摘要
烟碱样乙酰胆碱受体 ( n ACh R)是由 4种亚基组成的五聚体 .哺乳类动物出生后γ亚基由ε亚基取代 ,迄今为止鸡 n ACh Rγ基因是否存在上述置换规律尚无定论 .为探索发育过程鸡骨骼肌n ACh R基因表达是否存在γ/ε亚基的置换及其机制 ,采用 RT- PCR技术和凝胶阻滞试验检测了鸡胚发育 9d至出生后 6周小鸡 γ基因表达的动力学及骨骼肌核抽提物的 DNA结合活性 .RT-PCR检测结果显示 ,在鸡胚发育 9d至出生后 6周的雏鸡骨骼肌组织均检出有 γ亚基 m RNA转录 .提示与哺乳类不同 ,出生前后鸡骨骼肌组织 ACh Rγ亚基基因持续表达 ,不存在 γ/ε亚基的置换表达规律 .以 γ基因 - 2 60 /- 2 4 0 (含 E盒与 M- CAT盒重叠序列 )和 - 2 39/- 50 (含 M- CAT盒及 GC富含区 )片段为探针 ,分别与鸡胚发育 9d至出生后 2周小鸡骨骼肌的核抽提物进行凝胶阻滞试验 .在发育各阶段的骨骼肌核抽提物中均有识别 - 2 39/- 50片段的结合活性存在 ,但在出生后 2周小鸡骨骼肌的核抽提物中未检出 - 2 60 /- 2 4 0结合活性 .结果提示 ,在出生后第 1 4d的肌核抽提物中存在的、识别并结合 - 2 39/- 50片段的活性物质与鸡 ACh Rγ基因在出生后持续表达有关 .
Nicotinic acetylcholine receptor(AChR) is a pentomer composed of four subunits.The γ subunit gene is turned off after mammalian birth and thereafter ε subunit takes its place.Till now this expression substitution phenomenon has only been observed in mammalia.What happens to the birds remains unknown.To observe if there is an ε subunit that substitutes for γ subunit in AChR expression during chicken development,the AChR γ subunit gene transcripts in skeletal muscle from 9 day old embryo to 6 week old chicken were examined by reverse transcription polymerase chain reaction (RT PCR),and the binding activities of nuclear extracts from the same embryonic and chicken muscle to DNA were analyzed by gel mobility shift assay.The RT PCR experiments showed that AChR γ subunit gene expressed in all of the selected time points during chicken development suggested mammalian γ/ε expression substitution mechanism did not apply to chicken.The gel mobility shift assays using chicken γ subunit gene upstream sequences -260/-240(containing an overlap of E box and M CAT box)and -239/-50(containing M CAT box and GC rich) as probes showed that the sustained expression of γ subunit gene was related to binding activities of nuclear extracts to -239/-50 fragment.
出处
《中国生物化学与分子生物学报》
CSCD
2000年第5期669-673,共5页
Chinese Journal of Biochemistry and Molecular Biology
基金
国家自然科学基金!( 3 9770 42 2 )
教育部博士点科研基金!(972 8)资助
