The tRNA methylome.

Herein, the procyclic form and bloodstream form tRNA methylome is reported as revealed by small RNA bisulfite sequencing. 5-Methylcytosines were identified at six unique positions with 54 total 5-methylcytosines revealed. The main hot spot for 5-methylcytosine in tRNA is the junction between the variable region and the T-arm.

A role for a Trypanosoma brucei cytosine RNA methyltransferase homolog in ribosomal RNA processing.

In Trypanosoma brucei, gene expression is primarily regulated posttranscriptionally making RNA metabolism critical. T. brucei has an epitranscriptome containing modified RNA bases.

An Undergraduate Laboratory Exploring Mutational Mechanisms in Escherichia coli Based on the Luria-Delbrück Experiment.

Understanding the mechanism for DNA mutations is a key concept in most genetics and microbiology courses. In addition, understanding that most mutations occur prior to exposure to selection is an important yet often difficult concept for students to grasp. We developed an undergraduate laboratory activity on mutation mechanisms based on the classic experiment from Luria and Delbrück.

An undergraduate laboratory to detect viruses in human DNA samples using qPCR.

The COVID-19 pandemic has necessitated the need to reliably detect the presence of viral genomes in human clinical samples. The most accurate viral tests involve the use of qPCR. Thus, it is important for students to understand the mechanism to detect viral genomes by qPCR including critical qPCR controls and how to properly interpret qPCR data.

DNA cytosine methyltransferase enhances viability during prolonged stationary phase in Escherichia coli.

In Escherichia coli, DNA cytosine methyltransferase (Dcm) methylates the second cytosine in the sequence 5'CCWGG3' generating 5-methylcytosine. Dcm is not associated with a cognate restriction enzyme, suggesting Dcm impacts facets of bacterial physiology outside of restriction-modification systems. Other than gene expression changes, there are few phenotypes that have been identified in strains with natural or engineered Dcm loss, and thus Dcm function has remained an enigma.

An undergraduate laboratory on RNA sequencing analysis of bacterial gene expression.

Next generation sequencing has revolutionized molecular biology and has provided a mechanism for rapid DNA and RNA sequence analysis. Yet, there are few resources to introduce next generation sequencing into the undergraduate biochemistry and molecular biology curriculum. Herein, we describe the design, execution, and assessment of a four-week laboratory for junior and senior undergraduate students that focuses on bacterial gene expression changes detected by RNA sequencing (RNA-seq).

Discovery of Escherichia coli CRISPR sequences in an undergraduate laboratory.

Clustered regularly interspaced short palindromic repeats (CRISPRs) represent a novel type of adaptive immune system found in eubacteria and archaebacteria. CRISPRs have recently generated a lot of attention due to their unique ability to catalog foreign nucleic acids, their ability to destroy foreign nucleic acids in a mechanism that shares some similarity to RNA interference, and the ability to utilize reconstituted CRISPR systems for genome editing in numerous organisms. In order to introduce CRISPR biology into an undergraduate upper-level laboratory, a five-week set of exercises was designed to allow students to examine the CRISPR status of uncharacterized Escherichia coli strains and to allow the discovery of new repeats and spacers.

5-azacytidine induces transcriptome changes in Escherichia coli via DNA methylation-dependent and DNA methylation-independent mechanisms.

Background: Escherichia coli K-12 strains contain DNA cytosine methyltransferase (Dcm), which generates 5-methylcytosine at 5'CCWGG3' sites. Although the role of 5-methylcytosine in eukaryotic gene expression is relatively well described, the role of 5-methylcytosine in bacterial gene expression is largely unknown.

Results: To identify genes that are controlled by 5-methylcytosine in E.

Blue genes: An integrative laboratory to differentiate genetic transformation from gene mutation for underclassmen.

The ability of students to understand the relationship between genotype and phenotype, and the mechanisms by which genotypes and phenotypes can change is essential for students studying genetics. To this end, we have developed a four-week laboratory called Blue Genes, which is designed to help novice students discriminate between two mechanisms by which the genetic material can be altered: genetic transformation and gene mutation. In the first week of the laboratory, students incubate a plasmid DNA with calcium chloride-treated Escherichia coli JM109 cells and observe a phenotype change from ampicillin sensitive to ampicillin resistant and from white color to blue color on plates containing 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) and isopropyl β-D-thiogalactopyranoside (IPTG).

A map of 5-methylcytosine residues in Trypanosoma brucei tRNA revealed by sodium bisulfite sequencing.

In protozoan parasites, there is little information on the presence of covalent RNA modifications which comprise the epitranscriptome. Therefore, we determined if T. brucei tRNA(Asp(GUC)), tRNA(Gly(GCC)), tRNA(Val(AAC)), and tRNA(Tyr(GUA)) contain 5-methylcytosines via RNA bisulfite sequencing.

