A Genomics Education Alliance

Scientists are sequencing new genomes at an increasing rate with the goal of associating genome contents with phenotypic traits. After a new genome is sequenced and assembled, structural gene annotation is often the first step in analysis. Despite advances in computational gene prediction algorithms, most eukaryotic genomes still benefit from manual gene annotation. Undergraduates can become skilled annotators, and in the process learn both about genes/genomes and about how to utilize large datasets. Data visualizations provided by a genome browser are essential for manual gene annotation, enabling annotators to quickly evaluate multiple lines of evidence (e.g., sequence similarity, RNA-Seq, gene predictions, repeats). However, creating genome browsers requires extensive computational skills; lack of the expertise required remains a major barrier for many biomedical researchers and educators. To address these challenges, the Genomics Education Partnership (GEP; https://gep.wustl.edu/) has partnered with the Galaxy Project (https://galaxyproject.org) to develop G-OnRamp (http://g-onramp.org), a web-based platform for creating UCSC Assembly Hubs and JBrowse genome browsers. G-OnRamp can also convert a JBrowse instance into an Apollo instance for collaborative genome annotations in research and educational settings. G-OnRamp enables researchers to easily visualize their experimental results, educators to create Course-based Undergraduate Research Experiences (CUREs) centered on genome annotation, and students to participate in genomics research. Development of G-OnRamp was guided by extensive user feedback from in-person workshops. Sixty-five researchers and educators from over 40 institutions participated in these workshops, which produced over 20 genome browsers now available for research and education. For example, genome browsers for four parasitoid wasp species were used in a CURE engaging 142 students taught by 13 faculty members —producing a total of 192 gene models. G-OnRamp can be deployed on a personal computer or on cloud computing platforms, and the genome browsers produced can be transferred to the CyVerse Data Store for long-term access

Diacylglycerol-Stimulated Endocytosis of Transferrin in Trypanosomatids Is Dependent on Tyrosine Kinase Activity

Small molecule regulation of cell function is an understudied area of trypanosomatid biology. In Trypanosoma brucei diacylglycerol (DAG) stimulates endocytosis of transferrin (Tf). However, it is not known whether other trypanosomatidae respond similarly to the lipid. Further, the biochemical pathways involved in DAG signaling to the endocytic system in T. brucei are unknown, as the parasite genome does not encode canonical DAG receptors (e.g. C1-domains). We established that DAG stimulates endocytosis of Tf in Leishmania major, and we evaluated possible effector enzymes in the pathway with multiple approaches. First, a heterologously expressed glycosylphosphatidylinositol phospholipase C (GPI-PLC) activated endocytosis of Tf 300% in L. major. Second, exogenous phorbol ester and DAGs promoted Tf endocytosis in L. major. In search of possible effectors of DAG signaling, we discovered a novel C1-like domain (i.e. C1_5) in trypanosomatids, and we identified protein Tyr kinases (PTKs) linked with C1_5 domains in T. brucei, T. cruzi, and L. major. Consequently, we hypothesized that trypanosome PTKs might be effector enzymes for DAG signaling. General uptake of Tf was reduced by inhibitors of either Ser/Thr or Tyr kinases. However, DAG-stimulated endocytosis of Tf was blocked only by an inhibitor of PTKs, in both T. brucei and L. major. We conclude that (i) DAG activates Tf endocytosis in L. major, and that (ii) PTKs are effectors of DAG-stimulated endocytosis of Tf in trypanosomatids. DAG-stimulated endocytosis of Tf may be a T. brucei adaptation to compete effectively with host cells for vertebrate Tf in blood, since DAG does not enhance endocytosis of Tf in human cells

A Ser/Thr kinase Inhibitor does not block DAG-activated endocytosis of Tf in <i>T. brucei</i> or <i>L. major</i>.

<p>Bloodstream <i>T. brucei</i> (5×10⁶ cells) <i>(</i><b><i>A</i></b><i>)</i> or <i>L. major</i> promastigotes (1×10⁶ cells) <i>(</i><b><i>C</i></b><i>)</i> were incubated with DMSO (vehicle) or different amounts of Ro32-0432 for 10 min at 37°C (for <i>T. brucei</i>) or 27°C (for <i>L. major</i>). Subsequently, endocytosis of Tf was measured as described earlier. <i>T. brucei (</i><b><i>B</i></b><i>)</i> or <i>L. major (</i><b><i>D</i></b><i>)</i> was incubated in medium containing Ro32-0432 (500 nM) for 10 min (i.e., Stage I). Cells were then exposed to PMA (500 nM) (Stage II) for another 10 min, and endocytosis of Tf was measured. A representative experiment is presented. Data plotted are means (with standard deviations) of triplicate determinations.</p

