Doran Tim, Tizard Mark, Millar Douglas, Ford Jon, Sumar Nazira, Loughlin Mark, Hermon-Taylor John
Department of Surgery St Georges Hospital Medical School Cranmer Terrace London SW17 ORE UK.
Microbiology (Reading). 1997 Feb;143 ( Pt 2):547-552. doi: 10.1099/00221287-143-2-547.
The Mycobacterium avium subsp. paratuberculosis (formerly Mycobacterium paratuberculosis) atypical insertion sequence, IS900, encodes a novel gene on the complementary strand to the putative transposase, p43. This gene requires a promoter, ribosome binding site (RBS) and termination codon to be acquired upon insertion into the M. avium subsp. paratuberculosis genome and hence is designated the hed (host expression-dependent) gene of IS900. Analysis of IS900 insertion sites suggests that this element targets translation initiation signals in M. avium subsp. paratuberculosis, specifically inserting between the RBS and start codon of a putative gene sequence. This aligns the hed initiation codon adjacent to a functional RBS and possibly downstream of an active promoter, driving expression of Hed protein. We have confirmed this unique targeting process by detecting expression of hed in M. avium subsp. paratuberculosis at the level of transcription by reverse transcription-PCR. Further, two Hed-specific antibodies detected Hed translation products in Western blots of protein extracts from M. avium subsp. paratuberculosis. A recombinant form of Hed expressed and purified from Escherichia coli will facilitate studies of IS900 transposition and will also be assessed as a diagnostic antigen for M. avium subsp. paratuberculosis disease. Implications of IS900 insertion in M. avium subsp. paratuberculosis pathogenicity are discussed.
鸟分枝杆菌副结核亚种（以前称为副结核分枝杆菌）的非典型插入序列IS900，在与推定转座酶p43互补的链上编码一个新基因。该基因在插入鸟分枝杆菌副结核亚种基因组时需要获得启动子、核糖体结合位点（RBS）和终止密码子，因此被指定为IS900的hed（宿主表达依赖性）基因。对IS900插入位点的分析表明，该元件靶向鸟分枝杆菌副结核亚种中的翻译起始信号，特别是插入到一个推定基因序列的RBS和起始密码子之间。这使得hed起始密码子与一个功能性RBS相邻，并可能位于一个活性启动子的下游，从而驱动Hed蛋白的表达。我们通过逆转录PCR在转录水平检测鸟分枝杆菌副结核亚种中hed的表达，证实了这一独特的靶向过程。此外，两种Hed特异性抗体在鸟分枝杆菌副结核亚种蛋白提取物的Western印迹中检测到了Hed翻译产物。从大肠杆菌中表达和纯化的重组形式的Hed将有助于IS900转座的研究，也将作为鸟分枝杆菌副结核亚种疾病的诊断抗原来评估。本文讨论了IS900插入对鸟分枝杆菌副结核亚种致病性的影响。
