Molecular Analysis of Photoregulated Development

丝状真菌构巢曲霉的分生孢子形成（无性孢子形成）涉及多细胞分化结构的形成，这些结构在精确预定的时间产生有色素的单倍体分生孢子。研究表明，光对于分生孢子形成是必要的，但只有在发育的关键时期进行照射时才会引发发育。 此外，分生孢子形成是由红光诱导的，并被立即转向远红光所抑制，这是在植物中观察到的光敏色素介导的反应的特征。已经鉴定出影响光依赖性分生孢子形成的几种不同类别的突变体。一些突变体消除了光敏感性，允许在没有光的情况下发生分生孢子，而其他突变体则影响在特定波长下分生孢子的能力。因此，构巢曲霉中的光信号似乎是由类似光敏色素的光感受器感知的，该光感受器通过未知的机制将信息传递给控制无性发育的基因。该提案的目的是详细了解调节该生物体中光介导的分生孢子形成的机制。这项工作的主要目的是通过光敏色素相关序列的分子克隆来识别和表征红光感光器。已经分离出类似光敏色素的克隆，并建议进行实验来比较构巢曲霉光敏色素基因与已知的植物光敏色素基因的序列和功能。这些实验包括确定基因组和 cDNA 克隆的 DNA 序列，推断转录表达模式，通过使用基因融合和光敏色素特异性抗体来跟踪翻译表达模式，以及检查基因破坏体的表型。这项工作的另一个目的是确定光转导途径中发生的相互作用。 已在影响红光敏感性的克隆基因 (acoD) 中分离出突变体。实验旨在检查 acoD 在不同生长和光照条件下的转录和翻译活性，在核苷酸水平上识别导致红光不敏感的突变缺陷，并通过抑制突变的分离和分析来识别基因相互作用。这些研究将有助于阐明影响真核生物细胞分化的复杂调节机制。

Conidiation (asexual sporulation) in the filamentous fungus, Aspergillus nidulans, involves the formation of multicellular differentiated structures that produce pigmented, haploid conidia at precisely scheduled times. It has been shown that light is necessary for conidiation, but will only elicit development if irradiation occurs during a critical period of development. Furthermore, conidiation is induced by red light and suppressed by an immediate shift to far red light, a property that is characteristic of phytochrome mediated responses observed in plants. Several different classes of mutants that affect light dependent conidiation have been identified. Some mutants abolish light sensitivity, allowing conidiation to occur in the absence of light, while others affect the ability to conidiate at specific wavelengths. Thus, it appears that light signals are perceived in A. nidulans by a phytochrome like photoreceptor that transmits information by an unknown mechanism to genes that control asexual development. The objective of this proposal is to develop a detailed understanding of the mechanisms that regulate light mediated conidiation in this organism. The major aim of this work is to identify and characterize the red light photoreceptor through the molecular cloning of phytochrome related sequences. A phytochrome like clone has been isolated and experiments are proposed to compare the sequences and function of the A. nidulans phytochrome gene with known phytochrome genes from plants. These experiments include determining the DNA sequence of both genomic and cDNA clones, deducing the pattern of transcriptional expression, following the pattern of translational expression through the use of gene fusions and phytochrome specific antibodies, and examining the phenotype of gene disruptants. Another aim of this work is to identify interactions that occur within the photo transduction pathway. Mutants have been isolated in a cloned gene (acoD) that affect sensitivity to red light. Experiments are proposed to examine the transcriptional and translational activity of acoD in different growth and illumination conditions, to identify at the nucleotide level the mutational defects that cause red light insensitivity, and to identify gene interactions through the isolation and analysis of suppressor mutations. These studies will help elucidate the complex regulatory mechanisms that affect cellular differentiation is this eukaryote.