Biology subjects

Hill, A. M.

Publications and source records attributed to Hill, A. M..

9 recordsLinked to original sources

bioRxiv2025

Programmed DNA elimination drives rapid genomic innovation in two thirds of all bird species

Bird genomes are among the most stable in terms of synteny and gene content across vertebrates. However, germline-restricted chromosomes (GRCs) represent a striking exception where programmed DNA elimination confines large-scale genomic changes to the germline. GRCs are known to occur in songbirds (oscines), but have been studied only in a few species of Passerides such as the zebra finch, the key model for passerine genomics. Their presence and evolutionary dynamics in most major passerine lineages remain largely unexplored, with suboscines entirely unexamined by cytogenetic or genomic methods. Here, we present the most comprehensive comparative analysis of GRCs to date, spanning 44 million years of passerine evolution. By generating the first germline reference genomes of an oscine and a suboscine, 22 novel germline draft genomes spanning nearly all major passerine lineages and a germline draft genome of a parrot outgroup, we show that the GRC is likely present in 6,700 passerine species. Our results reveal that the GRC evolves rapidly and distinctly from the standard A chromosomes (autosomes and sex chromosomes), yet retains functionally important, selectively maintained genes. We observed gene and repeat turnover occuring orders of magnitude faster than on the A chromosomes. Some GRC genes, such as cpeb1 and pim1, are widespread from an ancient duplication. In contrast, other GRC genes, like mfsd2b and bmp15, have been independently duplicated onto the GRC multiple times, suggesting adaptive constraints. The discovery of zglp1 on the zebra finch GRC, initially copied from chromosome 30 and subsequently lost from it, indicates functional replacement, where the GRC permits gene loss from the standard genome. As the GRC harbors the only zglp1 copy in most of the [~]4000 Passerides species, GRC loss would compromise essential germline functions. Our findings establish the GRC as a genomic innovator driving rapid germline evolution. This fact highlights its evolutionary significance for passerine diversification and suggests that programmed DNA elimination may be an overlooked yet phylogenetically widespread mechanism in many understudied animal lineages.

genomics↗

bioRxiv2025

Availability of charged tRNAs drives maximal protein synthesis at intermediate levels of codon usage bias

Synonymous codon usage can influence protein expression, since codons with high numbers of corresponding tRNAs are naturally translated more rapidly than codons with fewer corresponding tRNAs. Although translation efficiency ultimately depends on the concentration of aminoacylated (charged) tRNAs, many theoretical models of translation have ignored tRNA dynamics and treated charged tRNAs as fixed resources. This simplification potentially limits these models from making accurate predictions in situations where charged tRNAs become limiting. Here, we derive a mathematical model of translation with explicit tRNA dynamics and tRNA re-charging, based on a stochastic simulation of this system that was previously applied to investigate codon usage in the context of gene overexpression. We use the mathematical model to systematically explore the relationship between codon usage and the protein expression rate, and find that in the regime where tRNA charging is a limiting reaction, it is always optimal to match codon frequencies to the tRNA pool. Conversely, when tRNA charging is not limiting, using 100% of the preferred codon is optimal for protein production. We also use the tRNA dynamics model to augment a wholecell simulation of bacteriophage T7. Using this model, we demonstrate that the high expression rate of the T7 major capsid gene causes rare charged tRNAs to become entirely depleted, which explains the sensitivity of the major capsid gene to codon deoptimization.

biochemistry↗

bioRxiv2024

Modeling and measuring how codon usage modulates the relationship between burden and yield during protein overexpression in bacteria

Excess utilization of translational resources is a critical source of burden on cells engineered to over-express exogenous proteins. To improve protein yields and genetic stability, researchers often use codon optimization strategies that improve translational efficiency by matching an exogenous genes codon usage with that of the host organisms highly expressed genes. Despite empirical data that shows the benefits of codon optimization, little is known quantitatively about the relationship between codon usage bias and the burden imposed by protein overexpression. Here, we develop and experimentally evaluate a stochastic gene expression model that considers the impact of codon usage bias on the availability of ribosomes and different tRNAs in a cell. In agreement with other studies, our model shows that increasing exogenous protein expression decreases production of native cellular proteins in a linear fashion. We also find that the slope of this relationship is modulated by how well the codon usage bias of the exogenous gene and the hosts genes match. Strikingly, we predict that an overoptimization domain exists where further increasing usage of optimal codons worsens yield and burden. We test our model by expressing sfGFP and mCherry2 from constructs that have a wide range of codon optimization levels in Escherichia coli. The results agree with our model, including for an mCherry2 gene sequence that appears to lose expression and genetic stability from codon overoptimization. Our findings can be leveraged by researchers to predict and design more optimal cellular systems through the use of more nuanced codon optimization strategies.

