Biology subjects

Hall, A. N.

Publications and source records attributed to Hall, A. N..

5 recordsLinked to original sources

bioRxiv2025

Phenotypic tolerance for rDNA copy number variation within the natural range of C. elegans

The genes for ribosomal RNA (rRNA) are encoded by ribosomal DNA (rDNA), whose structure is notable for being present in arrays of tens to thousands of tandemly repeated copies in eukaryotic genomes. The exact number of rDNA copies per genome is highly variable within a species, with differences between individuals measuring in potentially hundreds of copies and megabases of DNA. The extent to which natural variation in rDNA copy number impacts whole-organism phenotypes such as fitness and lifespan is poorly understood, in part due to difficulties in manipulating such large and repetitive tracts of DNA even in model organisms. Here, we used the natural resource of copy number variation in C. elegans wild isolates to generate new tools and investigated the phenotypic consequences of this variation. Specifically, we generated a panel of recombinant inbred lines (RILs) using a laboratory strain derivative with [~]130 haploid rDNA copies and a wild isolate with [~]417 haploid rDNA copies, one of the highest validated C. elegans rDNA copy number arrays. We find that rDNA copy number is stable in the RILs, rejecting prior hypotheses that predicted copy number instability and copy number reversion. To isolate effects of rDNA copy number on phenotype, we produced a series of near isogenic lines (NILs) with rDNA copy numbers representing the high and low end of the rDNA copy number spectrum in C. elegans wild isolates. We find no correlation between rDNA copy number and phenotypes of rRNA abundance, competitive fitness, early life fertility, lifespan, or global transcriptome under standard laboratory conditions. These findings demonstrate a remarkable ability of C. elegans to tolerate substantial variation in a locus critical to fundamental cell function. Our study provides strain resources for future investigations into the boundaries of this tolerance.

genomics↗

bioRxiv2023

Tools for Genetic Engineering and Gene Expression Control in Novosphingobium aromaticivorans and Rhodobacter sphaeroides

Alphaproteobacteria have a variety of cellular and metabolic features that provide important insights into biological systems and enable biotechnologies. For example, some species are capable of converting plant biomass into valuable biofuels and bioproducts have the potential to form the backbone of the sustainable bioeconomy. Among the Alphaproteobacteria, Novosphingobium aromaticivorans, Rhodobacter sphaeroides, and Zymomonas mobilis, show particular promise as organisms that can be engineered to convert extracted plant lignin or sugars into bioproducts and biofuels. Genetic manipulation of these bacteria is needed to introduce engineered pathways and modulate expression of native genes with the goal of enhancing bioproduct output. Although recent work has expanded the genetic toolkit for Z. mobilis, N. aromaticivorans and R. sphaeroides still need facile, reliable approaches to deliver genetic payloads to the genome and to control gene expression. Here, we expand the platform of genetic tools for N. aromaticivorans and R. sphaeroides to address these issues. We demonstrate that Tn7 transposition is an effective approach for introducing engineered DNA into the chromosome of N. aromaticivorans and R. sphaeroides. We screen a synthetic promoter library to identify inducible promoters with strong, regulated activity in both organisms. Combining Tn7 integration with promoters from our library, we establish CRISPR interference systems for N. aromaticivorans and R. sphaeroides that can target essential genes and modulate engineered pathways. We anticipate that these systems will greatly facilitate both genetic engineering and gene function discovery efforts in these industrially important species and other Alphaproteobacteria. IMPORTANCEIt is important to increase our understanding of the microbial world to improve health, agriculture, the environment and biotechnology. For example, building a sustainable bioeconomy depends on the efficient conversion of plant material to valuable biofuels and bioproducts by microbes. One limitation in this conversion process is that microbes with otherwise excellent properties for conversion are challenging to genetically engineer. Here, we report systems to overcome that barrier in the Alphaproteobacteria, Novosphingobium aromaticivorans and Rhodobacter sphaeroides, by producing genetic tools that allow easy insertion of engineered pathways into their genomes and to precisely control gene expression by inducing genes with synthetic promoters or repressing genes using CRISPR interference. These tools can be used in future work to gain additional insight into these and other Alphaproteobacteria and to optimize yield of biofuels and bioproducts.

