In situ reverse transcription, an approach to characterize genetic diversity and activities of prokaryotes
In situ reverse transcription, an approach to characterize genetic diversity and activities of prokaryotes
复制标题
原位逆转录，一种表征原核生物遗传多样性和活性的方法
DOI：
10.1128/aem.63.12.4907-4913.1997
复制
发表时间：
1997
影响因子：
4.4
通讯作者：
R. Hodson
中科院分区：
文献类型：
--
作者：
F. Chen;J. M. González;W. Dustman;M. Moran;R. Hodson
利用与特定16S rRNA或mRNA序列互补的单寡核苷酸逆转录酶，对完整细菌细胞内的RNA分子进行逆转录。荧光标记的核苷酸被纳入细胞内每个转录的cDNA。这种方法被称为原位逆转录（ISRT）。在本研究中，通过针对16S rRNA序列独特区域的物种特异性引物，ISRT成功地检测和枚举了两种降解木质素的细菌Microbulbifer hydrolyticus IRE-31和Sagittula stellata E-37，并选择了用于降解木质素的培养混合物和复杂富集群落。图像分析显示，M. hydrolyticus IRE-31和S. stellata E-37分别占木质素富集群落细菌细胞总数的约30%和2%。ISRT估计的种群数量与原位杂交（ISH）技术估计的种群数量以及与提取的群落DNA杂交估计的种群数量相当。ISRT还成功地检测了甲苯蒸汽暴露海水中表达todC1基因的恶臭假单胞菌F1。ISRT提供了比ISH更高的信号强度，特别是当靶向mRNA时。ISRT计算出的像素强度是ISH的4.2倍。这表明，将荧光标记的核苷酸多次掺入到cDNA中，为细菌种群的系统发育鉴定以及检测复杂细菌群落中表达特定功能基因的细胞提供了高灵敏度。
Reverse transcription of RNA molecules inside intact bacterial cells was carried out by using reverse transcriptase with a single oligonucleotide complementary to specific 16S rRNA or mRNA sequences. Fluorescently labeled nucleotides were incorporated into each transcribed cDNA inside cells. This protocol is termed in situ reverse transcription (ISRT). In this study, by using species-specific primers targeting unique regions of the 16S rRNA sequences, ISRT was used successfully to detect and enumerate the two lignin-degrading bacteria Microbulbifer hydrolyticus IRE-31 and Sagittula stellata E-37 in culture mixtures and complex enrichment communities selected for lignin degradation. Image analysis revealed that M. hydrolyticus IRE-31 and S. stellata E-37 accounted for approximately 30 and 2%, respectively, of the total bacterial cells in lignin enrichment communities. Populations estimated by ISRT were comparable to those estimated by in situ hybridization (ISH) techniques and to those estimated by hybridization against extracted community DNA. ISRT was also successfully used to detect Pseudomonas putida F1 expressing the todC1 gene in seawater exposed to toluene vapor. ISRT provided a higher signal intensity than ISH, especially when targeting mRNA. The calculated pixel intensities resulting from ISRT were up to 4.2 times greater than those from ISH. This suggests that multiple incorporation of fluorescently labeled nucleotides into cDNA provides a high sensitivity for phylogenetic identification of bacterial populations as well as detection of cells expressing a specific functional gene within complex bacterial communities.
影响因子：
56.9
作者：
PATTERSON, BK;TILL, M;WOLINSKY, SM
通讯作者：
WOLINSKY, SM
影响因子：
56.9
作者：
DELONG, EF;WICKHAM, GS;PACE, NR
通讯作者：
PACE, NR
铜绿假单胞菌 lasA 基因的核苷酸序列和在大肠杆菌中的表达。
影响因子：
3.2
作者：
Schad,PA;Iglewski,BH
通讯作者：
Iglewski,BH