Plasmid DNA is released from nanosized acicular material surface by low molecular weight oligonucleotides: exogenous plasmid acquisition mechanism for penetration intermediates based on the Yoshida effect
Plasmid DNA is released from nanosized acicular material surface by low molecular weight oligonucleotides: exogenous plasmid acquisition mechanism for penetration intermediates based on the Yoshida effect
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DOI：
10.1007/s00253-008-1637-5
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发表时间：
2008-10-01
影响因子：
5
通讯作者：
Ide, K.
中科院分区：
文献类型：
--
作者：
Yoshida, N.;Ide, K.
当纳米针状材料与细菌细胞组成的胶体溶液在水凝胶与界面形成材料的界面处受到滑动摩擦刺激时，摩擦系数迅速增大，伴随细菌细胞的纳米针状材料形成渗透中间体。这个效应被称为吉田效应，以纪念它的发现者。通过吉田效应，一个新的特性，其中渗透中间体纳入外源质粒DNA已被确定。本文提出了吉田效应中通过渗透中间体获取外源质粒的可能机制。分别以大肠杆菌细胞、pUC18和温石棉作为受体细胞、质粒DNA和纳米针状材料。即使反复洗涤由pUC18和温石棉组成的混合物，pUC18的转化效率也很稳定。因此，将吸附在温石棉上的pUC18引入受体大肠杆菌细胞。饱和时，温石棉吸附的pUC18量为0.8 ~ 1.2 μ g/mg。为了研究吸附在温石棉上的pUC18是否被聚合酶复制，我们对温石棉进行了聚合酶链反应（PCR）。pUC18编码的β -内酰胺酶基因的扩增被强烈抑制，该基因被吸附在温石棉上。这表明吸附在温石棉上的DNA在体内不能复制。当我们搜索释放吸附在温石棉上的pUC18的物质时，我们发现300 bp的DNA单链或双链片段可以从温石棉中释放pUC18。然后通过吉田效应研究了双链DNA和pUC18在温石棉上的竞争吸附。310- bp和603-bp双链核苷酸与pUC18的摩尔比相同，产生50%的竞争性抑制。因此，pUC18的吸附区长度约为300 bp。随着受体细胞培养时间的延长，转化效率降低，300- 600bp小RNA的表达量也降低。这些结果表明，吸附在温索石上的pUC18可以被300 bp的小RNA释放，并被DNA聚合酶复制，转移到子细胞中。
When a colloidal solution consisting of nanosized acicular material and bacterial cells is stimulated with sliding friction at the interface between the hydrogel and interface-forming material where the frictional coefficient increases rapidly, the nanosized acicular material accompanying the bacterial cells forms a penetration intermediate. This effect is known as the Yoshida effect in honor of its discoverer. Through the Yoshida effect, a novel property in which penetration intermediates incorporate exogenous plasmid DNA has been identified. This report proposes a possible mechanism for exogenous plasmid acquisition by penetration intermediates in the Yoshida effect. Escherichia coli cells, pUC18, and chrysotile were used as recipient cells, plasmid DNA, and nanosized acicular material, respectively. Even when repeatedly washing the mixture consisting of pUC18 and chrysotile, transformation efficiency by pUC18 was stable. Accordingly, pUC18 adsorbed onto chrysotile was introduced into recipient E. coli cells. At saturation, the amount of pUC18 adsorbed onto chrysotile was 0.8-1.2 mu g/mg. To investigate whether pUC18 adsorbed on chrysotile is replicated by polymerase, polymerase chain reaction (PCR) was carried out with the chrysotile. Amplification of the beta-lactamase gene coded in pUC18, which was adsorbed onto chrysotile, was strongly inhibited. This suggests that DNA adsorbed onto chrysotile is not replicated in vivo. When we searched for substances to release pUC18 adsorbed onto chrysotile, we found that a 300-bp single- or double-stranded segment of DNA releases pUC18 from chrysotile. Competitive adsorption onto chrysotile between double-stranded DNA and pUC18 was then examined through the Yoshida effect. The 310- and 603-bp double-stranded nucleotides caused 50% competitive inhibition at the same molar ratio with pUC18. Hence, the adsorbed region of pUC18 is about 300 bp in length. As the culture period for recipient cells increases, transformation efficiency decreases while the expression levels of small RNA of 300-600 bp also decrease. These results suggest that pUC18 adsorbed onto chrysotile can be released by 300-bp small RNA, replicated by DNA polymerase, and transferred to daughter cells.