Function of an Unconventional Myosin in Tetrahymena

Gavin博士的实验室在模式生物嗜热四膜虫（Tetrahymena thermophila）中发现了一种新型的非传统肌球蛋白Myo 1 p。迄今为止，已克隆并测序了约2.7 kb的MYO 1。 为了揭示Myo 1 p的功能，使用靶向基因破坏来创建缺乏功能性MYO 1基因的四膜虫菌株。转化MYO 1细胞的食物泡形成率降低，和大核经常无法正常伸长无丝分裂细胞分裂过程中导致DNA的后代的严重不平等的分离。为了测试Myo 1 p在内吞作用和核分离中功能的其他假设，必须获得全长MYO 1序列并开发新试剂。这个为期一年的项目包括MYO 1克隆和测序的完成，以及GFP-肌动蛋白，GFP-微管蛋白和GFP-Myo 1 p融合蛋白的表达构建体的创建。基因组DNA和cDNA文库的基于PCR的文库筛选将用于完成MYO 1的克隆和测序。 筛选工作正在进行中，明年将继续进行。 此外，RACE将用于确认克隆的MYO 1的5'和3'末端。涉及标准重组方案的两种不同方法将用于产生用于在四膜虫中表达的GFP构建体。 在一种方法中，GFP标记的基因（MYO 1、肌动蛋白或微管蛋白）将与pVGF-1（一种基于rDNA的复制载体）连接。 另一种用于产生GFP构建体的方法是在目的基因序列的5 ′端插入GFP标记构建体。常规肌球蛋白，如骨骼肌肌球蛋白的功能和结构-功能关系多年来一直是深入研究的主题，并且被合理地充分理解。 相比之下，各种非传统的非肌肉肌球蛋白最近才开始引起研究的重视，我们对它们在不同类型细胞的生命和功能中所起作用的理解才刚刚开始慢慢出现。 该项目预计将增加非常规肌球蛋白的知识基础和我们对单细胞生物体生物学的理解，如这些研究中使用的纤毛原生动物。

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.