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目的寻找钾-氯协同转运子(KCC)1新的同分异构体及分析其意义。方法抽提正常人肾组织RNA,用已发表KCCl序列设计引物,进行cDNA末端快速放大(RACE)及RT-PCR,以Northern印迹分析证实新的同分异构体的存在,并用生物信息学方法比较新序列。结果RACE和Northern印迹分析结果证实在人肾组织中有3种KCCl的新的同分异构体,与野生型KCCl不同的是,新的KCCl同分异构体2含部分内含子1的序列(外显子1b),作为其外显子1.蛋白翻译起始密码子也位于该外显子中。新的KCCl同分异构体3含外显子1b、外显子2、内含子2及以后与野生型KCCl相同的序列,蛋白翻译起始密码子位于外显子4,导致-长的5’端非翻译区,内含子2靠外显子3的位置有-翻译终止密码子。新的KCCl同分异构体4含部分内含子3作为其5’端非翻译区。结论新的异构体的发现为KCC家族增添了新的成员,并为KCCl这一重要分子的转录和功能调节提供了新的线索,其特异性序列可用于分析它们在人类疾病中的病理意义。由于启动子类型的不同对同分异构体基因转录的选择起主动的调节作用,多个同分异构体及多个启动子的存在对理解KCCl复杂的生理功能、调节过程和致病机制具有重要意义。
Aim To search for new isomers of potassium-chloride co-transporter (KCC) 1 and to analyze its significance. Methods The RNA of normal human renal tissues was extracted and the primers were designed by published KCCl sequences for rapid amplification of cDNA ends (RACE) and RT-PCR. The presence of new isoforms was confirmed by Northern blot analysis and bioinformatics methods Compare new sequences. Results RACE and Northern blot analysis confirmed the presence of three new isoforms of KCCl in human kidney tissue, in contrast to wild-type KCCl, the new KCCl isomer 2 with partial intron 1 Sequence (exon 1b) as its exon 1. The protein translation initiation codon is also located in this exon. The new KCCl isoform 3 contains the same sequence as exon 1b, exon 2, intron 2 and later, and wild-type KCCl, with the protein translation initiation codon at exon 4, resulting in a long 5 ’untranslated region, intron 2 by exon 3 position - translation stop codon. The new KCCl isomer 4 contains part of intron 3 as its 5 ’untranslated region. Conclusion The discovery of new isomers adds new members to the KCC family and provides new clues to the transcriptional and functional regulation of KCCl, an important molecule that can be used to analyze their pathological significance in human diseases . Due to the different types of promoters play an active regulatory role in the transcription of isoforms, the presence of multiple isoforms and multiple promoters has important implications for understanding the complex physiological functions, regulatory processes and pathogenesis of KCCl It is of great significance.