Biology subjects

Bubnell, J. E.

Publications and source records attributed to Bubnell, J. E..

4 recordsLinked to original sources

bioRxiv2025

Comparative functional and evolutionary analysis of essential germline stem cell genes across the genus Drosophila and two outgroup species

In Drosophila melanogaster, bag of marbles (bam) encodes a protein essential for germline stem cell daughter (GSC) differentiation in early gametogenesis. Despite its essential role in D. melanogaster, direct functional evaluation of bam in other closely related Drosophila species reveal this essential function is not necessarily conserved. In D. teissieri, for example, bam is not essential for GSC daughter differentiation. Here, we generated bam null alleles using CRISPR-Cas9 in a species more distantly related to D. melanogaster, D. americana, to interrogate whether bams essential GSC differentiation function is novel to the melanogaster species group or a function more basal to the Drosophila genus. To further characterize the extent of the functional flexibility of other GSC regulating genes, we generated a gene ortholog dataset for 366 GSC regulating genes essential in D. melanogaster across 15 additional Drosophila and two outgroup species. We find that bams essential GSC function is conserved between D. melanogaster and D. americana and therefore originated prior to the formation of the melanogaster species group. Additionally, we find that [~]8% of the 366 GSC genes essential in D. melanogaster are absent in at least one of the 17 species in our ortholog dataset. These results indicate that developmental systems drift (DSD), in which the specific genes regulating a function may change, but the final phenotype is retained, occurs in stem cell regulation and the production of gametes across Drosophila species. Article summaryResults from CRISPR induced bam null mutants in D. americana and comparative ortholog analysis of essential GSC regulating genes indicate that the evolutionary origin of bams essential GSC differentiation function is likely basal to the Drosophila genus, and there is functional flexibility in at least [~]8% of the 366 GSC regulating genes across the 17 included species.

genetics↗

bioRxiv2021

Functional divergence of the bag of marbles gene in the Drosophila melanogaster species group

In Drosophila melanogaster, a key germline stem cell (GSC) differentiation factor, bag of marbles (bam) shows rapid bursts of amino acid fixations between sibling species D. melanogaster and D. simulans, but not in the outgroup species D. ananassae. Here, we test the null hypothesis that bams differentiation function is conserved between D. melanogaster and four additional Drosophila species in the melanogaster species group spanning approximately 30 million years of divergence. Surprisingly, we demonstrate that bam is not necessary for oogenesis or spermatogenesis in D. teissieri nor is bam necessary for spermatogenesis in D. ananassae. Remarkably bam function may change on a relatively short time scale. We further report tests of neutral sequence evolution at bam in additional species of Drosophila and find a positive, but not perfect, correlation between evidence for positive selection at bam and its essential role in GSC regulation and fertility for both males and females. Further characterization of bam function in more divergent lineages will be necessary to distinguish between bams critical gametogenesis role being newly derived in D. melanogaster, D. simulans, D. yakuba, and D. ananassae females or it being basal to the genus and subsequently lost in numerous lineages.

genetics↗

bioRxiv2021

High rates of plasmid cotransformation in E. coli overturn the clonality myth and reveal colony development

The concept of DNA transfer between bacteria was put forth by Griffith in 1928. During the dawn of molecular cloning of DNA in the 1980s, Hanahan described how the transformation of DNA plasmids into bacteria would allow for cloning of DNA fragments. Through this foundational work, it is widely taught that a typical transformation produces clonal bacterial colonies. Using low concentrations of several plasmids that encode different fluorescent proteins, under the same selective antibiotic, we show that E. coli bacteria readily accept multiple plasmids, resulting in widespread aclonality and reveal a complex pattern of colony development. Cotransformation of plasmids occurs by either CaCl2 or by electroporation methods. A bacterium rod transformed with three plasmids - each expressing a high level of a unique fluorescent protein - and replated on agar, appears to reassign a random number of the three fluorescent plasmids to its daughter cell during cell division. The potential to simultaneously follow multiple lineages of clonally related bacteria in a bacteria colony would allow for mosaic analysis of gene function. We show that clonally related bacterium rods self-organize in a fractal growth pattern and can remain linked during colony development revealing a potential target against microbiota growth.

molecular biology↗

bioRxiv2021

Genetic variation among wMel strains of Wolbachia pipientis differentially rescues a bag of marbles partial loss of function mutant in Drosophila melanogaster

In Drosophila melanogaster, the maternally inherited endosymbiont Wolbachia pipientis interacts with germline stem cell genes during oogenesis. One such gene, bag of marbles (bam) is the key switch for differentiation and also shows signals of adaptive evolution for protein diversification. These observations have led us to hypothesize that W. pipientis could be driving the adaptive evolution of bam for control of oogenesis. To test this hypothesis, we must understand the specificity of the genetic interaction between bam and W. pipientis. Previously, we documented that the W. pipientis variant, wMel, rescued the fertility of the bamBW hypomorphic mutant as a transheterozygote over a bam null. However, bamBW was generated more than 20 years ago in an uncontrolled genetic background and maintained over a balancer chromosome. Consequently, the chromosome carrying bamBW accumulated mutations that have prevented controlled experiments to further assess the interaction. Here, we used CRISPR/Cas9 to engineer the same single amino acid bam hypomorphic mutation (bamL255F) and a new bam null disruption mutation into the w1118 isogenic background. We assess the fertility of wildtype bam, bamL255F/bamnull hypomorphic, and bamL255F/ bamL255F mutant females, each infected individually with ten W. pipientis wMel variants representing three phylogenetic clades. Overall, we find that all of the W. pipientis variants tested here rescue bam hypomorphic fertility defects with wMelCS-like variants exhibiting the strongest rescue effects. Additionally, these variants did not increase wildtype bam female fertility. Therefore, both bam and W. pipientis interact in genotype-specific ways to modulate female fertility, a critical fitness phenotype.

genetics↗