Yu G L, Hasson M, Blackburn E H
Department of Molecular Biology, University of California, Berkeley 94720.
Proc Natl Acad Sci U S A. 1988 Jul;85(14):5151-5. doi: 10.1073/pnas.85.14.5151.
We transformed the ciliate Tetrahymena thermophila by microinjection of circular plasmids containing the ribosomal RNA gene (rDNA). In the somatic macronucleus of Tetrahymena, the rDNA is in the form of linear palindromic molecules. The rDNA molecules from the C3 strain have a replication advantage over rDNA from both B strain and the C3 rDNA mutant rmm1. We constructed two circular plasmids carrying replication origin sequences from C3 rDNA and a point mutation (Pmr) in the 17S rRNA gene that confers resistance to the antibiotic paromomycin. One plasmid contained a single complete copy of the rRNA gene and its flanking sequences, while the other had an additional rDNA origin of replication. In all B or rmm1 Tetrahymena cell lines transformed with the plasmids, rDNA sequences from the plasmid were found in palindromic rDNA molecules. In one transformant line, a small amount of the plasmid was also retained in a form with the original circular restriction map. Our results show that the plasmids underwent homologous recombination with one arm of the endogenous rDNA to give heteropalindromic rDNA, or with both arms of the palindrome to form homopalindromic rDNA. The resulting recombinant molecules were able to replace the recipient's original rDNA completely, providing strong evidence that C3 rDNA sequences in the donor DNAs confer a replication advantage over recipient rDNA. Thus microinjection of circular plasmids provides a method for replacement of an endogenous gene or gene fragment with exogenous sequences.
我们通过显微注射含有核糖体RNA基因（rDNA）的环状质粒，对嗜热四膜虫进行了转化。在四膜虫的体细胞大核中，rDNA呈线性回文分子形式。来自C3菌株的rDNA分子比来自B菌株和C3 rDNA突变体rmm1的rDNA具有复制优势。我们构建了两个环状质粒，它们携带来自C3 rDNA的复制起始序列以及17S rRNA基因中的一个点突变（Pmr），该突变赋予对抗生素巴龙霉素的抗性。一个质粒包含rRNA基因的单个完整拷贝及其侧翼序列，而另一个则有额外的rDNA复制起点。在用这些质粒转化的所有B或rmm1四膜虫细胞系中，在回文rDNA分子中发现了来自质粒的rDNA序列。在一个转化细胞系中，少量质粒也以具有原始环状限制图谱的形式保留下来。我们的结果表明，这些质粒与内源性rDNA的一条臂进行了同源重组，产生异源回文rDNA，或者与回文的两条臂进行重组，形成同源回文rDNA。产生的重组分子能够完全取代受体的原始rDNA，有力地证明了供体DNA中的C3 rDNA序列比受体rDNA具有复制优势。因此，环状质粒的显微注射提供了一种用外源序列替换内源基因或基因片段的方法。
