Biology subjects

Kruglyak, L.

Publications and source records attributed to Kruglyak, L..

At least 19 recordsLinked to original sources

bioRxiv2026

Microscale bulk segregant analysis

The traits of an organism depend on its genetic background and environment. Bulk segregant analysis (BSA) maps quantitative trait loci (QTL) by measuring parental allele frequencies in pools of genetically recombined progeny (segregants). Conventional BSA requires laborious separate sample handling for each cross and environmental condition. Here, we describe a 96-well liquid format for extreme QTL mapping (X-QTL) in Saccharomyces cerevisiae. We crossed 40 isolates to a common parent carrying mating-type-specific drug-resistance markers at HO, which enabled us to select diploids and haploid segregants in 1 mL cultures. Each well held one segregant pool, which we then grew in deep wells in four conditions: the standard glucose medium, sodium chloride or caffeine added to the standard medium, or acetate replacing glucose as a carbon source. We barcoded DNA samples from each of 384 wells and sequenced them in one Illumina Nova-seq X run. Sequencing data for 181 segregant pools passed quality control steps and was used to map 1,472 QTL in 35 crosses. We replicated a subset of QTL previously mapped in these growth conditions by conventional X-QTL. The 96-well approach increases the throughput of every step of X-QTL, enabling genetic analysis of many more cross and conditions.

genetics↗

bioRxiv2026

Large-scale pooled selection enables mapping of modifiers of RNAi gene knockdown effects in C. elegans

Genetic interactions contribute to phenotypic variation, yet most studies characterize them in a small number of reference backgrounds rather than across natural populations. Studying genetic interactions in Caenorhabditis elegans is facilitated by RNAi by feeding, which enables gene-specific knockdowns across many genetic backgrounds without the need to construct individual mutant lines. However, individually screening hundreds of isolates across many RNAi conditions is laborious. Here, we developed a pooled platform that uses whole-genome sequencing to quantify the fitness of hundreds of genetically distinct C. elegans strains competing in a single flask. We leveraged this platform to evaluate RNAi responses in a pooled population of RNAi-sensitive C. elegans strains and identified two classes of QTL that influence RNAi responses. The first class influences responses to knockdowns of specific genes and is exemplified by a QTL that is specific to knockdown of mig-6. The second class influences general RNAi-response across multiple knockdowns. One QTL in this class localized to the right arm of chromosome III and was detected by both GWA and xQTL mapping, which suggests that a common large-effect variant underlies this QTL. We identified a T96K missense change in the dsRNA receptor SID-2 as the underlying causal variant. The 96K allele resides in the extracellular domain of SID-2 and confers increased RNAi sensitivity relative to the 96T allele. We hypothesize that the 96K allele enables greater uptake of ingested lumenal dsRNA through higher-affinity binding to the dsRNA phosphate backbone. The platform we developed enables systematic mapping of genetic interactions across natural populations at a previously impractical scale.

genetics↗

bioRxiv2026

The genetics of transcriptional responses to stress in yeast

Alteration of gene expression levels is a key cellular strategy for responding to internal and external challenges. While expression quantitative trait loci (eQTLs) are known to influence steady-state transcript levels, their role in mediating rapid physiological transitions remains poorly understood. Using a high-throughput, "one-pot" single-cell RNA-seq approach, we mapped eQTLs in yeast immediately before and after two acute environmental shifts: salt perturbation of actively cycling cells and nutrient repletion of cells after one week of starvation. We defined and characterized major cell populations in different physiological states and identified context-specific eQTLs as well as genetic determinants of state occupancy. We focused on distant eQTL hotspots, which capture the majority of expression heritability in yeast crosses. We found that the effects of hotspots on gene expression are highly dynamic in response to acute environmental perturbations, and that they influence biological processes directly relevant to adaptation to the new conditions. By integrating gene set enrichment analysis with computational prediction of variant effects, we prioritized candidate causal genes and variants underlying the hotspots. We observed extensive overlaps between hotspot loci, physiological state QTL, and fitness QTL. These findings reveal how regulatory variation rapidly modulates cellular responses to environmental changes, suggesting a mechanistic bridge between genetic variation, gene expression, and organismal fitness.

