Identification and analysis of internal promoters in Caenorhabditis elegans operons

Abstract

The current Caenorhabditis elegans genomic annotation has many genes organized in operons. Using directionally stitched promoterGFP methodology, we have conducted the largest survey to date on the regulatory regions of annotated C. elegans operons and identified 65, over 25% of those studied, with internal promoters. We have termed these operons "hybrid operons." GFP expression patterns driven from internal promoters differ in tissue specificity from expression of operon promoters, and serial analysis of gene expression data reveals that there is a lack of expression correlation between genes in many hybrid operons. The average length of intergenic regions with putative promoter activity in hybrid operons is larger than previous estimates for operons as a whole. Genes with internal promoters are more commonly involved in gene duplications and have a significantly lower incidence of alternative splicing than genes without internal promoters, although we have observed almost all trans-splicing patterns in these two distinct groups. Finally, internal promoter constructs are able to rescue lethal knockout phenotypes, demonstrating their necessity in gene regulation and survival. Our work suggests that hybrid operons are common in the C. elegans genome and that internal promoters influence not only gene organization and expression but also operon evolution.

Figure 1.

Schematic diagrams and expression patterns of the CEOPX040 (klp-8) operon and the CEOP3332 operon. (A) The klp-8 operon. Both the operon promoter (PG-L) for the leading gene C15C7.2 and the internal promoter (PG-D) for the downstream gene C15C7.1 have been extracted. (Green rectangles) Exons; (thin lines) introns; (thick red lines) intergenic regions; (gray areas) UTRs. (PO) The promoter of an operon; (PG-L) a promoter∷GFP constructed from the leading gene of an operon; (PG-D) a promoter∷GFP constructed from the corresponding operon downstream gene. (B) I and II show that the promoter extracted from leading gene C15C7.2 is able to drive GFP expression in the excretory cell, pharynx, pharyngeal neurons, and head neurons, while the promoter extracted from downstream gene C15C7.1 directs GFP expression in the seam cells (III and IV). (C) The CEOP3332 operon. Both the operon promoter and the internal promoter for downstream gene B0336.2 are used to construct GFP fusions. (D) I and II show that the promoter extracted from leading gene B0336.3 is able to drive GFP expression in hypodermis, pharyngeal gland cell, gut, and nerve ring, while that of downstream gene B0336.2 is able to drive GFP expression in the pharynx, gut, and head neurons (III and IV).

Figure 2.

Gene cDNA coverage. Percent of genes with corresponding sequenced ESTs or full-length cDNAs.