Reshmi Radhakrishnan, TA: Jared Tues Rm 24 Experiment 6 Purpose: To analyze if diploid yeast strain heterozygous for ARG8 ADE2 HIS3/arg8 ade2 his3 on chromosome XV and TRP1/trp1 on chromosome IV irradiated with UV light underwent mitotic recombination to form homozygous recessive ade2/ade2 mutants. The ade2/ade2 mutant are selected then plated on different media to observe if the homozygous recessive mutants were due to mitotic recombination. Controls: If all the red cells grew on the CSM-Trp plates, than it would show that they are wild type for the TRP1 gene.
All of the plates showed thick white colonies for the CSM-Trp plates. They should have been red because the ade2 colony streaked on the plate was red. They were still white because the adenine from the plate had not been exhausted. However, the cells are ade2- because they are red on all the other replica plates. The CSM-Ade plates are also a control. In order to ensure that the cells are ade2-, they would not be able to grown on this plate or have sparse red colonies. Because the cells have the dysfunctional form of the ade2 gene, they would not be able to survive with adenine present in the plate.
Since YPD plate was the last replica plate, it was used as a control to make sure that the transfer was effective for all the other plates. Since red colonies appear red on all the other media plates, it is concluded that the transfer was effective. Results: Growth on the replica plating Plate| 1| 2| 3| 4| CSM-Arg| Red colonies| Red| Red| Red| CSM-Ade| Sparse Red| No growth| Sparse Red | Sparse Red| CSM-His| Sparse Red| Red| Red| Red| CSM-Trp| White| White| White | White| YPD| Red| Red | Red| Red| Conclusion: We plated the red colonies or sectors on CSM-Arg, CSM-His, and CSM-His to iscern the genotype of each colony or sector. Colony 1 is ade1 his3 because of the sparse red growth seen on the CSM-Ade and CSM-His plate. Colony 2, 3 and 4 are ade2. The colonies on the CSM-Trp plates were white but should have been red. They were white because they haven’t exhausted the adenine present in the plate. Many of the ade2 cells were also his3 because the recombination occurred between the ade2 gene and centromere. Since mitotic recombination is rare, the chance of double recombination is improbable so the cells would ARG8.
Further Questions: i. Yeast can also exist in haploid form. So the ade2 his3 arg8 genotype is possible if there is only one copy of the chromosome and there is loss of the chromosome that was wild type for all the genes. ii. Red sectors in white colonies arise mitotic recombination, by loss of the entire chromosome containing the ade2+ allele, by a deletion of the portion of the chromosome containing the ade2+ allele, or by spontaneous mutation of the ade2+ allele to an ade2- allele. ii. Red colonies were ade2 before the first division on the plate. Red sectors arise from successive divisions after on the plate. Red colonies can also arise from loss of chromosome with the ADE2 allele, mutation of the ADE2 allele, or deletion of the ADE2 allele. iv. If it is a red sector, it is possible to be due to recombination. However reversion from ade2 to ADE2, cannot be distinguished from the white colonies because they look they same phenotypically.