Nucleic Acids Research, 1990, Vol. 18, No. 24 7349-7355
© 1990
Articles |
Site-directed, recombination-mediated mutagenesis of a complex gene locus
Regulatory Biology Laboratory, The Salk Institute for Biological Studies 10010 N. Torrey Pines Road, La Jolla, CA 92037, USA 1Molecular Biology and Virology Laboratory, The Salk Institute for Biological Studies 10010 N. Torrey Pines Road, La Jolla, CA 92037, USA
*To whom correspondence should be addressed
Received September 4, 1990. Revised October 29, 1990. Accepted October 29, 1990.
We have generated a site-specific 17 bp insertion within a 38 kb chick globin gene cluster by employing the recombination abilities of Saccharomyces cerevisiae. This gene cluster contains four ß-type globin genes which share a high degree of sequence homology. In this procedure, a small fragment of ßA-globin DNA containing a 17 bp insertion is subcloned into a URA3-based yeast integrating vector (YIp). This mutated globin subclone is introduced into cells that carry the 38 kb globin cluster clone on a single-copy, circular vector derived from a yeast artificial chromosome (YAC). Insertion of the 17 bp oligomer is achieved by targeted integration of the YIp subclone. The recombinant contains the normal ßA-globin gene, the mutant gene and YIp vector sequences between the two copies. Excision of the vector sequences and one copy of the duplicated globin sequences by homologous recombination is required for cell survival when exposed to the selective agent 5-fluoroorotic acid, which is toxic to ura+ yeast cells. Depending upon the point of the cross-over, a ura– yeast cell bearing either a wild-type globin gene or a 17 bp insertion mutation will result. By restriction mapping and in vitro transcription analysis, the ßA-globin gene containing the 17 bp insert has no nonspecific mutations generated during the recombination and selection procedures. Specific mutations of regulatory regions, including protein-DNA binding sites, can be accurately targeted within extensive DNA clones by this method.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  L. Heidet, D.-B. Borza, M. Jouin, M. Sich, M.-G. Mattei, Y. Sado, B. G. Hudson, N. Hastie, C. Antignac, and M.-C. Gubler A Human-Mouse Chimera of the {alpha}3{alpha}4{alpha}5(IV) Collagen Protomer Rescues the Renal Phenotype in Col4a3-/- Alport Mice Am. J. Pathol., October 1, 2003; 163(4): 1633 - 1644. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. P. Oksvold, E. Skarpen, J. Widerberg, and H. S. Huitfeldt Fluorescent Histochemical Techniques for Analysis of Intracellular Signaling J. Histochem. Cytochem., March 1, 2002; 50(3): 289 - 303. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. C. Barton, N. Madani, and B. M. Emerson Distal enhancer regulation by promoter derepression in topologically constrained DNA in vitro PNAS, July 8, 1997; 94(14): 7257 - 7262. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M C Barton and B M Emerson Regulated expression of the beta-globin gene locus in synthetic nuclei. Genes & Dev., October 15, 1994; 8(20): 2453 - 2465. [Abstract] [PDF]
|
|