title: Use of DNA end joining activity of a Xenopus laevis egg extract for construction of deletions and expression vectors for HIV-1 Tat and Rev proteins.
abstract: We have developed a cloning strategy which combines conventional T4 DNA ligation with the highly efficient nonhomologous DNA end joining (EJ) activity of an extract from Xenopus laevis eggs. The nonhomologous EJ activity allowed the rapid construction of deletion mutants by the intramolecular rejoining of nonhomologous DNA ends generated for the purpose of deleting restriction fragments from the vector. The combined use of T4 DNA ligase for intermolecular ligation and X. laevis egg extracts for intramolecular nonhomologous EJ proved to be a powerful tool, as demonstrated here for the construction of expression vectors for HIV-1 Tat and Rev.
authors.affiliation: Institut für Medizinische Mikrobiologie und Virologie, Heinrich-Heine-Universität, Düsseldorf, Germany.
authors.forename: H
authors.initials: H
authors.lastname: Schaal
authors.forename: P
authors.initials: P
authors.lastname: Pfeiffer
authors.forename: M
authors.initials: M
authors.lastname: Klein
authors.forename: P
authors.initials: P
authors.lastname: Gehrmann
authors.forename: A
authors.initials: A
authors.lastname: Scheid
mesh_terms: D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D003001:Cloning, Molecular / Q000379:methods*; D004279:DNA, Viral; D015694:Gene Products, rev / Q000235:genetics*; D015696:Gene Products, tat / Q000235:genetics*; D005822:Genetic Vectors; D015497:HIV-1 / Q000235:genetics*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009865:Oocytes; D015183:Restriction Mapping; D017384:Sequence Deletion; D014982:Xenopus laevis; D054321:rev Gene Products, Human Immunodeficiency Virus; D054322:tat Gene Products, Human Immunodeficiency Virus
chemical_list: D004279:DNA, Viral; D015694:Gene Products, rev; D015696:Gene Products, tat; D054321:rev Gene Products, Human Immunodeficiency Virus; D054322:tat Gene Products, Human Immunodeficiency Virus