双元载体提供组蛋白H3/H4拷贝1的elp3Δ菌株的构建

Construction of elp3Δ Strain Containing one Copy H3/H4 Provided by Binary Recombinant Expression Vector

人Elongator是在转录中有功能的组蛋白乙酰转移酶(HAT)复合物,为研究其催化亚基Elp3功能,构建了酵母组蛋白H3/H4拷贝1的双元表达载体pRS316CFT,通过PCR介导的基因敲除,获酵母基因组H3/H4缺失由双元载体提供单拷贝H3/H4的elp3Δ菌株.敏感性实验表明该载体提供H3/H4可维持酵母正常生长.本工作为构建H3/H4乙酰化位点突变菌株,用功能互补在体内研究人Elp3 HAT活性功能奠定了基础.

Human Elongator has histone acetyltransferase （HAT） activity and involvs in transcription. For further study the function of Elp3 catalytic subunit, yeast histone H3/H4 copy I （FT1） and transcription terminator sequence CYC1 are amplified from yeast genomic DNA and pYES2, respectively, to create a FT1-CYC1 chimera. The entire insert is then cloned into pRS313 to create the binary recombinant expression vector pRS313-CFT. Through gene knocked out mediated by PCR, yeast elp3A mutant containing H3/H4 copy I provided by pRS313-CFT are constructed. Results from complementation tests show that one copy H3/H4 provided by pRS313-CFT could sustain the normal growth of Yeast cells. The work has laid down the basis for further in vivo studies into roles of HAT activity of Elp3 in transcription by creating yeast mutant strain containing acetylation sites mutations in H3/H4.