Kurvari V, Qian F, Snell W J
Department of Cell Biology and Neuroscience, University of Texas Southwestern Medical Center, Dallas 75235-9039, USA.
Plant Mol Biol. 1995 Dec;29(6):1235-52. doi: 10.1007/BF00020465.
Chlamydomonas gametes of opposite mating types interact through flagellar adhesion molecules called agglutinins leading to a signal transduction cascade that induces cell wall loss and activation of mating structures along with other cellular responses that ultimately result in zygote formation. To identify molecules involved in these complex cellular events, we have employed subtractive and differential hybridization with cDNA from mt+ gametes activated for fertilization and non-signaling, vegetative (non-gametic) cells. We identified 55 cDNA clones whose transcripts were regulated in activated gametes. Here we report the molecular cloning and characterization of the complementary DNA (cDNA) for one clone whose transcripts in activated gametes were several-fold higher than in normal gametes. Regulation of the transcript was not related simply to protein synthesis because it was not increased in cells synthesizing new cell wall proteins. The cDNA contained a single open reading frame (ORF) of 815 amino acids encoding a polypeptide of calculated relative mass of 87 kDa. Database search analysis and sequence alignment indicated that the deduced amino acid sequence exhibited 42% identity and 62% similarity to a class of prokaryotic methyl transferases (5-methyltetrahydrofolate-homocysteine methyl transferase; EC 2.1.1.14) known to be involved in the terminal step of de novo biosynthesis of methionine. This enzyme catalyzes transfer of a methyl group from 5-methyltetrahydrofolate to homocysteine resulting in methionine formation. Affinity-purified polyclonal antibodies raised against a bacterially produced GST-fusion protein identified a 85 kDa soluble protein in Chlamydomonas gametes. Southern blot hybridization indicated that the enzyme is encoded by a single-copy gene. The evidence presented in this paper raises the possibility that, in addition to its participation in de novo biosynthesis and regeneration of methionine, Chlamydomonas methionine synthase may play a role in adhesion-induced events during fertilization.
不同交配型的衣藻配子通过称为凝集素的鞭毛粘附分子相互作用，引发信号转导级联反应，该反应诱导细胞壁丧失和交配结构激活，以及其他最终导致合子形成的细胞反应。为了鉴定参与这些复杂细胞事件的分子，我们利用了扣除杂交和差异杂交技术，使用来自激活用于受精的mt+配子和无信号的营养（非配子）细胞的cDNA。我们鉴定出55个cDNA克隆，其转录本在激活的配子中受到调控。在此，我们报告其中一个克隆的互补DNA（cDNA）的分子克隆和特征，该克隆在激活的配子中的转录本比正常配子中的高出几倍。转录本的调控并非简单地与蛋白质合成相关，因为在合成新细胞壁蛋白的细胞中它并未增加。该cDNA包含一个815个氨基酸的单一开放阅读框（ORF），编码一个计算相对分子量为87 kDa的多肽。数据库搜索分析和序列比对表明，推导的氨基酸序列与一类已知参与甲硫氨酸从头生物合成终末步骤的原核甲基转移酶（5-甲基四氢叶酸-同型半胱氨酸甲基转移酶；EC 2.1.1.14）具有42%的同一性和62%的相似性。该酶催化从5-甲基四氢叶酸向同型半胱氨酸转移甲基，从而形成甲硫氨酸。针对细菌产生的GST融合蛋白产生的亲和纯化多克隆抗体在衣藻配子中鉴定出一种85 kDa的可溶性蛋白。Southern印迹杂交表明该酶由单拷贝基因编码。本文提供的证据增加了一种可能性，即除了参与甲硫氨酸的从头生物合成和再生外，衣藻甲硫氨酸合酶可能在受精过程中的粘附诱导事件中发挥作用。
