The Quantitative Analysis Of Allantoicase And Related Genes During Dictyostelium Discoideum Development

The social amoeba Dictyostelium discoideum has a unique life cycle consisting of a unicellular growth phase and a multicellular developmental phase. Under starvation conditions, the solitary amoeboid cells initiate a developmental process and then undergo a series of morphogenesis, including the stage of aggregation, mound, slug and fruiting body. Though the multi-cellular development process is relatively short (24h), it presents the cell differentiation and collaboration which is similar to higher organisms. So Dictyostelium discoideum is a well-established model arganism for the study of basic aspects of cell development, differentiation, signal transduction, apoptosis, phagocytosis and cell motility.gp150which encoded by lagC gene functions as a cell-cell adhesion molecules that is required for multicellular development. The mutant strain AK127, in which lagC is disrupted, couldn't express the cell adhesion molecule gp150, as a result, the AK127cells can't complete multicellular development and the development is arrested at the loose aggregate stage. Recently, allantoicase (allC gene fragment was screened using mRNA differential display when compared the wild type KAx-3strain and the mutant AK127strain. This suggests that allantoicase also exists in D.discoideum. Allantoicase is an important enzyme of purine degradation, which catalyzes the hydrolysis of allantoic acid to ureidoglycolate. In animals, the end products of purine degradation vary greatly from species to species. To study the function of allantoicase in D.discoideum, the RNAi expression vector pAct15Gal-allCi was constructed and then transfected into the wild type KAx-3by electorporation. Positive clones were obtained by stable transfection KAx-3using pActl5Gal-allCi and G418screening. Transfected cells, morphogenesis in this allC RNAi mutant strain is blocked at the loose aggregate stage too, which is similar to the morphogenesis of AK127. Therefore, allantoicase is required for the development of D. discoideum. It is still unknown the interacting proteins of allantoicase, the signaling pathway and how does the allantoicase work in D.discoideum.To test proteins interacted with allantoicase in D.discoideum by the Co-immunoprecipitation, mass spectrometer detected two clear protein bands and the results showed that the two proteins were40S ribosomal protein S3(rpS3) and40S ribosomal protein S16(rpS16). It also had been found gp150had close relation with rpS3by the same way.Real-time quantitative PCR analyzed the expression of the genes allC, lagC, rpS3and rpS16during the development of D.discoideum. In KAx-3cells, the genes allC and lagC had similarly expression patterns, each of them reached the highest expression and then decreased.The expression of allC reached peak at16h, while the gene of lagC started to expression at8h and reached the highest level at12h. Whereas the expression patterns of rpS3and rpS16decreased substantially during all morphogenesis. However, all the gene expression maintained stable pattern and had no significant fluctuations in the mutant strain AK127and RNAi-allC. Obviously, the genes expression level in KAx-3cells was different from those in mutant cells during the multicellular development. Disrupted lagC and interference allC affected the related gene expression. What's more, the allantoicase was studied in stalk cells and spore cells respectively. The results from Western blot showed that the expression of allantoicase in prestalk cells was more than in prespore cells, which was consistent with the gp150. It is also implied allantoicase involved in the cell differentiation and had the closely relationship with gp150.To sum up, these results demonstrated that allC has some relationship with lagC, rpS3and rpS16.All of them may contribute to the regulation of cell differentiation and the multicellular development of D.discoideum. This represents a significant step forward in our understanding of protein function and has significance in the signal conduction during the development of D.discoideum.While we still unknow the gene allC interects with lagC, rpS3and rpS16directly or indirectly, and what is the detailed signal pathways.It is should be further study them at the molecular level.