Progression and Spacing of Heterocyst Differentiation

在结合氮的存在下，光合细菌水藻的细丝完全由营养细胞组成。当被剥夺组合氮时，5%至10%的细胞以半规则的间隔分化为固定n2的异囊。Anaebena提供了一个难得的机会来研究原核生物是如何形成多细胞模式的（异囊的间隔），以及一个罕见的机会来分析原核细胞是如何分化的。模式形成的机制一般被认为是：成熟和发育中的异囊通过形成一种沿丝向外移动的分化抑制物质来抑制附近细胞向异囊的分化。迄今为止，很少有基因和过程有助于间隔异囊分化是已知的。Anabaena PCC 7120的基因组序列已接近完成，为阐明调控分化的详细机制和总体策略提供了极具价值的工具。该工具将被用来试图识别大约100-200个基因，这些基因是模式形成和细胞分化所需要的。一旦确定了这些基因，对它们的分析将对理解分化的机制至关重要。诱变和通过DNA阵列杂交的转录物丰度全局分析构成了鉴定所需基因集的互补方法。杂交有可能同时显示大量RNA转录物的增加或减少。然而，由于杂交是相关的，它需要诱变来证明哪些基因是发育所必需的。此外，诱变可以，但杂交不能，认识到组成表达基因的发育重要性，这是发育所需要的特异性。其产物参与细胞间分化抑制的基因突变可能导致所有细胞启动分化。因为这样的突变可能会阻止植物生长，所以他们正在通过条件诱变单独寻找。异囊通常占总细胞的5-10%，可能也贡献了总mRNA的相同百分比。杂交测量通常可以解决两种条件下转录物丰度的2倍以上差异。因此，除非发育中的异囊能够在不丢失mRNA的情况下被分离出来，否则它们产生的mRNA必须是普通营养细胞的20倍以上，这样才能通过杂交检测到差异。由于这些原因，我们的主要方法将是诱变，但杂交分析也将进行。这个项目建立在已经确定的少数发育特异性基因的基础上。特别是，发育阶段的可用突变体。重点将放在杂交分析基因的特异性表达，因此可能特别需要，这些阶段。例如，在hetR突变体中激活的基因是参与氮剥夺的非发育性反应的候选基因，而在hetC突变体中激活但在hetR突变体中未激活的基因是参与模式形成和分化起始的候选基因。同样，在hepK和devA突变体以及突变体a71中激活的基因，而在hetC突变体中不激活的基因，除了最早的形态分化阶段外，都是参与所有阶段的候选基因。转座子Tn5的衍生物可以非常有效地诱变水藻，并且迄今为止没有发现位点特异性。这些转座子将用于分离可以在组合氮上生长但不能在N2上生长的突变体，通过测序绘制它们的图谱，并通过与绘制的克隆互补突变来测试它们的插入位点是否与发育相关。所鉴定的基因的发育作用将被识别和整合。

In the presence of combined nitrogen, filaments of the photosynthetic bacterium Anabaena are comprised wholly of vegetative cells. When deprived of combined nitrogen, 5 to 10% of the cells, at semi-regular intervals, differentiate into N2-fixing heterocysts. Anaebena offers a rare opportunity to study how a prokaryote forms a multicellular pattern (the spacing of heterocysts), and an infrequent opportunity to analyze how prokaryotic cells differentiate. The following mechanism of pattern formation is generally agreed on: mature and developing heterocysts inhibit the differentiation of nearby cells into heterocysts by elaborating a differentiation-inhibiting substance that moves outward along a filament. To date, few of the genes and processes that contribute to spaced heterocyst differentiation are known. The genomic sequence of Anabaena PCC 7120, nearly finished, provides a tool of great value to help elucidate the detailed mechanisms, and overall strategy, that regulate differentiation. That tool will be used to try to identify the estimated 100-200 genes that are required specifically for pattern formation and cellular differentiation. Analysis of these genes, once they are identified, will be crucial for understanding the mechanisms that underlie differentiation. Mutagenesis and global analysis of transcript abundance by hybridization to DNA arrays constitute complementary approaches to identifying the desired set of genes. Hybridization has the potential to show increases or decreases in a great number of RNA transcripts simultaneously. However, because hybridization is correlative, it requires mutagenesis to demonstrate which genes are necessary for development. Also, mutagenesis can, but hybridization cannot, recognize the developmental importance of constitutively expressed genes that are required specifically for development. Mutation of genes whose products participate in the intercellular inhibition of differentiation could lead all cells to initiate differentiation. Because such mutations might prevent vegetative growth, they are being sought separately by conditional mutagenesis. Heterocysts, normally 5-10% of total cells, presumably contribute a like percentage of total mRNA. Measurements of hybridization normally resolve differences of over 2-fold in transcript abundance between two conditions. Therefore, unless developing heterocysts can be isolated without loss of mRNAs, they must produce more than 20-fold more of an mRNA than does an average vegetative cell for the difference to be detectable by hybridization. For these reasons, our primary approach will be by mutagenesis, but hybridization analysis will also be performed. This project builds on the small number of development-specific genes already identified. In particular, available mutants bracket developmental stages. Focus will be on hybridization analysis on genes that are expressed specifically during, and so may be specifically required for, these stages. For example, whereas genes activated in a hetR mutant are candidates for involvement in non-developmental responses to nitrogen deprivation, genes activated in a hetC mutant but not in a hetR mutant are candidates for genes involved in pattern formation and the initiation of differentiation. Similarly, genes activated in hepK and devA mutants and in mutant a71 but not in a hetC mutant are candidates for involvement in all but the earliest stages of morphological differentiation. Derivatives of transposon Tn5 mutagenize Anabaena highly effectively, and without heretofore discerned site-specificity. Such transposons will be used to isolate mutants that can grow on combined nitrogen but not on N2, map them by sequencing, and test their sites of insertion for developmental relevance by complementing the mutations with mapped clones. The developmental roles of the genes identified will be discerned and integrated.