DNA shuffling method for generating estrogen receptor alpha and beta chimeras in yeast.
To facilitate our study of the molecular basis for the estrogen receptor (ER) subtype selectivity of novel ligands, we used DNA shuffling to construct chimeric ERs having ligand binding domains derived from both ER alpha and ER beta. The efficiency of chimera generation was low with traditional DNA shuffling protocols. Furthermore, ER ligand binding domain sequences lack convenient restriction sites for introducing chimeric ligand binding domain sequences into expression vectors. To overcome these problems, we developed a modified strategy whereby chimeric sequences were exclusively amplified from among the reassembled products from DNA shuffling using a special pair of PCR primers whose 3' ends specifically match the alpha and beta sequences, respectively, and whose 5' ends match sequences outside the ER beta ligand binding domain. When chimeric ligand binding domain DNA sequences, amplified with these primers, were co-transformed into a yeast strain with a linearized expression vector for ER beta, an active expression vector was produced by homologous recombination. Twenty-two different crossover sites were found; most occurred when there was a stretch of eight or more identical base pairs in both sequences, and many were concentrated in the regions important for studying ligand binding and transactivation. This method should prove to be useful for generating chimeric gene products from parent templates that share relatively low sequence identity.
Sun
Jun
J
University of Illinois, 407 South Goodwin Avenue, Urbana, IL 61801-3704, USA.
Katzenellenbogen
John A
JA
Zhao
Huimin
H
Katzenellenbogen
Benita S
BS
D000595:Amino Acid Sequence; D001483:Base Sequence; D002678:Chimera / Q000235:genetics* / Q000378:metabolism; D032621:DNA Shuffling / Q000379:methods*; D047628:Estrogen Receptor alpha; D047629:Estrogen Receptor beta; D006801:Humans; D008969:Molecular Sequence Data; D011960:Receptors, Estrogen / Q000235:genetics* / Q000378:metabolism; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment / Q000379:methods; D015003:Yeasts / Q000235:genetics* / Q000378:metabolism
D047628:Estrogen Receptor alpha; D047629:Estrogen Receptor beta; D011960:Receptors, Estrogen; D011994:Recombinant Proteins