Browsing by Subject "Phenotype"

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Development of imaging-based high-throughput genetic assays and genomic evaluation of yeast gene function in cell cycle progression
(2007-12) Niu, Wei; Marcotte, Edward M.
Systems biology studies the complex interactions between components of biological systems. One major goal of systems biology is to reconstruct the network of interactions between genes in response to normal and perturbed conditions. In order to accomplish this goal, large-scale data are needed. Accordingly, diverse powerful and high-throughput methods must be developed for this purpose. We have developed novel high-throughput technologies focusing on cellular phenotype profiling and now provide additional genome-scale analysis of gene and protein function. Few high-throughput methods can perform large-scale and high-throughput cellular phenotype profiling. However, analyzing gene expression patterns and protein behaviors in their cellular context will provide insights into important aspects of gene function. To complement current genomic approaches, we developed two technologies, the spotted cell microarray (cell chip) and the yeast spheroplast microarray, which allow high-throughput and highly-parallel cellular phenotype profiling including cell morphology and protein localization. These methods are based on printing collections of cells, combined with automated high-throughput microscopy, allowing systematic cellular phenotypic characterization. We used spotted cell microarrays to identify 15 new genes involved in the response of yeast to mating pheromone, 80 proteins associated with shmoo-tip 'localizome' upon pheromone stimulation and 5 genes involved in regulating the localization pattern of a group II intron encoded reverse transcriptase, LtrA, in Escherichia coli. Furthermore, in addition to morphology assays, yeast spheroplast microarrays were built for high-throughput immunofluorescence microscopy, allowing large-scale protein and RNA localization studies. In order to identify additional cell cycle genes, especially those difficult to identify in loss-of-function studies, we performed a genome-scale screen to identify yeast genes with overexpression-induced defects in cell cycle progression. After measuring the fraction of cells in G1 and G2/M phases of the cell cycle via high-throughput flow cytometry for each of ~5,800 ORFs and performing the validation and secondary assays, we observed that overexpression of 108 genes leads to reproducible and significant delay in the G1 or G2/M phase. Of 108 genes, 82 are newly implicated in the cell cycle and are likely to affect cell cycle progression via a gain-of-function mechanism. The G2/M category consists of 87 genes that showed dramatic enrichment in the regulation of mitotic cell cycle and related biological processes. YPR015C and SHE1 in the G2/M category were further characterized for their roles in cell cycle progression. We found that the G2/M delay caused by the overexpression of YPR015C and SHE1 likely results from the malfunction of spindle and chromosome segregation, which was supported by the observations of highly elevated population of large-budded cells in the pre-M phase, super-sensitivity to nocodazole, and high chromosome loss rates in these two overexpression strains. While the genes in the G2/M category were strongly enriched for cell cycle associated functions, no pathway was significantly enriched in the G1 category that is composed of 21 genes. However, the strongest enrichment for the G1 category consists of the genes involved in negative regulation of transcription. For instance, the overexpression of SKO1, a transcription repressor, resulted in strong cell cycle delay at G1 phase. Moreover, we found that the overexpression of SKO1 results in cell morphology changes that resembles mating yeast cells (shmoos) and activates the mating pheromone response pathway, thus explaining the G1 cell cycle arrest phenotype of SKO1 ORF strains.
