| Description |
The vertebrate pro-opiomelanocortin (POMC) gene codes for the large protein precursor to a number of small peptides with highly conserved sequence, including adrenocorticotropin (ACTH), melanocyte stimulating hormone (MSH), and $/beta$-endorphin. previous evidence suggests that structurally and possibly functionally related sequences (homologs) are present in unicellular organisms, and that these may be encoded within a similarly organized genes. This work attempted to identify a POMC homolog in the protozoan, Tetrahymena pyriformis, and in fission yeast, Saccharomyces cerevisiae. Using two different equilibrium-type radioimmunoassays with polyclonal antibodies, I initially identified ACTH-like immunoactivity in extracts of both species. However, the same extracts showed no reaction in an immunoradiometric assay requiring greater structural similarity to vertebrate ACTH. The low concentration meant purification of adequate amounts of material to sequence was impractical. Southern analysis of Tetrahymena genomic DNA was unfruitful in identifying POMC-related sequences. However, yeast genomic DNA showed hybridizing bands when probed at low stringency with the rat POMC gene. Screening a yeast genomic DNA library under low stringency resulted in identification of one of the DNA sequences responsible. Analysis of hybridizing DNA showed a long open reading frame coding for a putative protein of 610 amino acids, showing 18% identity and 45% chemical similarity to the rat POMC precursor, with 30% identity to the vertebrate ACTH sequence. There was no significant homology to other vertebrate POMC-derived peptides, and the protein showed no features suggesting cleavage into smaller peptides. The presence of a leucine zipper motif and multiple phosphorylation sites indicated an ability to dimerize and possibly bind DNA. A second open reading frame was also found on the opposite DNA strand 500 bases upstream, showing strong homology to DNA-unwinding proteins. A poly(dA-dT) stretch between both genes may function as a common regulatory sequence. Northern analysis indicated the ORF1gene was expressed. However, deletion and overexpression analysis showed it was not likely to be responsible for the measured immunoactivity, that it was not essential, and that it had no effect on growth rate under normal aerobic conditions. Significance of the sequence similarity to POMC and possible functions are discussed. |
