UBC Theses and Dissertations
The developmental expression of the Dictyostelium discoideum ras gene and preliminary detection of a second ras-homologous sequence in its genome Gray, Virginia Elaine
The expression of a mammalian ras gene analog was previously found by Reymond et al. to be developmentally regulated in Dictyostelium discoideum using Northern analysis of strain AX-3 RNA (1984, Cell 39;141) and by Pawson et al. using specific immunoprecipitation of in vivo synthesized proteins from strain V12M2 (1985, Mol. Cell Biol. 5;33). Due to differences in the results of the two studies, it was decided to further examine ras expression by applying both protein and RNA techniques to a single strain of D.discoideum, V12M2. RNA samples from strain V12M2 cells at different stages of development were analyzed using Northern blotting. The same RNAs were translated in vitro, and the ras proteins synthesized were immunoprecipitated and analysed by polyacrylamide gel electrophoresis. In agreement with the findings of Reymond et al. (1984, Cell 39;141), Northern analysis with the cDNA ras probe revealed that the highest levels of the 1.2 and 0.9 kb ras mRNAs were present in the total RNA of V12M2 cells at the pseudoplasmodial stage of development, and very little ras mRNA was present in early developing cells. In contrast to the Northern analysis the greatest amount of ras protein was in vitro translated from the RNA of vegetative and 2 hour cells. Hence this work confirms in a single strain of Dictyostelium that the greatest amount of ras protein is synthesized at those developmental stages that contained the lowest levels of mRNA detectable by the cDNA probe. Possible reasons for this phenomena are discussed. In vitro RNA translation was also used to study the relationship between the two ras proteins of 23 and 24 kd. The proteins did not appear to be derived from one another by degradation or by post-translational modification. This result suggested that the two ras proteins of strain V12M2 must be derived from two different mRNAs. High stringency Southern blots of AX-3 DNA showed the expected restriction fragments detected by Reymond et al. (1984, Cell 39;141) . Low stringency blots showed three faint additional restriction fragments in Eco RI digests of AX-3 DNA. No additional restriction fragments were generated by an Eco RI-Bg1 II digest, but two of the three faint bands were smaller. This suggested that at least two of the Eco RI ras fragments are non-contiguous, and hence two to three ras genes may be present in addition to the one characterized by Reymond et al. (1984, Cell 39;141). All Northern and Southern bolts were probed with antisense RNA probes in order to gain greater sensitivity of detection as described by Cox et al. (1984, Dev. Biol. 101;485).