Engineering programmable platforms in mammalian cells for the rapid characterization of viral proteins
(2022-10-28) Javanmardi, Kamyab (Mohammad); Ellington, Andrew D.; Finkelstein, Ilya J.; Xhemalce, Blerta; Maynard, Jennifer A; Barrick, Jeffrey E
Pressures to combat the coronavirus disease 2019 (COVID-19) pandemic have led to accelerated efforts to develop vaccines and therapeutics. The spike (S) protein, which is a key target of these efforts is metastable and challenging to recombinantly generate. Using a structure-guided approach, we characterized 100 spike designs, 26 of which improved protein yields. Combinations of the beneficial substitutions led to the development of HexaPro, a S protein variant with a 10-fold higher expression than the parental construct enhanced thermal-stability, due to six proline substitutions. Further structural and antigenic characterization of HexaPro confirmed a prefusion stabilized S protein with conserved ACE2 and antibody binding. The improved yield and stability of HexaPro has accelerated the development of serological diagnostics and vaccines for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Spike is also undergoing immunogenic selection with variants that increase infectivity and partially escape convalescent plasma. I developed Spike Display, a high-throughput platform to rapidly characterize glycosylated S ectodomains across multiple coronavirus-family proteins. I assayed ∼200 variant SARS-CoV-2 spikes for their expression, ACE2 binding, and recognition by 13 nAbs. An alanine scan of all five N-terminal domain (NTD) loops highlights a public epitope in the N1, N3, and N5 loops recognized by most NTD-binding nAbs. NTD mutations in the alpha, beta, gamma, epsilon, and delta impact spike expression and escape most NTD-targeting nAbs. Finally, beta and gamma completely escaped a potent ACE2 mimic. The continuous spread and evolution of SARS-CoV-2 has led to the repeated emergence of variants of concern. For the Omicron variant, sub-lineages BA.1 and BA.2 respectively contain 33 and 29 nonsynonymous and indel spike protein mutations. These amino acid substitutions and indels are implicated in increased transmissibility and enhanced immune evasion. By reverting individual Spike mutations of BA.1 or BA.2, I characterized the molecular effects of the Omicron spike mutations on expression, ACE2 receptor affinity, and neutralizing antibody recognition. I identified key mutations enabling escape from neutralizing antibodies at a variety of epitopes. Stabilizing mutations in the N-terminal and S2 domains of the spike protein can compensate for destabilizing mutations in the receptor binding domain, enabling the record number of mutations in Omicron. My results provide a comprehensive account of the mutational effects in the Omicron spike protein and illuminate previously uncharacterized mechanisms of host evasion. I anticipate that Spike Display will accelerate antigen design, deep scanning mutagenesis, and antibody epitope mapping for SARS-CoV-2 and other emerging viral threats.
The functional network in predictive biology : predicting phenotype from genotype and predicting human disease from fungal phenotype
(2008-12) McGary, Kriston Lyle; Marcotte, Edward M.
The ability to predict is one of the hallmarks of successful theories. Historically, the predictive power of biology has lagged behind disciplines like physics because the biological world is complex, challenging to quantify, and full of exceptions. However, in recent years the amount of available data has expanded exponentially and biological predictions based on this data become a possibility. The functional gene network is a quantitative way to integrate this data and a useful framework for making biological predictions. This study demonstrates that functional networks capture real biological insight and uses the network to predict both subcellular protein localization and the phenotypic outcome of gene knockouts. Furthermore, I use the functional network to evaluate genetic modules shared between diverse organisms that lead to orthologous phenotypes, many that are non-obvious. I show that the successful predictions of the functional network have broad applicability and implications that range from the design of large-scale biological experiments to the discovery of genes with potential roles in human disease.
Lysis time, optimality, and the genetics of evolution in a T7 phage model system
(2007) Heineman, Richard Hugh, 1978-; Bull, James J.
The ability of traits to adapt in response to change is one of the most fundamental aspects of evolution. Optimality models used to predict adaptation frequently make simplifying assumptions about the ability of traits to evolve freely within simple tradeoffs. However, we frequently have little understanding of genomic mechanisms underlying phenotypic evolution. Genetic constraints clearly limit phenotypic change, but the extent to which they do so is unclear. I will explore molecular and phenotypic responses to genomic and environmental perturbations through experimental evolution in T7 bacteriophage. First, I studied evolutionary robustness of the lysis time phenotype when lysin gene lysozyme was deleted. This deletion profoundly delayed lysis and thus decreased fitness. Evolved phages recovered much of the lost fitness and mostly restored lysis timing. The recovery was mediated by changes in a tail fiber gene (gene 16) with muralytic activity that is generally used in genome entry. Next, I extended the work on lysozyme to observe the effect of increasing constraint on evolutionary recovery. The effects of various combinations of deletions of lysozyme, 17.5 (which plays a role in lysis) and 16 suggested that another gene played a role similar to 17.5 in lysis. The phage defective in both lysozyme and 16 did not lyse hosts thoroughly even after long periods of infection, suggesting that these were the only effective lysin genes. Adaptation of this phage on cells expressing the essential gp16 constrained the primary adaptive pathway of recovery from lysozyme deletion. A mutually exclusive alternative pathway involving a variety of different genes evolved. The line recovered the ability to lyse normal hosts, by a mechanism involving multiple mutations. Finally, I tested the ability of T7 to adapt to an optimum lysis time. Based on empirical results from other phages, mature phage virions accumulate linearly inside the cell over time. This assumption underlies a model suggesting that availability of hosts determines optimal lysis time. While adaptation to different host densities caused the expected qualitative evolutionary changes, adaptation to conditions expected to select for slow lysis did not lead to the quantitative optimum. This is probably due to nonlinear virion accumulation.
