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.