Transcriptional regulatory network of genes for carbon concentrating mechanisms

莱茵衣藻（Chlamydomonas reinhardtii）可以通过信号转导途径诱导一组碳浓缩机制（CCM）基因来适应低co_2供应、高光等环境胁迫。降低CO_2诱导ccm相关基因的表达，包括编码外质碳酸酐酶（CA）的Cahl、编码线粒体碳酸酐酶的Mca和编码叶绿体包膜蛋白lp -36的Ccpl。这种对CO_2限制条件的适应表明存在细胞感知CO_2缺乏的感觉机制和信号转导到特定基因调控的途径。我们克隆了一个核调控基因Ccml，它补充了多性高co_2需求突变体C16的突变（Fukuzawa等）。2001)。Ccml在高co_2和低co_2条件下均本构表达。CCM1锌指基序中的His-54对其调控功能至关重要。为了分离受CCM1控制的靶基因，我们开发了包含10,368 EST的cDNA macroarray （Fukuzawa et al.）。在高co_2、低co_2环境下培养的WT细胞和低co_2环境下驯化的C16细胞的RNA样品用^<33>P-dCTP标记，并与cDNA阵列膜杂交。59个低co_2诱导EST克隆未被C16突变体诱导，表明这些克隆受CCM1l的调控。EST克隆的时间依赖性表达模式使我们能够检测到含有光呼吸或离子转运蛋白的低co_2诱导（Lci）基因。讨论了co_2限制胁迫与光呼吸或强光胁迫的关系，以及从调控突变体的阵列分析中推断出的co_2信号转导途径。

Chlamydomonas reinhardtii can acclimate to environmental stresses such as low CO_2-supply, high-light by inducing a set of genes for a carbon-concentrating mechanism (CCM) through putative signal transduction pathways. Expression of several CCM-related genes, Cahl coding for a periplasmic carbonic anhydrase (CA), Mca for a mitochondrial CA and Ccpl for a chloroplast envelope protein LIP-36, is induced by lowering CO_2. This acclimation to CO_2-limiting conditions suggests the existence of sensory mechanisms by which cells perceive the shortage of CO_2 and pathways by which the signal is transduced into the specific gene regulation. We have cloned a nuclear regulatory gene, Ccml, which complements the mutation in the pleiotropic high-CO_2 requiring mutant C16 (Fukuzawa et.al. 2001). Ccml is constitutively expressed both in high- and low-CO_2 conditions. His-54 in the zinc-finger motif of CCM1 is essential to its regulatory function. To isolate target genes, which are controlled by CCM1, we have developed cDNA macroarray containing 10,368 EST (Fukuzawa et al.). RNA samples from WT cells cultured in high-CO_2 or low-CO_2 and also from the low-CO_2-acclimated C16 were labeled with ^<33>P-dCTP and hybridized with cDNA array membranes. Fifty-nine low-CO_2 inducible EST clones were not induced in the C16 mutant, suggesting that these are regulated by CCM1l. Time-dependent expression patterns of EST clones enabled us to detect low-CO_2 inducible (Lci) genes containing those for photorespiration or ion-transporters. Relationship between CO_2-limiting stress and photorespiration or high-light stress will be discussed as well as CO_2-signal transduction pathways deduced from array analyses of regulatory mutants.