Mutation: lessons from RNA models
(2008-05) Cowperthwaite, Matthew Cranston, 1973-; Meyers, Lauren Ancel
Mutation is a fundamental process in evolution because affects the amount of genetic variation in evolving populations. Molecular-structure models offer significant advantages over traditional population-genetics models for studying mutation, mainly because such models incorporate simple, tractable genotype-to-phenotype maps. Here, I use RNA secondary structure models to study four basic properties of mutation. The first section of this thesis studies the statistical properties of beneficial mutations. According to population genetics theory, the fitness effects of new beneficial mutations will be exponentially distributed. I show that in RNA there is sufficient correlation between a genotype and its point mutant neighbors to produce non-exponential distributions of fitness effects of beneficial mutations. These results suggest that more sophisticated statistical models may be necessary to adequately describe the distribution of fitness effects of new beneficial mutations. The second section of this thesis addresses the dynamics of deleterious mutations in evolving populations. There is a vast body of theoretical work addressing deleterious mutations that almost universally assumes that the fitness effects of deleterious mutations are static. I use an RNA simulation model to show that, at moderately high mutation rates, initially deleterious mutations may ultimately confer beneficial effects to the individuals harboring them. This result suggests that deleterious mutations may play a more important role in evolution than previously thought. The third section of this thesis studies the global patterns of mutations connecting phenotypes in fitness landscapes. I developed a network model to describe global characteristics of the relationship between sequence and structure in RNA fitness landscapes. I show that phenotype abundance varies in a predictable manner and critically influences evolutionary dynamics. A study of naturally occurring functional RNA molecules using a new structural statistic suggests that these molecules are biased towards abundant phenotypes. These results are consistent with an "ascent of the abundant" hypothesis, in which evolution yields abundant phenotypes even when they are not the most fit. The final section of this thesis addresses the evolution of mutation rates infinite asexual populations. I developed an RNA-based simulation model in which each individual's mutation rate is controlled by a neutral modifier locus. Using this model, I show that smaller populations maintain higher mutation rates than larger populations. I also show that genome length and shape of the fitness function do not significantly determine the evolved mutation rate. Lastly, I show that intermediate rates of environmental change favor evolution of the largest mutation rates.
Neural correlates and modulators of social plasticity
(2002) Sakata, Jon Tatsuya; Crews, David.; González-Lima, Francisco
Phenotypic plasticity is the process by which external and internal factors alter the phenotype of the individual, and such factors include social experience and hormonal milieu. For example, sociosexual experience in adulthood can increase the retention of sexual behavior following gonadectomy. Though there exists substantial documentation of the various types of social plasticity, few studies highlight the neural correlates of these changes. Here I present a series of experiments linking social experience to changes in cytochrome oxidase activity and in social behavior in male rats, leopard geckos and whiptail lizards. Cytochrome oxidase (CO) is a rate-limiting enzyme in oxidative phosphorylation and a valuable marker of metabolic capacity. I focused on species differences in the degree to which social experience in adulthood changes the retention of courtship behavior following gonadectomy and CO activity. I found that social experience in adulthood enhanced retention of courtship behavior following castration in male whiptail lizards but not in male geckos. In other words, whiptail lizards resemble rats in this behavioral plasticity. I also found that species that show experience-dependent increases in the retention of courtship behavior following castration also show experience-dependent increases in CO activity in the preoptic area and medial amygdala. Cytochrome oxidase activity decreases following castration in limbic brain areas, and this decrease is likely to be linked to the post-castration decline in sexual behavior. Therefore, I propose that experience-dependent elevations in CO activity in the limbic system allow for the greater display of sexual behavior following castration. In summary, increases in CO activity in the preoptic area and amygdala are neural correlates of social plasticity. With regard to species and individual differences in social plasticity, I found that experience-dependent changes in post-castration behavior were correlated with the capacity for progesterone (P) to induce sexual behavior (i.e., P-sensitivity). I also found that, in male leopard geckos, embryonic incubation temperature can affect behavioral change following social experience. In other words, both P-sensitivity and embryonic incubation temperature were found to modulate social plasticity. Finally, these modulators are also likely to affect the degree to which CO activity changes following social experience.
