CHITIN SYNTHASE GENES FROM ASPERGILLUS

描述：几丁质合成是抗真菌药物的一个有吸引力的靶点 因为几丁质是真菌生长和生存所必需的， 聚合物不存在于宿主细胞中。 真菌几丁质合成复杂 并且需要多种几丁质合成酶同工酶， 用于在特定细胞位置或在特定细胞中的几丁质合成 在生命周期中。 特异性几丁质合成酶同工酶是 营养（菌丝）生长所必需的蛋白质是新的 毒品 因此，每个几丁质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。 五甲壳素 来自构巢曲霉的合成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， 菌丝生长或细胞裂解。 chsB同工酶合成几丁质 正常菌丝组织和形态所必需的亚组分 而不是渗透完整性。 chsD同工酶合成几丁质 渗透完整性所必需的亚组分，但不是正常的 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 了解丝状菌的生长形态发生和发育 真菌和评估特定的几丁质酶作为药物靶标。 概述了三个具体目标。 具体目标1是完成 五个已知几丁质合成酶基因的测序和破坏。 我们将 尝试鉴定另外的几丁质酶基因。 相当大 已经在实现这一目标方面取得了进展，并已提交。 具体目标2涉及几丁质表型的表征 合成酶破坏菌株，以评估发育的作用， 每一种酶 具体目标3是评估N-末端的功能， chsD同工酶，特别强调N-末端的能力 与细胞骨架蛋白结合的序列。 具体目标4： chsB和chsD酶的酶学将被检查和方面的 将检查这两种酶的时间和空间调节。 的 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 被修饰以编码表位标记的酶，所述表位标记的酶保留 补充相应的几丁质合成酶缺失菌株。 酶将 将被纯化，并将使用 表位的单克隆抗体。 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.