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1.
Front Immunol ; 10: 1516, 2019.
Article in English | MEDLINE | ID: mdl-31417541

ABSTRACT

Although structural studies of individual T cell receptors (TCRs) have revealed important roles for both the α and ß chain in directing MHC and antigen recognition, repertoire-level immunogenomic analyses have historically examined the ß chain alone. To determine the amount of useful information about TCR repertoire function encoded within αß pairings, we analyzed paired TCR sequences from nearly 100,000 unique CD4+ and CD8+ T cells captured using two different high-throughput, single-cell sequencing approaches. Our results demonstrate little overlap in the healthy CD4+ and CD8+ repertoires, with shared TCR sequences possessing significantly shorter CDR3 sequences corresponding to higher generation probabilities. We further utilized tools from information theory and machine learning to show that while α and ß chains are only weakly associated with lineage, αß pairings appear to synergistically drive TCR-MHC interactions. Vαß gene pairings were found to be the TCR feature most informative of T cell lineage, supporting the existence of germline-encoded paired αß TCR-MHC interaction motifs. Finally, annotating our TCR pairs using a database of sequences with known antigen specificities, we demonstrate that approximately a third of the T cells possess α and ß chains that each recognize different known antigens, suggesting that αß pairing is critical for the accurate inference of repertoire functionality. Together, these findings provide biological insight into the functional implications of αß pairing and highlight the utility of single-cell sequencing in immunogenomics.


Subject(s)
CD4-Positive T-Lymphocytes/immunology , CD8-Positive T-Lymphocytes/immunology , Complementarity Determining Regions , Machine Learning , Receptors, Antigen, T-Cell, alpha-beta , Sequence Analysis, Protein , Antigens/genetics , Antigens/immunology , Complementarity Determining Regions/genetics , Complementarity Determining Regions/immunology , Humans , Receptors, Antigen, T-Cell, alpha-beta/genetics , Receptors, Antigen, T-Cell, alpha-beta/immunology
2.
PLoS Biol ; 17(3): e3000147, 2019 03.
Article in English | MEDLINE | ID: mdl-30835725

ABSTRACT

Strains of Saccharomyces cerevisiae used to make beer, bread, and wine are genetically and phenotypically distinct from wild populations associated with trees. The origins of these domesticated populations are not always clear; human-associated migration and admixture with wild populations have had a strong impact on S. cerevisiae population structure. We examined the population genetic history of beer strains and found that ale strains and the S. cerevisiae portion of allotetraploid lager strains were derived from admixture between populations closely related to European grape wine strains and Asian rice wine strains. Similar to both lager and baking strains, ale strains are polyploid, providing them with a passive means of remaining isolated from other populations and providing us with a living relic of their ancestral hybridization. To reconstruct their polyploid origin, we phased the genomes of two ale strains and found ale haplotypes to both be recombinants between European and Asian alleles and to also contain novel alleles derived from extinct or as yet uncharacterized populations. We conclude that modern beer strains are the product of a historical melting pot of fermentation technology.


Subject(s)
Polyploidy , Saccharomyces cerevisiae/genetics , Asia , Beer , Europe , Fermentation/physiology , Haplotypes/genetics , Saccharomyces cerevisiae/classification , Wine
3.
Genetics ; 209(3): 725-741, 2018 07.
Article in English | MEDLINE | ID: mdl-29724862

ABSTRACT

In vitro studies suggest that stress may generate random standing variation and that different cellular and ploidy states may evolve more rapidly under stress. Yet this idea has not been tested with pathogenic fungi growing within their host niche in vivo Here, we analyzed the generation of both genotypic and phenotypic diversity during exposure of Candida albicans to the mouse oral cavity. Ploidy, aneuploidy, loss of heterozygosity (LOH), and recombination were determined using flow cytometry and double digest restriction site-associated DNA sequencing. Colony phenotypic changes in size and filamentous growth were evident without selection and were enriched among colonies selected for LOH of the GAL1 marker. Aneuploidy and LOH occurred on all chromosomes (Chrs), with aneuploidy more frequent for smaller Chrs and whole Chr LOH more frequent for larger Chrs. Large genome shifts in ploidy to haploidy often maintained one or more heterozygous disomic Chrs, consistent with random Chr missegregation events. Most isolates displayed several different types of genomic changes, suggesting that the oral environment rapidly generates diversity de novo In sharp contrast, following in vitro propagation, isolates were not enriched for multiple LOH events, except in those that underwent haploidization and/or had high levels of Chr loss. The frequency of events was overall 100 times higher for C. albicans populations following in vivo passage compared with in vitro These hyper-diverse in vivo isolates likely provide C. albicans with the ability to adapt rapidly to the diversity of stress environments it encounters inside the host.