synthetic biology↗

bioRxiv2024

Low-pass nanopore sequencing for measurement of global methylation levels in plants

Nanopore sequencing enables detection of DNA methylation at the same time as identification of canonical sequence. A recent study validated low pass nanopore sequencing to accurately estimate global methylation levels in vertebrates with sequencing coverage as low as 0.01x. We investigated the applicability of this approach to plants by testing three plant species and analysed the effect of technical and biological parameters on estimate precision and accuracy. Our results indicate that a higher coverage (0.1x) is required to assess plant global methylation at an equivalent accuracy to vertebrates. Shorter read length and a closer sequence match between sample and reference genome improved measurement accuracy. Application of this method in Vitis vinifera showed consistent global methylation levels across different leaf sizes, and different sample preservation and DNA extraction methods, whereas different varieties and tissue types did exhibit methylation differences. Similarly, distinct methylation patterns could be observed in different genomic features. Our findings suggest the suitability of this method as a low-cost screening tool for validation of experimental parameters, developmental time courses and to assess methylation status for different modification types and sequence contexts at the level of whole genome or for abundant genomic features such as transposable elements.

genomics↗

bioRxiv2024

A computational model for bacteriophage {varphi}X174 gene expression

Bacteriophage{phi} X174 has been widely used as a model organism to study fundamental processes in molecular biology. However, several aspects of{phi} X174 gene regulation are not fully resolved. Here we construct a computational model for{phi} X174 and use the model to study gene regulation during the phage infection cycle. We estimate the relative strengths of transcription regulatory elements (promoters and terminators) by fitting the model to transcriptomics data. We show that the specific arrangement of a promoter followed immediately by a terminator, which occurs naturally in the{phi} X174 genome, poses a parameter identifiability problem for the model, since the activity of one element can be partially compensated for by the other. We also simulate{phi} X174 gene expression with two additional, putative transcription regulatory elements that have been proposed in prior studies. We find that the activities of these putative elements are estimated to be weak, and that variation in{phi} X174 transcript abundances can be adequately explained without them. Overall, our work demonstrates that{phi} X174 gene regulation is well described by the canonical set of promoters and terminators widely used in the literature.

microbiology↗

bioRxiv2022

Transcriptional profiling of Hutchinson-Gilford Progeria syndrome fibroblasts reveals deficits in mesenchymal stem cell commitment to differentiation related to early events in endochondral ossification.

Hutchinson-Gilford Progeria Syndrome results from a mutation in Lamin A, and it is characterized by the incorporation of progerin into the nuclear lamina. Progerin expression leads to alterations in genome architecture, nuclear morphology, and epigenetic states, which in turn cause altered phenotypes in all cells of the mesenchymal lineage. Here, we report a comprehensive analysis of the transcriptional status of patient derived HGPS fibroblasts, including nine cell lines not previously reported. We observe that these fibroblasts carry abnormal transcriptional signatures, centering around five main functional hubs: DNA maintenance and epigenetics, bone development and homeostasis, blood vessel maturation and development, fat deposition and lipid management, and processes related to muscle growth. Stratification of patients by age revealed that a cohort of genes related to endochondral ossification and chondrogenic commitment show altered expression patterns in children aged four to seven years old, where this differentiation program starts in earnest, related to the growth of long bones. We further report changes in lamin associated domains and 3D genome organization around a cohort of genes of interest, identified in this study.

genomics↗

bioRxiv2021

Metagenomic Discovery of CRISPR-Associated Transposons

CRISPR-associated transposons (CASTs) co-opt Cas genes for RNA-guided transposition. CASTs are exceedingly rare in genomic databases; recent surveys have reported Tn7-like transposons that co-opt Type I-F, I-B, and V-K CRISPR effectors. Here, we expand the diversity of reported CAST systems via a bioinformatic search of metagenomic databases. We discover new architectures for all known CASTs, including novel arrangements of the Cascade effectors, new self-targeting modalities, and minimal V-K systems. We also describe new families of CASTs that have co-opted the Type I-C and Type IV CRISPR-Cas systems. Our search for non-Tn7 CASTs identifies putative candidates that co-opt Cas12a for horizontal gene transfer. These new systems shed light on how CRISPR systems have co-evolved with transposases and expand the programmable gene editing toolkit.

