Molecular mechanisms controlling complex traits in yeast

Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Biology, 2012.Cataloged from PDF version of thesis.Includes bibliographical references.A fundamental goal in biology is to understand how the information stored in DNA results in a cellular function. However, it is insufficient to study one variant of a particular DNA sequence because most people do not share identical genome sequences, and the differences in DNA sequence have functional consequences. In this thesis, I examine how natural variation in the Saccharomyces cerevisiae genome can affect cellular processes. This is done using deletion libraries to examine how mutations in the same gene but in two different genetic backgrounds of S. cerevisiae, S288c and [summation]1278b, can lead different phenotypes for two traits: gene essentiality and agar adhesion. We found that the genomes of the S288c and [summation]1278b strains are only as divergent as two humans in the population. However, analyses of deletion libraries in each strain revealed 57 genes have functions that are essential in one strain but not the other. Strain specific phenotypes are more pronounced for the trait of agar adhesion where 553 deletions have phenotypes that are specific to one strain or the other. Part of the difference is because the [summation]1278b strain requires the filamentation mitogen activated kinase pathway (fMAPK) for agar adhesion but the S288c strain does not. I found that S288c is able to bypass the fMAPK pathway because it contains an allele of the transcription factor RPI1 that promotes transcription of the gene FLO11. Characterization of the sequence differences between the S288c and 11278b alleles of RPIJ revealed that they differ in the number of intragenic tandem repeats. Examination of the genomes of both strains uncovered the possibility that expansions and contractions of intragenic repeats may be a general mechanism to quickly introduce genomic and phenotypic variation.by Brian L. Chin.Ph.D

FASTER MT: Isolation of Pure Populations of a and α Ascospores from Saccharomyces cerevisiae

The budding yeast Saccharomyces cerevisiae has many traits that make it useful for studies of quantitative inheritance. Genome-wide association studies and bulk segregant analyses often serve as first steps toward the identification of quantitative trait loci. These approaches benefit from having large numbers of ascospores pooled by mating type without contamination by vegetative cells. To this end, we inserted a gene encoding red fluorescent protein into the MATa locus. Red fluorescent protein expression caused MATa and a/α diploid vegetative cells and MATa ascospores to fluoresce; MATα cells without the gene did not fluoresce. Heterozygous diploids segregated fluorescent and nonfluorescent ascospores 2:2 in tetrads and bulk populations. The two populations of spores were separable by fluorescence-activated cell sorting with little cross contamination or contamination with diploid vegetative cells. This approach, which we call Fluorescent Ascospore Technique for Efficient Recovery of Mating Type (FASTER MT), should be applicable to laboratory, industrial, and undomesticated, strains

p97/VCP promotes degradation of CRBN substrate glutamine synthetase and neosubstrates

Glutamine synthetase (GS) plays an essential role in metabolism by catalyzing the synthesis of glutamine from glutamate and ammonia. Our recent study showed that CRBN, a direct protein target for the teratogenic and antitumor activities of immunomodulatory drugs such as thalidomide, lenalidomide, and pomalidomide, recognizes an acetyl degron of GS, resulting in ubiquitylation and degradation of GS in response to glutamine. Here, we report that valosin-containing protein (VCP)/p97 promotes the degradation of ubiquitylated GS, resulting in its accumulation in cells with compromised p97 function. Notably, p97 is also required for the degradation of all four known CRBN neo-substrates [Ikaros family zinc finger proteins 1 (IKZF1) and 3 (IKZF3), casein kinase 1α (CK1α), and the translation termination factor GSPT1] whose ubiquitylation is induced by immunomodulatory drugs. Together, these data point to an unexpectedly intimate relationship between the E3 ubiquitin ligase CRL4^(CRBN) and p97 pathways

Long-term culture captures injury-repair cycles of colonic stem cells

The colonic epithelium can undergo multiple rounds of damage and repair, often in response to excessive inflammation. The responsive stem cell that mediates this process is unclear, in part because of a lack of in vitro models that recapitulate key epithelial changes that occur in vivo during damage and repair. Here, we identify a Hop

Amyloid-beta/Fyn–Induced Synaptic, Network, and Cognitive Impairments Depend on Tau Levels in Multiple Mouse Models of Alzheimer’s Disease

Alzheimer\u27s disease (AD), the most common neurodegenerative disorder, is a growing public health problem and still lacks effective treatments. Recent evidence suggests that microtubule-associated protein tau may mediate amyloid-β peptide (Aβ) toxicity by modulating the tyrosine kinase Fyn.Weshowed previously that tau reduction prevents, and Fyn overexpression exacerbates, cognitive deficits in human amyloid precursor protein (hAPP) transgenic mice overexpressing Aβ. However, the mechanisms by which Aβ, tau, and Fyn cooperate in AD-related pathogenesis remain to be fully elucidated. Here we examined the synaptic and network effects of this pathogenic triad. Tau reduction prevented cognitive decline induced by synergistic effects of Aβ and Fyn. Tau reduction also prevented synaptic transmission and plasticity deficits in hAPP mice. Using electroencephalography to examine network effects, we found that tau reduction prevented spontaneous epileptiform activity in multiple lines of hAPP mice. Tau reduction also reduced the severity of spontaneous and chemically induced seizures in mice overexpressing both Aβ and Fyn. To better understand these protective effects, we recorded wholecell currents in acute hippocampal slices from hAPP mice with and without tau. hAPP mice with tau had increased spontaneous and evoked excitatory currents, reduced inhibitory currents, and NMDA receptor dysfunction. Tau reduction increased inhibitory currents and normalized excitation/inhibition balance and NMDA receptor-mediated currents in hAPP mice. Our results indicate that Aβ, tau, and Fyn jointly impair synaptic and network function and suggest that disrupting the copathogenic relationship between these factors could be of therapeutic benefit

