Fabiani L, Aragona M, Frontali L
Department of Cell and Developmental Biology, University of Rome La Sapienza, Italy.
Yeast. 1990 Jan-Feb;6(1):69-76. doi: 10.1002/yea.320060108.
We have undertaken a search for autonomously replicating (ARSs) from Kluyveromyces lactis chromosomal DNA able to sustain plasmid replication in K. lactis and in Saccharomyces cerevisiae. The discovery of such sequences might be interesting for the comparison of ARSs from different sources and possibly useful for the construction of multivalent vectors. HindIII fragments from K. lactis chromosomal DNA were inserted in the YIp5 plasmid (lacking an origin of replication) and the resulting chimaeric plasmids were selected for the ability to transform S. cerevisiae. Four plasmids were identified and further analysed. Two contained the same 1.8 kb K. lactis fragment and transformed both K. lactis and S. cerevisiae with the same efficiency and stability, whereas the third transformed only S. cerevisiae and the fourth transformed K. lactis with a higher efficiency than S. cerevisiae. A detailed study was performed on the 1.8 kb fragment which exhibited ARS function in both yeasts. The fragment was subcloned using different restriction enzymes and Bal31 exonuclease. Subclones were tested for ARS function. ARS activities in the two yeasts were localized in the same 100 bp region. Sequencing demonstrated the presence in this region of the dodecanucleotide 5'ATTTATTGTTTT3' differing from the ARS core consensus of S. cerevisiae only by a T insertion. A similar nucleotide sequence is present in the putative replication origin of the 2 mu-like plasmid pKD1 which stably replicates in K. lactis. Homologies with ARSs from S. cerevisiae were also found in the regions flanking the above-mentioned dodecanucleotide.
我们已着手从乳酸克鲁维酵母染色体DNA中寻找能够在乳酸克鲁维酵母和酿酒酵母中维持质粒复制的自主复制序列（ARSs）。发现此类序列可能有助于比较不同来源的ARSs，并且可能对构建多价载体有用。将乳酸克鲁维酵母染色体DNA的HindIII片段插入YIp5质粒（缺乏复制起点），并选择所得的嵌合质粒转化酿酒酵母的能力。鉴定并进一步分析了四个质粒。其中两个含有相同的1.8 kb乳酸克鲁维酵母片段，并且以相同的效率和稳定性转化乳酸克鲁维酵母和酿酒酵母，而第三个仅转化酿酒酵母，第四个转化乳酸克鲁维酵母的效率高于酿酒酵母。对在两种酵母中均表现出ARS功能的1.8 kb片段进行了详细研究。使用不同的限制酶和Bal31核酸外切酶对该片段进行亚克隆。测试亚克隆的ARS功能。两种酵母中的ARS活性定位于相同的100 bp区域。测序表明该区域存在十二核苷酸5'ATTTATTGTTTT3'，与酿酒酵母的ARS核心共有序列仅相差一个T插入。在乳酸克鲁维酵母中稳定复制的2μ样质粒pKD1的推定复制起点中存在相似的核苷酸序列。在上述十二核苷酸侧翼区域也发现了与酿酒酵母ARSs的同源性。