Subject(s)
Candida albicans/physiology , Candidiasis/microbiology , DNA, Fungal/genetics , Genetic Variation , Mouth/microbiology , Aneuploidy , Animals , Candida albicans/genetics , Candida albicans/isolation & purification , Fungal Proteins/genetics , Galactokinase/genetics , Gene Frequency , Genotype , Host-Pathogen Interactions , Loss of Heterozygosity , Male , Mice , Phenotype , Sequence Analysis, DNA
4.
G3 (Bethesda) ; 7(8): 2845-2854, 2017 08 07.
Article in English | MEDLINE | ID: mdl-28673928

ABSTRACT

Biofilm formation by microorganisms is a major cause of recurring infections and removal of biofilms has proven to be extremely difficult given their inherent drug resistance . Understanding the biological processes that underlie biofilm formation is thus extremely important and could lead to the development of more effective drug therapies, resulting in better infection outcomes. Using the yeast Saccharomyces cerevisiae as a biofilm model, overexpression screens identified DIG1, SFL1, HEK2, TOS8, SAN1, and ROF1/YHR177W as regulators of biofilm formation. Subsequent RNA-seq analysis of biofilm and nonbiofilm-forming strains revealed that all of the overexpression strains, other than DIG1 and TOS8, were adopting a single differential expression profile, although induced to varying degrees. TOS8 adopted a separate profile, while the expression profile of DIG1 reflected the common pattern seen in most of the strains, plus substantial DIG1-specific expression changes. We interpret the existence of the common transcriptional pattern seen across multiple, unrelated overexpression strains as reflecting a transcriptional state, that the yeast cell can access through regulatory signaling mechanisms, allowing an adaptive morphological change between biofilm-forming and nonbiofilm states.


Subject(s)
Biofilms , Gene Expression Profiling , Genetic Testing , Saccharomyces cerevisiae/genetics , Gene Deletion , Gene Expression Regulation, Fungal , MAP Kinase Signaling System/genetics , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Untranslated/genetics , Transcription Factors/metabolism
5.
G3 (Bethesda) ; 7(1): 233-246, 2017 01 05.
Article in English | MEDLINE | ID: mdl-27836908

ABSTRACT

Aneuploidy, a state in which the chromosome number deviates from a multiple of the haploid count, significantly impacts human health. The phenotypic consequences of aneuploidy are believed to arise from gene expression changes associated with the altered copy number of genes on the aneuploid chromosomes. To dissect the mechanisms underlying altered gene expression in aneuploids, we used RNA-seq to measure transcript abundance in colonies of the haploid Saccharomyces cerevisiae strain F45 and two aneuploid derivatives harboring disomies of chromosomes XV and XVI. F45 colonies display complex "fluffy" morphologies, while the disomic colonies are smooth, resembling laboratory strains. Our two disomes displayed similar transcriptional profiles, a phenomenon not driven by their shared smooth colony morphology nor simply by their karyotype. Surprisingly, the environmental stress response (ESR) was induced in F45, relative to the two disomes. We also identified genes whose expression reflected a nonlinear interaction between the copy number of a transcriptional regulatory gene on chromosome XVI, DIG1, and the copy number of other chromosome XVI genes. DIG1 and the remaining chromosome XVI genes also demonstrated distinct contributions to the effect of the chromosome XVI disome on ESR gene expression. Expression changes in aneuploids appear to reflect a mixture of effects shared between different aneuploidies and effects unique to perturbing the copy number of particular chromosomes, including nonlinear copy number interactions between genes. The balance between these two phenomena is likely to be genotype- and environment-specific.


