期刊文献+

钾-氯协同转运子1新的同分异构体的发现及其转录调控意义

Discovery of spliced isoforms of KCC1 and their implications in the transcriptional regulation
原文传递
导出
摘要 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。 Objective To look for new spliced isoforms of human K-Cl cotransporter 1 (KCC1) and to investigate their implications in the transcriptional regulation. Methods RNA from human kidney was amplified by 5' rapid amplification of cDNA endings (RACE)and RT-PCR.Northern blot was used to confirm the existance of the new spliced isoforms.New sequences were compared with published sequences of KCC1 EST database using bioinformatics method. Results Three novel spliced isoforms of KCC1,named KCC1 isoform 2, 3, and 4, were discovered by RACE and confirmed by Northern blot. New isoform 2 used part of intron 1 as its exon 1 (exon 1b). Its start codon was in exon 1b. The new isoform 3 utilized exon 1b, exon 2, intron 2 and the same downstream sequences as that of wild KCC1. The start codon was in the exon 4. This leaded to a long 5' untranslated region.A stop codon was in its intron 2 near exon 3. New spliced isoform 4 included part of intron 3 as its 5' untranslated region.Considering the report about the arbitrary truncations of KCC1 cDNA,new found isoforms could significantly reduce or abolish the function of KCC1 cotransport and inhibit the activity of wild type of KCC1 and KCC3. The existence of different 5' spliced isoforms implied that different first exons were used and therefore there were different promoters.Conclusions The discovery of new isoforms of KCCl increases new members of KCC family and provides new insights into the transcriptional and functional regulation of KCC1. Their specific sequences can be used in the analyses of human diseases. Because the types of core promoters are active contributors to combinatorial gene regulation, findings of this study have significant implications for the complicated KCC1 function, process of regulation and mechenisms of related diseases.
出处 《中华肾脏病杂志》 CAS CSCD 北大核心 2005年第1期47-51,共5页 Chinese Journal of Nephrology
关键词 钾-氯协同转运子 同分异构体 转录 调控 基因调节 Sodium-potassium-chloride cotransporter RNA splicing Stereoisomers Genes, regulator
  • 相关文献

参考文献12

  • 1Mercado A,Song L,Vazquez N, et al.Functional comparison of the K^+-Cl^- cotransporters KCCI and KCC4. J Biol Chem,2000, 275 : 30326-30334. 被引量:1
  • 2Boettger T, Hubner CA, Maier H, et al. Deafness and renal tubular acidosis in mice lacking the K-Cl co-transporter KCC4.Nature, 2002, 416:874-878. 被引量:1
  • 3Johnson JM, Castle J, Garrett-Engele P, et al. Genome-wide survey of human alternative pre-mRNA splicing with exon junction microarrays. Science, 2003, 302 : 2141-2144. 被引量:1
  • 4Bracco L, Kearsey J. The relevance of alternative RNA splicing to pharmacogenomics. Trends Biotechnol. 2003,21 :346-353. 被引量:1
  • 5Sazani P, Ryszard K. Therapeutic potential of antisense oligonucleotides as modulators of alternative splicing. J Clin Invest, 2003,112 : 481-486. 被引量:1
  • 6Auboeuf D, Honig A, Berget SM, et al. Coordinate regulation of transcription and splicing by steroid receptor coregulators. Science, 2002, 298:416-419. 被引量:1
  • 7Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thioeyanate-phenol-chloroform extraction. Anal Biochem, 1987,162:156-159. 被引量:1
  • 8Edwards JB, Delort J, Mallet J. Oligodeoxyribonucleotide ligation to single-stranded cDNAs: a new tool for cloning ends of mRNAs and for constructing eDNA libraries by in vitro amplification. Nucleic Acids Res, 1991,19 : 5227-5232. 被引量:1
  • 9Casula S, Shmukler BE, Wilhelm S, et al. A dominant negative mutant of the KCC1 K-C1 cotransporter: both N-and C-terminal cytoplasmic domains are required for K-Cl cotransport activity. J Biol Chem, 2001, 276:41870-41878. 被引量:1
  • 10Zhou GP, Wong C, Su R, et al. Human potassium chloride cotransporter 1 (SLC12A4) promoter is regulated by AP-2 and contains a functional downstream promoter element. Blood, 2004, 103:4302-4309. 被引量:1

相关作者

内容加载中请稍等...

相关机构

内容加载中请稍等...

相关主题

内容加载中请稍等...

浏览历史

内容加载中请稍等...
;
使用帮助 返回顶部