CLONING AND SEQUENCING OF THE GENES ENCODING ESCHERICHIA-COLI CYTOLETHAL DISTENDING TOXIN
CLONING AND SEQUENCING OF THE GENES ENCODING ESCHERICHIA-COLI CYTOLETHAL DISTENDING TOXIN
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DOI：
10.1128/iai.62.1.244-251.1994
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发表时间：
1994-01-01
影响因子：
3.1
通讯作者：
KAPER, JB
中科院分区：
文献类型：
--
作者：
SCOTT, DA;KAPER, JB
表达细胞致死性膨大蛋白(CDT)的EscherichiaCall菌株在24 h可引起CHO细胞伸长，随后细胞逐渐膨胀并死亡长达120 h。在HeLa、Hep-2和Vero细胞中也观察到类似的膨胀和细胞毒性。CHO细胞的初始伸长与E.称为不耐热毒素(LT)引起的伸长没有区别。与LT株相比，这些株的培养上清液对Y-1肾上腺细胞没有影响。将TnPhoA导入CDT阳性的E6468/62(086：H34)中，鉴定出13个CDT阴性的接合子。从CDT阴性突变体的TnPhoA插入位点两侧构建的DNA探针用于从插入5.5kb的E6468/62基因组文库中鉴定CDT阳性克隆。在CHO细胞中产生核酸外切酶缺失并进行检测。以这种方式，定义了2.3kb的CDT活性区域，并确定了核苷酸序列。序列分析确定了三个开放阅读框(ORF)，分别命名为cdtA、cdtB和cdtC。它们分别含有711、819和570个核苷酸，分别编码25.5、29.8和20.3 kDa的多肽。每个ORF都有一个推测的信号序列，在cdtA和cdtB之间以及cdtB和cdtC之间有4个碱基的重叠。核苷酸和预测的氨基酸序列与以前报道的任何基因或蛋白质没有显著的同源性。通过体外转录翻译和抗碱性磷酸酶免疫印迹，鉴定了与每个ORF对应的天然蛋白和/或融合蛋白。
Escherichia call strains expressing cytolethal distending to?rin (CDT) cause elongation of CHO cells at 24 h, followed by progressive cellular distention and death for up to 120 h. Similar distention and cytotoxicity are seen in HeLa, HEp-2, and, to a lesser extent, Vero cells. The initial elongation in CHO cells is indistinguishable from that caused by E. call heat-labile toxin (LT). In contrast to those from LT strains, supernatants from these strains have no effect on Y-1 adrenal cells. TnphoA was introduced into CDT-positive E. coli E6468/62 (086:H34), isolated from a child with diarrhea, and 13 CDT-negative transconjugants were identified. DNA probes constructed from DNA flanking the TnphoA insertion sites of CDT-negative mutants were used to identify a CDT-positive clone from an E6468/62 genomic library with a 5.5-kb insert. Exonuclease deletions were created and assayed in CHO cells. In this manner, a 2.3-kb CDT-active region was defined, and the nucleotide sequence was determined. Sequence analysis identified three open reading frames (ORFs), designated cdtA, cdtB, and cdtC. These contain 711, 819, and 570 bp, respectively, and encode polypeptides with predicted molecular masses of 25.5, 29.8, and 20.3 kDa, respectively. Each ORF has a putative signal sequence, end there are 4-bp overlaps between cdtA and cdtB and between cdtB and cdtC. The nucleotide and predicted amino acid sequences have no significant homology with those of any previously reported genes or proteins. By in vitro transcription-translation and an anti-alkaline phosphatase immunoblot, native proteins and/or fusion proteins corresponding to each ORF were identified.