Molecular Analysis Of The Role Of The Fem Proteins In Caenorhabditis Elegans Sex Determination Microform PDF Download

The C. elegans sex determination hierarchy specifies either one of two sexes: the hermaphrodite or the male. The tra-1 gene is the global terminal regulator of the somatic sex determination pathway. Active TRA-1 promotes female fates in the hermaphrodite by repressing the transcription of genes that direct male development. The objective of the work presented here is to understand how the activity of TRA-1 is regulated. The tra-1 gene encodes two transcripts, tra-1a and tra-1b. Males and hermaphrodites have similar levels of tra-1 transcripts, suggesting that tra-1 is regulated post transcriptionally. Using a new antibody against TRA-1 in whole mount immunostaining I observed that TRA-1 appears to be much more abundant in hermaphrodites than in males. Western blot analysis revealed similar levels of TRA-1A and TRA-1B in lysates from males and hermaphrodites. Additional TRA-1-specific species (90-110 kDa) were present in the hermaphrodite lysates but absent from male lysates. These isoforms are the most abundant form of TRA-1 protein in hermaphrodites. The hermaphrodite-specific TRA-1 isoforms appear to be phosphoisoforms of a single TRA-1 band. Here I also describe the identification of proteins that specifically interact with FEM-2 in affinity chromoatography experiments. Loss-of-function mutants and RNAi silencing of three of these genes appear to partially phenocopy fem-2 mutants. I show evidence that the hermaphrodite-specific isoform is generated by proteolytic processing of TRA-1A. Since the truncated TRA-1A isoforms are present in embryos before sexual differentiation is apparent, they are unlikely to be a result of female differentiation. Therefore I propose that these isoforms are responsible for the feminizing activity of the tra-1 gene. The truncation mapped to the region between amino acids 842 and 921. The observations that both a nonsense mutation that results in the production of a C-terminally truncated TRA-1A protein and truncated TRA-1A transgenes are feminizing, support my hypothesis that the truncated TRA-1 isoforms are responsible for the feminizing activity of the tra-1 gene. Genetic analyses indicate that the fem gene products regulate, directly or indirectly, the activity of TRA-1. Therefore, characterization of these putative FEM-2 binding proteins may identify the mechanism by which the FEMs regulate TRA-1 activity.