Bacterial DNA methylation and gene transfer efficiency
Bacterial DNA methylation and gene transfer efficiency
复制标题
DOI：
10.1006/bbrc.2000.3603
复制
发表时间：
2000-10-05
影响因子：
3.1
通讯作者：
Wion, D
中科院分区：
文献类型：
--
作者：
Allamane, S;Jourdes, P;Wion, D
目前用于DNA免疫或基因治疗的任何质粒在细菌中的必要扩增步骤在基因转移中使用的质粒DNA的核苷酸序列中引入修饰。这些变化分别影响所有GATC和CC（A/T）GG序列中的腺嘌呤和内部胞嘧啶。这些在质粒DNA中引入6-甲基腺嘌呤和5-甲基胞嘧啶的修饰是细菌修饰系统Dam和Dcm存在的结果。在真核生物中，在二核苷酸-CG-上存在B-甲基胞嘧啶参与沉默基因表达，但在基因转移实验中使用的质粒中存在细菌G（m）ATC和（CC）-C-m（A/T）GG序列的可能后果目前尚不清楚。由于存在通过使用（dam（-），dcm（-））细菌获得缺乏这种特定细菌甲基化模式的质粒DNA的可能性，我们进行了实验以比较在JM 109（dam（+），dcm（+））或JM 110（dam（-），dcm（-））细菌中扩增的pCMV-luc报告质粒的体外和体内基因转移效率。获得的数据表明，在GATC序列中存在6-甲基腺嘌呤和在CC（A/T）GG基序的第二个C中存在5-甲基胞嘧啶不会降低体外或体内基因转移后检测到的荧光素酶活性水平。相反，用在JM 109（dam（+），dcm（+））细菌中扩增的pCMV-luc进行的基因转移比用在突变的JM 110（dam（-），dcm（-））对应物中扩增的质粒进行的相同转染产生更大量的荧光素酶。因此，这些数据并不表明使用（dam（-）、dcm（-））细菌扩增质粒DNA可能会增加基因转移效率。然而，为了扩增质粒DNA而持续使用（dam（+）dcm（+））细菌引起了在真核细胞或生物体中引入细菌G（m）ATC和（CC）-C-m（A/T）GG序列的可能生物学后果的问题。(C)北京大学出版社.
The necessary amplification step in bacteria of any plasmid currently used in DNA immunization or gene therapy introduces modification in the nucleotide sequence of plasmid DNA used in gene transfer. These changes affect the adenine and the internal cytosine in respectively all of the GATC and CC(A/T)GG sequences. These modifications which introduce 6-methyladenine and 5-methylcytosine in plasmidic DNA are the consequence of the existence of the bacterial modification systems Dam and Dcm. In eucaryotes, the presence of B-methylcytosine at dinucleotides -CG- is involved in silencing gene expression, bud the possible consequences of the presence of the bacterial G(m)ATC and (CC)-C-m(A/T)GG sequences in the plasmids used in gene transfer experiments are presently unknown. Since the possibility exists to obtain plasmid DNA lacking this specific bacterial pattern of methylation by using (dam(-), dcm(-)) bacteria we performed experiments to compare in vitro and in vivo gene transfer efficiency of a pCMV-luc reporter plasmid amplified either in the JM109 (dam(+), dcm(+)) or JM110 (dam(-), dcm(-)) bacteria. Data obtained demonstrated that the presence of 6-methyladenine in GATC sequences and 5-methylcytosine in the second C of CC(A/T)GG motifs does not reduce the levels of luciferase activity detected following in vitro or in vivo gene transfer. On the contrary, gene transfer with a pCMV-luc amplified in JM109 (dam(+), dcm(+)) bacteria gives greater amounts of luciferase than the same transfection performed with a plasmid amplified in the mutated JM110 (dam(-), dcm(-)) counterpart. Therefore, these data do not suggest that the use of (dam(-), dcm(-)) bacteria to amplify plasmid DNA may increase gene transfer efficiency. However, the persistence of the use of (dam(+) dcm(+)) bacteria in order to amplify plasmid DNA raises the question of the possible biological consequences of the introduction of the bacterial G(m)ATC and (CC)-C-m(A/T)GG sequences in eukaryotic cells or organisms. (C) 2000 Academic Press.