Gagné G, Guertin M
Département de Biochimie, Faculté des Sciences et de Génie, Université Laval, Québec, Canada.
Plant Mol Biol. 1992 Feb;18(3):429-45. doi: 10.1007/BF00040659.
In the green unicellular alga Chlamydomonas eugametos, cellular division is readily synchronized by light/dark cycles. Under these conditions, light initiates photosynthetic growth in daughter cells and begins the G1 phase. Genes whose expression is regulated upon illumination are likely to be important mechanisms controlling cell proliferation. To identify some of those genes, two cDNA libraries were prepared with poly(A)+ extracted from cells either stimulated with light for 1 h or held in darkness (quiescent cells) during the same period. To restrict our analysis to those genes that are part of the primary response, cells were incubated in presence of cycloheximide. Differential screening of approximately 40,000 clones in each library revealed 44 clones which hybridize preferentially with a [32P] cDNA probe derived from RNA of light-stimulated cells and 15 clones which react selectively with a [32P] cDNA probe synthesized from poly(A)+ RNA of quiescent cells. Cross-hybridization of these clones identified 4 independent sequences in the light-induced (LI) collection and 2 in the uninduced (LR) library. Four of these cDNAs correspond to mRNAs that are positively or negatively regulated upon activation of photosynthesis. One clone represents a mRNA that accumulates transitorily at both transitions. Finally, LI818 cDNA identifies a new chlorophyll a/b-binding (cab) gene family whose mRNA accumulation is controlled by light and a circadian oscillator. The endogenous timing system controls LI818 mRNA accumulation so that it precedes the onset of illumination by a few hours. On the other hand, light affects LI818 mRNA levels independently of active photosynthesis.
在绿色单细胞藻类莱茵衣藻中,细胞分裂很容易通过光/暗循环实现同步。在这些条件下,光启动子细胞中的光合生长并开始G1期。其表达在光照时受到调控的基因可能是控制细胞增殖的重要机制。为了鉴定其中一些基因,制备了两个cDNA文库,分别从在光照下刺激1小时的细胞或在此期间处于黑暗中的细胞(静止细胞)中提取的聚腺苷酸加尾(poly(A)+)RNA构建。为了将我们的分析限制在那些属于初级反应的基因上,细胞在放线菌酮存在的情况下进行孵育。对每个文库中约40,000个克隆进行差异筛选,发现44个克隆优先与源自光刺激细胞RNA的[32P] cDNA探针杂交,15个克隆与从静止细胞的聚腺苷酸加尾(poly(A)+)RNA合成的[32P] cDNA探针选择性反应。这些克隆的交叉杂交在光诱导(LI)文库中鉴定出4个独立序列,在未诱导(LR)文库中鉴定出2个。这些cDNA中有4个对应于在光合作用激活时受到正向或负向调控的mRNA。一个克隆代表一种在两个转变阶段都短暂积累的mRNA。最后,LI818 cDNA鉴定出一个新的叶绿素a/b结合(cab)基因家族,其mRNA积累受光和昼夜振荡器控制。内源性计时系统控制LI818 mRNA的积累,使其在光照开始前几个小时出现。另一方面,光独立于活跃的光合作用影响LI818 mRNA水平。