Louis S A, Weeks G, Spiegelman G B
Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, V6T 1Z3, Canada.
Dev Biol. 1997 Oct 15;190(2):273-83. doi: 10.1006/dbio.1997.8675.
One of the Dictyostelium ras genes, rasD, is expressed preferentially in prestalk cells at the slug stage of development and overexpression of this gene containing a G12T activating mutation causes the formation of aberrant multitipped aggregates that are blocked from further development (Reymond et al., 1986, Nature, 323, 340-343). The ability of the Dictyostelium rap1 gene to suppress this abnormal developmental phenotype was investigated. The rap1 gene and G12V activated and G10V negative mutant forms of the rap1 gene were independently linked to the rasD promoter and each construct used to transform M1, a Dictyostelium cell line expressing RasD[G12T]. Transformants of M1 that expressed Rap1 or Rap1[G12V] protein still formed multitipped aggregates, but most tips were able to complete development and form fruiting bodies. Cell lines showing this modified phenotype were designated ME (multitipped escape). The rap1[G10V] construct did not modify the M1 phenotype. These data suggest that overexpression of RasD[G12T] has two effects, the formation of a multitipped aggregate and a block in subsequent differentiation and that the expression of Rap1 or Rap1[G12V] reverses only the latter. Differentiation of ME cells in low density monolayers showed the identical low level of stalk and spore cell formation seen for M1 cells under the same conditions. Thus the cell autonomous defect in monolayer differentiation induced in the M1 strain was not corrected in the ME strain. Cell type-specific gene expression during the development of M1 cells is dramatically altered: prestalk cell-specific gene expression is greatly enhanced, whereas prespore-specific gene expression is almost suppressed (Louis et al., 1997, Mol. Biol. Cell, 8, 303-312). During the development of ME cells, ecmA mRNA levels were restored to those seen for Ax3, and tagB mRNA levels were also markedly reduced, although not to Ax3 levels. cotC expression in ME cells was enhanced severalfold relative to M1, although levels were still lower than those observed during the development of Ax3. The low expression of car1 mRNA during early development of the M1 strain remained low during the development of ME cells. These data are consistent with the idea that the expression of RasD[G12T] affects two independent and temporally separated events and that only the later defect is reversed by rap1.
盘基网柄菌的一种Ras基因rasD，在发育的蛞蝓阶段优先在柄细胞前体细胞中表达，并且该含有G12T激活突变的基因的过表达导致形成异常的多尖端聚集体，这些聚集体被阻止进一步发育（雷蒙德等人，1986年，《自然》，323卷，340 - 343页）。研究了盘基网柄菌rap1基因抑制这种异常发育表型的能力。rap1基因以及rap1基因的G12V激活突变体和G10V阴性突变体形式分别与rasD启动子相连，并且每个构建体用于转化M1，一种表达RasD[G12T]的盘基网柄菌细胞系。表达Rap1或Rap1[G12V]蛋白的M1转化体仍然形成多尖端聚集体，但大多数尖端能够完成发育并形成子实体。表现出这种修饰表型的细胞系被命名为ME（多尖端逃逸）。rap1[G10V]构建体没有改变M1的表型。这些数据表明RasD[G12T]的过表达有两个作用，形成多尖端聚集体和随后分化的阻滞，并且Rap1或Rap1[G12V]的表达仅逆转后者。ME细胞在低密度单层中的分化显示出与相同条件下M1细胞所见相同的低水平的柄细胞和孢子细胞形成。因此，M1菌株中诱导的单层分化中的细胞自主缺陷在ME菌株中未得到纠正。M1细胞发育过程中的细胞类型特异性基因表达发生了显著改变：柄细胞前体细胞特异性基因表达大大增强，而孢子前体细胞特异性基因表达几乎被抑制（路易斯等人，1997年，《分子生物学细胞》，8卷，303 - 312页）。在ME细胞的发育过程中，ecmA mRNA水平恢复到Ax3所见水平，并且tagB mRNA水平也显著降低，尽管未降至Ax3水平。相对于M1，ME细胞中cotC的表达增强了几倍，尽管其水平仍低于Ax3发育过程中观察到的水平。M1菌株早期发育过程中car1 mRNA的低表达在ME细胞发育过程中仍然很低。这些数据与以下观点一致，即RasD[G12T]的表达影响两个独立且在时间上分开的事件，并且只有后期缺陷被rap1逆转。
