Biology subjects

Porter, C.

Publications and source records attributed to Porter, C..

3 recordsLinked to original sources

bioRxiv2018

xMD-miRNA-seq to generate near in vivo miRNA expression estimates in colon epithelial cells

Accurate, RNA-seq based, microRNA (miRNA) expression estimates from primary cells have recently been described. However, this in vitro data is mainly obtained from cell culture, which is known to alter cell maturity/differentiation status, significantly changing miRNA levels. What is needed is a robust method to obtain in vivo miRNA expression values directly from cells. We introduce expression microdissection miRNA small RNA sequencing (xMD-miRNA-seq), a method to isolate cells directly from formalin fixed paraffin-embedded (FFPE) tissues. xMD-miRNA-seq is a low-cost, high-throughput, immunohistochemistry-based method to capture any cell type of interest. As a proof-of-concept, we isolated colon epithelial cells from two specimens and performed low-input small RNA-seq. We generated up to 600,000 miRNA reads from the samples. Isolated epithelial cells, had abundant epithelial-enriched miRNA expression (miR-192; miR-194; miR-200b; miR-200c; miR-215; miR-375) and overall similar miRNA expression patterns to other epithelial cell populations (colonic enteroids and flow-isolated colon epithelium). xMD-derived epithelial cells were generally not contaminated by other adjacent cells of the colon as noted by t-SNE analysis. xMD-miRNA-seq allows for simple, economical, and efficient identification of cell-specific miRNA expression estimates. Further development will enhance rapid identification of cell-specific miRNA expression estimates in health and disease for nearly any cell type using archival FFPE material.

molecular biology↗

bioRxiv2017

Compensatory evolution via cryptic genetic variation: Distinct trajectories to phenotypic and fitness recovery

Populations are constantly exposed to deleterious alleles, most of which are purged via natural selection. However, deleterious fitness effects of alleles can also be suppressed by compensatory adaptation. Compensatory mutations can act directly to reduce deleterious effects of an allele. Alternatively, compensation may also occur by altering other aspects of an organisms phenotype or performance, without suppressing the phenotypic effects of the deleterious allele. Moreover, the origin of allelic variation contributing to compensatory adaptation remains poorly understood. Compensatory evolution driven by mutations that arise during the selective process are well studied. However less is known about the role standing (cryptic) genetic variation plays in compensatory adaptation. To address these questions, we examined evolutionary trajectories of natural populations of Drosophila melanogaster fixed for mutations that disrupt wing morphology, resulting in deleterious effects on several components of fitness. Lineages subjected only to natural selection, evolved modifications to courtship behavior and several life history traits without compensation in wing morphology. Yet, we observed rapid phenotypic compensation of wing morphology under artificial selection, consistent with segregating variation for compensatory alleles. We show that alleles contributing to compensation of wing morphology have deleterious effects on other fitness components. These results demonstrate the potential for multiple independent avenues for rapid compensatory adaptation from standing genetic variation, which ultimately may reveal novel adaptive trajectories.

evolutionary biology↗

bioRxiv2017

How Well Do You Know Your Mutation? Complex Effects Of Genetic Background On Expressivity, Complementation, And Ordering Of Allelic Effects

For a given gene, different mutations influence organismal phenotypes to varying degrees. However, the expressivity of these variants not only depends on the DNA lesion associated with the mutation, but also on factors including the genetic background and rearing environment. The degree to which these factors influence related alleles, genes, or pathways similarly, and whether similar developmental mechanisms underlie variation in the expressivity of a single allele across conditions and variation across alleles is poorly understood. Besides their fundamental biological significance, these questions have important implications for the interpretation of functional genetic analyses, for example, if these factors alter the ordering of allelic series or patterns of complementation. We examined the impact of genetic background and rearing environment for a series of mutations spanning the range of phenotypic effects for both the scalloped and vestigial genes, which influence wing development in Drosophila melanogaster. Genetic background and rearing environment influenced the phenotypic outcome of mutations, including intra-genic interactions, particularly for mutations of moderate expressivity. We examined whether cellular correlates (such as cell proliferation during development) of these phenotypic effects matched the observed phenotypic outcome. While cell proliferation decreased with mutations of increasingly severe effects, surprisingly it did not co-vary strongly with the degree of background dependence. We discuss these findings and propose a phenomenological model to aid in understanding the biology of genes, and how this influences our interpretation of allelic effects in genetic analysis.

genetics↗