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|Title:||In Vivo Analysis of Pre-Mrna Splicing in Dicot Plant Nuclei|
|Doctoral Committee Chair(s):||Schuler, Mary A.|
|Department / Program:||Plant Biology|
|Degree Granting Institution:||University of Illinois at Urbana-Champaign|
|Abstract:||In vivo gene transfer experiments have suggested that the elements mediating intron recognition differ in monocot and dicot nuclei.
To locate elements which modulate efficient recognition of introns in dicot nuclei, two monocot genes (wheat rbcS and maize Adh1), and one dicot gene (pea Adh1) have been expressed in Nicotiana benthamiana nuclei using an autonomously replicating Agrobacterium/geminivirus plant expression vector. Quantitative PCR-Southern assays indicate that introns from these genes were spliced at widely differing efficiencies in tobacco cells, which roughly correlate with the AU content of these introns. Inefficient splicing of one of these introns, maize Adh1 intron 1, in tobacco nuclei was dramatically enhanced by increasing the degree of U1 snRNA complementarity at the 5$\sp\prime$ splice site demonstrating that the 5$\sp\prime$ splice site plays a significant role in defining the splicing efficiency of an intron in dicot nuclei. Root transfection experiments using both maize and pea Adh1 full-length genomic DNA constructs in same expression vectors suggested that tissue-specific splicing differences exist for introns 1, 2 and 3 in both of these transcripts.
Using a series of intron deletions, I demonstrate that two cryptic 3$\sp\prime$ splice sites within the maize Adh1 intron 3 can be efficiently activated. Rearrangement of the normal and cryptic 3$\sp\prime$ splice site segments demonstrates that the 3$\sp\prime$ splice site is defined by its position relative to the AU transition point and that internal cryptic sites are masked by the presence of downstream AU elements. Disruption of the AU-rich elements upstream from the normal 3$\sp\prime$ splice site demonstrates that multiple AU-elements cooperatively define the 3$\sp\prime$ boundary of this intron. U-rich sequences in the region immediately preceding the 3$\sp\prime$ acceptor site serve as the critical signal for recognition of that site. These results are consistent with a model for plant intron recognition in which AU-rich elements spread throughout the length of the intron roughly define the intron boundaries by generating strong AU-transition points. Potential 3$\sp\prime$ splice sites are then selected if they are located downstream from this transition point and not if they are embedded within AU-rich sequences.
Thesis (Ph.D.)--University of Illinois at Urbana-Champaign, 1992.
|Date Available in IDEALS:||2014-12-17|