Site-specific mutagenesis of T4 gene 32: the role of tyrosine residues in protein-nucleic acid interactions.
Site-specific mutagenesis of T4 gene 32: the role of tyrosine residues in protein-nucleic acid interactions.
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T4 基因 32 的定点突变：酪氨酸残基在蛋白质-核酸相互作用中的作用。
DOI：
10.1021/bi00444a039
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发表时间：
1989
期刊：
影响因子：
2.9
通讯作者：
Konigsberg,WH
中科院分区：
文献类型：
--
作者：
Shamoo,Y;Ghosaini,LR;Keating,KM;Williams,KR;Sturtevant,JM;Konigsberg,WH
材料与方法聚[d（AT）]、聚（dT）、p（dT）8和p（dT）16购自Pharmacia;[y-32 P] ATP和定点诱变试剂盒购自阿默舍姆。所有DNA修饰酶购自新英格兰Biolabs或Boehringer-Mannheim。TPCK处理的胰蛋白酶购自库珀生物医学公司。Apo-gp 32通过使用Giedroc等的技术制备。（1986年）。基因的定点突变32.通过使用Gillam和Smith（1979）或Taylor等人的方法，（1985）在含有基因32的M13构建体上进行体外定点诱变[命名为pYS 5并描述于Shamoo et al.（1986）]。在阿尔伯特爱因斯坦医学院DNA合成实验室或耶鲁医学院蛋白质和核酸化学实验室合成了在该程序中用作诱变引物的寡脱氧核苷酸。通过使用Nucleogen DEAE 60-7柱和制造商的方案对寡核苷酸进行HPLC纯化。通过使用Gillam和Smith（1979）描述的技术制备Gp 32 ser 73（在位置73处酪氨酸至丝氨酸的取代）、lgp 32 serl 15和gp 32 serl 37。将突变寡核苷酸用作pYS 5 ssDNA上DNA聚合酶I大片段的引物，并将所得突变/亲本异源双链体与T4 DNA连接酶连接。用异源双链体转染大肠杆菌JM 103，并通过使用32* P标记的诱变寡核苷酸作为探针在硝酸纤维素滤膜上进行体外杂交来筛选所得噬斑（Mark等人，1984年）。通过重新筛选噬斑以引入新的限制性位点来实现突变的最终确认，所述新的限制性位点可诊断特定的取代（Prigodich等人，1986年）。利用Taylor等的技术制备突变体gp 32ala 84、gp 321 eu 92、gp 32 val 99、gp 321 eu 106和gp 32 ilel 86。（1985），其可作为试剂盒从Am-ersham商购获得。按照制造商的方案，将pYS 5 ssDNA退火至诱变寡核苷酸。然后在2 mM dATP、dGTP、dTTP和dCTPaS存在下，使用DNA聚合酶I的大片段延伸引发的模板。将thioclitide掺入突变链中允许核酸内切酶Adi在亲本链内产生切口，然后可以通过使用核酸外切酶III去除其中的大部分。然后用DNA聚合酶I进行另一个延伸反应，去除大部分亲本链，以产生突变体/突变体双链DNA。然后将突变体/突变体DNA转染到E. coliTG 1中的酶切位点，并直接筛选新的酶切位点或用桑格等的技术进行测序。（1977年）。
Materials and MethodsMaterials. Poly [d (AT)], poly (dT), p (dT) 8, and p (dT) 16 were purchased from Pharmacia;[y-32P] ATP and site-directed mutagenesis kits were obtained from Amersham. All DNA modification enzymes were purchased from either New England Biolabs or Boehringer-Mannheim. TPCK-treated trypsin was purchased from Cooper Biomedical. Apo-gp32 was made by using the technique of Giedroc et al.(1986). Site-Directed Mutagenesis of Gene 32. By use of the methods of Gillam and Smith (1979) or Taylor et al.(1985) in vitro site-directed mutagenesis was performed on Ml3 constructs containing gene 32 [designated pYS5 and described in Shamoo et al.(1986)]. Oligodeoxynucleotides used as mutagenic primers in the procedure were synthesized at either the Albert Einstein College of Medicine DNA Synthesis Fa-cility or the Yale School of Medicine Protein and Nucleic Acid Chemistry Facility. Oligonucleotides were HPLC purified by using a Nucleogen DEAE 60-7 column and the manufacturer’s protocol. Gp32ser73 (a tyrosine to serine substitution at position 73), 1 gp32serl 15, and gp32serl37 were made by using techniques described by Gillam and Smith (1979). The mu-tagenic oligonucleotides were used as primers on pYS 5 ssDNA for the large fragment of DNA polymerase I and the resulting mutant/parental heteroduplex ligated with T4 DNA ligase. Escherichia coli JM103 were transfected with the heteroduplex and the resulting plaques screened by in vitro hybridization on nitrocellulose filters by using 32* P-labeled mutagenic oligonucleotides as the probe (Mark et al., 1984). Final con-firmation of the mutation was achieved by rescreening plaques for the introduction of a new restriction site that was diagnostic for the particular substitution (Prigodich et al., 1986). Mutants gp32ala84, gp321eu92, gp32val99, gp321eul06, and gp32ilel 86 were made byusing the technique of Taylor et al.(1985), which is commercially available as a kit from Am-ersham. Following the manufacturer’s protocols pYS 5 ssDNA was annealed to a mutagenic oligonucleotide. The primed template was then extended by using the large frag-ment of DNA polymerase I in the presence of 2 mM dATP, dGTP, dTTP, and dCTPaS. Incorporation of thionucleotide into themutant strand allows endonuclease Adi to generate nicks within the parental strand, the majority of which can then be removed by using exonuclease III. Removal of most of the parental strand is then followedwith another extension reaction using DNA polymerase I to create a mutant/mutant duplex DNA. The mutant/mutant DNA was then transfected into E. coli TG 1 and either directly screened for the incor-poration of a new restriction site or sequenced by the technique of Sanger et al.(1977).