[自然科學]techniqueofmolecularbiology(留存版)

　　

【正文】 found Escherichia coli Species category R13 strain How to name a restriction endonuclease? EcoRI ? a linear DNA molecule with 6 copies of GAATTC ? it will be cut into 7 fragments which could be separated by the gel electrophoresis. (The largest fragment) (The smallest fragment) Digestionof a DNA fragment with endonuclease EcoRI 22 (1) Restriction enzymes differ in the recognition specificity: target sites are different. (2) Restriction enzymes differ in the length they recognized, and thus the frequencies differ: Sau3AI, 5’GATC3’。s own DNA from being cut by the restriction endonucleases? ? Modification ? One of the nucleotides in bacteria DNA is modified by an methylase enzyme (甲基化酶， modification enzyme) that attaches a methyl group to one of the bases ? The restriction enzyme doesn39。t cut randomly. They bind to specific sequences and only cut at those sites識別回文結構 ) ? Smith, . and Wilcox, . A restriction enzyme from Hemophilus influenzae. 1. Purification and general properties. J. Mol. Biol. 51, 379 (1970). ? Kelly, ., Jr. and Smith, . A restriction enzyme from Hemophilus influenzae. 11. Base sequence of the recognition site. J. Mol. Biol. 51, 393 (1970). ? Roy, . and Smith, . The DNA methylases of Hemophilus influenzae Rd. 1. Purification and properties. J. Mol. Biol. 81, 427 (1973). ? Roy, . and Smith, . The DNA methylases of Hemophilus influenzae Rd. 11. Partial recognition site base sequences. J. Mol. Biol. 81, 445 (1973). ?Papers Daniel Nathans, 1978 Nobel Prize in Physiology or Medicine The last step in this development was taken by Dan Nathans He pioneered the application of restriction enzymes in geics and his work has been a source of inspiration for scientists who subsequently started to use restriction enzymes He constructed the first geic map using restriction enzymes by cleaving the DNA from a monkey virusSV40 ? As I look back on the last few decades of my life, I am struck by the good fortune that came my way. ? Throughout my schooling there was an abundance of opportunity and encouragement. ? Several of my teachers were remarkable individuals who had a lasting influence on me. ? At every stage of my career I have had interesting and cordial colleagues, some of whom are close friends. ? My field of research is as exciting to me as ever, and it remains essentially a cottage industry effort. ? I have had talented students who are a source of much enjoyment, and I anticipate more to e as their careers develop. ? And most important, my wife and sons have created in our home an atmosphere of joy and harmony, so essential to everything else. ? Arber discovered restriction enzymes. He postulated that these enzymes bind to DNA at specific sites containing recurring structural elements made up of specific basepair sequences. ? Smith verified Arber39。s Nobel Prize in medicine or physiology is awarded for discoveries with far reaching consequences for geics. The task of geics is to describe and explain how genes are anized and expressed in cells and in living anisms. The discovery of restriction enzymes provided new tools for the detailed chemical analysis of the mechanism of gene action. Even though these enzymes have been available only during a few years their application to geics has already led to new and far reaching results, in particular concerning the anisation and expression of genes (= DNA) of higher animals. All work in this area carried out by many research groups all over the world, is based on the discoveries made by the three laureates. Restriction enzymes are used as tools to dissect DNA into smaller defined fragments. These can be used to determine the order of genes on chromosomes, to analyse the chemical structure of genes and to rebine genes by chemical means. Most important restriction enzymes are used to analyse the function of regions of DNA which regulate gene expression. This opens up new areas of research to study the connection between heredity and function. We can now begin to answer questions of central biological importance in developmental biology: how do genes direct the evolution of a single fertilized egg to a plete individual with many different ans? What determines that the cells within one an normally retain their specialized functions? Different diseases are expressions of disturbances in normal functions and increased knowledge in molecular geics should aid in preventing and treating malformations, hereditary diseases and cancer. Werner Arber started this field of research in Geneva during the 196039。s. Nathans uses in his experiments the small DNA from a simian virus, called SV40, but his results are of general significance. In his first munication from 1971 he showed that the restriction enzyme discovered by Smith cleaves SV40 DNA into 11 well defined fragments. In this munication Nathans also discussed other possible applications of restriction enzymes in geics and in a brilliant way predicted much of the later development. Nathan39。 NotI, 5’GCGGCCGC3’ (3) Restriction enzymes differ in the nature of the DNA ends they generate: blunt/flush ends (平末端 ), sticky/staggered ends (粘性末端 ). sticky ends (粘性末端 ) blunt ends (平末端 ) Recognition sequences and cut sites of various endonucleases 24 Cleavage of an EcoRI site: The 5’ protruding ends are said to be “sticky” because they readily anneal (退火 ) through basepairing to DNA molecules cut with the same enzyme Gene B Gene A Discovery of restriction endonucleases Research strategy: from phenomenon to mechanism Phenomenon Hypothesis Experimental proof Mechanism Practical use of restriction endonucleases Salvador E. Luria, 1969 Nobel prize in P