Biology subjects

Shoura, M. J.

Publications and source records attributed to Shoura, M. J..

3 recordsLinked to original sources

bioRxiv2024

CGC1, a new reference genome for Caenorhabditis elegans

The original 100.3 Mb reference genome for Caenorhabditis elegans, generated from the wild-type laboratory strain N2, has been crucial for analysis of C. elegans since 1998 and has been considered complete since 2005. Unexpectedly, this long-standing reference was shown to be incomplete in 2019 by a genome assembly from the N2-derived strain VC2010. Moreover, genetically divergent versions of N2 have arisen over decades of research and hindered reproducibility of C. elegans genetics and genomics. Here we provide a 106.4 Mb gap-free, telomere-to-telomere genome assembly of C. elegans, generated from CGC1, an isogenic derivative of the N2 strain. We used improved long-read sequencing and manual assembly of 43 recalcitrant genomic regions to overcome deficiencies of prior N2 and VC2010 assemblies, and to assemble tandem repeat loci including a 772-kb sequence for the 45S rRNA genes. While many differences from earlier assemblies came from repeat regions, unique additions to the genome were also found. Of 19,972 protein-coding genes in the N2 assembly, 19,790 (99.1%) encode products that are unchanged in the CGC1 assembly. The CGC1 assembly also may encode 183 new protein-coding and 163 new ncRNA genes. CGC1 thus provides both a completely defined reference genome and corresponding isogenic wild-type strain for C. elegans, allowing unique opportunities for model and systems biology.

genomics↗

bioRxiv2019

Loop-closure Kinetics Reveal a Stable, Right-handed DNA Intermediate in Cre Recombination

In Cre site-specific recombination, the synaptic intermediate is a recombinase homotetramer containing a pair of DNA target sites. The strand-exchange mechanism proceeds via a Holliday-junction (HJ) intermediate; however, the geometry of the DNA segments in the synapse has remained highly controversial. In particular, all crystallographic structures are consistent with an achiral planar Holliday-junction (HJ) structure, whereas topological assays based on Cre-mediated knotting of plasmid DNAs are consistent with a right-handed chiral junction. Here we use the kinetics of loop closure involving closely spaced (131-151 bp), directly repeated loxP sites to investigate the in-aqueo ensemble of conformations for the longest-lived looped DNA intermediate. Fitting the experimental site-spacing dependence of the loop-closure probability, J, to a statistical-mechanical theory of DNA looping provides evidence for substantial out-ofplane HJ distortion. This result unequivocally stands in contrast to the square-planar intermediate geometry determined from crystallographic data for the Cre-loxP system and other int-superfamily recombinases. J measurements carried out with an isomerization-deficient Cre mutant suggest that the apparent geometry of the wild-type complex may result from the temporal averaging of diverse right-handed and achiral structures. Applied to Cre recombinase, and other biological systems, our approach bridges the static pictures provided by crystal structures and the natural dynamics of macromolecules in vivo. This approach thus advances a more comprehensive dynamic analysis of large nucleoprotein structures and their mechanisms.

biophysics↗

bioRxiv2019

Deconvolution of Nucleic-acid Length Distributions: A Gel Electrophoresis Analysis Tool and Applications

Next-generation DNA-sequencing (NGS) technologies, which are designed to streamline the acquisition of massive amounts of sequencing data, are nonetheless dependent on various preparative steps to generate DNA fragments of required concentration, purity, and average size (molecular weight). Current automated electrophoresis systems for DNA- and RNA-sample quality control, such as Agilents Bioanalyzer (R)and TapeStation (R) products, are costly to acquire and use; they also provide limited information for samples having broad size distributions. Here we describe a software tool that helps determine the size distribution of DNA fragments in an NGS library, or other DNA sample, based on gel-electrophoretic line profiles. The software, developed as an ImageJ plug-in, allows for straightforward processing of gel images, including lane selection and fitting of univariate functions to intensity distributions. The user selects the option of fitting either discrete profiles in cases where discrete gel bands are visible, or continuous profiles, having multiple bands buried under a single broad peak. The method requires only modest imaging capabilities and is a cost-effective, rigorous alternative characterization method to augment existing techniques for library quality control.

genomics↗