Gileva I P, Karginova E A, Mikriukov N N, Serpinskiĭ O I, Kravchenko V V
Mol Biol (Mosk). 1988 Jan-Feb;22(1):44-54.
The promoter-probing vector (pSK plasmid) was explored for cloning of the fragments from lambda cI857 and lambda b2 DNAs containing different regions of the att site. We have constructed all-tet fusions where the fusions are: 1) HindIII/BamHI-491 base pairs (b. p.) fragment of lambda cI857 DNA containing POP' site (plasmid pSK-PP'); 2) AluI-242 b. p. fragment of lambda cI857 DNA containing the left arm of the POP' site (plasmid pSK-P); 3) AluI-242 b. p. fragment of lambda cI857 DNA with opposite orientation (plasmid pSK-P); 4) EcoRI/BamHI-750 b. p. fragment of lambda b2 DNA containing the right arm of the POP' site (plasmid pSK-P'). These fusions permit us to analyse the effect of various pieces of the attachment site on the expression tet gene as the result of reparation of this gene promoter. We find that expression of tet (tetracycline resistant phenotype) takes place in the pSK-PP' and pSK-P but not in the pSK-P' and pSK-P. These facts permit us to conclude that the left arm of the att site contains a rightward promoter functioning in vivo. We postulate that this promoter activity might correspond to the promoter patt, which was described in previous experiments in vitro.
为了克隆来自λcI857和λb2 DNA中包含att位点不同区域的片段,对启动子探测载体(pSK质粒)进行了研究。我们构建了全tet融合体,其中的融合体为:1)包含POP'位点的λcI857 DNA的HindIII/BamHI - 491碱基对(b.p.)片段(质粒pSK - PP');2)包含POP'位点左臂的λcI857 DNA的AluI - 242 b.p.片段(质粒pSK - P);3)具有相反方向的λcI857 DNA的AluI - 242 b.p.片段(质粒pSK - P);4)包含POP'位点右臂的λb2 DNA的EcoRI/BamHI - 750 b.p.片段(质粒pSK - P')。这些融合体使我们能够分析附着位点的各个片段对tet基因表达的影响,这是该基因启动子修复的结果。我们发现tet(四环素抗性表型)在pSK - PP'和pSK - P中表达,但在pSK - P'和pSK - P中不表达。这些事实使我们能够得出结论,att位点的左臂包含一个在体内起作用的向右启动子。我们推测这种启动子活性可能对应于先前体外实验中描述的启动子patt。