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首页> 外文期刊>Journal of bacteriology >Detection of a Group II Intron without an Open Reading Frame in the Alpha-Toxin Gene of Clostridium perfringens Isolated from a Broiler Chicken
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Detection of a Group II Intron without an Open Reading Frame in the Alpha-Toxin Gene of Clostridium perfringens Isolated from a Broiler Chicken

机译：肉鸡产气荚膜梭菌α-毒素基因中没有开放阅读框的II组内含子的检测

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A DNA insertion of 834 bp, designated CPF-G2Im, was identified within the alpha toxin gene (cpa) of Clostridium perfringens strain CPBC16ML, isolated from a broiler chicken. Sequence analysis of CPF-G2Im indicated that it was integrated 340 nucleotides downstream of the start codon of cpa. However, the insertion did not abolish the phospholipase C and hemolytic activities of CPBC16ML. To investigate the expression of its alpha toxin, the intact copy of cpa was cloned into an expression vector and transformed into Escherichia coli M15 cells. Immunoblotting analysis showed that the protein expressed from the transformant as well as in the culture supernatant of C. perfringens strain CPBC16ML had the expected molecular weight detected in reference strains of C. perfringens. Northern hybridization and reverse transcriptase PCR (RT-PCR) analysis revealed that the entire CPF-G2Im insertion was completely spliced from the cpa precursor mRNA transcripts. The sequence of the insertion fragment has 95% and 97% identity to two noncoding regions corresponding to sequences that flank a predicted group II RT gene present in the pCPF4969 plasmid of C. perfringens. However, an RT was not encoded by the CPF-G2Im fragment. Based on the secondary structure prediction analysis, CPF-G2Im revealed typical features of group II introns. The present study shows that CPF-G2Im is capable of splicing in both C. perfringens and E. coli. To our knowledge, this is the first report that a group II intron without an open reading frame (ORF) is located in the cpa ORF of C. perfringens.
机译：在产自肉仔鸡的产气荚膜梭状芽孢杆菌菌株CPBC16ML的α毒素基因（ cpa ）中鉴定出了834 bp的DNA插入，​​称为CPF-G2Im。 CPF-G2Im的序列分析表明，它整合了 cpa 起始密码子下游340个核苷酸。但是，插入并没有消除CPBC16ML的磷脂酶C和溶血活性。为了研究其α毒素的表达，将完整的 cpa 克隆到表达载体中，并转化到大肠杆菌 M15细胞中。免疫印迹分析表明该蛋白从转化子以及在 C的培养上清中表达。 perfringens 菌株CPBC16ML具有在 C参考菌株中检测到的预期分子量。 perfringens 。 Northern杂交和逆转录酶PCR（RT-PCR）分析表明，整个CPF-G2Im插入物完全是从 cpa 前体mRNA转录物中剪接的。插入片段的序列与两个非编码区具有95％和97％的同一性，所述两个非编码区对应于位于 C的pCPF4969质粒中的预测的II型RT基因侧翼的序列。 perfringens 。但是，RT并非由CPF-G2Im片段编码。基于二级结构预测分析，CPF-G2Im揭示了II组内含子的典型特征。本研究表明，CPF-G2Im能够在两个C中剪接。 perfringens 和 E。大肠杆菌。据我们所知，这是第一个报告，其中没有开放阅读框（ORF）的II组内含子位于 C的 cpa ORF中。 perfringens 。

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