MAP KINASE SIGNAL SPEICIFICITY IN C ELEGANS GERMLINE

这一建议的总体目标是阐明信号特异性的分子和机制。实现细胞对信号的适当反应对有机体的健康至关重要，因为错误的反应可能导致发育缺陷或癌症。在线虫的多种组织中，包括外阴和生殖系，MAP激酶信号级联起促进分化的作用。已经确定了确定外阴信号特异性的机制，并涉及到组织特异性转录因子LIN-31的激活。这项建议侧重于通过识别组织中作用于MAP激酶下游的分子，并将它们与外阴中使用的分子进行比较，来研究生殖系中信号特异性的机制。简而言之，1)分离MAP激酶下游靶点的遗传筛选设计正在进行中；2)DNA微阵列将用于精确定位MAP激酶信号在胚系中发生的转录变化；以及3)将对1和2中确定的靶点进行分子克隆，并分析其表达模式和作为MAP激酶磷酸化底物的可能作用。遗传筛查的设置如下：MAP激酶突变MPK-1(Ga111)具有胚系特有的表型，并且对温度敏感。在20摄氏度时，MPK-1(Ga111)表现得像野生型，动物是可育的；在25摄氏度，MPK-1(Ga111)表现得像突变体，粗线虫中的生殖核停止，动物不育。通过在25摄氏度筛选不育的抑制子，MAP激酶的下游效应将被分离出来。效应器将使用标准的遗传程序进行分析。含有部分线虫cDNA的DNA微阵列将用从生长在20摄氏度和25摄氏度的MPK-1(Ga111)菌株中提取的荧光标记的cDNA进行探测。这项实验确定了两个菌株之间差异调控的基因。将包括对温差和生殖系形成的控制，以帮助识别最相关的目标。不久，包含所有线虫基因的微阵列将被制作出来，并用于上述实验。在抑制子筛选和微阵列分析中出现的作为MAPK的潜在靶点的基因将在进一步的研究中被优先考虑。这些基因将被克隆，并通过RNA原位分析和抗体研究确定它们的表达模式。这些基因将作为可能的MAPK底物进行测试。识别这些基因在胚系MAP激酶信号传递中的作用将决定在该组织中实现特异性的机制。用于产生生殖系减数分裂进程的组织特异性结果的分子和机制将与用于外阴的分子和机制进行比较，以增加对此类机制的一般性的理解。

The general objective of this proposal is the elucidation of the molecules and mechanisms of signaling specificity. Achieving the appropriate cellular response to a signal is vital to the well- being of an organism, as the wrong response can result in developmental defects or cancer. The MAP kinase signal cascade functions to promote differentiation in multiple tissues in C. elegans, including the vulva and the germline. A mechanism for determining signalling specificity in the vulva has already been determined, and involves activation of a tisssue-specific transcription factor, LIN-31. This proposal focuses on investigating the mechanism of signal specificity in the germline by identifying molecules acting downstream of MAP kinase in that tissue, and comparing them to those utilized in the vulva. Briefly, 1) a genetic screen design to isolate downstream targets of MAP kinase is being performed; 2) DNA microarrays will be used to pinpoint transcriptional changes that occur in the germline upon MAP kinase signaling; and 3) the targets identified in 1 and 2 will be molecularly cloned, and analyzed for expression pattern and a possible role as substrates of MAP kinase phosphorylation. The genetic screen is set up as follows: a mutation in map kinase, mpk-1(ga111), has a phenotype specific to the germline and is temperature-sensitive. At 20 degrees C, mpk-1(ga111) acts like wild type and the animals are fertile; at 25 degrees C, mpk- 1(ga111) acts like mutant, and the germ nuclei arrest in pachytene and the animal is sterile. By screening for suppressors of sterility at 25 degrees C, downstream effectors of MAP kinase will be isolated. The effectors will be analyzed using standard genetic procedures. DNA microarrays containing a portion of all the C. elegans cDNAs will be probed with fluorescently labelled cDNA extracted from the mpk-1(ga111) strain grown at 20 degrees C and at 25 degrees. This experiment identifies genes that are differentially regulated between the two strains. Controls will be included for temperature differences and germline formation, to aid in identification of the most relevant targets. Soon, microarrays containing every C. elegans cDNA will be made, and used in the above experiment. Genes which appear in both the suppressor screen and the microarray analysis as potential targets of MAP kinase will be given top priority in further studies. These genes will be cloned and their expression patterns determined by both RNA in situ analysis and antibody studies. These genes will be tested as possible MAP kinase substrates. Identification of the roles of these genes in MAP kinase signalling in the germline will determine the mechanism by which specificity is achieved in this tissue. The molecules and mechanisms used to generate the tissue-specific outcome of meiotic progression in the germline will be compared with those used in the vulva to increase the understanding of the generality of such mechanisms.