Subject(s)
Aneuploidy , Gene Expression Regulation/genetics , Saccharomyces cerevisiae/genetics , Stress, Physiological/genetics , Chromosomes, Fungal/genetics , Gene Dosage/genetics , Haploidy , Humans , Karyotype
6.
Curr Biol ; 26(7): 965-71, 2016 Apr 04.
Article in English | MEDLINE | ID: mdl-27020745

ABSTRACT

Modern transportation networks have facilitated the migration and mingling of previously isolated populations of plants, animals, and insects. Human activities can also influence the global distribution of microorganisms. The best-understood example is yeasts associated with winemaking. Humans began making wine in the Middle East over 9,000 years ago [1, 2]. Selecting favorable fermentation products created specialized strains of Saccharomyces cerevisiae [3, 4] that were transported along with grapevines. Today, S. cerevisiae strains residing in vineyards around the world are genetically similar, and their population structure suggests a common origin that followed the path of human migration [3-7]. Like wine, coffee and cacao depend on microbial fermentation [8, 9] and have been globally dispersed by humans. Theobroma cacao originated in the Amazon and Orinoco basins of Colombia and Venezuela [10], was cultivated in Central America by Mesoamerican peoples, and was introduced to Europeans by Hernán Cortés in 1530 [11]. Coffea, native to Ethiopia, was disseminated by Arab traders throughout the Middle East and North Africa in the 6(th) century and was introduced to European consumers in the 17(th) century [12]. Here, we tested whether the yeasts associated with coffee and cacao are genetically similar, crop-specific populations or genetically diverse, geography-specific populations. Our results uncovered populations that, while defined by niche and geography, also bear signatures of admixture between major populations in events independent of the transport of the plants. Thus, human-associated fermentation and migration may have affected the distribution of yeast involved in the production of coffee and chocolate.


Subject(s)
Cacao/microbiology , Coffee/microbiology , Saccharomyces cerevisiae/classification , Saccharomyces cerevisiae/isolation & purification , Fermentation , Geography , Saccharomyces cerevisiae/genetics , Saccharomyces cerevisiae/metabolism , Transportation
7.
Genetics ; 199(1): 247-62, 2015 Jan.
Article in English | MEDLINE | ID: mdl-25398792

ABSTRACT

Clinically relevant features of monogenic diseases, including severity of symptoms and age of onset, can vary widely in response to environmental differences as well as to the presence of genetic modifiers affecting the trait's penetrance and expressivity. While a better understanding of modifier loci could lead to treatments for Mendelian diseases, the rarity of individuals harboring both a disease-causing allele and a modifying genotype hinders their study in human populations. We examined the genetic architecture of monogenic trait modifiers using a well-characterized yeast model of the human Mendelian disease classic galactosemia. Yeast strains with loss-of-function mutations in the yeast ortholog (GAL7) of the human disease gene (GALT) fail to grow in the presence of even small amounts of galactose due to accumulation of the same toxic intermediates that poison human cells. To isolate and individually genotype large numbers of the very rare (∼0.1%) galactose-tolerant recombinant progeny from a cross between two gal7Δ parents, we developed a new method, called "FACS-QTL." FACS-QTL improves upon the currently used approaches of bulk segregant analysis and extreme QTL mapping by requiring less genome engineering and strain manipulation as well as maintaining individual genotype information. Our results identified multiple distinct solutions by which the monogenic trait could be suppressed, including genetic and nongenetic mechanisms as well as frequent aneuploidy. Taken together, our results imply that the modifiers of monogenic traits are likely to be genetically complex and heterogeneous.


Subject(s)
Aneuploidy , Genes, Modifier , Genetic Variation , Quantitative Trait Loci , Saccharomyces cerevisiae/genetics , Alleles , Chromosome Mapping/methods , Galactose/metabolism , Galectins/deficiency , Galectins/genetics
8.
Proc Natl Acad Sci U S A ; 110(30): 12367-72, 2013 Jul 23.
Article in English | MEDLINE | ID: mdl-23812752

ABSTRACT

Although microorganisms are traditionally used to investigate unicellular processes, the yeast Saccharomyces cerevisiae has the ability to form colonies with highly complex, multicellular structures. Colonies with the "fluffy" morphology have properties reminiscent of bacterial biofilms and are easily distinguished from the "smooth" colonies typically formed by laboratory strains. We have identified strains that are able to reversibly toggle between the fluffy and smooth colony-forming states. Using a combination of flow cytometry and high-throughput restriction-site associated DNA tag sequencing, we show that this switch is correlated with a change in chromosomal copy number. Furthermore, the gain of a single chromosome is sufficient to switch a strain from the fluffy to the smooth state, and its subsequent loss to revert the strain back to the fluffy state. Because copy number imbalance of six of the 16 S. cerevisiae chromosomes and even a single gene can modulate the switch, our results support the hypothesis that the state switch is produced by dosage-sensitive genes, rather than a general response to altered DNA content. These findings add a complex, multicellular phenotype to the list of molecular and cellular traits known to be altered by aneuploidy and suggest that chromosome missegregation can provide a quick, heritable, and reversible mechanism by which organisms can toggle between phenotypes.


