Progression and Spacing of Heterocyst Differentiation

在化合态氮存在下，光合细菌鱼腥藻的丝状体完全由营养细胞组成。当剥夺结合氮时，5%至10%的细胞以半规则的间隔分化成固氮异形胞。Anaebena提供了一个难得的机会来研究原核生物如何形成多细胞模式（异形胞的间距），以及一个罕见的机会来分析原核细胞如何分化。以下模式形成的机制通常是一致的：成熟和发育中的异形胞通过产生一种沿沿着细丝向外移动的分化抑制物质来抑制附近细胞分化为异形胞。迄今为止，很少的基因和进程，有助于间隔异形胞分化是已知的。 鱼腥藻PCC 7120的基因组序列，几乎完成，提供了一个非常有价值的工具，以帮助阐明详细的机制，和整体战略，调节分化。该工具将用于尝试识别模式形成和细胞分化所需的估计100 - 200个基因。一旦这些基因被识别出来，对其进行分析将对理解分化的机制至关重要。通过与DNA阵列杂交的转录本丰度的诱变和全局分析构成了鉴定所需基因组的互补方法。杂交具有同时显示大量RNA转录物的增加或减少的潜力。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，认识到组成型表达的基因，是专门为发展所需的发展的重要性。其产物参与细胞间分化抑制的基因的突变可导致所有细胞启动分化。因为这样的突变可能会阻止营养生长，他们正在寻找单独的条件诱变。异形胞，通常占总细胞的5 - 10%，大概占总mRNA的相似百分比。杂交的测量通常分辨两种条件之间转录物丰度超过2倍的差异。因此，除非发育中的异形胞可以在不损失mRNA的情况下分离，否则它们必须产生比平均营养细胞多20倍的mRNA，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，但也将进行杂交分析。 该项目建立在已经确定的少数发育特异性基因的基础上。特别地，可用的突变体包括发育阶段。重点将是杂交分析的基因，表达具体期间，因此可能是特别需要的，这些阶段。例如，在hetR突变体中激活的基因是参与对氮剥夺的非发育反应的候选基因，而在hetC突变体中激活但在hetR突变体中不激活的基因是参与模式形成和分化起始的基因的候选基因。类似地，在hepK和devA突变体和突变体a71中激活的基因，但在hetC突变体中不激活的基因是参与形态分化的所有阶段的候选者，但最早阶段除外。转座子Tn5的衍生物高度有效地诱变鱼腥藻，并且迄今为止没有识别的位点特异性。这样的转座子将被用来分离突变体，可以生长在结合氮，但不是在N2，映射它们通过测序，并测试其插入位点的发育相关性，通过补充突变与映射克隆。 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.