Biology subjects

Karpinski, E.

Publications and source records attributed to Karpinski, E..

4 recordsLinked to original sources

bioRxiv2024

Repeated climate-driven dispersal and speciation in peripheral populations of Pleistocene mastodons

Recent ancient DNA work has shed some light on the responses of mastodons to Pleistocene glacial/interglacial cycling but focused primarily on their expansion into Beringia. However, genetics has complicated our understanding of the relationships within Mammut, specifically between Pacific and American mastodon phylogeography and questioned whether these are in fact two separate species or regionally localized morphotypes. Here we expand on both avenues by sequencing and contextualizing the mitochondrial genome of a Pacific mastodon, as well as from several North American eastern specimens throughout the last 800 thousand years. We show that Pacific mastodons fall within a previously established, and deeply divergent mitochondrial clade, extending the range of this species into western Canada and potentially Mexico. We also present evidence for at least three discrete expansion events into northeastern coastal regions (i.e. Nova Scotia and the eastern continental shelf), and identify two new mastodon clades, which contain temporally distinct, but geographically co-occurrent specimens. This work sheds further light on mastodon taxonomy and phylogeography across North America throughout the Pleistocene, highlighting interglacial range expansion into northeastern America mirroring the effects on the western side of the continent (Beringia).

evolutionary biology↗

bioRxiv2024

Engineering Asian elephant TP53: TP53 retrogene knockouts activate common and unique cancer-relevant pathways

Proboscideans have developed a suite of genetic changes including those responsible for tumor suppression and environmental adaptation. Here we examine a handful of woolly mammoth-specific deletions, and their potential contributions to arctic adaptation, as well as the expanded TP53 genetic repertoire in elephants. We use CRISPR-Cas9 to introduce mammoth-specific noncoding deletions as well as knockouts of TP53, all 29 TP53 retrogenes, or both in combination in Asian elephant cell lines, and examine the transcriptomic response. We find that many of the mammoth-specific deletions likely contribute to various arctic phenotypes including vascular development, metabolism and thermogenesis, and hair and skin adaptations. We also find that while there is considerable overlap in DNA damage responses of the TP53 and retrogene knockouts, retrogenes knockouts also exhibit strong enrichment of many extracellular pathways suggesting they may play a role in the tumor microenvironment and mitigating metastatic growth.

genetics↗

bioRxiv2024

Plasmid2MC: Efficient cell-free recombination of plasmids into high-purity minicircle DNA for use in genome editing applications

AbstractDNA plasmids are widely used for delivering proteins and RNA in genome editing. However, their bacterial components can lead to inactivation, cell toxicity, and reduced efficiency compared to minicircle DNA (mcDNA), which lacks such bacterial sequences. Existing commercial kits that recombine plasmids into mcDNA within proprietary bacterial strains are labor-intensive, yield inconsistent results, and often produce endotoxin-contaminated low-quality mcDNA. To address this challenge, we developed Plasmid2MC, a novel cell-free method utilizing{Phi} C31 integrase-mediated recombination to efficiently excise the bacterial backbone from conventionally prepared plasmids, followed by digestion of the bacterial backbone and all other DNA contaminants, resulting in highly pure and virtually endotoxin-free mcDNA. We demonstrated the application of mcDNA to express CRISPR-dCas9 for base editing in HEK293T cells and mouse embryonic stem cells, as well as for homology-independent targeted insertion (HITI) genome editing. The methods ease of preparation, high efficiency, and the high purity of the resulting mcDNA make Plasmid2MC a valuable tool for applications requiring bacterial backbone-free circular DNA.

genetics↗

bioRxiv2019

PalaeoChip Arctic1.0: An optimised eDNA targeted enrichment approach to reconstructing past environments

Ancient environmental DNA has been established as a viable biomolecular proxy for tracking taxonomic presence through time in a local environment, even in the total absence of primary tissues. It is thought that sedimentary ancient DNA (sedaDNA) survives through mineral binding. And while these organo-mineral complexes likely facilitate long-term preservation, they also challenge our ability to release and isolate target molecules. Two limitations in sedaDNA extraction impede many palaeoenvironmental reconstructions: the post-extraction carryover of enzymatic inhibitors, and sedaDNA loss when attempting to reduce inhibitor co-elution. Here, we present an optimised eDNA targeted enrichment approach for reconstructing past environments. Our new extraction protocol with targeted enrichment averages a 14.6-fold increase in on-target plant and animal DNA compared to a commercial soil extraction kit, and a 22.6-fold increase compared to a PCR metabarcoding approach. To illustrate the effectiveness of the PalaeoChip Arctic1.0 protocol, we present results of plant and animal presence from permafrost samples and discuss new potential evidence for the late survival (ca. 9685 BP) of mammoth (Mammuthus sp.) and horse (Equus sp.) in the Klondike Region of Yukon, Canada. This approach translates to a more diverse and sensitive dataset with increased sequencing efficiency of ecologically informative sedaDNA.

ecology↗