Date of Award
Doctor of Philosophy (PhD)
ABSTRACT OF THE DISSERTATIONAn Investigation of Second-Site Non-Complementation among Motile Cilia Genes in Chlamydomonas reinhardtii by Gervette M. Penny Doctor of Philosophy in Biology and Biomedical Sciences Molecular Genetics and Genomics Washington University in St. Louis, 2023 Professor Susan K. Dutcher, Chair
Motile cilia are complex microtubule-based organelles used by eukaryotic cells for locomotion or directing fluid flow. In humans, motile cilia defects cause primary ciliary dyskinesia (PCD), characterized by neonatal respiratory distress, recurrent lung infections, bronchiectasis, and low nitric oxide. Situs inversus and/or male infertility are present in about 50% of cases. PCD inheritance is recessive; both copies (alleles) of a single gene have a deleterious variant, known as homozygosity. The result is loss-of-function and disease. In recessive inheritance, a variant in one allele, or heterozygosity, is insufficient to cause a disease phenotype. Despite identification of over 50 PCD genes, approximately 30% of clinically diagnosed patients have unknown genetic etiology using exome sequencing. Explanations for this finding are: 1) causative PCD genes remain undiscovered, 2) patient variants may be non-coding, or 3) an alternative inheritance mechanism exists. One alternative is digenic inheritance (DI) or second-site non-complementation (SSNC). Co-occurring heterozygous mutations in two recessive genes often produce a normal phenotype; they complement. Abnormal phenotypes are absent because each gene has one functional copy that provides sufficient gene product. In rare instances, double heterozygosity in two genes causes a mutant phenotype and disease; this is the definition of DI/SSNC. In SSNC, the gene products often interact. Motile cilia proteins are organized into interacting structures or complexes and are good candidates for SSNC studies. Chlamydomonas reinhardtii is a haploid algal cell with two motile cilia; most human motile cilia proteins are conserved. I assessed whether SSNC can occur among motile cilia genes in Chlamydomonas diploids. I screened 231 strains with mutations in dynein arm structural proteins, dynein adapters, dynein maturation factors, dynein assembly proteins, and a radial spoke protein. Initial assessment revealed no obvious phenotypes and indicated that these genes are recessive. In a sensitized screen, double heterozygous diploids show a cilia regeneration phenotype under stress conditions. Chlamydomonas cilia were removed using pH shock and allowed to regenerate in medium with cycloheximide, a protein synthesis inhibitor that requires the cells to use the existing pool of ciliary proteins. A subset of double heterozygous diploids shows SSNC and regenerate shorter cilia than the wild-type control; each of these diploids contains at least one mutation in a dynein arm assembly factor gene. To assess whether assembly factors show gene dosage effects, I generated a null strain of dynein assembly factor PF23 (pf23-4) using CRISPR/Cas9 targeted insertional mutagenesis. Mass spectrometry analysis shows a more severe loss of dynein arm components in the cilia than the pf23-1 in-frame deletion strain with a shorter PF23 protein. Immunoblot analysis of steady-state pf23-4/PF23; wdr92/WDR92 and pf23-4/PF23; oda8/ODA8 diploids shows that PF23 protein is reduced compared to wild-type controls. In the pf23/PF23 single heterozygous strain, PF23 protein is also reduced. I suggest that PF23 shows a dosage-dependent phenotype. When heterozygous with other dynein assembly factors, the proteins in the dynein assembly pathway may be reduced below a critical functional threshold during stress. Analysis of patients for SSNC is an important novel approach for the future.
Chair and Committee
Susan K. Dutcher
Timothy Schedl, Steven Brody, Douglas Chalker, Sheng Chih Jin,
Penny, Gervette M., "An Investigation of Second-Site Non-Complementation among Motile Cilia Genes in Chlamydomonas reinhardtii" (2023). Arts & Sciences Electronic Theses and Dissertations. 2905.
Available for download on Thursday, April 25, 2024