Dynes J L, Firtel R A
Department of Biology, University of California, San Diego, La Jolla 92093.
Proc Natl Acad Sci U S A. 1989 Oct;86(20):7966-70. doi: 10.1073/pnas.86.20.7966.
We constructed a partial Sau3A Dictyostelium genomic DNA library in a shuttle vector that replicates extrachromosomally in Dictyostelium cells. This library was used to complement Dictyostelium strain HPS400, which lacks thymidylate synthase activity and requires exogenous thymidine for growth. We have used a modified high-frequency transformation protocol that allows the introduction of the library into a sufficient number of Dictyostelium cells to select complementing plasmids. Using this approach, we have isolated a gene (Thy1) that complements the thymidine growth requirement of HPS400. The gene encodes a 1.2-kilobase RNA and the derived amino acid sequence shows no homology to thymidylate synthase, a protein highly conserved throughout evolution, or any other protein sequence in the data base examined. Thy1 provides an important selectable marker for transforming Dictyostelium cells. In addition, this work suggests that it will be possible to isolate genes that are essential for developmental processes in Dictyostelium by complementation.
我们构建了一个部分Sau3A盘基网柄菌基因组DNA文库，该文库存在于一种穿梭载体中，可在盘基网柄菌细胞中进行染色体外复制。这个文库用于互补盘基网柄菌HPS400菌株，该菌株缺乏胸苷酸合成酶活性，生长需要外源性胸苷。我们使用了一种改良的高频转化方案，该方案可将文库导入足够数量的盘基网柄菌细胞中，以筛选互补质粒。通过这种方法，我们分离出了一个可互补HPS400对胸苷生长需求的基因（Thy1）。该基因编码一个1.2千碱基的RNA，推导的氨基酸序列与胸苷酸合成酶（一种在整个进化过程中高度保守的蛋白质）或所检测数据库中的任何其他蛋白质序列均无同源性。Thy1为转化盘基网柄菌细胞提供了一个重要的选择标记。此外，这项工作表明，通过互补作用有可能分离出盘基网柄菌发育过程中必需的基因。
