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Cloning and Characterization of Yeast LEU4, One of Two Genes Responsible for α-Isopropylmalate Synthesis 总被引:4,自引:0,他引:4 下载免费PDF全文
Li-fen L. Chang Thomas S. Cunningham Paula R. Gatzek Wen-Ji Chen Gunter B. Kohlhaw 《Genetics》1984,108(1):91-106
By complementation of an alpha-isopropylmalate synthase-negative mutant of Saccharomyces cerevisiae (leu4 leu5), a plasmid was isolated that carried a structural gene for alpha-isopropylmalate synthase. Restriction mapping and subcloning showed that sequences sufficient for complementation of the leu4 leu5 strain were located within a 2.2-kilobase SalI-PvuII segment. Southern transfer hybridization indicated that the cloned DNA was derived intact from the yeast genome. The cloned gene was identified as LEU4 by integrative transformation that caused gene disruption at the LEU4 locus. When this transformation was performed with a LEU4fbr LEU5 strain, the resulting transformants had lost the 5',5',5'-trifluoro-D,L-leucine resistance of the recipient strain but were still Leu+. When it was performed with a LEU4 leu5 recipient, the resulting transformants were Leu-. The alpha-isopropylmalate synthase of a transformant that carried the LEU4 gene on a multicopy plasmid (in a leu5 background) was characterized biochemically. The transformant contained about 20 times as much alpha-isopropylmalate synthase as wild type. The enzyme was sensitive to inhibition by leucine and coenzyme A, was inactivated by antibody generated against alpha-isopropylmalate synthase purified from wild type and was largely confined to the mitochondria. The subunit molecular weight was 65,000-67,000. Limited proteolysis generated two fragments with molecular weights of about 45,000 and 23,000. Northern transfer hybridization showed that the transformant produced large amounts of LEU4-specific RNA with a length of about 2.1 kilonucleotides. The properties of the plasmid-encoded enzyme resemble those of a previously characterized alpha-isopropylmalate synthase that is predominant in wild-type cells. The existence in yeast of a second alpha-isopropylmalate synthase activity that depends on the presence of an intact LEU5 gene is discussed. 相似文献
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苏林娜刘向强吕丽芬聂勇战时永全 《现代生物医学进展》2014,14(10):1875-1878
目的:研究FXR在胃炎,胃粘膜肠化生及胃癌组织中的表达,分析其在胃癌发生中的意义。方法:采用免疫组化方法检测FXR在55例胃炎组织,61例胃黏膜肠化生组织及61例胃癌组织中的表达,利用统计学方法 SPSS17.0软件分析其在三种组织中的表达变化,结合文献回顾,分析FXR在胃癌发生中的意义。结果:FXR在胃黏膜肠化生中的表达明显高于胃炎组织(P0.05),而在胃癌组织中,FXR的表达显著低于胃粘膜肠化生组织(P0.05)。结论:FXR是一个潜在的胃癌发生生物标记物,其具体机制有待于进一步探索。 相似文献
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Expressionofmilkproteingenesisinvolvedinahugenetworkofregulatorycircuitswhicharelinkedtotheintactdevelopingmammarygland,andhomeostasisduringpuberty,pregnancy,lactationandinvolution.Analysisofputativeregulatoryelementsandhybridgeneintissueculturesystems… 相似文献
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酪酪肽(peptide tyrosine tyrosine, PYY)是存在于机体肠道的肽类激素,有 PYY1-36和PYY3-36两种形式,后者可以降低个体食欲并减少食物摄入。分别通过人工合成基因和PCR定点突变的方法,得到PYY3-36衍生物PYY3-36-Gly37及PYY3-36的基因,然后克隆到pET32a(+)表达载体中,转化大肠杆菌并进行诱导表达。通过亲和层析得到融合蛋白,经肠激酶酶切和二次亲和层析得到目的多肽。在昆明鼠体内检测二者生物活性,结果显示剂量为800μg/kg时PYY3-36及PYY3-36-Gly37均可抑制昆明鼠的摄食,且抑制作用可达9h,而PYY3-36-Gly37组的平均抑制率可达50%,明显高于PYY3-36。 相似文献
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根据鳜鱼传染性脾肾坏死病毒(Infectious spleen and kidney necrosis virus,ISKNV)丰要衣壳蚩白(Major Capsid protein,MCP)基因(mcp)序列设计引物,PCR扩增得到一长约1400bp的DNA片段,将其克隆到pGEM-T Easy Vector。氨基酸亲水性分析表明,在150—250位氨基酸之间亲水性很高,可构成主要抗原决定簇及形成跨膜区。mcp基因经PCR改造后克隆至原核表达裁体pBV220,构建表达MCP的大肠杆菌基因工程菌,该工程菌经42℃诱导,SDS—PAGE检测,在约50kDa处有一特异蛋白带,含量约为菌体总蛋白的23%。用纯化和复性后的蛋白免疫新西兰大白兔制备抗血清,Western—blotting分忻显示,重组MCP制备的抗血清能与ISKNV MCP特异结合,说明表达产物具有与ISKNV MCP相似的抗原特性。 相似文献
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玉米幼苗地上部/根间氮的循环及其基因型差异 总被引:8,自引:0,他引:8
以两个玉米(ZeamaysL.)自交系原引1号(YY1)和综31(Z31)为研究材料,采用盆栽土培的培养方法,在正常供氮(HN,0.15gN/kg干土)和低氮量供应(LN,0.038gN/kg干土)培养条件下对玉米幼苗植株体内氮的循环量及其在地上部/根间的分配量进行了定量地测定、计算。结果表明,在玉米幼苗地上部/根间氮的循环量很高。低氮量供应使玉米幼苗植株吸氮量下降,根中氮的分配比例增加,同时地上部/根间氮的循环量也随之减少。与氮低效自交系Z31相比,氮高效自交系YY1幼苗中地上部/根间的氮循环量大、氮向根的分配量高,因而有利于其根系的生长,表现为根/地上部之比和总根长较高。这可能有利于其中后期对氮素的高效吸收与利用。 相似文献
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在南亚热带的广州对荷兰引进的紫花凤梨的生物学习性观察研究。考察了紫花凤梨的分芽发生与叶片生长;花序生长发育与开花;耐性、抗性表现等多个方面的生物学习性,并进行综合评价,并提出掌握和利用其习性的建议。 相似文献
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