第11章基因表达与功能研究.pptVIP

* Gene Gene product Assay lacZ β-Galactosidase Histochemical test neo Neomycin phosphotransferase kanamycin resistance cat Chloramphenicol acetyltransferase chloramphenicol resistance dhfr Dihydrofolate reductase Methotrexate resistance aphlV Hygromycin phosphotransferase Hygromycin resistance lux Luciferase Bioluminescence uidA β-Glucuronidase Histochemical test A few example of reporter genes used in studies of gene regulation in higher organisms * 11.3 Identifying and studying the translation product of a cloned gene HRT and HART can identify the translation product of a cloned gene HRT: hybrid-release translation(杂交释放翻译) HART: hybrid-arrest translation(杂交俘获翻译) Analysis of proteins by in vitro mutagenesis Studying protein-protein interactions * 11.3.1 HRT and HART can identify the translation product of a cloned gene Two related techniques, hybrid-release translation (HRT) and hybrid-arrest translation (HART), are used to identify the translation product encoded by a gene. Both depend on the ability of purified mRNA to direct synthesis of proteins in cell-free systems. Both of them work best when a cDNA clone prepared directly from the mRNA sample is available. * Fig 11.19 Cell-free translation * H R T H A R T * 11.3.2 Analysis of proteins by in vitro mutagenesis In vitro mutagenesis(体外突变), a technique that produce a specified mutation at a predetermined position in a DNA molecule. * Different types of in vitro mutagenesis techniques A restriction fragment can be deleted. The gene can be opened at a unique restriction site, a few nucleotides removed with a double-strand-specific endunuclease such as Bal31, and the gene religated. A short oligonucleotide can be inserted at a restriction site. The sequence of the oligonucleotide can be such that the additional stretch of amino acids inserted into the protein produces, for example, a new structure such as an α-helix, or destabilizes an existing structure. * Fig 11.23 Various in vitro mutagenesis techn