Cell morphology in bacteria is a crucial factor for survival in that it affects evading predators and acquiring nutrition leading to growth and development. In order to grow, bacteria divide through binary fission to create two daughter cells identical in size, shape, and genome. One of the main contributors to accurate and efficient cell division is the Min system. Composed of proteins, MinC, MinD, and MinE, the following work together to place the FtsZ ring in the middle of the cell for septum formation. While the Min system and its effects have been heavily studied in other model organisms, little is known about its function and mechanism in the Gram-negative soil bacteria, Acinetobacter baylyi (ADP1). Bioinformatics tools such as sequence alignments, protein and operon predictions demonstrated evolutionary similarities between ADP1 and other rod-shaped organisms such as Bacillus subtilis, Escherichia coli, and Pseudomonas aeruginosa. In this study, we utilized ADP1’s high transforming capabilities to create individual knockout mutants of minC, minD, and minE. Based on bioinformatics, it is predicted that the min mutants would exert similar compromised growth and division morphologies of filamentation and minicell production as seen in other organisms. To test this hypothesis, cells were imaged under atomic force microscopy (AFM), and we acquired detailed nanoscopic data that showcased many filamented cells with few minicells. Features such as indents, side, and through bites also appeared on the surface of the mutant cells. Indents are shallow dips that appear on the cell surface, while bites are deeper features that either depress through the width of the cell (through bite) or asymmetrically along the side of the cell (side bite). Due to the mutation in division, bites and indent distribution were random as expected in the min mutants. This preliminary finding into the morphology effects of the Min system provides further insight into the complex mechanism of bacterial cell division.
In the past decade, RNA sequencing has become a notable method for identifying transcript alterations that correlate to disease development and progression. RNA-seq allows researchers to pinpoint mutated sequence elements that hinder transcriptional regulation, ultimately altering cellular functioning, and specify research questions around disease advancement. To use RNA-seq as a reliable molecular tool for mapping RNAs in diseased cells, researchers have focused on optimizing the step in which an adaptor is ligated to the RNA to be sequenced, prior to cDNA development. Although wildtype T4 RNA Ligase 2 (Rnl2) has been previously utilized for this step, its usage develops a mixture of ligated products and circularized RNA, making it an unreliable tool. C-terminus truncated T4 RNA Ligase 2 (1-249) with double mutations K227Q and R55K (DM Rnl2trunc) has been identified as an efficient tool for ligating known pre-adenylated adaptors to any RNA with reduced amounts of unwanted side products. In this study, we present the experimental parameters utilized to successfully purify a high yield of DM Rnl2trunc that is both soluble and enzymatically active from E. coli cultures. Growth media volume was identified as the largest contributing factor for solubilizing DM Rnl2trunc. Further, SDS-PAGE analysis suggests the recombinant protein was eluted from nickel columns using 100mM imidazole. Most importantly, our purification protocol yielded abundant DM Rnl2trunc with enzymatic activity comparable to commercially available product. Bioanalyzer data indicates both homemade and commercial DM Rnl2trunc is capable of ligating desired RNA sequences and pre-adenylated adaptors with very high efficiency and unwanted side products.
Reproductive mechanisms play a vital role in a species’ ability to proliferate and evolve. The complex and dynamic mating strategies that yeast species employ to effectively proliferate provide insight into how various reproductive models operate—comparing species with these unique capabilities can illuminate how sex and reproduction have evolved over time. The methylotrophic yeast Ogataea polymorpha, like many yeast species, exhibits asexual and sexual reproductive capabilities and can undergo mating-type switching before mating. Mating-type switching is a genetic process governed by the MAT locus, and recent investigations have characterized the structure and function of the locus in several species. However, unlike other species, the molecular mechanisms and specific environmental conditions required for mating and mating-type switching in methylotrophic yeast are poorly understood and contemporary testing protocols are time consuming. Here, we began creation of a high throughput assay to quantify mating and mating-type switching frequencies with flow cytometry. We designed three flow cytometry-based assays, including (1) utilizing nuclear DNA staining and cell cycle arrest to identify variations in ploidy indicative of mating, (2) bilateral mating with N- and C-terminus ends of Green Fluorescent Protein (GFP) in mating partners to track mating frequencies, and (3) molecular fluorescent tagging at mating-type specific genes of the MAT locus with genes for GFP and Red Fluorescent Protein (RFP) such that mating-type specific gene expression can be indicated via fluorescence to observe mating-type switching frequencies. Nuclear DNA staining protocols in O. polymorpha produced indistinguishable cell cycle histogram plots, indicating a need for an adapted DNA staining protocol for the species. Bilateral mating is effective at quantifying mating frequencies in Saccharomyces cerevisiae but fails to work effectively in O. polymorpha. Transformations for MAT locus molecular fluorescent tagging are in progress and have yet to be tested on the flow cytometer. Complete development of these assays will streamline the process of studying the genetic and environmental conditions in which yeast reproduce. Establishing more efficient methods to investigate the molecular dynamics of mating and mating-type switching will further our understanding of how reproduction has evolved across yeast species.
