Construction and use of E. coli Nonsense Suppressor Bank
{'Code': '08070100', 'Directorate': {'Abbreviation': 'BIO', 'LongName': 'Direct For Biological Sciences'}, 'Division': {'Abbreviation': 'MCB', 'LongName': 'Div Of Molecular and Cellular Bioscience'}}
{'SignBlockName': 'Philip Harriman', 'PO_EMAI': None, 'PO_PHON': None}
This proposal is aimed at constructing synthetic tRNA nonsense                  suppressor genes, which will direct the in vivo synthesis of a                  series of nonsense suppressors.  Each suppressor will insert a                  different amino acid in response to a nonsense (in most cases                   UAG) codon.  The goal is to derive a set of suppressors capable                 of inserting each of the 20 amino acids at a UAG site.  This                    would greatly aid systematic studies of amino acid substitutions                in proteins, since UAG mutations can be introduced by in vivo and               in vitro methods at any position in a protein.  The information                 gained from virtual unlimited amino acid substitutions would                    facilitate protein engineering experiments.  The synthetic genes                are put together from a series of oligonucleotides and introduced               into E. coli cells by a plasmid vector.  Additional modifications               are introduced by site-directed mutagenesis, by random                          mutagenesis followed by selection, and by constructing hybrids of               different tRNA molecules.  The amino acid inserted by each                      suppressor is determined by protein sequencing in a system that                 employs an amber mutation in the gene encoding dihydrofolate                    reductase.  Mutants will be sought which improve the specificity                of certain suppressors by limiting the mischarging by non-cognate               synthetases, such as glutamine tRNA synthetase.  Different vector               systems will be used to carry the suppressor genes to increase                  the general utility of the suppressors to different                             investigators.                                                                                                                                                  The goal of this proposal is to facilitate the engineering of                   specific amino acid replacements in proteins.  In principle, new                amino acids can be inserted at any site in the product of a                     cloned gene by a chemical procedure known as site-directed                      mutagenesis.  However, this procedure becomes laborious when                    large numbers of specific replacements are needed.  In                          collaboration with Dr. John Abelson at the California Institute                 of Technology, Dr. Miller intends to circumvent this problem by                 constructing transfer RNA genes that will perform the                           suppressors, will be constructed so as to allow insertion of any                one of the 20 different amino acids whenever the nonsense codon                 UAG occurs.  Since this codon can be created at any location in a               cloned gene, virtually all amino acid replacement s will be                     possible.  This collection of suppressor genes will be extremely                useful to scientists studying the relationships between the                     structure and function of proteins.  Important information on the               functioning of transfer RNA molecules will also be obtained.
{'FirstName': 'John', 'LastName': 'Abelson', 'PI_MID_INIT': 'N', 'PI_SUFX_NAME': None, 'PI_FULL_NAME': 'John N Abelson', 'EmailAddress': None, 'NSF_ID': '000412572', 'StartDate': '06/01/1988', 'EndDate': None, 'RoleCode': 'Principal Investigator'}
{'Name': 'California Institute of Technology', 'CityName': 'PASADENA', 'ZipCode': '911250001', 'PhoneNumber': '6263956219', 'StreetAddress': '1200 E CALIFORNIA BLVD', 'StreetAddress2': None, 'CountryName': 'United States', 'StateName': 'California', 'StateCode': 'CA', 'CONGRESSDISTRICT': '28', 'CONGRESS_DISTRICT_ORG': 'CA28', 'ORG_UEI_NUM': 'U2JMKHNS5TG4', 'ORG_LGL_BUS_NAME': 'CALIFORNIA INSTITUTE OF TECHNOLOGY', 'ORG_PRNT_UEI_NUM': None}
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{'Code': '1112', 'Text': 'Genetic Mechanisms'}
{'url': 'https://www.nsf.gov/awardsearch/download?DownloadFileName=1987&All=true', 'xml': '8716260.xml'}