CHITIN SYNTHASE GENES FROM ASPERGILLUS

描述：甲壳素合成是抗真菌药物的一个有吸引力的靶点。 因为甲壳素是真菌生长和存活所必需的，而且因为 宿主细胞中不存在聚合物。真菌甲壳素的合成是复杂的 并需要多种甲壳素合成酶同工酶，其中每一种都是必需的 在特定细胞位置或在特定细胞中合成甲壳素 在生命周期中键入。特定的甲壳素合成酶同工酶有 营养(菌丝)生长所必需的是小说的潜在目标 毒品。因此，每个甲壳素合成酶的精确发育功能是 确定哪些同工酶是有用的药物靶点的关键。五种甲壳素 来自尼杜拉曲霉的合酶基因已经被鉴定和破坏。 至少有两种同工酶，chsB和chsd，是有希望的药物靶点，因为 通过基因破坏使酶失活会导致速度极慢 菌丝生长或向细胞裂解。ChsB同工酶合成甲壳素 正常菌丝组织和形态所必需的亚组分 但不是为了渗透的完整性。CHSD同工酶合成甲壳素 渗透完整性所必需但不是正常所必需的亚组分 菌丝形态。CHSD同工酶的N-末端与 肌球蛋白。从拟议研究中获得的数据将有助于 对丝状菌生长、形态发生和发育的认识 真菌和作为药物靶标的特定甲壳素合成酶的评估。 概述了三个具体目标。具体目标1是完成 已知的五个甲壳素合成酶基因的测序和破坏。我们会 尝试鉴定其他几丁质合成酶基因。相当可观 实现这一目标的进展已经完成，现已提出。 具体目标2涉及甲壳素表型的表征 合酶干扰菌株，以评估在发育过程中的作用 每种酶。具体目标3是评估N-末端的功能 侧重N-末端能力的CHSD同工酶 与细胞骨架蛋白结合的序列。在具体目标4中， ChsB和CHSD酶的酶学将被检查，并且 这两种酶的时间和空间调节将被检测。这个 几丁质合成酶基因的转录水平将被确定 在有性和无性的生命周期中。ChsB和chsd基因将 被修改为编码表位标记的酶，这些酶保留了 补充相应的甲壳素合成酶缺失株。这些酶将会 将被提纯，并将使用 针对该表位的单抗。ChsB和CHSD酶的加工 将在体内和体外进行检查。

DESCRIPTION: Chitin synthesis is an attractive target for antifungal drugs because chitin is necessary for fungal growth and viability and because the polymer is not present in host cells. Fungal chitin synthesis is complex and requires multiple chitin synthase isozymes each of which is necessary for chitin synthesis at a specific cellular location or in a particular cell type during the life cycle. Specific chitin synthase isozymes hat are necessary for vegetative (hyphal) growth are potential targets for novel drugs. Thus the precise developmental function of each chitin synthase is the key to determining which isozymes are useful drug targets. Five chitin synthase genes from Aspergillus nidulans have been identified and disrupted. At least two isozymes, chsB and chsD, are promising drug targets because inactivation of the enzymes by gene disruption leads to extremely slow hyphal growth or to cell lysis. The chsB isozyme synthesizes a chitin subfraction that is necessary for normal hyphal organization and morphology but not for osmotic integrity. The chsD isozyme synthesizes a chitin subfraction that is necessary for osmotic integrity but not for normal hyphal morphology. The N-terminus of the chsD isozyme is homologous to myosin. The data obtained from the proposed studies will contribute to the understanding of the growth morphogenesis and development of filamentous fungi and to the assessment of specific chitin synthases as drug targets. Three specific aims are outlined. Specific Aim 1 is to complete the sequencing and disruption of the five known chitin synthase genes. We will attempt to identify additional chitin synthases genes. Considerable progress completion toward this goal has already been made and is presented. Specific Aim 2 involves the characterization of the phenotypes of the chitin synthase disruptant strains in order to assess the developmental role for each enzyme. Specific Aim 3 is to assess the functions of the N-terminus of the chsD isozyme with special emphasis on the ability of N-terminal sequences to bind to cytoskeletal proteins. In Specific Aim 4, the enzymology of the chsB and chsD enzymes will be examined and aspects of the temporal and spatial regulation of these two enzymes will be examined. The transcriptional levels of chitin synthase genes will be determined throughout the sexual and asexual life cycles. The chsB and chsD genes will be modified to encode epitope tagged enzymes that retain the ability to complement the corresponding chitin synthase null strain. The enzymes will be purified and their cellular localization will be determined using monoclonal antibody to the epitope. The processing of chsB and chsD enzymes will examined both in vivo and in vitro.