Browsing by Subject "gene duplication"

Floral color polymorphisms are pervasive in nature. Understanding the genetic basis of such polymorphisms allows us to address major evolutionary questions including: what types of genetic changes contribute to the evolution of phenotypic diversification? Have they arisen from new or pre-existing genetic material? And are certain types of genetic changes disproportionately involved? This dissertation address these questions by investigating two polymorphisms involving loss of anthocyanin pigmentation in some of the individuals in the populations. In Chapter 1, I characterized the genetic basis of the “white cup” phenotype in Clarkia gracilis ssp. sonomensis, where no anthocyanins are produced in the basal region of the petal. I demonstrated that the cup pigmentation is controlled by an R2R3-MYB transcription factor using transcriptome analysis, gene expression assays and cosegregation examination. I also found that petal pigmentation requires at least four R2R3-MYB genes, each gene exhibiting a spatiotemporal expression pattern that is different from each other, and each color pattern element is controlled by a different R2R3-MYB gene. In Chapter 2, I examined the evolution of petal pigmentation patterning by analyzing the phylogenetic relationship of these petal R2R3-MYB genes from C. g. ssp. sonomensis, its closely related subspecies, C. g. ssp. albicaulis and their progenitor species, C. amoena ssp. huntiana and C. lassenensis. I also compared the expression domains of these R2R3-MYB genes in these four (sub)species. I found that the R2R3-MYB genes that have region-specific expression patterns are derived from duplication events occurred before polyploidization of C. gracilis. These findings suggest that gene duplication of the R2R3-MYB genes plays an important role in the evolution of petal pigmentation patterning in C. gracilis. In Chapter 3, I identified an R2R3-MYB gene involved in producing purple or yellow anthers in Erythronium umbilicatum using transcriptome analysis, gene expression assays and a likelihood-estimation procedure. This R2R3-MYB gene regulates the expression of three anthocyanin enzyme-coding genes coordinately. However, this R2R3-MYB is present in multiple, highly similar copies. Isolating causal genetic changes from these copies has not been successful, as it would require a better knowledge of the Erythronium genome. In sum, this work shows that R2R3-MYB transcription factors are the primary determinants of floral-color polymorphisms and the R2R3-MYB gene family generated by gene duplication facilitates the evolution of novel characters.

Determining the genetic basis of adaptation has become a central focus of evolutionary biology, and the incorporation of increasingly sophisticated analytical tools from molecular biology has made identifying causal genes a practical reality. The work presented herein addresses the effects of pleiotropic constraint on evolutionary change at the level of individual genes and genetic networks. In the first chapter, I combine molecular phylogenetic analyses and direct assays of enzymatic function to determine the evolutionary processes following a gene duplication in the anthocyanin pathway. My results show that, prior to duplication, the DFR gene was constrained from functional improvement by its multiple enzymatic roles. Following duplication, this constraint was released and adaptive evolution proceeded along both paralog lineages. In the second chapter, I determine the molecular genetic basis of a flower color transition that is associated with change in pollinator attraction in morning glories. A regulatory change in a branching gene in the flavonoid biosynthetic pathway restricted flux down the cyanidin-producing branch, conferring nearly exclusive production of red pelargonidin pigment in flowers. I further demonstrate that this regulatory change was restricted to floral tissue, and that ancestral pathway flux predominates in vegetative tissues. I propose that deleterious pleiotropic effects prevented evolutionary change via enzymatic changes in the pathway due to the numerous essential products downstream of this branching point. Together, these two results show that evolutionary change may be constrained by the molecular genetic context in which prospective adaptive mutations occur.

Gene duplication is an important evolutionary tool for fostering diversification and expanding gene families. However, while this concept is well understood and accepted in a theoretical capacity, the particular changes that lead to the functional diversification of gene duplicates are less well understood and documented. Additionally, little work has been done to understand how functions are gained or lost, which leads to the diversification of orthologous genes. The Sir2 family of NAD+-dependent deacetylases is an excellent gene family to study questions of duplication and diversification as it is ubiquitous throughout all kingdoms of life, and it has expanded through a number of gene duplications so that while most bacteria have a single sirtuin/species, mammals have seven sirtuins/species. Sirtuins also have a wide array of biological functions and targets, but some of these functions are conserved in eukaryotes.

In this study, Sir2 is used to investigate the principles behind gene duplication and functional diversification in a molecular context. Sir2 function is studied in multiple species of budding yeast, the model organism Saccharomyces cerevisiae, Kluyveromyces lactis, and Candida lusitaniae using a combination of genetic, biochemical, and high-throughput methods. Sir2 and its paralog Hst1 from S. cerevisiae were used with their non-duplicated ortholog Sir2 from K. lactis to examine the type of molecular changes that occur after gene duplication and lead to subfunctionalization. Then Sir2 from the more divergent C. lusitaniae was used to study how functions are gained or lost.

To study the molecular mechanism of subfunctionalization in the duplicated deacetylases ScSir2 and ScHst1 with the non-duplicated KlSir2 used as a proxy for the ancestral state, we hypothesized that the basis for subfunctionalization in this case was in the interaction domains. ScSir2 and ScHst1 act in distinct complexes that target them to the genomic loci they regulate. KlSir2 interacts with the same complexes as both ScSir2 and ScHst1. Therefore, we first identified the minimal regions of ScSir2 and ScHst1 necessary for each to interact with its respective complex. Then we identified mutations in those interaction domains that eliminated those interactions. Those mutations were then tested in KlSir2 for their impact on its interactions with the same complexes. We found that the interaction domains in ScSir2 and ScHst1 were conserved in KlSir2, demonstrating that Sir2 and Hst1 subfunctionalized by acquiring complementary inactivating mutations in these interaction domains.

To understand better how Sir2 has gained or lost functions, we studied the Sir2 function in C. lusitaniae to serve as an intermediate between the fission yeast Schizosaccharomyces pombe Sir2, whose functions have been identified, and K. lactis and S. cerevisiae. Interestingly, ClSir2 was localized to the rDNA, which is also the case in S. pombe, K. lactis, and S. cerevisiae, but not at the telomeres, which is another locus at which Sir2 is found in other yeast. Additionally, ClSir2 was not found to have an impact on gene expression unlike Sir2 and Hst1 in other yeast where they repress transcription.

Although much research has been done on how new genes arising from gene duplications has been done, little is still known on how these genes arose and by what mechanisms they evolved their new function. Here I address a case study on how the evolution of a novel defensive gene in the Solanaceae family arose by examining the defensive copy of threonine deaminase (TD2) in tomato (Solanum lycopersicum). This gene evolved by gene duplication from an ancestor involved in biochemical gene synthesis to become an exceptionally stable anti-nutritive defensive gene in insect guts. This stability is hypothesized to have evolved due to three critical ion pairs, ionically bonded amino acids that stabilize proteins. Here, I test whether these three critical ion pairs stabilize the protein and what effects it has the activity of TD2. The removal of the second critical ion pair reduces the Kcat of the enzyme, indicating that it affects activity in a positive manner. Removal of the first and third critical ion pairs increase activity during the temperature and pH stability assays. The results suggest that the interactions and effects of each critical ion pair is complex within the enzyme, but are likely to be stabilizing without sacrificing much activity and in some cases increasing activity as well.