genetics↗

bioRxiv2024

A maternal-effect toxin-antidote element causes larval arrest in C. elegans

Toxin-antidote elements (TAs) are selfish DNA sequences that bias their transmission to the next generation. TAs typically consist of two linked genes: a toxin and an antidote. The toxin kills progeny that do not inherit the TA, while the antidote counteracts the toxin in progeny that inherit the TA. We previously discovered two TAs in C. elegans that follow the canonical TA model of two linked genes: peel-1/zeel-1 and sup-35/pha-1. Here, we report a new TA that exists in three distinct states across the C. elegans population. The canonical TA, which is found in isolates from the Hawaiian islands, consists of two genes that encode a maternally deposited toxin (TMRL-1) and a zygotically expressed antidote (AMRL-1). The toxin induces larval lethality in embryos that do not inherit the antidote gene. A second version of the TA has lost the toxin gene but retains a partially functional antidote. Most C. elegans isolates, including the standard laboratory strain N2, carry a highly divergent allele of the toxin that has retained its activity, but have lost the antidote through pseudogenization. Multiple lines of evidence suggest that the N2 tmrl-1 allele is recognized by piRNAs, leading to MUT-16-dependent 22G siRNA production and post-transcriptional silencing of the transcript. The N2 haplotype represents the first naturally occurring unlinked toxin-antidote system where the toxin is post-transcriptionally suppressed by endogenous small RNA pathways.

evolutionary biology↗

bioRxiv2023

Single-cell eQTL mapping in yeast reveals a tradeoff between growth and reproduction

Expression quantitative trait loci (eQTLs) provide a key bridge between noncoding DNA sequence variants and organismal traits. The effects of eQTLs can differ among tissues, cell types, and cellular states, but these differences are obscured by gene expression measurements in bulk populations. We developed a one-pot approach to map eQTLs in Saccharomyces cerevisiae by single-cell RNA sequencing (scRNA-seq) and applied it to over 100,000 single cells from three crosses. We used scRNA-seq data to genotype each cell, measure gene expression, and classify the cells by cell-cycle stage. We mapped thousands of local and distant eQTLs and identified interactions between eQTL effects and cell-cycle stages. We took advantage of single-cell expression information to identify hundreds of genes with allele-specific effects on expression noise. We used cell-cycle stage classification to map 20 loci that influence cell-cycle progression. One of these loci influenced the expression of genes involved in the mating response. We showed that the effects of this locus arise from a common variant (W82R) in the gene GPA1, which encodes a signaling protein that negatively regulates the mating pathway. The 82R allele increases mating efficiency at the cost of slower cell-cycle progression and is associated with a higher rate of outcrossing in nature. Our results provide a more granular picture of the effects of genetic variants on gene expression and downstream traits.

genetics↗

bioRxiv2023

Regeneration leads to global tissue rejuvenation in aging sexual planarians

An ability to delay aging--or to reverse the negative effects of aging--could prevent age-related disease and greatly enhance quality of life in old age. However, whether it is possible to globally reverse the physiological effects of aging in order to extend healthspan is unknown. The freshwater planarian Schmidtea mediterranea has been considered "immortal" due to its exceptional tissue regeneration capabilities. Here, we report that a sexually reproducing lineage of S. mediterranea exhibits age-associated physiological decline 12 months after birth. Age-associated changes include alterations in sensory organs, loss of neurons and muscle, loss of fertility, and impaired motility, but no reduction in stem cells at the age of 3 years. Differential gene expression analysis, comparing young and old planarian cells, furthermore revealed cell-type-specific changes in transcription as well as changes in classical aging pathways (e.g., insulin signaling). Remarkably, amputation followed by regeneration of lost tissues led to a global reversal of these age-associated changes. Older individuals that underwent regeneration showed restored youthful patterns of gene expression, stem cell states, tissue composition and rejuvenation of whole-animal physiology. Our work reveals a naturally evolved solution to age reversal in planaria that may provide insights into anti-aging strategies in humans.

developmental biology↗

bioRxiv2023

Cas9-induced nonhomologous recombination in C. elegans

Identification of the genetic basis of phenotypic variation within species remains challenging. In species with low recombination rates, such as Caenorhabditis elegans, genomic regions linked to a phenotype of interest by genetic mapping studies are often large, making it difficult to identify the specific genes and DNA sequence variants that underlie phenotypic differences. Here, we introduce a method that enables researchers to induce targeted recombination in C. elegans with Cas9. We demonstrate that high rates of targeted recombination can be induced by Cas9 in a genomic region in which naturally occurring recombination events are exceedingly rare. We anticipate that Cas9-induced nonhomologous recombination (CINR) will greatly facilitate high-resolution genetic mapping in this species.

