Cdh1和Num1过表达菌株的构建
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摘要
[目的]研究Cdh1和Num1之间是否存在相互作用,构建用于Cdh1和Num1免疫共沉淀试验的过表达菌株。[方法]首先在酵母菌株YWL630中用GAL1和3HA对CDH1进行标记,构建GAL1-3HA-CDH1 NUM1-GFP菌株,再利用酵母四分体解离的方法使菌株YWL63和YWL490分别与该菌株杂交并进行四分体解离,从而得到所需菌株。[结果]成功构建不同基因型和配型的重组酵母菌株,其中YT52成功过表达了约340 kD的Num1,YT53成功过表达了340 kD的Num1和75 kD的Cdh1,YT57成功过表达了75 kD的Cdh1。[结论]经过同源重组的方法成功标记CDH1并诱导蛋白过表达,通过酵母四分体解离的方法构建用于Cdh1和Num1互作试验的过表达菌株,为揭示APC/C是否介导Num1的降解及机制奠定基础。
[Objective] To study whether there is an interaction between Cdh1 and Num1, and construct an over-expression strain for Cdh1 and Num1 co-immunoprecipitation experiments. [Method] Firstly, CDH1 was labeled with GAL1 and 3HA in yeast strain YWL630 to construct GAL1-3HA-CDH1 NUM1-GFP strain, and then yeast YWL63 and YWL490 were hybridized with the strain by yeast tetrad dissection method. The tetrad dissection was carried out to obtain the desired strain. [Result] The recombinant yeast strains with different genotypes and mating types were successfully constructed. YT52 successfully over-expressed Num1 with a size of about 340 kD. YT53 successfully over-expressed 340 kD Num1 and 75 kD Cdh1, and YT57 successfully over-expressed 75 kD of Cdh1. [Conclusion]The method of homologous recombination successfully labeled CDH1 and induced protein over-expression, and constructed an over-expression strain for Cdh1 and Num1 interaction experiments by yeast tetrad dissection thereby laying the foundation for revealing whether APC/C mediates the degradation of Num1 and its mechanism.
[Objective] To study whether there is an interaction between Cdh1 and Num1, and construct an over-expression strain for Cdh1 and Num1 co-immunoprecipitation experiments. [Method] Firstly, CDH1 was labeled with GAL1 and 3HA in yeast strain YWL630 to construct GAL1-3HA-CDH1 NUM1-GFP strain, and then yeast YWL63 and YWL490 were hybridized with the strain by yeast tetrad dissection method. The tetrad dissection was carried out to obtain the desired strain. [Result] The recombinant yeast strains with different genotypes and mating types were successfully constructed. YT52 successfully over-expressed Num1 with a size of about 340 kD. YT53 successfully over-expressed 340 kD Num1 and 75 kD Cdh1, and YT57 successfully over-expressed 75 kD of Cdh1. [Conclusion]The method of homologous recombination successfully labeled CDH1 and induced protein over-expression, and constructed an over-expression strain for Cdh1 and Num1 interaction experiments by yeast tetrad dissection thereby laying the foundation for revealing whether APC/C mediates the degradation of Num1 and its mechanism.
引文
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[16] HUANG J N,PARK I,ELLINGSON E,et al.Activity of the APCCdh1 form of the anaphase-promoting complex persists until S phase and prevents the premature expression of Cdc20p[J].The journal of cell biology,2001,154(1):85-94.
[17] SETHI N,MONTEAGUDO M C,KOSHLAND D,et al.The CDC20 gene product of Saccharomyces cerevisiae,a beta-transducin homolog,is required for a subset of microtubule-dependent cellular processes[J].Molecular & cellular biology,1991,11(11):5592-5602.
[18] QIN L,GUIMAR?ES D S,MELESSE M,et al.Substrate recognition by the Cdh1 destruction box receptor is a general requirement for APC/CCdh1-mediated proteolysis[J].Journal of biological chemistry,2016,291(30):15564-15574.
[2] FARKASOVSKY M,KüNTZEL H.Yeast Num1p associates with the mother cell cortex during S/G2 phase and affects microtubular functions[J].The journal of cell biology,1995,131(4):1003-1014.
[3] BLOOM K.Nuclear migration:Cortical anchors for cytoplasmic dynein[J].Current biology,2001,11(8):326-329.
[4] FARKASOVSKY M,KüNTZEL H.Cortical Num1p interacts with the dynein intermediate chain Pac11p and cytoplasmic microtubules in budding yeast[J].The journal of cell biology,2001,152(2):251-262.
[5] KORMANEC J,SCHAAFF-GERSTENSCHL?GER I,ZIMMERMANN F K,et al.Nuclear migration in Saccbaromyces cerevisiae is controlled by the highly repetitive 313 kDa NUM1 protein[J].Molecular and general genetics,1991,230(1/2):277-287.
[6] TANG X Y,GERMAIN B S,LEE W L.A novel patch assembly domain in Num1 mediates dynein anchoring at the cortex during spindle positioning[J].The journal of cell biology,2012,196(6):743-756.
[7] LACKNER L L,PING H,GRAEF M,et al.Endoplasmic reticulum-associated mitochondria-cortex tether functions in the distribution and inheritance of mitochondria[J].Proceedings of the national academy of sciences,2013,110(6):458-467.
[8] PING H A,KRAFT L M,CHEN W T,et al.Num1 anchors mitochondria to the plasma membrane via two domains with different lipid binding specificities[J].The journal of cell biology,2016,213(5):513-524.
[9] TANG X Y,PUNCH J J,LEE W L.A CAAX motif can compensate for the PH domain of Num1 for cortical dynein attachment[J].Cell cycle,2009,8(19):3182-3190.
[10] STEGMEIER F,RAPE M,DRAVIAM V M,et al.Anaphase initiation is regulated by antagonistic ubiquitination and deubiquitination activities[J].Nature,2007,446(7138):876-881.
[11] STEVERMANN L,LIAKOPOULOS D.Molecular mechanisms in spindle positioning:Structures and new concepts[J].Current opinion in cell biology,2012,24(6):816-824.
[12] BOCHIS O V,FETICA B,VLAD C,et al.The importance of ubiquitin E3 ligases,SCF and APC/C,in human cancers[J].Clujul medical,2015,88(1):9-14.
[13] PESIN J A,ORR-WEAVER T L.Regulation of APC/C activators in mitosis and meiosis[J].Annu Rev Cell Dev Biol,2008,24:475-499.
[14] ZACHARIAE W,NASMYTH K.Whose end is destruction:Cell division and the anaphase-promoting complex[J].Genes Dev,1999,13(16):2039-2058.
[15] PFLEGER C M,KIRSCHNER M W.The KEN box:An APC recognition signal distinct from the D box targeted by Cdh1[J].Genes & development,2000,14(6):655-665.
[16] HUANG J N,PARK I,ELLINGSON E,et al.Activity of the APCCdh1 form of the anaphase-promoting complex persists until S phase and prevents the premature expression of Cdc20p[J].The journal of cell biology,2001,154(1):85-94.
[17] SETHI N,MONTEAGUDO M C,KOSHLAND D,et al.The CDC20 gene product of Saccharomyces cerevisiae,a beta-transducin homolog,is required for a subset of microtubule-dependent cellular processes[J].Molecular & cellular biology,1991,11(11):5592-5602.
[18] QIN L,GUIMAR?ES D S,MELESSE M,et al.Substrate recognition by the Cdh1 destruction box receptor is a general requirement for APC/CCdh1-mediated proteolysis[J].Journal of biological chemistry,2016,291(30):15564-15574.