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鉴定猪 MYL1 基因的新型转录本及其启动子的初步特征分析。

Identification of novel transcripts from the porcine MYL1 gene and initial characterization of its promoters.

机构信息

College of Animal Science, South China Agricultural University, Guangzhou, People's Republic of China.

出版信息

Mol Cell Biochem. 2010 Oct;343(1-2):239-47. doi: 10.1007/s11010-010-0519-1. Epub 2010 Jun 19.

Abstract

The fast skeletal alkali myosin light polypeptide 1 (MYL1) gene is one of three mammalian alkali MLC genes and encodes two isoforms, 1f and 3f, which play a vital role in embryonic, fetal, and adult skeletal muscle development. We isolated the MYL1 gene from a pig BAC library with the goal of characterizing its promoter and identifying its transcripts. Genes and isoforms were identified by reverse transcriptase-PCR, northern blot and RACE (Rapid Amplification of cDNA Ends). Potential MYL1 gene promoters were characterized using a luciferase reporter assay and electrophoretic mobility shift assays (EMSA). MLC1f, MLC3f, and three additional isoforms of porcine MYL1, MLC5f-A, -B, and -C were identified. Up to now, the three novel isoforms had not been reported in human or mouse. Northern blot analysis indicated that MLC1f, MLC3f, and MLC5fs were expressed only in longissimus dorsi muscles. Two transcription initiation and termination sites were identified by RACE. Promoter analysis and EMSA demonstrated the presence of a MEF3 (skeletal muscle-specific transcriptional enhancer) binding site (+384 to +481), which might be essential for porcine MYL1 transcription. Our results suggested that five transcript variants were generated using alternative promoters, two transcription start sites, and polyA sites, as well as variable splicing of the pig MYL1 exon 5. The identification of alternative promoters and splice variants, the expression of the splice variants in different muscle tissues, and the definition of regulatory elements provide important molecular genetic knowledge concerning the MYL1 gene.

摘要

快速骨骼肌碱肌球蛋白轻链 1(MYL1)基因是三种哺乳动物碱肌球蛋白 MLC 基因之一,编码两种同工型,1f 和 3f,它们在胚胎、胎儿和成年骨骼肌发育中起着至关重要的作用。我们从猪 BAC 文库中分离出 MYL1 基因,旨在对其启动子进行特征分析并鉴定其转录本。通过反转录 PCR、 northern blot 和 RACE(快速扩增 cDNA 末端)鉴定基因和同工型。使用荧光素酶报告基因测定和电泳迁移率变动分析(EMSA)对潜在的 MYL1 基因启动子进行了特征分析。鉴定了 MLC1f、MLC3f 和猪 MYL1 的另外三个同工型,即 MLC5f-A、-B 和 -C。到目前为止,这三种新同工型在人类或小鼠中尚未报道过。northern blot 分析表明,MLC1f、MLC3f 和 MLC5fs 仅在背最长肌中表达。通过 RACE 鉴定了两个转录起始和终止位点。启动子分析和 EMSA 表明存在 MEF3(骨骼肌特异性转录增强子)结合位点(+384 至+481),这可能对猪 MYL1 转录至关重要。我们的研究结果表明,使用替代启动子、两个转录起始位点和 polyA 位点以及猪 MYL1 外显子 5 的可变剪接,产生了五种转录变体。替代启动子和剪接变体的鉴定、剪接变体在不同肌肉组织中的表达以及调节元件的定义为 MYL1 基因提供了重要的分子遗传知识。

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