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Dissertation
The fission yeast prp4 + gene involved in pre-mRNA splicing codes for a predicted serine/threonine kinase and is essential for growth
Doctor of Philosophy (Ph.D.), Drexel University
1994
DOI:
https://doi.org/10.17918/00000377
Abstract
Pre-mRNA splicing is the removal of introns from nuclear precursor-messenger RNA (pre-mRNA) genes. The splicing event is carried out by the interaction of several trans-factors. One method of identifying the trans-factors is by making conditional mutants. We made a bank of temperature sensitive (ts) Schizosacchromyces pombe mutants using the mutagen nitrosoguanidine. 150 of these mutants were transformed with a ura4 gene containing an artificial intron. These ts mutants were screened for ura4 intron splicing defects. This analysis revealed that three mutants cannot splice out the introns resulting in accumulating pre-mRNA at the restrictive temperature. Complementation analysis revealed that two of these mutants fall into the prpl complementation group and one is a new locus mutation which has been referred to as prp4 (pre-mRNA processing mutation). The prp4 mutant was rescued by complementation using a wild type genomic library. Our RNA studies showed that the rescued clones were able to splice the introns, consequently produce mRNA at both the permissive and restrictive temperatures. Sequence analysis of the prp4 suppressing gene revealed that it contains all kinase sub-domains which are characteristic of serine/threonine kinase family members. Significant similarities with three different kinases were observed. This is the first kinase gene identified whose product is involved in pre-mRNA splicing. Sequence analysis also revealed that this gene has a putative intron within the reading frame. Presence of the intron was confirmed by isolating and sequencing the cDNA of the prp4 suppressing gene. Gene replacement studies provided evidence that this gene is essential for growth. Genetic analysis of the prp4 integrated strain indicated that the cloned gene is allelic to the prp4 locus. Isolation and sequence analysis of the prp4 mutant allele revealed that at position 704 a guanine has been mutated to a adenine. As a result of this mutation, cysteine has been changed to tyrosine.
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Details
- Title
- The fission yeast prp4 + gene involved in pre-mRNA splicing codes for a predicted serine/threonine kinase and is essential for growth
- Creators
- Venkata Suresh Kumar Alahari
- Contributors
- Norbert F. Kaufer (Advisor)
- Awarding Institution
- Drexel University
- Degree Awarded
- Doctor of Philosophy (Ph.D.)
- Publisher
- Drexel University; Philadelphia, Pennsylvania
- Number of pages
- xi, 98 pages
- Resource Type
- Dissertation
- Language
- English
- Academic Unit
- Bioscience and Biotechnology [Historical]; College of Arts and Sciences; Drexel University
- Other Identifier
- 991014970306604721