NULL MUTATIONS OF VERTEBRATE NONMUSCLE MYOSIN HEAVY CHAINS

非肌肉肌球蛋白重链是一种普遍存在的蛋白质， 真核细胞 它的真实的功能不是很清楚，但它是 很明显，它不仅参与细胞的运动， 也存在于细胞质分裂和封盖中。 这个实验室有 在人和鸡组织中分离到两种不同的CDNAS， 非肌肉肌球蛋白重链（NMMHCs）。 这两种异构体被称为 NMMHC-A和-B位于两条不同的染色体上。 到 为了了解NMMHCs的两种亚型的功能，我们正在尝试 使用同源的基因敲除两个基因中的一个， 在胚胎干细胞和后来的转基因小鼠中进行重组。 的 该项目的目的是观察当两种蛋白质中的一种 由两种同种型编码的基因被消除。 为了克隆NMMHC-A同种型， 使用以下筛选在粘粒载体中构建的小鼠基因组文库： 5'部分CDNA人探针（HA-302，1.2 kb）。 一个阳性克隆是 被定性。 对于NMMHC-B，小鼠基因组文库 使用5 ′ cDNA人探针（HB）筛选在λ噬菌体中构建的 5.12，1.7 kb）。 在这种情况下，40个斑块呈阳性，其中， 一个是被定性。 人寡核苷酸探针用于 确认基因的5'上游序列是否存在于我们的 殖民地 然后，将含有ATG的约5 kb的片段 序列在Bluescript SBK+质粒中亚克隆，以用于 同源重组 这两个片段的限制性图谱 基因进行杂交，并将消化产物与 含有ATG序列的寡核苷酸。 这些基因组小鼠 片段将用于在ATG之后引入新霉素序列 位点以破坏NMMHC的蛋白质翻译。 这 构建体将被引入胚胎干细胞，然后在 转基因小鼠

The nonmuscle myosin heavy chain is an ubiquitous protein present in all eukaryotic cells. Its real function is not very well know, but it is clear that its is involved not only in the motility of the cells, but also in the cellular cytokinesis and capping. This laboratory had isolated two different CDNAS in human and chicken tissues encoding for nonmuscle myosin heavy chains (NMMHCs). These two isoforms are called NMMHC-A and -B and are located on two different chromosomes. To understand the function of the two isoforms of the NMMHCs, we are trying to perform a knockout of one of the two genes using homologous recombination in embryonic stem cells and later in transgenic mice. The aim of the project is to see what happens when one of the two proteins encoded by the two isoforms is eliminated. To clone the NMMHC-A isoform, a mouse genomic library constructed in a cosmid vector was screened using a 5' portion CDNA human probe (HA-302, 1.2 kb). One positive clone is being characterized. For the NMMHC-B, a mouse genomic library constructed in lambda phage was screened using a 5' CDNA human probe (HB 5.12, 1.7 kb). In this case, 40 plaques appeared positive and, of these, one is being characterized. Human oligonucleotide probes were used to confirm if the 5' upstream sequence of the genes was present in our colonies. After that, fragments of about 5 kb containing the ATG sequence were subcloned in the Bluescript SBK+ plasmid to be used for homologous recombination. Restriction maps of these fragments from both genes were performed and the digestion products were hybridized with oligonucleotides containing the ATG sequence. These genomic mouse fragments will be used to introduce a neomycin sequence after the ATG site in order to disrupt the protein translation of NMMHCs. This construct will be introduced into embryonic stem cells and then in transgenic mice.