Function of an Unconventional Myosin in Tetrahymena

加文博士的实验室在模式生物嗜热四膜虫（一种纤毛原生动物）中发现了一种新型的非常规肌球蛋白Myo1p。到目前为止，已经克隆和测序了大约2.7 kb的myl1。为了揭示Myo1p的功能，使用靶向基因破坏方法创建了缺乏功能性MYO1基因的四膜虫菌株。转化后的MYO1细胞食物液泡形成率降低，在无丝分裂细胞分裂过程中，巨核经常不能正常伸长，导致DNA向后代的分离严重不平等。为了验证Myo1p在胞吞作用和核分离中功能的其他假设，必须获得全长MYO1序列并开发新的试剂。这个为期一年的项目包括完成MYO1的克隆和测序，以及gfp -肌动蛋白、gfp -微管蛋白和GFP-Myo1p融合蛋白的表达构建。基于pcr的基因组DNA文库和cDNA文库筛选将用于完成MYO1的克隆和测序。筛查正在进行中，并将在明年继续进行。此外，RACE将用于确认克隆的MYO1的5‘和3’端。涉及标准重组协议的两种不同的方法将用于创建四膜虫中表达的绿色荧光蛋白结构。在一种方法中，gfp标记的基因（MYO1，肌动蛋白或微管蛋白）将连接到pVGF-1，一种基于rdna的复制载体。另一种创建绿色荧光蛋白构建体的方法是在感兴趣的基因序列的5'端插入绿色荧光蛋白标记构建体。传统的肌球蛋白，如骨骼肌肌球蛋白的功能和结构-功能关系，多年来一直是研究的热点，并得到了很好的理解。相比之下，各种非常规的非肌肉肌球蛋白直到最近才开始引起认真的研究关注，我们对它们在不同类型细胞的生命和功能中所起的作用的理解才慢慢开始出现。该项目有望增加非常规肌球蛋白的知识基础，并增加我们对单细胞生物（如这些研究中使用的纤毛原生动物）生物学的理解。

Dr. Gavin's laboratory has identified a novel unconventional myosin, Myo1p, in the model organism Tetrahymena thermophila, a ciliated protozoan. Approximately 2.7 kb of MYO1 have been cloned and sequenced thus far. To uncover the functions of Myo1p, targeted gene disruption was used to create a Tetrahymena strain that lacked a functional MYO1 gene. Transformed MYO1 cells had a reduced rate of food vacuole formation, and the macronucleus frequently failed to elongate properly during amitotic cell division resulting in grossly unequal segregation of DNA to progeny. In order to test additional hypotheses for Myo1p function in endocytosis and nuclear segregation, a full-length MYO1 sequence will have to be acquired and new reagents developed. This one-year project involves the completion of MYO1 cloning and sequencing and the creation of expression constructs for GFP-actin, GFP-tubulin, and GFP-Myo1p fusion proteins. PCR-based library screening of both genomic DNA and cDNA libraries will be used to complete the cloning and sequencing of MYO1. Screening is in progress and will be continued over the next year. In addition, RACE will be used to confirm the 5' and 3' ends of the cloned MYO1. Two different approaches involving standard recombinant protocols will be used to create GFP constructs for expression in Tetrahymena. In one approach, GFP-tagged genes (MYO1, actin, or tubulin) will be linked to pVGF-1, a rDNA-based replicative vector. Another approach for creating GFP constructs that will be used is to insert a GFP tagging construct at the 5' end of the gene sequence of interest.The functions and structure-function relationships of conventional myosins, such as skeletal muscle myosin, have been the subject of intense study for many years, and are reasonably well understood. In contrast, the various unconventional non-muscle myosins have only recently begun to attract serious research attention, and our understanding of the roles they play in the life and functionality of different types of cells is only slowly beginning to emerge. This project is expected to add to both the knowledge base of unconventional myosins and to our understanding of the biology of single-celled organisms such as the ciliated protozoan used in these studies.