Phenotyping perinatal depression : exploring interactions among biopsychosocial and behavioral determinants
(2023-08-16) Longoria, Kayla D.; Wright, Michelle (Michelle Lynn), 1979-; Walker, Lorraine Olszewski; Widen, Elizabeth; Kessler, Shelli; Hsu, Kean
Perinatal depression is complex, and the etiology remains poorly understood which likely contributes to its underdiagnosis and being a leading cause of maternal mortality. This dissertation used a mixed methods approach to explore biopsychosocial and behavioral factors influencing perinatal depression and aimed to generate evidence for the development of a comprehensive risk phenotype. The systematic review indicated a total of 14 categories of determinants were investigated: biological (5), behavioral (4), social and environmental (5). Though only 28% of studies simultaneously considered determinants under more than one domain, a pattern of interactions with the tryptophan pathway emerged once determinants across domains were aggregated. Methodological limitations related to depression measures and biospecimen collection were identified. Qualitative findings indicated a network of structural factors act as barriers or facilitators to psychological well-being during the perinatal period. Social support, physical health, and logistics were commonly perceived as barriers during pregnancy, while physical health, logistics, and physical activity were seen as facilitators. In the postpartum period, barriers included logistics, social support, and physical health while the most common facilitators were social support, logistics, and nutrition. Mothers reported feelings of loneliness, isolation, worry, anxiety, frustration, and mental fatigue when facing barriers, which were heightened postpartum due to unmet expectations of increased support. Conversely, facilitators improved mothers’ ability to cope with stress and helped mothers find positive aspects about their situation when faced with challenges in motherhood. The metabolomics pilot study found gut gamma-aminobutyric acid (GABA) to be significantly lower postpartum compared to levels in the second and third trimesters. However, correlations among changes in individual pregnancy metabolites and postpartum depression were weak and no predictive models were significant suggesting individual metabolites may not serve as robust predictors of postpartum depression. These findings support perinatal depression is a multifactorial condition, and exploration of complex interactions between biopsychosocial and behavioral factors may provide a more holistic understanding of the pathophysiology. Thus, future research considering a comprehensive panel of variables in large and diverse samples is needed to replicate and build upon the present findings and further refine the phenotypic characteristics of perinatal depression described in this dissertation.
Respiratory plasticity of red drum to chronic hypoxia
(2022-12-01) Negrete, Benjamin Jr.; Esbaugh, Andrew; Thomas, Peter; Brandl, Simon J; Richards, Jeffrey G
Ocean deoxygenation (hypoxia) is a pressing concern in the face of climate change as hypoxic areas increase in size, duration, and magnitude with each year. When a fish cannot escape hypoxia, it must be able to make the appropriate physiological adjustments to maintain fitness. Juveniles and adults can display reversible phenotypic changes in low oxygen (O₂), while embryos and larvae may producing fixed traits that are carried to adulthood through developmental plasticity. Across all life stages aerobic metabolism is the most efficient way that fish generate energy, and the most impacted pathway under hypoxia. In my dissertation, I explore the flexible responses that compensate for changes to aerobic metabolism across different life stages of the marine teleost red drum (Sciaenops ocellatus) exposed to chronic, sub-lethal hypoxia. Hypoxia-acclimated juvenile drum demonstrated significant changes in hemoglobin (Hb) isoform expression relative to control. Changes in Hb expression co-occurred with reduced pH sensitivity, and increased O₂ binding affinity. Additionally, this correlated with increased maximum metabolic rate and aerobic scope relative to controls in hypoxia. These results demonstrate an important role for Hb isoforms in maximizing respiratory performance in hypoxia with implications at the whole-animal level. Furthermore, I investigated how acclimated fish respond to exhaustive exercise, and their anaerobic swim performance. I found that hypoxia-acclimated juveniles decreased ATP in the red muscle and increased ATP and glycolytic potential in their white muscle. This phenotype recruited white muscle at lower swim speeds than control fish, indicating a prioritization of glycolytic white muscle swimming over aerobic red muscle. Finally, I assess whole animal respiratory and swim performance in fish exposed to hypoxia during a critical window in early development. These fish show increases in aerobic performance in normoxia, while becoming more vulnerable to hypoxia in later life. I sought to understand the mechanisms and implications of hypoxia-induced respiratory adaptations, and demonstrated the ability of a marine fish to adapt to environmental stress, and how these adaptations changed in different life stages. This work demonstrates the species-specific resiliency and limitations in environmental stress, and illustrates the need for more species-specific work in a changing ocean.