Subject(s)
Aneuploidy , Saccharomyces cerevisiae/genetics , Chromosomes, Fungal , Gene Dosage , Phenotype
9.
Nat Methods ; 10(7): 671-5, 2013 Jul.
Article in English | MEDLINE | ID: mdl-23666411

ABSTRACT

Tetrad analysis has been a gold-standard genetic technique for several decades. Unfortunately, the need to manually isolate, disrupt and space tetrads has relegated its application to small-scale studies and limited its integration with high-throughput DNA sequencing technologies. We have developed a rapid, high-throughput method, called barcode-enabled sequencing of tetrads (BEST), that uses (i) a meiosis-specific GFP fusion protein to isolate tetrads by FACS and (ii) molecular barcodes that are read during genotyping to identify spores derived from the same tetrad. Maintaining tetrad information allows accurate inference of missing genetic markers and full genotypes of missing (and presumably nonviable) individuals. An individual researcher was able to isolate over 3,000 yeast tetrads in 3 h, an output equivalent to that of almost 1 month of manual dissection. BEST is transferable to other microorganisms for which meiotic mapping is significantly more laborious.


Subject(s)
Algorithms , Chromosome Mapping/methods , DNA, Fungal/genetics , Genetic Markers/genetics , High-Throughput Nucleotide Sequencing/methods , Meiosis/genetics , Saccharomyces cerevisiae/genetics
10.
Leuk Res ; 35(12): 1597-604, 2011 Dec.
Article in English | MEDLINE | ID: mdl-21752465

ABSTRACT

Flow cytometric cell sorting combined with molecular gene rearrangement analysis was used to detect and to further characterize simultaneously occurring phenotypically distinct B cell monoclonal lymphoid and monoclonal plasma cell populations from 38 individual specimens. By sorting and subsequent gene rearrangement analysis, separate or identical monoclonality genotypes could be revealed and confirmed. In only 13 of 38 specimens, the B lymphoid cells and plasma cell populations showed an identical genotypic profile, while 25 had non-identical profiles (including 4 process control specimens). The majority of the genotypically identical group had a phenotype consistent with Waldenström's/lymphoplasmacytic lymphoma (WM/LPL), while WM/LPL phenotype was present in 16/25 of the non-identical cases. Proof of an identical monoclonal genotype for plasmacytic and B-lymphoid cell populations must be used to define WM/LPL as a distinct entity in the clinical setting of monoclonal lymphoid and plasma cells expressing the same light chains. Conversely, the confirmation of genotypically distinct populations can significantly improve confidence in diagnostic and prognostic decisions in specimens with B lymphoid lymphomas and a concurrent, possibly smoldering myeloma or multiple myeloma. These techniques are requisite in future clinical studies for diagnosis and prognosis in these diseases.


Subject(s)
Immunoglobulin Light Chains/metabolism , Lymphocytes/pathology , Plasma Cells/pathology , Waldenstrom Macroglobulinemia/genetics , Waldenstrom Macroglobulinemia/immunology , Adult , Aged , Aged, 80 and over , Bone Marrow/pathology , Cohort Studies , Cytogenetic Analysis , Female , Gene Frequency , Gene Rearrangement, B-Lymphocyte/physiology , Humans , Immunoglobulin Light Chains/genetics , Lymphocyte Count , Lymphocytes/immunology , Lymphocytes/metabolism , Male , Middle Aged , Plasma Cells/metabolism , Sequence Analysis, DNA , Waldenstrom Macroglobulinemia/pathology
11.
J Immunol ; 174(10): 6416-23, 2005 May 15.
Article in English | MEDLINE | ID: mdl-15879143

