ISOLATION AND CHARACTERIZATION OF MUTANTS IN ADENOVIRUS TYPE 5 THAT ARE ALTERED IN THE SPLICING OF REGION ELA TRANSCRIPTS
Abstract
Most eukaryotic genes contain non-coding intervening sequences. These sequences (called introns) are removed from the primary RNA transcripts by a process called mRNA splicing. DNA sequences around the 5' end of an intron (donor sequences) and 3' end of an intron (acceptor sequences) are important in the recognition and splicing of the primary transcript. The mechanism by which splicing occurs is not known. Whether splicing plays a role in the regulation of eukaryotic gene expression is also not understood. These studies have been undertaken in order to investigate the mechanism and regulation of splicing. The experimental system used is Adenovirus type 5. I have constructed various mutations in region Ela of Adenovirus type 5 and have investigated the effect of these mutations on the splicing of the corresponding region Ela transcripts. Specifically, the following questions are addressed: (1) how is splicing regulated when multiple splicing pathways are available? (2) are all donor and acceptor splice sites compatible? and (3) does the secondary structure of the primary RNA transcript play a role in splicing? Through the construction of two deletion mutants which remove donor sequences, I have shown that the temporal regulation of the region Ela transcripts can be altered. These deletion mutants also demonstrate that the gene products from one of the region Ela specific transcripts have an effect on the regulation of its own transcription, both on the rate and at the site of transcription initiation. The biological properties of these mutants have also been analyzed. Analysis of two insertion mutants has shown that not all donor and acceptor pairs are compatible. In fact, a normal donor/acceptor pair is not compatible when the location of the donor site is altered. These results, in addition to other mutant analyses, also suggest that the secondary structure of the RNA plays an important role in splicing. The results from the analysis of these splicing mutants will be discussed in conjunction with present hypotheses on the mechanism of splicing.
Degree
Ph.D.
Subject Area
Genetics
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