Cytosine DNA methylation influences drug resistance in Escherichia coli through increased sugE expression.

Escherichia coli K-12 strains contain the orphan cytosine-5 DNA methyltransferase enzyme Dcm (DNA cytosine methyltransferase). Two recent reports indicate that Dcm has an influence on stationary phase gene expression in E. coli.

Studying epigenetic DNA modifications in undergraduate laboratories using complementary bioinformatic and molecular approaches.

Epigenetic inheritance is the inheritance of genetic information that is not based on DNA sequence alone. One type of epigenetic information that has come to the forefront in the last few years is modified DNA bases. The most common modified DNA base in nature is 5-methylcytosine.

Conservation of Dcm-mediated cytosine DNA methylation in Escherichia coli.

In Escherichia coli, cytosine DNA methylation is catalyzed by the DNA cytosine methyltransferase (Dcm) protein and occurs at the second cytosine in the sequence 5'CCWGG3'. Although the presence of cytosine DNA methylation was reported over 35 years ago, the biological role of 5-methylcytosine in E. coli remains unclear.

Patterns of gene-specific and total transcriptional activity during the Plasmodium falciparum intraerythrocytic developmental cycle.

The relationships among gene regulatory mechanisms in the malaria parasite Plasmodium falciparum throughout its asexual intraerythrocytic developmental cycle (IDC) remain poorly understood. To investigate the level and nature of transcriptional activity and its role in controlling gene expression during the IDC, we performed nuclear run-on on whole-transcriptome samples from time points throughout the IDC and found a peak in RNA polymerase II-dependent transcriptional activity related to both the number of nuclei per parasite and variable transcriptional activity per nucleus over time. These differential total transcriptional activity levels allowed the calculation of the absolute transcriptional activities of individual genes from gene-specific nuclear run-on hybridization data.

African trypanosomes contain 5-methylcytosine in nuclear DNA.

It is currently unclear if there are modified DNA bases in Trypanosoma brucei other than J-base. We identify herein a cytosine-5 DNA methyltransferase gene and report the presence and location of 5-methylcytosine in genomic DNA. Our data demonstrate that African trypanosomes contain a functional cytosine DNA methylation pathway.

Antisense RNA and RNAi in protozoan parasites: working hard or hardly working?

The complex life cycles of many protozoan parasites require the ability to respond to environmental and developmental cues through regulated gene expression. Traditionally, parasitologists have investigated these mechanisms by identifying and characterizing proteins that are necessary for the regulated expression of the genetic material. Although often successful, it is clear that protein-mediated gene regulation is only part of a complex story in which RNA itself is endowed with regulatory functions.

Regulatory motifs uncovered among gene expression clusters in Plasmodium falciparum.

Control of gene expression is poorly understood in the Plasmodium system, where relatively few homologues to known eukaryotic transcription factors have been uncovered. Recent evidence suggests that the parasite may utilize a combinatorial mode of gene regulation, with multiple cis-acting sequences contributing to overall activity at individual promoters [1]. To further probe this mechanism of control, we first searched for over-represented sequence motifs among gene clusters sharing similar expression profiles in Plasmodium falciparum.

RNA polymerase II synthesizes antisense RNA in Plasmodium falciparum.

The recent identification of antisense RNA in the transcriptomes of many eukaryotes has generated enormous interest. The presence of antisense RNA in Plasmodium falciparum, the causative agent of severe malaria, remains controversial. Elucidation of the mechanism of antisense RNA in P.

Identification of regulatory elements in the Plasmodium falciparum genome.

There is little information regarding regulatory sequences in the newly sequenced genome of the malaria parasite, Plasmodium falciparum. Thus, for the first time, a bioinformatic strategy was utilized to identify regulatory elements in this genome using the P. falciparum heat shock protein (hsp) gene family as a model system.

Polyadenylation regulates the stability of Trypanosoma brucei mitochondrial RNAs.

Polyadenylation of RNAs plays a critical role in modulating rates of RNA turnover and ultimately in controlling gene expression in all systems examined to date. In mitochondria, the precise mechanisms by which RNAs are degraded, including the role of polyadenylation, are not well understood. Our previous in organello pulse-chase experiments suggest that poly(A) tails stimulate degradation of mRNAs in the mitochondria of the protozoan parasite Trypanosoma brucei (Militello, K.

A new reporter gene for transient transfection of Plasmodium falciparum.

Transfection of Plasmodium falciparum has remained difficult and laborious due to a lack of suitable reporter genes and low transfection efficiency. Therefore, the luciferase gene of Renilla reniformis, a sensitive mammalian reporter gene, was evaluated as a reporter gene in this system. Our studies indicate that the R.