Isolation and characterization of UV-sensitive mutants from Arabidopsis thaliana
|Doctor of Philosophy (Ph.D.)
|Our understanding of DNA repair and mutagenesis in higher plants is very limited, in comparison to other eukaryotes. This is primarily due to the fact that the genes which control DNA repair in plants have not yet been isolated and to the relative lack of repair-defective mutants. This study involves isolation and characterization of UV-sensitive mutants of the angiosperm Arabidopsis thaliana. UV-sensitive mutants were isolated by screening approximately 49,000 seeds which were T4 progeny of 4,900 independent Agrobacterium tumefaciens T-DNA transformed lines. A total of 55 UV-sensitive mutants were isolated on the basis of the sensitivity of their root tissue to UV-C. The mutants were named uvs1-55. Out of those 55 uvs mutants, in 11 cases the roots were UV-sensitive in the light (uvs1-11) and in 44 cases, the roots were sensitive in dark (uvs12-55). Based on 100% inhibition of root growth, uvs12-55 were further classified into: (1) mutants where the roots stopped growing at $\sim$600 J/m$\sp2$; (2) those which stopped growing at $\sim$800 J/m$\sp2$; and (3) those which stopped growing at $\sim$1,200 J/m$\sp2$. In all uvs mutants (uvs1-55), the mutation was found to be recessive. Results of segregation analysis helped in determining the pattern in which these UV-sensitive mutations segregate in successive generations. Complementation analysis, where UV-sensitivity of the roots was observed in the F$\sb1$ generation, placed the mutants in 48 separate complementation groups. Mutants were further characterized by exposing the leaves of 15 day old seedlings to a series of UV-B and UV-C doses and by assessing the sensitivity of the seeds to ionizing radiation. In contrast to UV, ionizing radiation is highly penetrating and causes DNA damage by predominantly single- and double-strand breaks. Thus, mutants sensitive to ionizing radiation are most likely DNA repair defective, potentially in recombinational repair. The uvs mutants showed characteristic patterns of UV and ionizing radiation sensitivity, quite similar to the yeast RAD mutants. Based on their sensitivity to UV and ionizing radiation the uvs mutants were grouped into six different classes sharing similar phenotypic characteristics with the three yeast epistasis groups (RAD3, RAD6 and RAD52). In the final part of this study, cosegregation analysis was used to determine whether the UV-sensitive mutation and the T-DNA were linked. In mutants, where the UV-sensitivity and the T-DNA was found to be linked, the copy number of T-DNA inserts was determined. This was done by Southern hybridization using probes made from the right and left border ends and the pBR322 origin of replication component of the T-DNA insert sequence. With the exception of one mutant (where the copy number of T-DNA could not be discerned), the copy number of T-DNA was proven in all cases to be more than one.
|Isolation and characterization of UV-sensitive mutants from Arabidopsis thaliana