Biology subjects

Heil, C. S.

Publications and source records attributed to Heil, C. S..

5 recordsLinked to original sources

bioRxiv2025

Elimination of myotonia improves myopathy in a muscleblind knockout model of myotonic dystrophy

A cardinal sign of myotonic dystrophy type 1 (DM1) is slow of muscle relaxation after voluntary contraction known as myotonia. Myotonia results from mis-regulated splicing of chloride channel 1 (ClC-1), leading to loss of channel function and runs of involuntary action potentials in muscle fibers. Heralding the onset of weakness, myotonia is often the first symptom of DM1, and raising the possibility that muscle hyperexcitability promotes the subsequent development of myopathy. We used genome editing to test this possibility by deleting the alternatively spliced and frameshift inducing ClC-1 exon 7a (E7a) in the Mbnl1 knockout model of DM1. Although several ClC-1 exons exhibit mis-regulated splicing in DM1, deletion of this single cryptic exon was sufficient to restore ClC-1 function and eliminate myotonia systemically and permanently. As determined by long-read sequencing, deletion of E7a reduced the frequency of other splicing defects in ClC-1 transcripts, likely as a passive consequence of restoring reading frame and nonsense surveillance. Furthermore, we observed significantly improved muscle force generation, fiber-type distribution, and histology, and partial restoration of the muscle transcriptome, including differential gene expression and alternative splicing, in non-myotonic Mbnl1 knockout mice. These results suggest that E7a inclusion is a lynchpin splice event that contributes to skeletal myopathy, highlighting myotonia as a therapeutic target and an outcome of interest in DM1. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=171 SRC="FIGDIR/small/677400v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@101b333org.highwire.dtl.DTLVardef@9be92corg.highwire.dtl.DTLVardef@1e95c88org.highwire.dtl.DTLVardef@1b9eeee_HPS_FORMAT_FIGEXP M_FIG C_FIG

physiology↗

bioRxiv2024

Mixed outcomes in recombination rates after domestication: Revisiting theory and data

The process of domestication has altered many phenotypes. Selection on these phenotypes has long been hypothesized to indirectly select for increases in recombination rate. This hypothesis is consistent with theory on the evolution of recombination rate, but empirical support has been unclear. We review relevant theory, lab-based experiments, and data comparing recombination rates in wild progenitors and their domesticated counterparts. We utilize population sequencing data and a deep learning method to infer genome-wide recombination rates for new comparisons of chicken/red junglefowl, sheep/mouflon, and goat/bezoar. We find evidence of increased recombination in domestic goats compared to bezoars, but more mixed results in chicken, and generally decreased recombination in domesticated sheep compared to mouflon. Our results add to a growing body of literature in plants and animals that finds no consistent evidence of an increase in recombination with domestication.

evolutionary biology↗

bioRxiv2020

Cell-free synthesis of natural compounds from genomic DNA of biosynthetic gene clusters

A variety of chemicals can be produced in a living host cell via optimized and engineered biosynthetic pathways. Despite the successes, pathway engineering remains demanding and partly impossible owing to the lack of specific functions or substrates in the host cell, its sensitivity in vital physiological processes to the heterologous components, or constrained mass transfer across the membrane. In this study, we demonstrate that cell-free systems can be useful in driving the characterization and engineering of biosynthetic pathways. We show that complex multidomain proteins involved in natural compound biosynthesis can be produced from encoding DNA in vitro in a minimal complex PURE system to directly run multistep reactions. We prove the concept of this approach on the direct synthesis of indigoidine and rhabdopeptides with the in vitro produced multidomain megasynthases BpsA and KJ12ABC. The in vitro produced proteins are analyzed in detail, i.e., in yield, quality, post-translational modification and specific activity, and compared to recombinantly produced proteins. Our study highlights cell-free PURE systems as suitable setting for the rapid engineering of biosynthetic pathways.

bioengineering↗

bioRxiv2018

Site-Specific Labelling of Multidomain Proteins by Amber Codon Suppression

Amber codon suppression is a powerful tool to site-specifically modify proteins to generate novel biophysical probes. Yet, its application on large and complex multidomain proteins is challenging, leading to difficulties during structural and conformational characterization using spectroscopic methods. The animal fatty acid synthase type I is a 540 kDa homodimer displaying large conformational variability. As the key enzyme of de novo fatty acid synthesis, it attracts interest in the fields of obesity, diabetes and cancer treatment. Substrates and intermediates remain covalently bound to the enzyme during biosynthesis and are shuttled to all catalytic domains by the acyl carrier protein domain. Thus, conformational variability of animal FAS is an essential aspect for fatty acid biosynthesis. We investigate this multidomain protein as a model system for probing amber codon suppression by genetic encoding of non-canonical amino acids. The systematic approach relies on a microplate-based reporter assay of low complexity, that was used for quick screening of suppression conditions. Furthermore, the applicability of the reporter assay is demonstrated by successful upscaling to both full-length constructs and increased expression scale. The obtained fluorescent probes of murine FAS type I could be subjected readily to a conformational analysis using single-molecule fluorescence resonance energy transfer.

biochemistry↗

bioRxiv2016

Experimental evolution reveals favored adaptive routes to cell aggregation in yeast

Yeast flocculation is a community-building cell aggregation trait that is an important mechanism of stress resistance and a useful phenotype for brewers; however, it is also a nuisance in many industrial processes, in clinical settings, and in the laboratory. Chemostat-based evolution experiments are impaired by inadvertent selection for aggregation, which we observe in 35% of populations. These populations provide a testing ground for understanding the breadth of genetic mechanisms Saccharomyces cerevisiae uses to flocculate, and which of those mechanisms provide the biggest adaptive advantages. In this study, we employed experimental evolution as a tool to ask whether one or many routes to flocculation are favored, and to engineer a strain with reduced flocculation potential. Using a combination of whole genome sequencing and bulk segregant analysis, we identified causal mutations in 23 independent clones that had evolved cell aggregation during hundreds of generations of chemostat growth. In 12 of those clones we identified a transposable element insertion in the promoter region of known flocculation gene FLO1, and in an additional five clones we recovered loss-of-function mutations in transcriptional repressor TUP1, which regulates FLO1 and other related genes. Other causal mutations were found in genes that have not been previously connected to flocculation. Evolving a flo1 deletion strain revealed that this single deletion reduces flocculation occurrences to 3%, and demonstrated the efficacy of using experimental evolution as a tool to identify and eliminate the primary adaptive routes for undesirable traits.

genetics↗