Chen M J, Locker J, Weiss S B
J Biol Chem. 1976 Jan 25;251(2):536-47.
Transfer RNAs, isolated from Escherichia coli F cells infected with T5 bacteriophage, were charged with radioactive amino acids and used in RNA-DNA hybridization studies to detect and locate T5 tRNA cistrons in the T5 DNA chromosome. Hybridization of 14 3H-aminoacyl-tRNA species, including purified T5 [35S]Met-tRNAm and [35S]Met-tRNAf, to the separated strands of T5+ DNA indicates that most, if not all, of the T5 tRNAs are transcribed from the continuous heavy strand of T5 DNA. Heteroduplex mapping of eight mutant T5 DNA deletions has enabled us to locate and determine the size of these deleted segments. By correlating this information with the presence and absence of specific tDNA sequences in these mutants, as determined by tRNA-DNA hybridization, we were able to define the physical limits of four tDNA-containing loci along the T5 DNA molecule. A physical map for 15 tRNA species examined indicates that the structural genes for these tRNAs are clustered within a segment length of T5 DNA that represents approximately 11.2% of the total wild type T5 DNA. The existence of the deletion mutants indicates that T5 tRNAs are dispensable for T5 replication under the growth conditions and for the host employed.
从感染了T5噬菌体的大肠杆菌F细胞中分离出转移RNA（tRNA），用放射性氨基酸对其进行负载，并用于RNA-DNA杂交研究，以检测和定位T5 DNA染色体中的T5 tRNA顺反子。14种3H标记的氨酰-tRNA，包括纯化的T5 [35S]甲硫氨酸-tRNAm和[35S]甲硫氨酸-tRNAf，与T5+ DNA的单链杂交，表明大部分（如果不是全部）T5 tRNA是从T5 DNA的连续重链转录而来。对八个突变型T5 DNA缺失进行异源双链图谱分析，使我们能够定位并确定这些缺失片段的大小。通过将这些信息与这些突变体中特定tDNA序列的有无相关联（通过tRNA-DNA杂交确定），我们能够确定沿着T5 DNA分子的四个含tDNA位点的物理界限。对15种tRNA进行的物理图谱分析表明，这些tRNA的结构基因聚集在T5 DNA的一个片段长度内，该片段约占野生型T5 DNA总量的11.2%。缺失突变体的存在表明，在所用的生长条件和宿主下，T5 tRNA对于T5复制是可有可无的。