genetics↗

bioRxiv2022

A minimal role for synonymous variation in human disease

Synonymous mutations change the DNA sequence of a gene without affecting the amino acid sequence of the encoded protein. Although emerging evidence suggests that synonymous mutations can impact RNA splicing, translational efficiency, and mRNA stability1, studies in human genetics, mutagenesis screens, and other experiments and evolutionary analyses have repeatedly shown that most synonymous variants are neutral or only weakly deleterious, with some notable exceptions. In their recent article, Shen et al. claim to have disproved these well-established findings. They perform mutagenesis experiments in yeast and conclude that synonymous mutations frequently reduce fitness to the same extent as nonsynonymous mutations2. Based on their findings, the authors state that their results "imply that synonymous mutations are nearly as important as nonsynonymous mutations in causing disease." An accompanying News and Views argues that "revising our expectations about synonymous mutations should expand our view of the genetic underpinnings of human health"3. Considering potential technical concerns with these experiments4 and a large, coherent body of knowledge establishing the predominant neutrality of synonymous variants, we caution against interpreting this study in the context of human disease.

genetics↗

bioRxiv2022

No evidence that synonymous mutations in yeast genes are mostly deleterious

In a recent paper1, Shen et al. reported that most mutations in the coding regions of 21 yeast genes were strongly deleterious, and that the distributions of fitness effects were similar for synonymous and nonsynonymous mutations. Taken at face value, these results would conflict with well-established findings from a broad range of fields and approaches. Here, we argue that these results arose from a lack of appropriate controls for the impacts of background genetic effects in edited strains. A re-examination of the data in Shen et al. strongly suggests that it is entirely consistent with the expectation that most nonsynonymous and nearly all synonymous mutations have no detectable effects on fitness. We present analyses which show that the data is inconsistent with the proposed explanation that pervasive fitness effects of synonymous mutations arise from their effects on mRNA levels, that the sequence-based fitness assay overestimates fitness effects compared to the growth-based fitness assay, and that the observed wide fitness distributions for nonsense mutations are consistent with off-target effects or other uncontrolled sources of biological variation contributing to measured fitness. We conclude by discussing the essential controls and other experimental design considerations that are required to produce interpretable results regarding the fitness effects of mutations in large-scale screens.

genetics↗

bioRxiv2022

Towards Synthetic PETtrophy: Engineering Pseudomonas putida for concurrent polyethylene terephthalate (PET) monomer metabolism and PET hydrolase expression

BackgroundBiocatalysis offers a promising path for plastic waste management and valorization, especially for hydrolysable plastics such as polyethylene terephthalate (PET). Microbial whole-cell biocatalysts for simultaneous PET degradation and growth on PET monomers would offer a one-step solution toward PET recycling or upcycling. We set out to engineer the industry-proven bacterium Pseudomonas putida for (i) metabolism of PET monomers as sole carbon sources, and (ii) efficient concurrent extracellular expression of PET hydrolases. We pursued this approach for both PET and the related polyester polybutylene adipate co-terephthalate (PBAT), aiming to learn about the determinants and potential applications of bacterial polyester-degrading biocatalysts. ResultsP. putida was engineered to metabolize the PET and PBAT monomer terephthalic acid (TA) through genomic integration of four tphII operon genes from Comamonas sp. E6. Efficient cellular TA uptake was enabled by a point mutation in the native P. putida membrane transporter mhpT. Metabolism of the PET and PBAT monomers ethylene glycol and 1,4-butanediol was achieved through adaptive laboratory evolution. We then used fast design-build-test-learn cycles to engineer extracellular PET hydrolase expression, including tests of (i) the three PET hydrolases LCC, HiC, and IsPETase; (ii) genomic versus plasmid-based expression, using expression plasmids with high, medium, and low cellular copy number; (iii) three different promoter systems; (iv) three membrane anchor proteins for PET hydrolase cell surface display; and (v) a 30-mer signal peptide library for PET hydrolase secretion. PET hydrolase surface display and secretion was successfully engineered but often resulted in host cell fitness costs, which could be mitigated by promoter choice and altering construct copy number. Plastic biodegradation assays with the best PET hydrolase expression constructs genomically integrated into our monomer-metabolizing P. putida strains resulted in various degrees of plastic depolymerization, although self-sustaining bacterial growth remained elusive. ConclusionOur results show that balancing extracellular PET hydrolase expression with cellular fitness under nutrient-limiting conditions is a challenge. The precise knowledge of such bottlenecks, together with the vast array of PET hydrolase expression tools generated and tested here, may serve as a baseline for future efforts to engineer P. putida or other bacterial hosts towards becoming efficient whole-cell polyester-degrading biocatalysts.

