LIN-14 mutants

A temporal gradient of the novel nuclear protein LIN-14 specifies the timing and sequence of stage-specific developmental events in Caenorhabditis elegans. The profound effects of lin-14 mutations on worm development suggest that LIN-14 directly or indirectly regulates stage-specific gene expression. We show that LIN-14 can associate with chromatin in vivo and has in vitro DNA binding activity. A bacterially expressed C-terminal domain of LIN-14 was used to select DNA sequences that contain a putative consensus binding site from a pool of randomized double-stranded oligonucleotides. To identify candidates for genes directly regulated by lin-14, we employed DNA microarray hybridization to compare the mRNA abundance of C. elegans genes in wild-type animals to that in mutants with reduced or elevated lin-14 activity. Five of the candidate LIN-14 target genes identified by microarrays, including the insulin/insulin-like growth factor family gene ins-33, contain putative LIN-14 consensus sites in their upstream DNA sequences. Genetic analysis indicates that the developmental regulation of ins-33 mRNA involves the stage-specific repression of ins-33 transcription by LIN-14 via sequence-specific DNA binding. These results reinforce the conclusion that lin-14 encodes a novel class of transcription factor. Set of arrays organized by shared biological context, such as organism, tumors types, processes, etc. Keywords: Logical Set新型核蛋白LIN-14的时间梯度，决定了秀丽隐杆线虫（Caenorhabditis elegans）阶段特异性发育事件的时序与顺序。lin-14突变对蠕虫发育具有显著影响，提示LIN-14可直接或间接调控阶段特异性基因表达。我们证实LIN-14可在体内结合染色质，并具备体外DNA结合活性。利用经大肠杆菌表达的LIN-14羧基端结构域，从随机化双链寡核苷酸池中筛选出带有推定共识结合位点的DNA序列。为鉴定lin-14直接调控的候选基因，我们采用DNA微阵列（DNA microarray）杂交技术，比较野生型秀丽隐杆线虫与lin-14活性降低或升高的突变体中各基因的mRNA丰度。通过微阵列筛选得到的5个候选LIN-14靶基因（包括胰岛素/胰岛素样生长因子家族基因ins-33），其上游DNA序列中均带有推定的LIN-14共识结合位点。遗传分析表明，ins-33 mRNA的发育调控涉及LIN-14通过序列特异性DNA结合，对ins-33转录进行阶段特异性抑制。上述结果进一步支持lin-14编码一类新型转录因子的结论。该阵列集合按共享的生物学背景（如生物体、肿瘤类型、生物学过程等）进行组织。关键词：逻辑集合（Logical Set）