Student Theses and Dissertations

Frej Tulin

Date of Award

2014

Document Type

Thesis

Degree Name

Doctor of Philosophy (PhD)

RU Laboratory

Cross Fred Laboratory

Abstract

The cell cycle encompasses all the steps required for cell proliferation, and is normally tightly coupled to growth and division in all organisms. Much research has resulted in a well-supported model of eukaryotic cell cycle control. However, since most of this research has been carried out in yeast and animals (opisthokonts), it could in principle apply poorly to early-diverging groups of organisms, such as the green plants. Plant cell cycle research has largely followed a candidate strategy based on reverse genetics. These studies have a provided insights into plant cell cycle control, but are generally dependent upon sequence conservation between plant and opisthokont genes. This thesis presents work from an ongoing screen to identify critical components of the plant cell cycle by forward genetic methods that are independent of prior knowledge of specific mechanisms of cell cycle control. The screen was carried out in the unicellular green alga Chlamydomonas, a microbial member of the Viridiplantae, which has wellestablished experimental Mendelian genetics, and many features that might facilitate identification of loss-of-function mutations. We have developed semi-automatic techniques for isolation of temperature-sensitive lethal mutants that are capable of cell growth at a near-wild-type rate, but that exhibit first-cycle failure of cell division (div phenotype). We developed efficient methods for identification of causative mutations by next-generation sequencing of bulked segregant pools. The normal cell division cycle in Chlamydomonas is characterized by a long period of G1 growth, followed by a series of rapidly alternating rounds of S phases and mitoses (S/M phase). Analysis of more than 50 div mutants identified two main phenotypic classes. One class showed somewhat reduced growth and arrested in a G1-like state. This class included genes with diverse molecular functions based on gene annotations, including transcription, translation, and membrane biogenesis. The other class exhibited wild-type cell growth rate, and entered the S/M program on time; mutant cells then developed various S/M-specific defects. This class included genes directly involved in DNA replication and chromosome segregation. Other mutations identified genes likely involved in cell cycle control, including the cyclin-dependent kinases CDKA and CDKB, two anaphase-promoting complex subunits, and the mitotic kinases Aurora B and MPS1. The phenotype of the cdka-1 mutant suggested a specific role for CDKA in the transition from cell growth to initiation of the S/M cell division program. CDKB, in contrast, functions specifically after DNA replication, in entry into the first mitosis. Although most DIV genes had clear homologues involved in cell cycle progression in opisthokonts, some genes had clear homologues in Viridiplantae but not in opisthokonts, including the BSL1 phosphatase, which we demonstrate to have a role in mitotic entry similar to that of CDKB. The div mutants isolated in this screen provide an opportunity to study the plant cell cycle in a simple microbial setting. Since a large majority of the mutants alter genes with clear Arabidopsis sequelogues, the results also suggest targeted candidates for cell cycle experiments in Angiosperms.

Comments

A Thesis Presented to the Faculty of The Rockefeller University in Partial Fulfillment of the Requirements for the degree of Doctor of Philosophy

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