporters via seamless counter-selection recombineering two resource sets were first constructed. The first of these contained FP coding sequences synthesized de novo and codon-optimized for expression in C. elegans. The second set comprised a series of constructs designed to streamline the recombineering method. A subset of these resources was used to insert FP coding sequences seamlessly into the 3’ ends of genes within three- (CEOP1312) and two- (CEOP1358) gene-containing operons each of which was located centrally on a different fosmid genomic clone insert. Comparative genomic analyses indicated that operon CEOP1312 was conserved across all available Caenorhabditis spp whereas, in contrast, operon CEOP1358 was not. Further investigation of the conservation of CEOP1312 within the Caenorhabditis spp lead tothe identification of a GC-rich non-coding sequence conserved in the intergenic region between the second and third genes in all the CEOP1312 orthologous operons. Making use of the available resource sets counter-selection recombineering was utilized to construct a series of fosmid-based translational reporters in which this conserved sequence was precisely, and seamlessly, manipulated. Although the absolute conservation of this non-coding sequence is strongly suggestive of functionality worms transgenic for these different constructs displayed essentially equivalent expression patterns for all CEOP1312 genes indicating that any such function may not, in this case, involve internal transcriptional regulation of one or more of the operon genes.
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