bioinformatics↗

bioRxiv2020

Generating complex patterns of gene expression without regulatory circuits

Synthetic biology has successfully advanced our ability to design and implement complex, time-varying genetic circuits to control the expression of recombinant proteins. However, these circuits typically require the production of regulatory genes whose only purpose is to coordinate expression of other genes. When designing very small genetic constructs, such as viral genomes, we may want to avoid introducing such auxiliary gene products while nevertheless encoding complex expression dynamics. To this end, here we demonstrate that varying only the placement and strengths of promoters, terminators, and RNase cleavage sites in a computational model of a bacteriophage genome is sufficient to achieve solutions to a variety of basic gene expression patterns. We discover these genetic solutions by computationally evolving genomes to reproduce desired gene expression time-course data. Our approach shows that non-trivial patterns can be evolved, including complex patterns where the relative ordering of genes by abundance changes over time. We find that some patterns are easier to evolve than others, and comparable expression patterns can be achieved via different genetic architectures. Our work opens up a novel avenue to genome engineering via fine-tuning the balance of gene expression and gene degradation rates. Author summaryViruses that infect bacteria, commonly called bacteriophages, typically have small genomes that encode as few as 10 genes. From the perspective of understanding genome design and regulation, these organisms are important model systems. Similar to cellular species, the genes encoded on a phage genome often must be expressed at different levels and at particular times during the phage lifecycle. Given their unique size constraints, it may be advantageous for phages to accomplish differential gene expression without having to produce a variety of dedicated regulatory molecules--which are frequently encoded on the larger and more complex genomes of free-living species. Here, we use a computational simulation of phage infection coupled with an evolutionary selection algorithm to illustrate that phage genomes can encode complex time-dependent gene expression patterns without the need for dedicated regulatory molecules. We anticipate that this simulation framework may additionally aid future phage genome design and engineering efforts.

synthetic biology↗

bioRxiv2020

Adolescent intermittent ethanol exposure induces sex-dependent divergent changes in ethanol drinking and motor activity in adulthood in C57BL/6J mice

With alcohol readily accessible to adolescents, its consumption leads to many adverse effects, including impaired learning, attention, and behavior. Adolescents report higher rates of binge drinking compared to adults. Adolescents are also more prone to substance use disorder during adulthood due to physiological changes during the adolescent developmental period. We used C57BL/6J male and female mice to investigate the long-lasting impact of binge ethanol exposure during adolescence on voluntary ethanol intake and open field behavior during later adolescence and in young adulthood. The present set of experiments were divided into four stages: (1) chronic intermittent vapor inhalation exposure, (2) abstinence, (3) voluntary ethanol intake, and (4) open field behavioral testing. During adolescence, male and female mice were exposed to air or ethanol using an intermittent vapor inhalation with repeated binge pattern ethanol exposure from postnatal day (PND) 28-42. Following this, mice underwent abstinence during late adolescence from PND 43-49 (Experiment 1) or PND 43-69 (Experiment 2). Beginning on PND 49-76 (Experiment 1) or PND 70-97 (Experiment 2), mice were assessed for intermittent voluntary ethanol consumption using a two-bottle drinking procedure over 28 days. Male mice that were exposed to ethanol during adolescence showed increased ethanol consumption during later adolescence (Experiment 1) and in emerging adulthood (Experiment 2), while the female mice showed decreased ethanol consumption. These data demonstrate a sexually divergent shift in ethanol consumption following binge ethanol exposure during adolescence and differences in open field behavior. These data highlight sex-dependent vulnerability to developing substance use disorders in adulthood. Significance StatementCurrently, it is vital to determine the sex-dependent impact of binge alcohol exposure during adolescence, given that until recently females have largely been ignored. Here we show that adolescent male mice that are exposed to binge ethanol during adolescence show long-term changes in behavior in adulthood. In contrast, female mice show a transient decrease in ethanol consumption in adulthood and decreased motor activity spent in the center zone of the open field test. Male mice appear to be more susceptible to the long-term changes in ethanol consumption following binge ethanol exposure during adolescence.

neuroscience↗