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.
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.
Schaal
H
H
Institut für Medizinische Mikrobiologie und Virologie, Heinrich-Heine-Universität, Düsseldorf, Germany.
Pfeiffer
P
P
Klein
M
M
Gehrmann
P
P
Scheid
A
A
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
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