SITE-SPECIFIC DELETION OF THE N-TERMINAL SEGMENTS FROM EcoRI ENDONUCLEASE USING THE POLYMERASE CHAIN REACTION

We developed a general and efficient method for directing the deletions of DNA sequences of any lengths using polymerase chain reaction (PCR). The method was based on in vitro amplification of target sequences with site-specific deletions, Klenow end-flushing and blunt-end cloning. As an example, it was used to delete the restriction gene encoding EcoRI endonuclease, resulting in plasmids expressing two truncated forms. Assays using SDS-PAGE and gel retardation revealed the important role of the amphipathic helix (29-43) of the EcoRI endonuclease in binding to its cognate substrate.     

AN EFFICIENT SITE-DIRECTED MUTAGENESIS USING POLYMERASE CHAIN REACTION

We report here a simple and efficient method for site-directed mutagenesis using polymer-ase chain reaction (PCR). In constructing a new expression plasmid for the EcoRI restrictiongene, we made two point mutations. While one created a new Sall site prior to the SDsequence, the other replaced Glu144 with Lys. A 1.5 kb Sall-PstI fragment isolated frompER101 was used as the template. Two 25 mer oligonucleotide primers containing the de-sired mutations were synthesized and used to direct PCR amplification with Taq DNA poly-merase. About 0.5μg of the 0.49 kb fragment was obtained from 0.05 μ of the 1.5 kb frag-ment by carrying out polymerase chain reaction for 30 cycles. As calculated theoretically,99% of the product contained the desired mutations. The product was cloned into pUC19using Sall and PstI, two of the transformed colonies were randomly chosen for sequence anal-ysis, and both of them were shown to contain the desired mutations. Finally, the amplifiedfragment was cloned into pER304 to place the     

胰泌素基因的化学合成和克隆

本文报道的内容为胰泌素基因的化学合成和克隆。胰泌素为一由2了个氨基酸组成的肠胃道多肽激素,该多肽合成基因的顺序由计算机辅助设计,共216个核苷酸碱基;合成基因选用酵母细胞偏爱的密码子,并在其中设置了一些便于今后用于基因改造、表达调控和产物分离所需的位点,排除了可能影响酶促连接反应的各种重复顺序;用化学法(固相磷酸三酯法和固相亚磷酸三酯法)合成了12个基因片段,长度为12寡聚核苷酸至24寡聚核苷酸,聚丙烯酰胺凝胶电泳分离纯化合成产物;利用T_4DNA连接酶组建完整的胰泌素基因,克隆于pWR 13质粒中,构建了一个含胰泌素基因的新质粒pWS 1。     

SYNTHESIS OF THE 5'-HALF MOLECULE OF YEAST ALANINE tRNA

In this paper, we report the synthesis of the 5'-half molecule of yeast alanine tRNA (tRNA_y~(Ala)) by ligating three oligonucleotide fragments corresponding to the nucleotide sequences 1-13, 14-22 and 23-35 respectively under the catalysis of T_4 RNA ligase (Fig. 1). Because of the high purity of the oligonuclcotide fragments and the excellent quality of T_4RNA ligase and polynucleotide kinase we prepared, the isolation steps were simplified and the overall yields were much higher. The ligating yield of the docosamer (Ⅳ) was 75%, that of the pentatriacontamer (Ⅴ), 90%, and the isolated yield of the final product was 21% calculated on the basis of the tridecamer (Ⅲ) used in the. first reaction. Under the action of T_4 RNA ligase the synthetic 5'-half molecule was joined with the natural 3'-half molecule forming a semi-synthetic tRNA_y~(Ala), which possessed the biological activities of both accepting (~3H)-alanine and incorpprating it into proteins. The correctness of the structure of the synthetic 5     

漫谈酵母丙氨酸转移核糖核酸的人工合成

1981年11月19日,由中国科学院上海生物化学研究所、细胞生物研究所、有机化学研究所、北京生物物理研究所、北京大学生物系和上海试剂二厂等六个单位共同协作完成了酵母丙氨酸转移核糖核酸(简称酵母丙氨酸 tRNA)的     

TOTAL SYNTHESIS OF YEAST ALANINE TRANSFER RIBONUCLEIC ACID

By a combination of chemical and enzymatic methods, small oligonucleotides with lengths varying from 2 to 8 nucleotides were synthesized from mononucleotides. The small oligonucleotides were then ligated with T_4 RNA ligase into six laxge ligonucleotides (9 to 19 nucleotides long) which were further ligated to form two half molecules with 35 and 41 nucleotides respectively, Finally, the two synthetic half molecules were annealed and ligated to obtain the whole molecule of yeast alanine tRNA (tRNA_y~(Ala)). Prior to this, two semi-syntheses were performed, i. e. ligation of the synthetic 5'-half molecule with the natural 3'-half molecule and that of the natural 5'-half molecule with the synthetic 3'-half molecule.     

SYNTHESIS OF THE LEU-ENKEPHALIN GENE

The synthesis of Leu-enkephalin gene by an alternative approach was described in thispaper. The sequence of the synthetic gene, 26 base-pairs in length, was derived from the ami-no acid sequence of the hormone peptide Leu-enkephalin. It bears single-stranded cohesive ter-mini for the restriction endonucleases EcoR I and BamH I so that it may be inserted into apBR 322 plasmid. The 4 deoxyoligonucleotide fragments, varying in length from octanucleotideto octadecanucleotide, were synthesized by an improved phosphotriester method developed inour laboratory. All chemically synthetic fragments were pure and had the correct sequences.The assembly of the Leu-enkephalin gene was carried out in a one-step ligation reaction cata-lysed by T_4-DNA ligase with good yield, Finally, the purified products from polyacrylamidegel electrophoresis had the correct joining-points as predicted.     

THE CLONING AND EXPRESSION OF THE SYNTHETIC LEU-ENKEPHALIN GENE IN E.coli

The synthetic leu-enkephalin (LEK) gene was joined with pBR322 and transformed toE. coli. The recombinant plasmids containing the LEK gene were selected by colony hybri-dization, and characterized by restriction mapping and Southern's technique. The lac operonwas used to control the expression of the LEK gene. A recombinant plasmid, pEL 103, inwhich the lac operon and LEK gene are transcribed in the same direction, produces LEK inE. coli. The level of LEK detected by radioimmunoassay reaches 426 ng per mg of bacte-rial protein.