A human $tRNA^{ser}$ gene was found in a sequenced human chromosomal DNA fragment, and secondary structure of $tRNA^{ser}$ was deduced from the gene. The human $tRNA^{ser}$ gene has anticodon of 5'-AGA-3', and sequence of the gene differs from the previously sequenced human $tRNA^{ser}$ gene in only a 5' position base of anticodon.
This gene had been isolated from human genomic library through plaque hybridization by using labeled human $tRNA^{ser}$ as probe. Here, 1.1Kb DNA fragment was isolated from the DNA of the recombinant λcharon 4A which was named λStlB. The DNA fragment was ligated with pUC9 DNA vector, and used to transform E. coli JM83 cells. Recombinant pUC9 DNA from transformant was confirmed to contain tRNA coding gene by dot hybridization with labeled human total tRNA. After restriction mapping, the restrictly digested 1.1Kb fragment was subcloned to M13 RF DNA and M13 single-stranded DNA was obtained for sequencing of DNA. The nucleotides of DNA fragments were sequenced by dideoxynucleotide chain termination sequencing method.
Labeled $tRNA^{Ser}$ 을 probe 로 사용한 plaque hybridization 을 통해 human genomic library 로 부터 $tRNA^{Ser}$ gene 을 갖는 λcharon 4A clone 을 isolation 했다. (14) 이 실험에서는 $\lambda$st 1B 라 명명된 이 λcharon 4A 로 부터 DNA 를 얻어 1.1 KB 길이의 Eco RI fragment 를 분리한 후 pUC 9 vector와 ligation 하여 E. coli JM 83 을 transformation 하였다. 여기서 얻은 recombinant pUC 9 DNA 를 $[\gamma-^{32}P]$ATP 로 label 된 human total tRNA 와 dot hybridization시켜 tRNA gene이 있음을 다시 확인한 다음 restriction mapping 을 통해 sequencing strategy를 수립하고 restriction digestion 한 1.1 Kb fragment 를 M13RF DNA vector 에 subcloning하여 E. coli JM 103 을 transformation 했다. 여기서 얻은 plaque로 부터 M13 phage를 amplify하여 single-stranded DNA 를 얻은 후 Sanger 의 dideoxynucleotide chain termination sequencing method 사용하여 sequencing 함으로써 $tRNA^{Ser}$을 coding하는 gene을 발견 하였다. 이 $tRNA^{Ser}$ gene 은 이미 밝혀진 다른 human $tRNA^{Ser}$ gene과는 anticodon의 5' position이 달랐으며 3'-flanking region 에서 tRNA structural gene 내의 7 개 nucleotide의 sequence 와 일치하는 sequence 가 발견되었다.
