The C. elegans heterochronic gene lin-4, a key regulator of temporal patterns of cell division and differentiation in diverse cell types of the worm has been cloned and sequenced in our laboratory. Certain features of the lin-4 sequence lead us to propose a somewhat unusual approach to the initial structural analysis of the lin-4 gene product. Experiments are designed to test the hypothesis that lin-4 does not encode a protein, but rather encodes a small regulatory RNA that controls the level of lin-14 protein during development. Probes from candidate transcribed regions will be used in Northern blot, nuclease protection and PCR experiments to identify an RNA product. The lin-4 gene will be isolated from diverse nematode species and the nucleotide sequences will be compared. Essential structural features of a lin-4 RNA product will be deduced from the conserved DNA sequences. The C. elegans lin-4 gene will be mutagenized in vitro to create frameshift and base substitution mutations designed to test the hypothesis that lin-4 does not encode a protein, and to test key elements of proposed RNA structures. DNA transformation will be used to test lin-4 genes from other nematode species for the ability to function in C. elegans. Regulatory and structural sequences required for function in C. elegans will be mapped by systematically constructing and testing in vivo interspecific recombinant genes.***//