ABSTRACT

Current evidence indicates that the chronic inflammation observed in the intestines of patients with inflammatory bowel disease is due to an aberrant immune response to enteric flora. We have developed a lipid A-mimetic, CRX-526, which has antagonistic activity for TLR4 and can block the interaction of LPS with the immune system. CRX-526 can prevent the expression of proinflammatory genes stimulated by LPS in vitro. This antagonist activity of CRX-526 is directly related to its structure, particularly secondary fatty acyl chain length. In vivo, CRX-526 treatment blocks the ability of LPS to induce TNF-alpha release. Importantly, treatment with CRX-526 inhibits the development of moderate-to-severe disease in two mouse models of colonic inflammation: the dextran sodium sulfate model and multidrug resistance gene 1a-deficient mice. By blocking the interaction between enteric bacteria and the innate immune system, CRX-526 may be an effective therapeutic molecule for inflammatory bowel disease.


Subject(s)
Adjuvants, Immunologic/pharmacology , Anti-Inflammatory Agents, Non-Steroidal/pharmacology , Glucosamine/analogs & derivatives , Glucosamine/pharmacology , Inflammatory Bowel Diseases/pathology , Inflammatory Bowel Diseases/prevention & control , Lipid A/analogs & derivatives , Lipid A/pharmacology , Receptors, Immunologic/antagonists & inhibitors , ATP Binding Cassette Transporter, Subfamily B, Member 1/deficiency , ATP Binding Cassette Transporter, Subfamily B, Member 1/genetics , Adjuvants, Immunologic/chemical synthesis , Animals , Caproates/chemistry , Cells, Cultured , Colitis/chemically induced , Colitis/genetics , Colitis/immunology , Colitis/prevention & control , Dextran Sulfate/toxicity , Disease Models, Animal , Female , Glucosamine/chemistry , HeLa Cells , Humans , Inflammatory Bowel Diseases/genetics , Inflammatory Bowel Diseases/immunology , Lipopolysaccharides/administration & dosage , Lipopolysaccharides/antagonists & inhibitors , Lipopolysaccharides/pharmacology , Mice , Mice, Inbred BALB C , Mice, Inbred C3H , Mice, Knockout , Monocytes/drug effects , Monocytes/immunology , Monocytes/metabolism , Receptors, Immunologic/metabolism , Toll-Like Receptor 4 , Tumor Necrosis Factor-alpha/antagonists & inhibitors , Tumor Necrosis Factor-alpha/metabolism
12.
J Biol Chem ; 279(6): 4440-9, 2004 Feb 06.
Article in English | MEDLINE | ID: mdl-14570885

ABSTRACT

Important questions remain regarding the impact of variations in the structure of the lipid A portion of lipopolysaccharide on activation of cells via the Toll-like receptor 4 complex. We have studied a series of synthetic lipid A mimetic compounds known as aminoalkyl glucosaminide phosphates in which the length of the secondary acyl chain has been systematically varied. Using transcriptional profiling of human monocytes and responses of Toll-like receptor 4 complex cell transfectants, we demonstrate a clear dependence of length on secondary acyl chain on Toll-like receptor 4 activation. Compounds with secondary acyl chains less than eight carbons in length have dramatically reduced activity, and substitutions of the left-sided secondary acyl chain had the most important effect on the Toll-like receptor 4 agonist activity of these molecules. The structure-function relationships of these compounds assessed via the induction of chemokines and cytokines following in vivo administration closely mirrored those seen with cell-based studies. This novel set of synthetic lipid A mimetics will be useful for Toll-like receptor 4-based investigations and may have clinical utility as stand-alone immunomodulators.


Subject(s)
Lipid A/analogs & derivatives , Membrane Glycoproteins/agonists , Receptors, Cell Surface/agonists , Adjuvants, Immunologic/chemistry , Adjuvants, Immunologic/pharmacology , Animals , Cytokines/biosynthesis , Cytokines/genetics , Gene Expression Profiling , Humans , In Vitro Techniques , Lipid A/chemistry , Lipid A/pharmacology , Macrophages/drug effects , Macrophages/immunology , Macrophages/metabolism , Mice , Mice, Inbred BALB C , Molecular Mimicry , Monocytes/drug effects , Monocytes/immunology , Monocytes/metabolism , Structure-Activity Relationship , Toll-Like Receptor 4 , Toll-Like Receptors , Transcriptional Activation/drug effects , Tumor Necrosis Factor-alpha/biosynthesis , Tumor Necrosis Factor-alpha/genetics
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