UNKEMPT CYS(3)HIS FINGERS

CYS(3)他的手指蓬乱

• 批准号: 2666594
• 负责人: JAMES P MOHLER
• 金额: $ 9.78万
• 依托单位: BARNARD COLLEGE
• 依托单位国家: 美国
• 财政年份: 1998
• 资助国家: 美国
• 起止时间: 1998-07-01 至 2001-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2666594
• 关键词: DNA binding protein; Escherichia coli; RNA binding protein; alleles; aminoacid; blood cells; cell differentiation; cell proliferation; cytogenetics; developmental genetics; embryogenesis; endoribonucleases; enzyme activity; eye; gel mobility shift assay; gene expression; genetic regulation; immunoprecipitation; oogenesis; posttranscriptional RNA processing; protein structure function

DESCRIPTION: The unkempt gene in Drosophila melanogaster encodes a product that contains five so-called Cys(3)His finger motifs thought to bind or cleave RNA's of several types. Dr. Mohler and his students will try to confirm the interaction with RNA and the presence of chelating zinc atoms in these motifs and, secondly, assess the role of the wild type unkempt gene product in ovarian and embryonic development, in the developing eye disc and in blood cell proliferation. In the first investigations, full length, and N and C terminal halves of the peptides will be expressed in and purified from E. coli. Their ability to bind DNA and RNA will be assessed by column retention and gel shift assays. RNase activity will also be examined using substrates with single 5' or 3' terminal hairpin loops to assess 5' and 3' exonucleolytic activity, or, in the case of an RNA substrate with both 5' and 3' hairpins, endonucleolytic activity. Zinc binding will be determined renatured peptides bound to nitrocellulose membranes in the presence of radiolabelled zinc atoms. Unkempt's role in eye development will be determined in mitotic clones with homozygous null alleles. Specific cell losses during development will be assessed both cytologically and by staining with cell type specific antibodies. To see if unkempt mutants affect blood cell proliferation, mutant lymph glands will be transplanted to wild type hosts. The cells from these mutant glands will be identified by LacZ staining, and their proliferative and differentiative responses assessed by host lethality and cytology respectively. By using the FLP-FRT system and the ovoD mutant, germ line clones of homozygous unkempt null mutants will be produced. If eggs are not laid, the status of the arrested germ line cells will be examined, after being identified immunologically with an antibody to a myc-epitope expressed by a transgene on the same chromosome as the unkempt null mutant allele. If eggs are laid but do not develop, the stage of developmental arrest will be determined and certain early embryonic structures will be examined cytologically.

描述：果蝇中蓬乱的基因编码一种产物 包含五个所谓的半胱氨酸（3）他的手指图案被认为可以结合或 切割多种类型的 RNA。 莫勒博士和他的学生将尝试 确认与 RNA 的相互作用以及螯合锌原子的存在 其次，评估野生型蓬乱基因的作用 产品在卵巢和胚胎发育、正在发育的眼盘和 在血细胞增殖中。 在第一次调查中，完整的长度和 肽的 N 和 C 末端一半将在 中表达并纯化 来自大肠杆菌。 它们结合 DNA 和 RNA 的能力将通过柱进行评估 保留和凝胶位移测定。 RNase 活性也将使用 具有单个 5' 或 3' 末端发夹环的底物以评估 5' 和 3' 核酸外切活性，或者，对于具有两个 5' 的 RNA 底物来说 和3'发夹，核酸内切活性。 锌结合将被测定 在存在的情况下，复性肽与硝酸纤维素膜结合 放射性标记的锌原子。 Unkempt 在眼睛发育中的作用将在有丝分裂克隆中确定 纯合无效等位基因。 发育过程中特定的细胞损失将是 通过细胞学和细胞类型特异性染色进行评估 抗体。 为了看看蓬乱的突变体是否会影响血细胞增殖， 突变的淋巴腺将被移植到野生型宿主中。 细胞来自 这些突变腺体将通过 LacZ 染色进行鉴定，并且它们的 通过宿主致死率评估增殖和分化反应 分别进行细胞学检查。 通过使用FLP-FRT系统和ovoD突变体，种系克隆 将产生纯合的蓬乱无效突变体。 如果不产卵， 被捕获的生殖系细胞的状态将在被检查后进行检查 用针对由 a 表达的 myc 表位的抗体进行免疫学鉴定 转基因与蓬乱的无效突变等位基因位于同一染色体上。 如果鸡蛋 产卵但不发育，发育停滞阶段 确定并检查某些早期胚胎结构 细胞学上。