Lluch-Senar Maria, Vallmitjana Miquel, Querol Enrique, Piñol Jaume
Institut de Biotecnologia i Biomedicina and Departament de Bioquímica i Biologia Molecular, Universitat Autònoma de Barcelona, 08193 Bellaterra, Barcelona, Spain.
Microbiology (Reading). 2007 Aug;153(Pt 8):2743-2752. doi: 10.1099/mic.0.2006/007559-0.
The mechanisms that promote and regulate transcription in mycoplasmas are poorly understood. Here, a promoter-probe vector based on the pMTnTetM438 minitransposon and containing a promoterless lacZ reporter gene was constructed to analyse Mycoplasma genitalium transcription in vivo. Recovered transposon insertions were in monocopy, with 16 % expressing enough beta-galactosidase (beta-Gal) to yield colonies exhibiting a detectable blue colour. A sample of 52 blue colonies was propagated and selected for further analyses. The beta-Gal activity of the corresponding cultures was measured to quantify, in a reproducible way, the transcription levels of the interrupted ORFs. Several insertions were found in sense with the interrupted ORF, but surprisingly there was also a number of insertions in non-coding regions, many of them in repetitive DNA regions known as MgPa islands. Moreover, 30 % of the analysed transposon insertions had the lacZ gene in the opposite orientation to the coding frame, suggesting the existence of antisense transcripts that may be involved in the control of gene expression in M. genitalium.
目前对支原体中促进和调节转录的机制了解甚少。在此，构建了一种基于pMTnTetM438微型转座子且含有无启动子lacZ报告基因的启动子探针载体，用于在体内分析生殖支原体的转录情况。回收的转座子插入为单拷贝，其中16%表达足够的β-半乳糖苷酶（β-Gal），从而产生呈现可检测蓝色的菌落。选取52个蓝色菌落样本进行传代培养并进一步分析。测量相应培养物的β-Gal活性，以可重复的方式量化中断的开放阅读框（ORF）的转录水平。在与中断的ORF方向一致的位置发现了一些插入，但令人惊讶的是，在非编码区也有许多插入，其中许多位于称为MgPa岛的重复DNA区域。此外，30%的分析转座子插入中，lacZ基因与编码框方向相反，这表明可能存在反义转录本，其可能参与生殖支原体基因表达的调控。