microbiology↗

bioRxiv2020

Copy numbers of 45S and 5S ribosomal DNA arrays lack meaningful correlation in humans

The ribosomal DNA genes are tandemly arrayed in most eukaryotes and exhibit vast copy number variation. There is growing interest in integrating this variation into genotype-phenotype associations. Here, we explored a possible association of rDNA copy number variation with autism spectrum disorder and found no difference between probands and unaffected siblings. However, rDNA copy number estimates from whole genome sequencing are error-prone, so we sought to use pulsed-field gel electrophoresis, a classic gold-standard method, to validate rDNA copy number genotypes. The electrophoresis approach is not readily applicable to the human 45S arrays due to their size and location on five separate chromosomes; however, it should accurately resolve copy numbers for the shorter 5S arrays that reside on a single chromosome. Previous studies reported tightly correlated, concerted copy number variation between the 45S and 5S arrays, which should enable the validation of 45S copy number estimates with CHEF-gel-verified 5S copy numbers. Here, we show that the previously reported strong concerted copy number variation is likely an artifact of variable data quality in the earlier published 1000 Genomes Project sequences. We failed to detect a meaningful correlation between 45S and 5S copy numbers in the large, high-coverage Simons Simplex Collection dataset as well as in the recent high-coverage 1000 Genomes Project sequences. Our findings illustrate the challenge of genotyping repetitive DNA regions accurately and call into question the accuracy of recently published studies of rDNA copy number variation in cancers and aging that relied on diverse publicly available resources for sequence data.Competing Interest StatementThe authors have declared no competing interest.View Full Text

genomics↗

bioRxiv2017

Head-on replication-transcription collisions lead to formation of life threatening R-loops

Encounters between transcription and DNA replication machineries lead to conflicts that shape genomes, influence evolution, and lead to genetic diseases in humans. Although unclear why, head-on transcription (lagging strand genes) is especially disruptive to replication, increases DNA breaks, and promotes mutagenesis. Here, we show that head-on replication-transcription conflicts lead to pervasive RNA:DNA hybrid formation in Bacillus subtilis. We find that replication beyond head-on conflict regions requires the activity of a RNA:DNA hybrid processing enzyme, RNase HIII. Remarkably, pervasive RNA:DNA hybrid formation at head-on genes completely stops replication and inhibits gene expression in a replication-dependent manner. Accordingly, we find that resolution of head-on conflicts by RNase HIII is crucial for survival upon exposure to various stresses, as many stress response genes are encoded head-on to replication. We conclude that R-loops, RNA:DNA hybrids formed outside of the transcription bubble, exacerbate head-on replication-transcription conflicts, thereby threatening life, especially upon exposure to environmental stresses.

molecular biology↗

bioRxiv2017

Short tandem repeats with massive variation and functional consequences across strains of Arabidopsis thaliana

Short tandem repeat (STR) mutations may be responsible for more than half of the mutations in eukaryotic coding DNA, yet STR variation is rarely examined as a contributor to complex traits. We assess the scope of this contribution across a collection of 96 strains of Arabidopsis thaliana by massively parallel STR genotyping. We found that 95% of examined STRs are polymorphic, with a median of six alleles per STR in these strains. Modest STR expansions are found in most strains, some of which have evident functional effects. For instance, three of six intronic STR expansions are associated with intron retention. Coding STRs are depleted of variation relative to non-coding STRs, consistent with the action of purifying selection, and some STRs show hypervariable patterns consistent with diversifying selection. Finally, we detect dozens of novel STR-phenotype associations that could not be detected with SNPs alone, validating several with follow-up experiments. Our results demonstrate that STRs comprise a large, unascertained reservoir of functionally relevant genomic variation.

genomics↗