TN5401, A NEW CLASS-II TRANSPOSABLE ELEMENT FROM BACILLUS-THURINGIENSIS
TN5401, A NEW CLASS-II TRANSPOSABLE ELEMENT FROM BACILLUS-THURINGIENSIS
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DOI：
10.1128/jb.176.10.2835-2845.1994
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发表时间：
1994-05-01
影响因子：
3.2
通讯作者：
BAUM, JA
中科院分区：
文献类型：
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作者：
BAUM, JA
从苏云金芽孢杆菌亚种孢子形成缺陷变体中回收了一种新的 II 类（Tn3 样）转座元件，命名为 Tn5401。 morrisoni EG2158 插入重组质粒后。插入片段的序列分析揭示了一个 4,837 bp 的转座子，具有两个大的开放阅读框，方向相同，编码 36 kDa（306 个残基）和 116 kDa（1,005 个残基）的蛋白质以及 53 bp 末端反向重复序列。推导的 36 kDa 蛋白氨基酸序列与苏云金芽孢杆菌转座子 Tn4430（位点特异性重组酶噬菌体整合酶家族的成员）的 TnpI 重组酶具有 24% 的序列同一性。推导的 116 kDa 蛋白氨基酸序列与 Tn3 转座酶有 42% 的序列同一性，但与 Tn4430 的 TnpA 转座酶只有 28% 的序列同一性。还鉴定了两个功能未知的小开放阅读框，命名为 orf1（85 个残基）和 orf2（74 个残基）。 Southern印迹分析表明，与Tn4430相比，Tn5401在苏云金芽孢杆菌的不同亚种中并不常见，并且通常与已知的杀虫晶体蛋白基因无关。使用质粒 pEG922（一种含有 Tn5401 的温度敏感穿梭载体）对苏云金芽孢杆菌进行转座研究。 Tn5401 转座到染色体和质粒靶位点，但表现出对质粒位点的明显偏好。转座是可复制的，并导致在目标位点产生 5 bp 的重复。通过引物延伸分析绘制 Tn5401 内的转录起始位点。两个启动子，命名为 P-L 和 P-R，分别指导 orf1 orf2 和 tnpI-tnpA 的转录，并受 TnpI 负调控。 Tn5401 和 Tn4430 启动子区域的序列比较表明保守序列元件 ATGTCCRCTAAY 介导 TnpI 结合和共整合解析。相同的元件包含在 53 bp 末端反向重复序列中，因此解释了它们不寻常的长度，并表明 TnpI 在调节 Tn5401 转座中具有额外的作用。
A new class II (Tn3-like) transposable element, designated Tn5401, was recovered from a sporulation-deficient variant of Bacillus thuringiensis subsp. morrisoni EG2158 following its insertion into a recombinant plasmid. Sequence analysis of the insert revealed a 4,837-bp transposon with two large open reading frames, in the same orientation, encoding proteins of 36 kDa (306 residues) and 116 kDa (1,005 residues) and 53-bp terminal inverted repeats. The deduced amino acid sequence for the 36-kDa protein shows 24% sequence identity with the TnpI recombinase of the B. thuringiensis transposon Tn4430, a member of the phage integrase family of site-specific recombinases. The deduced amino acid sequence for the 116-kDa protein shows 42% sequence identity with the transposase of Tn3 but only 28% identity with the TnpA transposase of Tn4430. Two small open reading frames of unknown function, designated orf1 (85 residues) and orf2 (74 residues), were also identified. Southern blot analysis indicated that Tn5401, in contrast to Tn4430, is not commonly found among different subspecies of B. thuringiensis and is not typically associated with known insecticidal crystal protein genes. Transposition was studied with B. thuringiensis by using plasmid pEG922, a temperature-sensitive shuttle vector containing Tn5401. Tn5401 transposed to both chromosomal and plasmid target sites but displayed an apparent preference for plasmid sites. Transposition was replicative and resulted in the generation of a 5-bp duplication at the target site. Transcriptional start sites within Tn5401 were mapped by primer extension analysis. Two promoters, designated P-L and P-R, direct the transcription of orf1 orf2 and tnpI-tnpA, respectively, and are negatively regulated by TnpI. Sequence comparison of the promoter regions of Tn5401 and Tn4430 suggests that the conserved sequence element ATGTCCRCTAAY mediates TnpI binding and cointegrate resolution. The same element is contained within the 53-bp terminal inverted repeats, thus accounting for their unusual lengths and suggesting an additional role for TnpI in regulating Tn5401 transposition.