Trypanosoma brucei: Reduction of GPI-phospholipase C protein during differentiation is dependent on replication of newly transformed cells

The protozoan parasite Trypanosoma brucei lives in the bloodstream of vertebrates or in a tsetse fly. Expression of a GPI-phospholipase C polypeptide (GPI-PLCp) in the parasite is restricted to the bloodstream form. Events controlling the amount of GPI-PLCp expressed during differentiation are not completely understood. Our metabolic “pulse-chase” analysis reveals that GPI-PLCp is stable in bloodstream form. However, during differentiation of bloodstream to insect stage (procyclic) T. brucei, translation GPI-PLC mRNA ceases within 8 h of initiating transformation. GPI-PLCp is not lost precipitously from newly-transformed procyclic trypanosomes. Nascent procyclics contain 400-fold more GPI-PLCp than established insect stage T. brucei. Reduction of GPI-PLCp in early-stage procyclics is linked to parasite replication. Sixteen cell divisions are required to reduce the amount of GPI-PLCp in newly-differentiated procyclics to levels present in the established procyclic. GPI-PLCp is retained in strains of T. brucei that fail to replicate after differentiation of the bloodstream to the procyclic form. Thus, at least two factors control levels of GPI-PLCp during differentiation of bloodstream T. brucei; (i) repression of GPI-PLC mRNA translation, and (ii) sustained replication of newly-transformed procyclic T. brucei. These studies illustrate the importance of repeated cell divisions in controlling the steady-state amount of GPI-PLCp during differentiation of the African trypanosome

A Working Model for DAG Activation of Tf Endocytosis in Trypanosomatids.

<p>Based on our biochemical (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008538#pone-0008538-g001" target="_blank">Figs. 1</a> through <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008538#pone-0008538-g006" target="_blank">Fig. 6</a>), bioinformatic (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008538#pone-0008538-g006" target="_blank">Fig. 6</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008538#pone-0008538-t001" target="_blank">Table 1</a>), and pharmacological data (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008538#pone-0008538-g007" target="_blank">Fig. 7</a>), we propose that DAG binds to a C1_5 domain of a PTK in <i>T. brucei</i> (or <i>L. major</i>). The trypanosomatid PTK is activated by DAG, and the enzyme phosphorylates components of the endosomal pathway to activate uptake of Tf.</p

A PTK inhibitor blocks phorbol ester-stimulated endocytosis of Tf in <i>T. brucei</i> and <i>L. major</i>.

<p><i>(</i><b><i>A</i></b><i>) T. brucei</i> (5×10⁶) were incubated with varying concentrations of Tyrphostin A47 for 10 min. Parasites were rinsed, and endocytosis of Tf measured (see legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008538#pone-0008538-g001" target="_blank">Fig. 1</a> for protocol). <i>(</i><b><i>B</i></b><i>) T. brucei</i> (5×10⁶ cells) was incubated in medium containing Tyrphostin A47 (TphA47) (7.5 µM) for 10 min (37°C) (i.e. Stage I). Cells were then exposed to PMA (500 nM) (Stage II) for another 10 min, and endocytosis of Tf was measured. <i>(</i><b><i>C</i></b><i>) L. major</i> (5×10⁶) were incubated with varying concentrations of Tyrphostin A47 for 10 min. Parasites were rinsed, and endocytosis of Tf measured (see legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008538#pone-0008538-g001" target="_blank">Fig. 1</a> for protocol). <i>(</i><b><i>D</i></b><i>) L major</i> (5×10⁶/ml) was treated with Tyrphostin A47 (5 µM) for 15 min in culture medium. Thereafter, cells were incubated with PMA (500 nM; final conc.) for 15 min, and endocytosis of Tf was measured as described the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0008538#pone-0008538-g001" target="_blank">Figure 1</a>.</p

Enzyme activity is important for GPI-PLCp stimulation of Tf Endocytosis in <i>L. major</i>.

<p><i>L. major</i> pUTK/GPIPLC-Q81L and pUTK-GPIPLC were cultured in 50 µg/ml G418 and allowed to endocytose transferrin-Alexa Fluor 594 at 27°C for indicated time intervals. Cell-associated transferrin is plotted as relative fluorescence units.</p