Li Fei, Holloway Stephen P, Lee Jaesung, Herrin David L
Molecular Cell and Developmental Biology Section and Institute for Cellular and Molecular Biology, Bio 311, University of Texas at Austin, Austin, TX 78712, USA.
Plant J. 2002 Nov;32(4):467-80. doi: 10.1046/j.1365-313x.2002.01437.x.
Single nucleotide substitutions were made in the core helices P4, P6, and P7, and in the metal-binding GAAA motif in the J4/5 region of the chloroplast group I rRNA intron of Chlamydomonas reinhardtii, Cr.LSU. In vitro assays showed that these substitutions had surprisingly strong effects on Cr.LSU self-splicing; however, splicing of all but the P6 mutations could be at least partially recovered by increasing the Mg2+ concentration. The mutant constructs were transformed into chloroplasts to replace the wild-type intron; however, only the P4 mutants became homoplasmic, indicating that the other mutations were lethal. The splicing-deficient P4125A mutant, which exhibited slow growth and light sensitivity, was used to isolate suppressor strains that showed a substantial restoration of Cr.LSU splicing. Genetic analysis of the 7151, 7120 and 71N1 suppressors indicated that these mutations are in at least two nuclear genes. The 7151 suppressor mutation, which defines the chloroplast-splicing suppressor (css1) gene, had no obviously altered growth phenotype with the wild-type intron, and was dominant in vegetative diploids containing the mutant intron. All three of the suppressor strains also suppressed a mutation in the P4 region of the fourth psbA intron, Cr.psbA4, indicating that these genes play a role in splicing of multiple group I introns in the chloroplast.
在莱茵衣藻叶绿体I组rRNA内含子（Cr.LSU）的核心螺旋P4、P6和P7以及J4/5区域的金属结合GAAA基序中进行了单核苷酸替换。体外实验表明，这些替换对Cr.LSU自我剪接具有惊人的强烈影响；然而，除P6突变外，所有突变的剪接都可以通过提高Mg2+浓度至少部分恢复。将突变构建体转化到叶绿体中以取代野生型内含子；然而，只有P4突变体变成了同质体，这表明其他突变是致死的。具有缓慢生长和光敏感性的剪接缺陷型P4125A突变体被用于分离显示Cr.LSU剪接显著恢复的抑制菌株。对7151、7120和71N1抑制子的遗传分析表明，这些突变存在于至少两个核基因中。定义叶绿体剪接抑制子（css1）基因的7151抑制子突变，与野生型内含子相比，其生长表型没有明显改变，并且在含有突变内含子的营养二倍体中是显性的。所有三个抑制菌株也抑制了第四个psbA内含子（Cr.psbA4）的P4区域中的一个突变，这表明这些基因在叶绿体中多个I组内含子的剪接中起作用。
