Date of Award

5-1-2022

Language

English

Document Type

Master's Thesis

Degree Name

Master of Science (MS)

College/School/Department

Department of Biomedical Sciences

Content Description

1 online resource (iv, 34 pages) : color illustrations.

Dissertation/Thesis Chair

Joan Curcio

Committee Members

Randall H Morse, Douglas Conklin

Keywords

Centrosomes, Scaffold proteins, Capsid proteins, Reverse transcriptase, Genetic transcription, Transposons, Saccharomyces cerevisiae, Protein-protein interactions

Subject Categories

Genetics

Abstract

Nearly all eukaryotic genomes harbor mobile genetic elements known as retrotransposons. The mRNA ofthese elements undergo reverse transcription, yielding cDNA that is inserted in a new location in the host genome. We study Saccharomyces cerevisiae Ty1 elements, an active family of retrovirus-like retrotransposons, and the mechanism behind its regulation of spindle pole body (SPB) inheritance. Ty1 retromobility is activated during growth at 20˚C. In a manuscript that includes my work (Murphy et al., 2022, manuscript in preparation), we report that essential SPB proteins Nud1 and Spc42 are sequestered in the Ty1 retrosome, the site of Ty1 nucleocapsid assembly, in a strain expressing a Gag:ORF fusion from a single chromosomal Ty1 allele. This observation led our lab to hypothesize that Nud1, a SPB fate determinant, interacts with the Ty1 nucleocapsid protein, Gag at low temperature, thereby diverting Nud1 from its role in SPB fate determination and causing loss of the distinguishing molecular mark on the old SPB. Thus, a temperature-dependent interaction between Nud1 at the old SPB and Gag could underlie the random fate of SPBs observed in one-half of cell divisions at 20˚C. Here, I used the yeast two-hybrid system to demonstrate interactions between Gag and Spc42 and Gag and Nud1. These interactions were observed when the assay was performed at 20˚C or at the retrotransposition non-permissive temperature of 30˚C. A mutant form of Spc42 with the Cdk1-phosphorylation sites S4 and T6 mutated interacted more strongly with Gag than wild-type Spc42 did. I also mapped the involvement of different structural domains of Gag in the interaction with Spc42, Spc42-S4A,T6A, and Nud1. Both p45-Gag and p49-Gag fused to the GAL4 binding domain (BD) as well as BD-Gag1-245 and Gag245-440 fusions, representing the N-terminal and C-terminal domains of Gag, respectively, interacted with Spc42, Spc42-S4A,T6A, and Nud1 fused to the GAL4 activation domain (AD). However, AD-Nud1, AD-Spc42 and AD-Spc42-s4a,t6a interacted more strongly with BD-Gag1-245 than BD-Gag245-440, whereas AD-p45-Gag and AD-p49-Gag interacted more strongly with BD-Gag245-440 than BD-Gag1-245. These findings raise the possibility that Gag multimers can bind Nud1 and Spc42. In addition to these results, I also performed retrotransposition assays that demonstrate that Nud1 interacts genetically with an intermediate in Ty1 retrotransposition. Overall, my findings support the hypothesis that there is crosstalk between Ty1 Gag and Nud1 that might cause a loss of Nud1 function in SPB fate determination. Mapping the interactions between regions of Gag and SPB proteins offers insight into the mechanism of retrotransposons effect on the canonical pattern of inheritance.

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