The aim of this proposal is to further the understanding of the heterochronic gene pathway of the nematode C. elegans. Heterochronic genes are global developmental timing genes. In gain-of-function and loss-of-function mutations for these genes, diverse developmental events occur at earlier or later stages than normal. Previous work of this investigator as well as others has defined a pathway comprising some 8 genes that give mutant phenotypes of this type. Present work is focussed on how the action of these genes is regulated, and how they in turn act to control development.
The aims of the present proposal fall into three broad areas: to continue to define the identities and properties of new and existing genes of the pathway, to study in detail the novel mechanism of action of one pathway component, lin-4, and to study how the heterochronic genes control two developmental events, development of the vulva and entrance into the dauer pathway. In the first area, Dr. Ambros proposes to clone three known genes of the heterochronic pathway, lin-41, lin-42, and lin-46, in order to determine the sequence of their gene products and to study their expression patterns. Two of the heterochronic genes already cloned, lin-14 and lin-28, are thought to act at the same or parallel steps of the pathway. One of these, lin-14, has a novel product while the other, lin-28, encodes an apparent transcription factor with both DNA and RNA binding motifs. Dr. Ambros will determine whether the gene products of lin-14 and lin-28 interact by means of the yeast 2-hybrid system, by attempting to co-immunoprecipitate them from nematode extracts, and by determining whether over-expression of either can cause a dominant mutant phenotype. He will further explore the mechanism of action of lin-28 by determining whether it is regulated by lin-4 in the same antisense manner that lin-14 is, by determining whether LIN-28 protein precipitates with an hnRNP fraction, suggesting the RNA-binding domain is functional, and by attempting to define DNA and RNA binding targets by the Selex PCR procedure. A variety of mutant screens are proposed to identify new genes of the heterochronic gene pathway. Finally, mosaic analysis of lin-4, lin-14, and lin-28 will be carried out to determine their focus of action. In the second area, Dr. Ambros wishes to understand in greater detail how the gene lin-4, the first known gene of the heterochronic gene pathway, regulates its apparent immediate downstream target gene, lin-14. Dr. Ambros has shown that lin-4 does not encode a protein product, but instead appears to encode a 22 nt antisense RNA product. Dr. Ambros proposes a variety of experiments to test the current working model for how lin-4 represses lin-14, namely, by an antisense mechanism involving base-pairing with a complementary region in the 3'UTR of lin-14 mRNA, thereby blocking elongation of translation. Dr. Ambros wishes to demonstrate the reality of the base-pairing interaction by isolating pairs of mutually suppressing mutations in lin-4 and lin-14 that result in complementary changes. He also proposes to search for such complementary changes by comparing lin-4 and lin-14 sequences from other nematode species. A variety of experiments are proposed to demonstrate basepairing between lin-4 and lin-14 RNA's, to measure the rate of lin-14 translation in vivo, to demonstrate the presence of lin-4 RNA in nematode polysomes, to identify proteins that interact with lin-4 and lin-14 RNA's, to demonstrate an effect of lin-4/lin-14 RNA interaction on translation in monkey kidney cells (in collaboration with Margaret Baron of Harvard), or in a nematode cell-free translation system, to define the biochemical structure of the lin-4 RNA product, and to look for genes with sequences that might suggest that they are also regulated by interaction with lin-4 RNA. Mutant screens are proposed taking advantage of a reporter gene regulated by lin-4 to identify other genes required for its antisense function. Finally, Dr. Ambros proposes to investigate regulation of transcription of lin-4 itself by determining which RNA polymerase is responsible for transcribing it, by carrying out a promoter dissection, by attempting to identify promoter binding proteins in a yeast 1-hybrid system, and by mutant screens for alterations in lin-4 regulation. In the last section, Dr. Ambros proposes to study regulation of vulva cell lineages and dauer larva formation by the heterochronic gene pathway. He will identify and clone a gene defined by a translocation breakpoint that appears to have heterochronic effects confined to the vulval pathway. To identify additional vulva-specific genes, he will express lin-14 and lin-28 from a vulva-specific promoter and determine whether this results in a retardation of vulva development. If it does, this will be reverted to define target genes. A recent observation that timing of vulva lineages is via regulation of their cell cycle will be followed up by determining whether two vulva pattern formation genes, lin-12 and let-23, are regulated by the cell cycle, and by determining whether blocking the cell cycle reprograms vulva development in the manner that heterochronic genes appear to do. Finally, two genetic screens are proposed to identify heterochronic genes unique to postdauer development, and to identify genes responsible for regulation of dauer formation by the heterochronic gene pathway.
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