ISSN:
1432-0983
Keywords:
Yeast
;
FLP-FRT
;
BFBC
;
Gene conversion
Source:
Springer Online Journal Archives 1860-2000
Topics:
Biology
Notes:
Summary A YEp chimaeric plasmid containing URA3 and SMR1 [sulfometuron methyl resistant (SMR) allele of ILV2] as selectable markers, and the 2 μm site-specific recombination FLP recognition target (FRT), was integrated at the ilv2-Δ1 site in chromosome XIII in a cir°] haploid. Southern analysis defined two integrant structures. Structure I had URA3 distal and SMR1 proximal to FRT whereas in structure II both markers were distal to FRT. Selectable markers were stably inherited in [cir°] haploids and [cir°] diploids heterozygous for the integrant and ILV2. Approximately 14% of heterozygous [cir +] diploid cells exhibited homozygotization for the distal (500 kb) ade4 marker in trans. In [cir +] diploids FLP-FRT recombination resulted in the simultaneous loss of both structure II markers, whereas the structure I distal URA3 marker loss always preceded the variable loss of the proximal SMR1 marker. URA− cells continued to segregate for loss of SMR1 until stable URA− SMR or URA−SMS cells were produced. Gene conversion was identified in stable URA−SMR cells that were homozygous SMR1/SMR1 but contained wild type ILV2 restriction endonuclease sites. These observations support a model based on concerted FLP-FRT action resulting from the secondary integration of native 2 μm DNA followed by unequal sister chromatid exchange (USCE) within inverted FRTs. The resultant chromatid bridge resulted in a double-stand break. Fusion of the broken ends of sister chromatids generated a breakage-fusion-bridge cycle (BFBC). Repeated rounds of the BFBC resulted in proximal marker loss and the generation of additional double-strand breaks. Recombinogenic properties of the double-strand break initiated events leading to homozygotization and gene conversion.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/BF00424420
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