Expression of Streptococcus mutans aspartate-semialdehyde dehydrogenase gene cloned into plasmid pBR322.
Expression of Streptococcus mutans aspartate-semialdehyde dehydrogenase gene cloned into plasmid pBR322.
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克隆到质粒pBR322中的变形链球菌天冬氨酸半醛脱氢酶基因的表达。
DOI：
10.1099/00221287-128-5-1135
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发表时间：
1982
期刊：
影响因子：
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通讯作者：
Curtiss3rd,R
中科院分区：
文献类型：
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作者：
Jagusztyn-Krynicka,EK;Smorawinska,M;Curtiss3rd,R
克隆到大肠杆菌中的载体质粒pBR322中的变形链球菌染色体DNA能够补充宿主菌株中天冬氨酸半醛脱氢酶(EC 1.2.1.11)基因(asd)缺失的代谢缺陷。我们构建了两个Asd+重组质粒pYA570和pYA571，分别含有4·7和4·5千碱基的S。将变异染色体DNA以相同方向插入pBR322的HindIII限制性内切酶位点。 TheS。 mutansUAB62 Asd+DNA 不与大肠杆菌杂交。含有完整asd基因的大肠杆菌DNA，但确实与链球菌杂交。 mutansUAB62 染色体 DNA。然后从pYA570构建衍生的Asd+质粒。其中一个，pYA574，有 4·5 千碱基。 mutans以与pYA570相反的方向插入DNA。在另一个中，pYA575，theS。 mutansinsert DNA 的大小减少至 1·3 千碱基。可以看出，S 的方向。插入pBR322四环素抗性(Tcr)基因启动子区域的mutansDNA片段影响Tcr的表达。 S 的方向。 mutansinsert也影响了某些大肠杆菌中质粒的稳定性。粘菌素。使用核酸内切酶 EcoRI、BamHI、HindIII、PstI 和 SalI 确定了 pYA570、pYA571、pYA574 和 pYA575 的限制性图谱。在大肠杆菌中研究了 Asd+质粒指导的蛋白质合成。大肠杆菌细胞。质粒pYA570、pYA574和pYA575各自产生大量的蛋白质，单体分子量约为45000，这与pBR322和E不同。大肠杆菌特异蛋白；这种蛋白质就是theS。突变体asdgene产品。重组质粒 pYA575 的较小衍生物 Asd- 允许定位 S。 mutans asdgene启动子和转录方向待确定。
Streptococcus mutanschromosomal DNA cloned into the vector plasmid pBR322 inEscherichia coliis able to complement the metabolic defect of an aspartate-semialdehyde dehydrogenase (EC 1.2.1.11) gene (asd) deletion in the host strain. We constructed two Asd+recombinant plasmids, pYA570 and pYA571, containing 4·7 and 4·5 kilobases, respectively, ofS. mutanschromosomal DNA inserted into theHindIII restriction endonuclease site of pBR322 in the same orientation. TheS. mutansUAB62 Asd+DNA did not hybridize withE. coliDNA which contained an intactasdgene, but did hybridize withS. mutansUAB62 chromosomal DNA. Derivative Asd+plasmids were then constructed from pYA570. One, pYA574, had a 4·5 kilobaseS. mutansinsert DNA in the opposite direction from pYA570. In another, pYA575, theS. mutansinsert DNA was reduced in size to 1·3 kilobases. It was seen that the orientation of theS. mutansDNA fragment inserted into the promoter region of the pBR322 tetracycline resistance (Tcr) gene affected expression of Tcr. Orientation of theS. mutansinsert also affected the stability of the plasmid in certainE. colistrains. Restriction maps for pYA570, pYA571, pYA574 and pYA575 using the endonucleasesEcoRI,BamHI,HindIII,PstI andSalI were determined. Asd+plasmid-directed protein synthesis was studied inE. coliminicells. The plasmids pYA570, pYA574 and pYA575 each produced large amounts of a protein, with a monomeric molecular weight of about 45000, that was distinct from both pBR322 andE. colispecified protein; this protein is theS. mutans asdgene product. Smaller derivatives of recombinant plasmid pYA575 that were Asd−allowed the location of theS. mutans asdgene promoter and the direction of transcription to be determined.