MAP KINASE SIGNAL SPEICIFICITY IN C ELEGANS GERMLINE

本建议的总体目标是阐明信号特异性的分子和机制。对信号作出适当的细胞反应对生物体的健康至关重要，因为错误的反应可能导致发育缺陷或癌症。MAP激酶信号级联在秀丽隐杆线虫的多种组织中起促进分化的作用，包括外阴和种系。确定外阴信号特异性的机制已经确定，涉及组织特异性转录因子LIN-31的激活。本研究的重点是通过识别MAP激酶下游的分子，并将其与外阴中使用的分子进行比较，来研究种系中信号特异性的机制。简而言之，1)正在进行基因筛选设计以分离MAP激酶的下游靶点；2) DNA微阵列将用于查明发生在种系中MAP激酶信号传导的转录变化；3)对1和2中鉴定的靶点进行分子克隆，分析其表达模式和可能作为MAP激酶磷酸化底物的作用。基因筛选如下：图激酶mpk-1（ga111）突变具有种系特异性表型，对温度敏感。在20摄氏度时，mpk-1（ga111）的行为与野生型相似，动物具有可育性；在25℃时，mpk- 1（ga111）表现为突变体，胚核停留在粗线期，动物不育。通过筛选25℃下的不育抑制因子，将分离出MAP激酶的下游效应物。效应器将使用标准的遗传程序进行分析。含有部分秀丽隐杆线虫cDNA的DNA微阵列将用从mpk-1（ga111）菌株中提取的荧光标记cDNA进行探针，这些菌株在20摄氏度和25摄氏度下生长。该实验确定了两种菌株之间受不同调控的基因。将包括对温差和种系形成的控制，以帮助确定最相关的目标。很快，包含所有秀丽隐杆线虫cDNA的微阵列将被制作出来，并用于上述实验。在抑制基因筛选和微阵列分析中出现的作为MAP激酶潜在靶点的基因将在进一步的研究中得到优先考虑。这些基因将被克隆，并通过RNA原位分析和抗体研究确定其表达模式。这些基因将作为可能的MAP激酶底物进行检测。确定这些基因在种系MAP激酶信号传导中的作用将决定在该组织中实现特异性的机制。用于产生生殖细胞减数分裂过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普遍性的理解。

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.