MAP KINASE SIGNAL SPEICIFICITY IN C ELEGANS GERMLINE

该提案的总体目标是阐明信号特异性的分子和机制。 对信号做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C.包括外阴和生殖细胞。 确定外阴信号特异性的机制已经确定，并涉及组织特异性转录因子LIN-31的激活。 该提案的重点是通过鉴定在该组织中作用于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）正在进行分离MAP激酶下游靶标的遗传筛选设计; 2）DNA微阵列将用于查明在MAP激酶信号传导后种系中发生的转录变化; 3）将对1和2中鉴定的靶标进行分子克隆，并分析其表达模式和作为MAP激酶磷酸化底物的可能作用。遗传筛选如下：map激酶中的突变mpk-1（ga 111）具有种系特异性表型，并且是温度敏感性的。 在20摄氏度，mpk-1（ga 111）的行为像野生型，动物是可育的;在25摄氏度，mpk- 1（ga 111）的行为像突变体，生殖核停滞在粗线期，动物是不育的。 通过在25 ° C下筛选不育抑制因子，将分离MAP激酶的下游效应物。 将使用标准遗传程序分析效应子。 DNA微阵列含有所有C.将用从在20 ℃和25 ℃下生长的mpk-1（ga 111）菌株中提取的荧光标记的cDNA探测线虫cDNA。 该实验鉴定了两种菌株之间差异调节的基因。 将纳入温度差异和生殖系形成的对照，以帮助识别最相关的靶标。 很快，包含每一个C.制备线虫cDNA并用于上述实验。在抑制筛选和微阵列分析中出现的MAP激酶潜在靶基因将在进一步的研究中优先考虑。 这些基因将被克隆，它们的表达模式将通过RNA原位分析和抗体研究来确定。 这些基因将作为可能的MAP激酶底物进行检测。 这些基因在胚系中MAP激酶信号传导中的作用的鉴定将确定在该组织中实现特异性的机制。 用于在生殖系中产生减数分裂进展的组织特异性结果的分子和机制将与外阴中使用的分子和机制进行比较，以增加对这些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.