Acquisition of a Confocal Microscope Imaging System

这项建议的具体目标是： 1)确定小麦胚eIF-（iso）4F和eIF-4F亚基的功能结构域。 eIF-（iso）4F亚基已在E.大肠杆菌中，并重组以形成酶活性复合物。将在eIF-（iso）4F和eIF-4F的亚基中进行突变，并在体外麦胚翻译系统和几种测量部分反应的体外测定中进行测试，即，ATP水解、RNA解旋、与m7 G帽交联以及mRNA与40 S核糖体亚基结合。 2)完成了小麦eIF-4 B的部分cDNA序列，并在E.杆菌 eIF-4 B cDNA的缺失部分（100-200个核苷酸）将通过RACE-PCR、反向PCR或基因组测序获得。该cDNA将在E.大肠杆菌中的酶活性和在上述测定中测定的酶活性（具体目的1）。 3)完成拟南芥eIF-4F、eIF-（iso）4F的p28和eIF-4 B亚基的cDNA序列，并在E.杆菌 我们将继续使用小麦因子和小麦cDNA序列的抗体进行筛选。将小麦cDNA的不同区域用于制备用于筛选的探针。拟南芥因子的cDNA将在E.杆菌将在上述测定中测量表达的拟南芥属因子的活性。这一目标的完成将为今后在体内探索控制起始因子的表达和调节植物蛋白质合成的工作奠定基础。

The specific objectives of this proposal are to: 1) Determine the functional domains of the subunits of wheat germ eIF-(iso)4F and eIF-4F. The subunits of eIF-(iso)4F have been expressed in E. coli and recombined to form an enzymatically active complex. Mutations will be made in the subunits of eIF-(iso)4F and eIF-4F and tested in an in vitro wheat germ translation system and several in vitro assays that measure partial reactions, i.e., ATP hydrolysis, RNA unwinding, cross-linking to the m7G cap and binding of mRNA to 40S ribosomal subunits. 2) Complete the partial cDNA sequence of wheat eIF-4B and express this protein in E. coli. The missing portion ( 100-200 nucleotides) of the cDNA for eIF-4B will be obtained by RACE-PCR, inverse PCR or genomic sequencing. The cDNA will be expressed in E. coli and the enzymatic activity determined in the assays described above (Specific Objective 1). 3) Complete the cDNA sequence for the subunits of Arabidopsis eIF-4F, p28 of eIF-(iso)4F and eIF-4B and express these proteins in E. coli. We will continue to screen using antibodies to wheat factors and wheat cDNA sequences. Different regions of the wheat cDNAs will be used to prepare probes for screening. The cDNAs for the Arabidopsis factors will be expressed in E. coli. The activity of the expressed Arabidopsis factors will be measured in the assays described above. The completion of this goal will set the stage for future work in vivo exploring the control of expression of the initiation factors and regulation of protein synthesis in plants.