Worm Breeder's Gazette 17(1): 42 (October 1, 2001)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
Department of Molecular Genetics, Ohio State University, Columbus, OH 43210
Comparative analysis shows that proteins can be conserved entities throughout the animal kingdom, but their regulation can change during the evolutionary process. Therefore, a meaningful investigation of the molecular changes underlying gene evolution can be provided by comparison of noncoding regulatory regions from closely related species. Previous studies have characterized the C.elegans ovo-related gene lin-48 as a downstream gene of the Pax factor EGL-38, and showed lin-48 functions with egl-38 in the development of hindgut (1). In C. elegans, lin-48 encodes a C2H2 zinc finger protein. It is expressed in head sensory neuron support cells and excretory duct cell as well as hindgut cells. In preliminary studies, we showed that in C. briggsae, the C. elegans lin-48 (Ce-lin-48) promoter was able to drive gfp (green fluorescent protein) expression in the same hindgut cells and neuron support cells as it does in C. elegans. However, expression in the excretory duct cell was essentially eliminated. This result indicated that there is a difference between C. elegans and C. briggsae in either the function or regulation of lin-48.
To investigate the molecular nature of this difference, we have isolated the C. briggsae lin-48 gene (Cb-lin-48) from a fosmid clone. A C. briggsae 6.2kb Sal I subclone containing the lin-48 gene could rescue the hindgut and male spicule defects in C. elegans lin-48(sa469) mutants. To test the fuction of lin-48 in C. briggsae, we used RNAi. RNAi treated animals from both species showed low but reproducible levels of lethality, which is similar to C. elegans lin-48 chromosomal mutations (2).
We constructed gfp reporter transgenes to investigate the expression pattern of Cb-lin-48. Cb-lin-48::gfp is expressed only in hindgut cells and neuronal support cells in C. briggsae animals, as was observed for the C. elegans gene. This result suggests the expression pattern of lin-48 is different between C. elegans and C. briggsae. Cb-lin-48::gfp in C. elegans animal is likewise expressed only in hindgut cells and neuronal support cells. Thus, at least part of the difference between lin-48 in C. elegans and C. briggsae results from differences in the regulatory sequences. To narrow down the sequences that are critical for the regulation differences between the two species, we did a series of swapping tests. The basic idea behind this is that if any sequences within the regulatory region are important for the regulation differences, the swapping of C.elegans sequences into C.briggsae lin-48 should recover the expression in the excretory duct cell. One swapping clone made up of C.briggsae distal region and C.elegans proximal region showed high excretory duct cell expression. To further narrow down the proximal region, we constructed another swapping clone including 2.4kb C. briggsae distal and about 500bp proximal C. elegans sequence. This clone is able to drive expression of gfp in the excretory duct cell, indicating that this 500bp piece of C. elegans sequence is sufficient for lin-48 transcriptional regulation in this cell. Currently, we are performing a deletion analysis of the C. elegans sequences in this clone. With deletion clones, it is our hope that we will be able to pinpoint the sequence changes that are responsive for the evolutionary changes in gene transcriptional regulation.