Junker V, Teichmann T, Hekele A, Fingerhut C, Beier H
Institut für Biochemie, Bayerische Julius-Maximilians-Universität, Biozentrum, Am Hubland, D-97074 Würzburg, Germany.
Nucleic Acids Res. 1997 Nov 1;25(21):4194-200. doi: 10.1093/nar/25.21.4194.
In the ciliated protozoa Tetrahymena thermophila introns have been detected in rRNA and mRNAs until now. We have isolated and sequenced seven tRNATyr genes from the T.thermophila nuclear genome. All of these genes contain introns of identical length and sequence. The 11 bp long intervening sequences are located 1 nt 3' to the anticodon as found in other eukaryotic nuclear tRNA genes. Tetrahymena tRNATyr genes are efficiently transcribed in HeLa cell nuclear extract. Moreover, processing and splicing occurred in HeLa as well as in wheat germ extracts, supporting the notion that Tetrahymena tRNATyr introns can be classified as authentic tRNA introns. We have also isolated cytoplasmic tRNATyr from Tetrahymena cells. This tRNATyr isoacceptor has a QPsiA anticodon and is not a UAG suppressor as shown in in vitro translation studies. Since UAG and UAA codons are used as glutamine codons in Tetrahymena macronuclear DNA, the presence of a strong natural UAG suppressor such as tRNATyr with GPsiA anticodon should cause misreading of the glutamine as tyrosine codons and the absence of the latter had thus been predicted. Furthermore we have studied the organization of tRNATyr genes in the genome of T.thermophila and have found two types of tRNATyr gene arrangement. A minimum of 12 tRNATyr genes are present as single copies in genomic DNA HindIII restriction fragments ranging in size from 0.6 to 7 kb. Additionally one cluster of tRNATyr genes consisting of six members has been detected in a 2.3 kb HindIII fragment.
在纤毛原生动物嗜热栖热四膜虫中，迄今为止已在rRNA和mRNA中检测到内含子。我们从嗜热栖热四膜虫的核基因组中分离并测序了7个酪氨酸tRNA基因。所有这些基因都含有长度和序列相同的内含子。与其他真核细胞核tRNA基因一样，11bp长的间隔序列位于反密码子的3'端1个核苷酸处。嗜热栖热四膜虫的酪氨酸tRNA基因在HeLa细胞核提取物中能高效转录。此外，在HeLa以及小麦胚芽提取物中都发生了加工和剪接，这支持了嗜热栖热四膜虫酪氨酸tRNA内含子可被归类为真正的tRNA内含子这一观点。我们还从嗜热栖热四膜虫细胞中分离出了细胞质酪氨酸tRNA。这种酪氨酸tRNA同工受体具有QPsiA反密码子，并且如体外翻译研究所表明的那样，它不是UAG抑制子。由于在嗜热栖热四膜虫大核DNA中UAG和UAA密码子被用作谷氨酰胺密码子，因此预计不存在具有GPsiA反密码子的强天然UAG抑制子如酪氨酸tRNA，因为后者的存在会导致谷氨酰胺被误读为酪氨酸密码子。此外，我们研究了嗜热栖热四膜虫基因组中酪氨酸tRNA基因的组织方式，发现了两种类型的酪氨酸tRNA基因排列。在大小从0.6到7kb的基因组DNA HindIII限制性片段中，至少有12个酪氨酸tRNA基因以单拷贝形式存在。此外，在一个2.3kb的HindIII片段中检测到了一个由六个成员组成的酪氨酸tRNA基因簇。
