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A Genetic and Biochemical Analysis of pre-mRNA Splicing in Saccharomyces cerevisiae


Vijayraghavan, Usha (1989) A Genetic and Biochemical Analysis of pre-mRNA Splicing in Saccharomyces cerevisiae. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/cxmk-nj42.


Pre-mRNA splicing requires interaction of cis- acting intron sequences with trans -acting factors: proteins and small nuclear ribonucleoproteins (snRNPs). The assembly of these factors into a large complex, the spliceosome, is essential for the subsequent two step splicing reaction. First, the 5' splice site is cleaved and free exon 1 and a lariat intermediate (intron- exon2) form. In the second reaction the 3' splice site is cleaved the exons ligated and lariat intron released. A combination of genetic and biochemical techniques have been used here to study pre-mRNA splicing in yeast.

Yeast introns have three highly conserved elements. We made point mutations within these elements and found that most of them affect splicing efficiency in vivo and in vitro, usually by inhibiting spliceosome assembly.

To study trans -acting splicing factors we generated and screened a bank of temperature-sensitive (ts) mutants. Eleven new complementation groups (prp17 to prp27) were isolated. The four phenotypic classes obtained affect different steps in splicing and accumulate either: 1) pre-mRNA, 2) lariat intermediate, 3) excised intron or 4) both pre-mRNA and intron. The latter three classes represent novel phenotypes. The excised intron observed in one mutant: prp26 is stabilized due to protection in a snRNP containing particle. Extracts from another mutant: prpl8 are heat labile and accumulate lariat intermediate and exon 1. This is especially interesting as it allows analysis of the second splicing reaction. In vitro complementation of inactivated prp18 extracts does not require intact snRNPs. These studies have also shown the mutation to be in a previously unknown splicing protein. A specific requirement for A TP is also observed for the second step of splicing. The PRP18 gene has been cloned and its polyadenylated transcript identified.

Item Type:Thesis (Dissertation (Ph.D.))
Subject Keywords:Biology, site-directed mutagenesis, yeast splicing pathway, spliceosome
Degree Grantor:California Institute of Technology
Major Option:Biology
Thesis Availability:Public (worldwide access)
Research Advisor(s):
  • Abelson, John N.
Thesis Committee:
  • Abelson, John N. (chair)
  • Campbell, Judith L.
  • Emr, Scott D.
  • Simon, Melvin I.
  • Wold, Barbara J.
Defense Date:1 October 1988
Record Number:CaltechTHESIS:10172013-115606044
Persistent URL:
Related URLs:
URLURL TypeDescription chapter adapted for Introduction. adapted for Chapter 1.
Default Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:7997
Deposited On:22 Oct 2013 16:15
Last Modified:06 Jan 2022 01:52

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