Nonsense-mediated mRNA decay (NMD) is a eukaryotic quality control mechanism for the dynamic regulation of gene expression. NMD degrades transcripts containing a premature termination codon (PTC) more than 50-55 nucleotides upstream of the final exon-exon junction. Although NMD is a ubiquitous mechanism for degrading RNA transcripts in all eukaryotes, there is great variety in the efficiency and specificity of the degradation mechanism. While most transcripts containing a PTC are degraded via NMD, transcripts containing a PTC can evade NMD and produce truncated or full-length proteins1. NMD efficiency may also vary based on gene sequence, intracellular location, tissue, or on an individual level. This study aimed to aid the understanding of NMD as an endogenous control for gene expression by evaluating NMD efficiency in homogenous cell cultures. We evaluated NMD efficiencies in human embryonic kidney cells by transfecting cell cultures with dual-fluorescing reporters for NMD. We measured fluorescent levels through flow cytometry, and surprisingly detected varying NMD efficiencies among cells of the same culture. To investigate the possible causes of the range in NMD efficiency, we sorted cell cultures based on NMD efficiency levels and evaluated cell populations for their concentrations of NMD factors through immunoblotting and RT-qPCR. Results revealed that NMD factor expression levels did not correlate with NMD efficiency, which proposes new questions for the role of NMD factors in NMD and other possible intracellular mechanisms affecting NMD efficiency. We hypothesized that cell cycle may be affecting NMD. To study the possible relationship, groups of cells with varying NMD efficiencies were evaluated through immunoblotting for cell cycle stage. Preliminary results did not indicate a relationship; however, the association must be further evaluated. Conclusively, we determined a range in NMD efficiency among individual cells in homogenous human embryonic kidney cell cultures. We aim to progress this research by determining key factors and mechanisms that may influence NMD efficiency. Implications for understanding the specificities of NMD activity are far-reaching in the medical field, as several severe human diseases, such as facioscapulohumeral muscular dystrophy, are strongly tied to NMD inhibition.
Saccharomyces cerevisiae has the ability to alter its growth based on how much of a particular carbon source is present in the environment; when glucose is depleted the cells will enter the post-diauxic phase. Based on previous evidence, we suspect that mRNA that encode for mitochondrial proteins are degraded by a novel autophagic decay pathway in the post-diauxic phase, requiring the ribonucleases Xrn1 and Rny1 for proper mitochondrial growth. First, we tested the localization of the ribonucleases Xrn1 and Rny1 to determine if their localization changes due to stressful conditions, and if these results can indicate a change in their function. Second, to test if an autophagic mRNA decay pathway exists, we used the MS2 labeling technique to fluorescently tag specific mRNAs within the cell in order to see if they associate with structures that maybe involved in autophagic mRNA decay, such as autophagosomes and stress granules. We tested three specific mRNAs for their localization: PGK1, DPI8 and COX5A. PGK1 encodes for a protein kinase that aids in metabolism, while DPI8 and COX5A both encode for mitochondrial proteins. We were able to determine that we can visualize mRNA using the MS2-CP-GFP fusion protein, however we believe that the MS2-CP-mCherry fusion did not detect MS2-tagged mRNAs. Using the MS2-CP-GFP assay, we were able to determine that PGK1 localizes to mitochondria in the post-diauxic phase.
The long noncoding RNA (lncRNA) HOTAIR acts in trans to epigenetically silence a 40-kb region of the HOXD gene cluster during development. In aggressive breast cancer, HOTAIR overexpression promotes metastasis, drug resistance, recurrence, and is a negative prognostic factor. However, the precise molecular mechanisms by which HOTAIR induces the formation of heterochromatin at specific sites in the genome to silence tumor suppressor genes remains widely unknown. To learn more about the molecular role of HOTAIR, we explored whether it was modified by the N6-methyladenosine RNA modification, which is dysregulated in breast cancer and has been proposed to play an essential role in gene repression by mediating lncRNA-protein interactions. Because we mapped m6A to a single nucleotide within the second domain of HOTAIR, we performed a quantitative proteomic analysis to identify additional HOTAIR-protein interactions that may be mediated by the post-transcriptional modification. We found that HOTAIR interacts with the YTH domain-containing protein 1 (YTHDC1), a member of a family of proteins known to recognize m6A residues. To determine the role of m6A in the HOTAIR-YTHDC1 interaction, we performed RNA pulldown assays with purified YTHDC1 and in vitro transcribed wild-type and m6A mutant HOTAIR in the presence or absence of methylation. Our data found that YTHDC1 preferentially interacts with wild-type methylated HOTAIR, suggesting that m6A mediates the HOTAIR-YTHDC1 interaction. However, it is not yet known if the m6A residue of HOTAIR recruits YTHDC1 to promote the transcriptional silencing of important suppressor genes. Thus, future research exploring the role of the m6A-mediated HOTAIR-YTHDC1 interaction in metastatic breast cancer will demonstrate whether a better understanding of the lncRNA can drive the development of breast cancer therapeutics. This is of utmost importance because invasive breast cancer remains a leading cause of death for women worldwide despite recent advancements in diagnosis and treatments.