Phosphoprotein SAK1 is a regulator of acclimation to singlet oxygen in Chlamydomonas reinhardtii

Abstract Singlet oxygen is a highly toxic and inevitable byproduct of oxygenic photosynthesis. The unicellular green alga Chlamydomonas reinhardtii is capable of acclimating specifically to singlet oxygen stress, but the retrograde signaling pathway from the chloroplast to the nucleus mediating this response is unknown. Here we describe a mutant, singlet oxygen acclimation knocked-out 1 (sak1), that lacks the acclimation response to singlet oxygen. Analysis of genome-wide changes in RNA abundance during acclimation to singlet oxygen revealed that SAK1 is a key regulator of the gene expression response during acclimation. The SAK1 gene encodes an uncharacterized protein with a domain conserved among chlorophytes and present in some bZIP transcription factors. The SAK1 protein is located in the cytosol, and it is induced and phosphorylated upon exposure to singlet oxygen, suggesting that it is a critical intermediate component of the retrograde signal transduction pathway leading to singlet oxygen acclimation

MESSENGER Observations of Large Flux Transfer Events at Mercury

Six flux transfer events (FTEs) were encountered during MESSENGER's first two flybys of Mercury (M1 and M2). For M1 the interplanetary magnetic field (IMF) was predominantly northward and four FTEs with durations of 1 to 6 s were observed in the magnetosheath following southward IMF turnings. The IMF was steadily southward during M2, and an FTE 4 s in duration was observed just inside the dawn magnetopause followed approx. 32 s later by a 7 s FTE in the magnetosheath. Flux rope models were fit to the magnetic field data to determine FTE dimensions and flux content. The largest FTE observed by MESSENGER had a diameter of approx. 1 R(sub M) (where R(sub M) is Mercury s radius), and its open magnetic field increased the fraction of the surface exposed to the solar wind by 10 - 20 percent and contributed up to approx. 30 kV to the cross-magnetospheric electric potential

A high-resolution map of human evolutionary constraint using 29 mammals.

The comparison of related genomes has emerged as a powerful lens for genome interpretation. Here we report the sequencing and comparative analysis of 29 eutherian genomes. We confirm that at least 5.5% of the human genome has undergone purifying selection, and locate constrained elements covering ∼4.2% of the genome. We use evolutionary signatures and comparisons with experimental data sets to suggest candidate functions for ∼60% of constrained bases. These elements reveal a small number of new coding exons, candidate stop codon readthrough events and over 10,000 regions of overlapping synonymous constraint within protein-coding exons. We find 220 candidate RNA structural families, and nearly a million elements overlapping potential promoter, enhancer and insulator regions. We report specific amino acid residues that have undergone positive selection, 280,000 non-coding elements exapted from mobile elements and more than 1,000 primate- and human-accelerated elements. Overlap with disease-associated variants indicates that our findings will be relevant for studies of human biology, health and disease

The ALMA Interferometric Pipeline Heuristics

We describe the calibration and imaging heuristics developed and deployed in the ALMA interferometric data processing pipeline, as of ALMA Cycle 9. The pipeline software framework is written in Python, with each data reduction stage layered on top of tasks and toolkit functions provided by the Common Astronomy Software Applications package. This framework supports a variety of tasks for observatory operations, including science data quality assurance, observing mode commissioning, and user reprocessing. It supports ALMA and VLA interferometric data along with ALMA and NRO45m single dish data, via different stages and heuristics. In addition to producing calibration tables, calibrated measurement sets, and cleaned images, the pipeline creates a WebLog which serves as the primary interface for verifying the data quality assurance by the observatory and for examining the contents of the data by the user. Following the adoption of the pipeline by ALMA Operations in 2014, the heuristics have been refined through annual development cycles, culminating in a new pipeline release aligned with the start of each ALMA Cycle of observations. Initial development focused on basic calibration and flagging heuristics (Cycles 2-3), followed by imaging heuristics (Cycles 4-5), refinement of the flagging and imaging heuristics with parallel processing (Cycles 6-7), addition of the moment difference analysis to improve continuum channel identification (2020 release), addition of a spectral renormalization stage (Cycle 8), and improvement in low SNR calibration heuristics (Cycle 9). In the two most recent Cycles, 97% of ALMA datasets were calibrated and imaged with the pipeline, ensuring long-term automated reproducibility. We conclude with a brief description of plans for future additions, including self-calibration, multi-configuration imaging, and calibration and imaging of full polarization data.Comment: accepted for publication by Publications of the Astronomical Society of the Pacific, 65 pages, 20 figures, 10 tables, 2 appendice