microbiology↗

bioRxiv2022

Genome-wide base editor screen identifies regulators of protein abundance in yeast

Abundance of proteins is extensively regulated both transcriptionally and post-transcriptionally. To systematically characterize how regulation of protein abundance is encoded in the genome and identify protein regulators on a genome-wide scale, we developed a genetic screen that uses a CRISPR base editor. We examined the effects of 16,452 genetic perturbations on the abundance of eleven yeast proteins representing a variety of cellular functions. Thereby, we uncovered hundreds of regulatory relationships, including a novel link between the GAPDH isoenzymes Tdh1/2/3 and the Ras/PKA pathway. Many of the identified regulators are specific to one of the eleven proteins, but we also found genes that, upon perturbation, affected the abundance of most of the tested proteins. While the more specific regulators usually act transcriptionally, broad regulators often have roles in protein translation. Our results provide unprecedented insights into the components, scale and connectedness of the protein regulatory network.

genetics↗

bioRxiv2021

Island-specific evolution of a sex-primed autosome in the planarian Schmidtea mediterranea

The sexual biotype of the planarian Schmidtea mediterranea is a hermaphrodite indigenous to Tunisia and several Mediterranean islands. Here, we isolated individual chromosomes and used sequencing, Hi-C and linkage mapping to assemble a chromosome-scale genome reference. The linkage map revealed an extremely low rate of recombination on chromosome 1. We confirmed suppression of recombination on chromosome 1 by quantifying recombination events in individual sperm and oocytes. We showed that the extensive heterozygous regions, previously designated as J and V haplotypes, comprise essentially all of chromosome 1. Genome sequencing of individuals isolated in the wild indicated that this heterozygosity has evolved specifically in populations from Sardinia and Corsica. We found that chromosome 1 acquired many genes that determine the development of female and male reproductive systems, along with haplotype-specific expression of such sex specific genes. These molecular signatures and restricted recombination in a hermaphrodite led us to propose that chromosome 1 is a sex-primed autosome, providing direct molecular evidence for the traditional model of how sex chromosomes may have evolved from autosomes.

genetics↗

bioRxiv2020

Genetics of white color and iridophoroma in "Lemon Frost" leopard geckos

The squamates (lizards and snakes) are close relatives of birds and mammals, with more than 10,000 described species that display extensive variation in a number of important biological traits, including coloration, venom production, and regeneration. Due to a lack of genomic tools, few genetic studies in squamates have been carried out. The leopard gecko1, Eublepharis macularius, is a popular companion animal, and displays a variety of coloration patterns. We took advantage of a large breeding colony and used linkage analysis, synteny, and homozygosity mapping to investigate a spontaneous semi-dominant mutation, "Lemon Frost", that produces white coloration and causes skin tumors (iridophoroma). We localized the mutation to a single locus which contains a strong candidate gene, SPINT12,3, a tumor suppressor implicated in human skin cutaneous melanoma (SKCM) and over-proliferation of epithelial cells in mice and zebrafish4-16. Our work establishes the leopard gecko as a tractable genetic system and suggests that a tumor suppressor in melanocytes in humans can also suppress tumor development in iridophores in lizards.

genetics↗

bioRxiv2020

Massively parallel identification of causal variants underlying gene expression differences in a yeast cross

Sequence variation in regulatory DNA alters gene expression and shapes genetically complex traits. However, the identification of individual, causal regulatory variants is challenging. Here, we used a massively parallel reporter assay to measure the cis-regulatory consequences of 5,832 natural DNA variants in the promoters of 2,503 genes in the yeast Saccharomyces cerevisiae. We identified 451 causal variants, which underlie genetic loci known to affect gene expression. Several promoters harbored multiple causal variants. In five promoters, pairs of variants showed non-additive, epistatic interactions. Causal variants were enriched at conserved nucleotides, tended to have low derived allele frequency, and were depleted from promoters of essential genes, which is consistent with the action of negative selection. Causal variants were also enriched for alterations in transcription factor binding sites. Models integrating these features provided modest, but statistically significant, ability to predict causal variants. This work revealed a complex molecular basis for cis-acting regulatory variation.

