The role of SPE-11 in C. elegans egg activation

在过去的一年里，我们继续通过结构功能分析来表征秀丽隐杆线虫中一种已知的父本效应致死（PEL）蛋白SPE-11。SPE-11是一个299个氨基酸的蛋白，无明显的蛋白结构域。SPE-11具有5个预测核定位序列（NLS），定位于雄性种系核。为了确定五个预测的NLS中哪一个对SPE-11的定位是重要的，以及正确的定位对SPE-11的功能是否重要，每个NLS将从我们实验室生成的GFP::SPE-11报告结构中系统地突变。突变体构建将用于创建转基因动物，然后将对其进行分析，以确定正确的SPE-11定位和挽救SPE-11缺失突变体。将评估显示定位错误的NLS突变体，以确定其对早期胚胎事件的影响。到目前为止，我们已经创建了两个单NLS突变结构和一个双NLS突变结构。已经产生了含有GFP::SPE-11 NLS1突变体的单一整合系。该突变株能够挽救sp11缺失突变株的胚胎致死性，并正确定位于雄性种系。我们还生成了GFP::SPE-11 NLS5突变体的单一整合系。该细胞系正确定位于雄性种系，但未能挽救spe-11缺失突变体。因为NLS5突变体定位正确，所以它并没有揭示定位对功能是否重要。然而，它无法挽救spe-11缺失突变体，这表明这些氨基酸残基对spe-11的功能很重要。我们正在继续努力制造其他NLS突变体的综合转基因系。

In the past year, we have continued to characterize the single known paternal-effect-lethal (PEL) protein in C. elegans, SPE-11, through structure function analysis. SPE-11 is a 299 amino acid protein with no obvious protein domains. SPE-11 does have five predicted nuclear localization sequences (NLS) and localizes to germ-line nuclei of the male. To determine which of the five predicted NLSs are important for SPE-11 localization and if proper localization is important for SPE-11 function, each NLS will be systematically mutated from the GFP::SPE-11 reporter construct that was generated in our lab. The mutant constructs will be used to create transgenic animals, which will then be analyzed for proper SPE-11 localization and rescue of a spe-11 deletion mutant. NLS mutants that show mislocalization will be evaluated to determine their effects on early embryonic events. Thus far, we have created two single NLS mutant constructs and one double NLS mutant construct. A single integrated line harboring the GFP::SPE-11 NLS1 mutant has been generated. This mutant line is able to rescue the embryonic lethality of the spe-11 deletion mutant and localizes correctly in the male germ line. We have also generated a single integrated line with the GFP::SPE-11 NLS5 mutant. This line localizes correctly in the male germ line but fails to rescue the spe-11 deletion mutant. Because the NLS5 mutant localizes correctly, it does not reveal whether localization is important for function. However, it is unable to rescue the spe-11 deletion mutant, suggesting that these amino acid residues are important for the function of SPE-11. We are continuing our efforts to make integrated transgenic lines of other NLS mutants. Interestingly, NLS4 and NLS5 reside in the C-terminus of the SPE-11 protein. Curiously, the majority of existing spe-11 mutant alleles result in C-terminal truncations of the SPE-11 protein, suggesting that the C-terminus is very important for function. To determine which part of the C-terminus is essential for early embryogenesis, we are creating constructs with truncated versions of SPE-11 and generating transgenic lines as mentioned previously. The mutant allele with the longest predicted SPE-11 protein has an early stop codon at amino acid 244. We have created two constructs with truncated versions of SPE-11 (GFP::SPE-11 1-289 and GFP::SPE-11 1-294) and generated transgenic lines. Analysis of these two mutant lines has found that both fail to rescue the embryonic lethality of the spe-11 deletion mutant suggesting that even the most C-terminal end of the protein (i.e. the last five amino acids) are essential for function. We are continuing to test additional SPE-11 NLS mutants and whether SPE-11 variants with specific regions deleted are capable of rescue. We have also pursued a study to identify additional PEL mutants. Previous genetic screens identified embryonic lethal mutants that could be rescued by mating with wild-type males, thus indicating a sperm defect. The ultimate test is to show that mutant males sire dead progeny when mated to wild type hermaphrodites (or females). We have found one such mutant, mel-15. Initial analysis confirms that sperm from mel-15 mutant males produce dead embryos, even when fertilizing wild-type oocytes. In addition, characterization of early embryonic events indicates fertilization is successful and that sperm components (e.g. centrosomes) are transmitted to the embryo, but that mel-15 male sperm lack DNA. Preliminary characterization of meiotic events in mel-15 male germ lines points to a defect in chromosome segregation during meiosis I. We are currently in the process of determining the molecular identity of mel-15. Once confirmed, we will further characterize the mel-15 phenotypes, determine expression patterns, and start suppression screens to identify other genes in the pathway. We hope to also determine whether mel-15 functions with spe-11 or is involved in a distinct process.