Davidson Mari K, Young Nathan P, Glick Gloria G, Wahls Wayne P
Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock, AR 72205, USA.
Nucleic Acids Res. 2004 Aug 17;32(14):4400-10. doi: 10.1093/nar/gkh767. Print 2004.
Identification of genes required for segregation of chromosomes in meiosis (scm) is difficult because in most organisms high-fidelity chromosome segregation is essential to produce viable meiotic products. The biology of fission yeast Schizosaccharomyces pombe facilitates identification of such genes. Insertional mutagenesis was achieved by electroporation of linear ura4+ DNA into cells harboring a ura4 deletion. Approximately 1000 stable transformants were screened individually for the production of elevated frequencies of aneuploid spore colonies. Twenty-two candidates were subjected to a secondary screen for cytological defects. Five mutants exhibited significant levels of aberrant meiotic chromosome segregation, but were proficient for mating and completion of meiosis. Each mutant's phenotype cosegregated with its respective ura4+ transgene. The mutations were recessive and defined five complementation groups, revealing five distinct genes (scm1, scm2, scm3, scm4 and scm5). Southern blotting revealed single-site integration in each transformant, indicating that insertional mutagenesis is useful for generating single-locus scm mutations linked to a selectable marker. The transgene insertion points were refractory to analysis by inverse-PCR. Molecular and real-time PCR analyses revealed the presence of multiple, truncated copies of ura4+ at each integration site. Thus, electroporation-mediated insertional mutagenesis in S.pombe is preceded by exonucleolytic processing and concatomerization of the transforming DNA.
鉴定减数分裂中染色体分离所需的基因（scm）很困难，因为在大多数生物体中，高保真染色体分离对于产生可存活的减数分裂产物至关重要。裂殖酵母粟酒裂殖酵母的生物学特性有助于鉴定此类基因。通过将线性ura4⁺ DNA电穿孔导入携带ura4缺失的细胞中来实现插入诱变。对大约1000个稳定转化体分别进行筛选，以检测非整倍体孢子菌落频率的升高。对22个候选者进行细胞学缺陷的二次筛选。五个突变体表现出明显水平的异常减数分裂染色体分离，但在交配和减数分裂完成方面表现正常。每个突变体的表型与其各自的ura4⁺转基因共分离。这些突变是隐性的，定义了五个互补群，揭示了五个不同的基因（scm1、scm2、scm3、scm4和scm5）。Southern印迹显示每个转化体中存在单一位点整合，表明插入诱变可用于产生与选择标记连锁的单基因座scm突变。转基因插入点难以通过反向PCR进行分析。分子和实时PCR分析表明，每个整合位点存在多个截短的ura4⁺拷贝。因此，在粟酒裂殖酵母中，电穿孔介导的插入诱变之前会发生转化DNA的核酸外切加工和多联体形成。