genetics↗

bioRxiv2020

Whole-organism mapping of the genetics of gene expression at cellular resolution

Genetic regulation of gene expression underlies variation in disease risk and other complex traits. The effect of expression quantitative trait loci (eQTLs) varies across cell types; however, the complexity of mammalian tissues makes studying cell-type eQTLs highly challenging. We developed a novel approach in the model nematode Caenorhabditis elegans that uses single cell RNA sequencing to map eQTLs at cellular resolution in a single one-pot experiment. We mapped eQTLs across cell types in an extremely large population of genetically distinct C. elegnas individuals. We found cell-type-specific trans-eQTL hotspots that affect the expression of core pathways in the relevant cell types. Finally, we found single-cell-specific eQTL effects in the nervous system, including an eQTL with opposite effects in two individual neurons. Our results show that eQTL effects can be specific down to the level of single cells.

genetics↗

bioRxiv2020

Rapid cost-effective viral genome sequencing by V-seq

Conventional methods for viral genome sequencing largely use metatranscriptomic approaches or, alternatively, enrich for viral genomes by amplicon sequencing with virus-specific PCR or hybridization-based capture. These existing methods are costly, require extensive sample handling time, and have limited throughput. Here, we describe V-seq, an inexpensive, fast, and scalable method that performs targeted viral genome sequencing by multiplexing virus-specific primers at the cDNA synthesis step. We designed densely tiled reverse transcription (RT) primers across the SARS-CoV-2 genome, with a subset of hexamers at the 3 end to minimize mis-priming from the abundant human rRNA repeats and human RNA PolII transcriptome. We found that overlapping RT primers do not interfere, but rather act in concert to improve viral genome coverage in samples with low viral load. We provide a path to optimize V-seq with SARS-CoV-2 as an example. We anticipate that V-seq can be applied to investigate genome evolution and track outbreaks of RNA viruses in a cost-effective manner. More broadly, the multiplexed RT approach by V-seq can be generalized to other applications of targeted RNA sequencing.

genomics↗

bioRxiv2020

Ubiquitous selfish toxin-antidote elements in Caenorhabditis species

Toxin-antidote elements (TAs) are selfish genetic dyads that spread in populations by selectively killing non-carriers. TAs are common in prokaryotes, but few examples are known in animals. We discovered five maternal-effect TAs in the nematode Caenorhabditis tropicalis and one in C. briggsae. Unlike previously reported TAs, five of these novel toxins do not kill embryos but instead cause larval arrest or developmental delay. We identified the genes underlying a TA causing developmental delay, slow-1/grow-1, and found that the toxin, slow-1, is homologous to nuclear hormone receptors. Last, we found that balancing selection of conflicting TAs hampers their ability to drive in populations, leading to more stable genetic incompatibilities. Our results show that TAs are common in Caenorhabditis species, target a wide range of developmental processes, and may act as barriers preventing gene flow.

genomics↗

bioRxiv2019

Ancient balancing selection maintains incompatible versions of a conserved metabolic pathway in yeast

Differences in nutrient availability have led to the evolution of diverse metabolic strategies across species, but within species these strategies are expected to be similar. Here, we discovered that the galactose metabolic pathway in the yeast Saccharomyces cerevisiae exists in two functionally distinct, incompatible states maintained by ancient balancing selection. We identified a genetic interaction for growth in galactose among the metabolic genes GAL2, GAL1/10/7, and PGM1. We engineered strains with all allelic combinations at these loci and showed that the reference allele of PGM1 is incompatible with the alternative alleles of the other genes. We observed a strong signature of ancient balancing selection at all three loci and found that the alternative alleles diverged from the reference alleles before the birth of the Saccharomyces sensu stricto species cluster 10-20 million years ago. Strains with the alternative alleles are found primarily in galactose-rich dairy environments, and they grow faster in galactose, but slower in glucose, revealing a tradeoff on which balancing selection may have acted.

evolutionary biology↗