Linked Life Data

11319828

Source:http://linkedlifedata.com/resource/pubmed/id/11319828

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
1
pubmed:dateCreated
2001-4-26
pubmed:abstractText
We describe a gene system allowing the facile production of multiply substituted reverse transcriptases (RTs), the enzymatic characterization of these purified RTs, and the study of these mutations in the defined genetic background of the macrophagetropic, non-laboratory-adapted human immunodeficiency virus type 1 (HIV-1) AD8 strain. Thirteen unique silent restriction sites were introduced in the pol gene encoding HIV-1 RT, allowing easy introduction of mutations. To simplify genetic manipulation and generate p66/p51 heterodimers in Escherichia coli, a gene construct of the viral protease alone was optimized for expression from a separate vector carrying a p15A origin of replication. Active-site titration experiments using pre-steady-state kinetics showed that our system yields a higher proportion of active enzyme than that obtained by alternate methods. To facilitate phenotype/genotype correlations, the modified RT gene was designed to be easily reintroduced into a recombinant proviral AD8 HIV-1 DNA. Infectious viruses made from this vector were undistinguishable from wild-type AD8 HIV-1, an isolate able to infect peripheral blood mononuclear cells and macrophages. Thus, the pol gene can tolerate many silent mutations in the polymerase domain without affecting the functionality of the HIV-1 genome. The system was validated biochemically and virologically using the V75T substitution associated with stavudine resistance.
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
May
pubmed:issn
0003-2697
pubmed:author
pubmed:issnType
Print
pubmed:day
1
pubmed:volume
292
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
139-47
pubmed:dateRevised
2007-11-15
pubmed:meshHeading
pubmed:year
2001
pubmed:articleTitle
An integrated system to study multiply substituted human immunodeficiency virus type 1 reverse transcriptase.
pubmed:affiliation
Architecture et Fonction des Macromolécules Biologiques (AFMB), UMR 6098, CNRS, Universités d'Aix-Marseille I et II, 163 Avenue de Luminy, Marseille Cedex 09, F-13288, France.
pubmed:publicationType
Journal Article, In Vitro, Research Support, Non-U.S. Gov't