ten Asbroek A L, Mol C A, Kieft R, Borst P
Division of Molecular Biology, The Netherlands Cancer Institute, Amsterdam.
Mol Biochem Parasitol. 1993 May;59(1):133-42. doi: 10.1016/0166-6851(93)90014-o.
We have further analyzed parameters affecting stable transformation of Trypanosoma brucei. Linear DNA was much more efficient than circular DNA and in the vast majority of transformants analyzed the plasmid DNA had inserted into the chromosomes by homologous recombination. The presence of non-homologous (vector) DNA at one or both ends of linear constructs inhibited transformation efficiency. Less than 1 kb of homologous flanking sequence was sufficient for efficient targeting of a marker gene into the tubulin gene array. When transformants with a single neomycin phosphotransferase (neo(r)) gene replacing a beta-tubulin gene were selected for higher levels of G418 resistance, the neo(r) gene was amplified and spread through the tubulin gene cluster. The additional neo(r) gene copies were adjacent in the tubulin gene array and were added to the array rather than replacing beta-tubulin genes. These results are compatible with asymmetric post-replication recombination (unequal sister chromatid exchange) as the mechanism for neo(r) gene amplification. Starting with a circular construct containing the neo(r) gene between tubulin intergenic regions, we obtained a single transformant that maintained the neo(r) genes as an extrachromosomal plasmid. We show this plasmid to consist of a circular pentamer of the input construct. All other attempts to derive a shuttle vector that replicates extrachromosomally in T. brucei were unsuccessful. Our experiments extend previous observations suggesting that T. brucei has a strong preference for chromosomal insertion of exogenous DNA by homologous recombination.
我们进一步分析了影响布氏锥虫稳定转化的参数。线性DNA比环状DNA效率更高，并且在绝大多数分析的转化体中，质粒DNA已通过同源重组插入染色体。线性构建体一端或两端存在非同源（载体）DNA会抑制转化效率。少于1 kb的同源侧翼序列足以将标记基因有效靶向微管蛋白基因阵列。当选择用单个新霉素磷酸转移酶（neo(r)）基因取代β-微管蛋白基因的转化体以获得更高水平的G418抗性时，neo(r)基因会扩增并扩散到微管蛋白基因簇中。额外的neo(r)基因拷贝在微管蛋白基因阵列中相邻，并添加到该阵列中而非取代β-微管蛋白基因。这些结果与不对称复制后重组（不等姐妹染色单体交换）作为neo(r)基因扩增的机制相符。从一个在微管蛋白基因间隔区之间含有neo(r)基因的环状构建体开始，我们获得了一个将neo(r)基因作为染色体外质粒维持的单个转化体。我们显示该质粒由输入构建体的环状五聚体组成。所有其他尝试在布氏锥虫中获得染色体外复制穿梭载体的实验均未成功。我们的实验扩展了先前的观察结果，表明布氏锥虫对外源DNA通过同源重组进行染色体插入有强烈偏好。
