In vitro expression and characterization of MYH9 mutant alleles linked to hereditary hearing loss.
In vitro expression and characterization of MYH9 mutant alleles linked to hereditary hearing loss.
复制标题
与遗传性听力损失相关的 MYH9 突变等位基因的体外表达和表征。
DOI：
10.1016/j.otohns.2009.12.030
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发表时间：
2010
期刊：
影响因子：
--
通讯作者：
Mhatre,AnandN
中科院分区：
文献类型：
--
作者：
Wei,CalvinC;Lalwani,AnilK;Mhatre,AnandN
目的：研究设计：将显性遗传MYH 9突变等位基因MYH 9 R702和MYH 9 R705 H整合到真核表达载体中，转染培养的人细胞系进行瞬时表达和分析。评估转染细胞的转基因诱导的细胞表型改变，包括NMHC-IIA依赖性细胞形状、肌动蛋白细胞骨架完整性和胞质分裂抑制。HeLa和MDA-MB-231培养的细胞系用携带与非综合征性和综合征性听力损失相关的野生型或突变型MYH 9等位基因的表达载体瞬时转染。外源转基因产物的表达用针对其N-末端HA标签的抗体检测，转染效率大于95%。通过免疫荧光检测转染前后宿主细胞的细胞形状、肌动蛋白-肌球蛋白细胞骨架的完整性和细胞核状态。发现用MYH 9 R705 H而不是MYH 9 R702 C转染的MDA-MB-231细胞中具有丝状伪足的细胞增加了2倍以上，具有多个核的细胞比例增加了10倍，表明胞质分裂受到抑制，相对于用野生型MYH 9转染的对照细胞。MDA-MB-231细胞内的肌动蛋白细胞骨架配置不受MYH 9 R702 CorMYH 9 R705 H表达的影响。结论：MYH 9 R705 H诱导的MDA-MB-231细胞表型改变支持MYH 9 R705 H突变的致病性，并可作为鉴定和表征MYH 9 R702 C功能障碍的合适检测系统。
Objective: To assess whetherMYH9mutant alleles linked to hereditary hearing loss induce disruption of cellular functions and associated phenotype following transient expression within cultured human cell lines.Study Design: Dominantly inheritedMYH9mutant alleles,MYH9R702CandMYH9R705H, were integrated within eukaryotic expression vector and then transfected into cultured human cell lines for transient expression and analysis. The transfected cells were assessed for transgene-induced alterations of the cellular phenotype, including NMHC-IIA-dependent cell shape, actin cytoskeleton integrity, and inhibition of cytokinesis.Setting: Laboratories of Molecular Otology and Molecular Genetics at the New York University School of Medicine.Subjects and Methods: HeLa and MDA-MB-231 cultured cell lines were transiently transfected with an expression vector carrying a wild type or mutantMYH9alleles, linked to nonsyndromic and syndromic hearing loss. Expression of exogenous transgene product was detected with antibodies directed toward its N-terminal HA tag, and transfection efficiency was greater than 95 percent. Host cells were characterized for cell shape, integrity of actin-myosin cytoskeleton, and nuclear status before and after transfections via immunofluorescence.Results: MDA-MB-231 cells transfected withMYH9R705Hbut notMYH9R702Cwere found to have a greater than two-fold increase in cells with filopodia and a ten-fold increase in proportion of cells with multiple nuclei, indicating inhibition of cytokinesis, relative to the control cells transfected with wild typeMYH9. Actin cytoskeleton configuration within MDA-MB-231 cells was unaffected by expression ofMYH9R702CorMYH9R705H. Unlike MDA-MB-231 cells, HeLa cells were refractory toMYH9R705HandMYH9R702C.Conclusions:MYH9R705H-induced altered phenotype of the MDA-MB-231 cell line supports the pathogenicity of the mutation and represents a suitable assay system for identification and characterization of its dysfunction.
DOI：
10.1002/j.1460-2075.1988.tb03045.x
发表时间：
1988-07
期刊：
The EMBO Journal
影响因子：
--
作者：
D. J. G. Rees;Ian M. Jones;P. Handford;S. J. Walter;M. P. Esnouf;K. J. Smith;George G. Brownlee
通讯作者：
D. J. G. Rees;Ian M. Jones;P. Handford;S. J. Walter;M. P. Esnouf;K. J. Smith;George G. Brownlee
DOI：
10.1111/j.1399-3011.1987.tb02269.x
发表时间：
1987
期刊：
International journal of peptide and protein research
影响因子：
--
作者：
Tam,JP
通讯作者：
Tam,JP
凝血因子 IX 的衍生物含有缺乏 γ-羧基谷氨酸的高亲和力 Ca2 结合位点。
DOI：
--
发表时间：
1984
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Morita,T;Isaacs,BS;Esmon,CT;Johnson,AE
通讯作者：
Johnson,AE