EXPRESSION OF NONMUSCLE MYOSIN ISOFORMS IN EUKARYOTIC CELLS

非肌肉肌球蛋白重链的两种形式(MHCIIA和MHCIIB)有 在这个实验室里被克隆了，并被证明是两个 不同的基因。这些异构体在组织依赖的 方式和，当出现在同一单元中时，表现出截然不同的 本地化。MHCIIB亚型还显示了可供选择的 在MHC的两个区域进行剪接：一个在氨基酸211之后，靠近 ATP结合结构域(环1中的PESPKPVKHQ)和靠近肌动蛋白的另一个结构域- 结合结构域(环路2)。 非肌肉肌球蛋白IIB片段HMM的生化特性 它包含酶活性和肌动蛋白结合的部位 对从杆状病毒表达系统中分离到的HMM进行了研究。 隐马尔可夫模型的能力没有太大差别 在环1中插入区域以移动肌动蛋白细丝或水解三磷酸腺苷。 平滑肌MHC也在相同的区域显示了不同的剪接 在ATP结合域附近。含有这七种成分的平滑肌肌球蛋白 氨基酸插入物(QGPSFSY)在体外移动肌动蛋白细丝 活力测定的速度比未插入的异构体快2.5倍。 HMM IIB已经被设计成含有来自于 通过将编码插入片段的21个核苷酸拼接到 非肌肉型HMM基因。包含HMM的ATPase活性 从平滑肌MHC插入的是非平滑肌MHC的两倍 插入HMM(0.268s-1对0.128s-1)。在体外运动试验中， 插入的HMM移动肌动蛋白细丝的速度比 未插入的HMM(0.275+/-.061 vs 0.054+/-.021)。当前 正在与Lee Sweeney合作进行实验，以研究 肌动蛋白序列突变对酶和肌动蛋白表达的影响 插入的隐马尔可夫模型的机械活动。 在两种形式的非肌肉肌球蛋白中，一种亚型，MHCII-B，已经被 目的是产生一种不表达该蛋白的基因敲除小鼠 由MHCII-B基因编码。MHCII-B基因的缺失导致 心肌细胞的破裂和心脏的畸形 作为大脑。心脏的表型变化类似于人类 法洛四联症和右室双出口疾病。这个 当前项目的目标是删除由 MHCII-A基因，产生MHCII-A基因敲除小鼠。 一个包含7.5kb MHCII-A基因的基因组克隆，包括 外显子3已亚克隆到载体pPNT中。在此构建中，外显子 3被新霉素抗性基因中断，该基因将用于 胚胎干细胞的阳性选择。胸苷激酶基因 遵循构建体3‘端的MHCII-A基因片段，并 将用于胚胎干细胞的负选择。 为了排除新霉素抗性基因对细胞生长的影响。 观察到的表型，相同的基因组片段，减去外显子3的BP，具有 已被改造成含lox P位点的载体pBS479(赠予 布莱恩·鲍尔，美国国立卫生研究院)。在交配时，一只携带此集成组件的鼠标 用表达Cre重组酶的小鼠构建，小鼠 不表达MHCII-A且不含新霉素 就会产生抗性基因。

Two forms of the nonmuscle myosin heavy chain (MHCIIA and MHCIIB) have been cloned in this laboratory and shown to be the products of two different genes. The isoforms are expressed in a tissue-dependent manner and, when present in the same cell, exhibit distinct localization. The MHCIIB isoform additionally shows alternative splicing in two areas of the MHC: one after amino acid 211, near the ATP-binding domain (PESPKPVKHQ in loop 1) and another near the actin- binding domain (loop 2). Biochemical characterization of the nonmuscle myosin IIB fragment, HMM which contains the sites of enzymatic activity and actin-binding has been carried out on HMM isolated from the Baculovirus expression system. There is no major difference in the ability of HMM with or without the inserted region in loop 1 to move actin filaments or to hydrolyze ATP. Smooth muscle MHC also shows alternative splicing in an identical area near the ATP-binding domain. Smooth muscle myosin containing this seven amino acid insert (QGPSFSY) moves actin filaments in the in vitro motility assay at a rate 2.5 times faster than the non-inserted isoform. HMM IIB has been engineered to contain the seven amino acid insert from smooth muscle by splicing the 21 nucleotides coding for the insert into the nonmuscle HMM cDNA. The ATPase activity of the HMM containing the insert from the smooth muscle MHC is two times higher than that of non- inserted HMM (0.268s-1 vs 0.128s-1). In the in vitro motility assay, the inserted HMM moves actin filaments at a five-times faster rate than the non-inserted HMM (0.275 +/- .061 vs 0.054 +/- .021). Current experiments, in collaboration with Lee Sweeney are underway to study the effect of mutations in the smooth muscle sequence on the enzymatic and mechanical activity of the inserted HMM. Of the two forms of nonmuscle myosin, one isoform, MHCII-B, has been targeted to generate a knockout mouse which does not express the protein encoded by the MHCII-B gene. The deletion of MHCII-B results in disruption of the cardiac myocytes and malformation of the heart as well as the brain. Phenotypic alterations in the heart resemble the human diseases tetralogy of Fallot and double outlet right ventricle. the goal of the current project is to delete the protein encoded by the MHCII-A gene, generating a knockout mouse for the MHCII-A. A genomic clone encompassing 7.5 kb of the MHCII-A gene and including exon 3 has been subcloned into the vector pPNT. In this construct, exon 3 is interrupted by the neomycin resistance gene which will be used for positive selection of embryonic stem cells. The thymidine kinase gene follows the MHCII-A gene fragment at the 3' end of the construct and will be used for negative selection in embryonic stem cells. In order to rule out effects of the neomycin resistance gene on the phenotype observed, the same genomic fragment, minus bp in exon 3, has been engineered into the lox P site-containing vector, pBS479 (gift of Brian Sauer, NIH). Upon mating a mouse carrying this integrated construct with a mouse expressing the Cre recombinase, a mouse which does not express MHCII-A and which does not contain the neomycin resistance gene will be generated.