In eukaryotes, 40S and 60S ribosomal subunits are assembled in the nucleus from rRNAs and ribosomal proteins, exported as premature complexes, and processed in final maturation steps in the cytoplasm. Ltv1 is a conserved 40S ribosome biogenesis factor that interacts with pre-40S complexes in vivo and is proposed to function in yeast in nuclear export. Cells lacking LTV1 grow slowly and are significantly impaired in mature 40S subunit production. Here we show that mutation or deletion of a putative nuclear export sequence in LTV1 is strongly dominant negative, but the protein does not accumulate in the nucleus, as expected for a mutation affecting export. In fact, most of the mutant protein is cytoplasmic and associated with pre-40S subunits. Cells expressing mutant Ltv1 have a 40S biogenesis defect, accumulate 20S rRNA in the cytoplasm as detected by FISH, and retain the late-acting biogenesis factor Tsr1 in the cytoplasm. Finally, overexpression of mutant Ltv1 is associated with nuclear retention of 40S subunit marker proteins, RpS2-GFP and RpS3-GFP. We suggest that the proximal consequence of these LTV1 mutations is inhibition of the cytoplasmic maturation of 40S subunits and that nuclear retention of pre-40S subunits is a downstream consequence of the failure to release and recycle critical factors back to the nucleus. R IBOSOME biogenesis is a major biosynthetic activity of eukaryotic cells and a significant ratelimiting factor in cell growth and proliferation. The pathway appears to be conserved in most respects in eukaryotes from yeast to humans and has been extensively analyzed in the model organism, Saccharomyces cerevisiae. Ribosome biogenesis begins cotranscriptionally in the nucleolus, continues in the nucleoplasm, and is completed in the cytoplasm where final maturation events must occur (for reviews, see More than 150 trans-acting factors have been identified by genetic and proteomic studies with roles in ribosome biogenesis. Generally, these factors are well conserved through eukaryotic evolution, but the specific function of many of the proteins remains largely unknown.In yeast, the 18S, 5.8S, and 25S ribosomal RNAs (rRNAs) are transcribed as a single 35S precursor rRNA. About 40 ribosome biogenesis factors, U3 snoRNA, and ribosomal proteins assemble cotranscriptionally on the nascent 35S pre-rRNA to form the 90S pre-ribosome (Dragon et al. 2002;Grandi et al. 2002;Schafer et al. 2003). Cleavage of the 35S pre-rRNA at site A2 releases a pre-40S particle containing 20S pre-rRNA, which then sheds most of the processing factors associated with it (Schafer et al. 2003). Pre-60S biogenesis factors and ribosomal proteins then assemble on the remaining transcript. The pre-60S subunit undergoes extensive remodeling and processing in the nucleolus and nucleoplasm, acquiring new processing factors and incorporating the independently transcribed 5S pre-rRNA before being exported through the nuclear pore to the cytoplasm (reviewed in Fromont-Racine et al. 2003;Tschochner and Hurt 2003). The pre-40S an...
Reports such as Vision and Change in Undergraduate Biology Education call for integration of course-based undergraduate research experiences (CUREs) into biology curricula and less emphasis on “cookbook” laboratories. CUREs, often characterized by a single open-ended research question, allow students to develop hypotheses, design experiments, and collaborate with peers. Conversely, “cookbook” labs incentivize task completion and have pre-determined experimental outcomes. While research comparing CUREs and “cookbook” labs is growing, there are fewer comparisons among CUREs. Here, we present a novel CURE built around an invasive grass, Bromus inermis. We evaluated this CURE’s effectiveness in improving students’ understanding of the Vision and Change competency relating to the application of the scientific process through development and testing of hypotheses. We did so by comparing changes in pre- and posttest scores on the Experimental Design Ability Test (EDAT) between Brome CURE students and students in a concurrent CURE, SEA-PHAGES. While students in both CUREs showed improvements at the end of the semester, Brome CURE students showed a greater increase in EDAT scores than did SEA-PHAGES CURE students. Additionally, Brome CURE students had significantly higher gains in 6 of the 10 EDAT criteria. We conclude that the Brome CURE is an effective ecological parallel to the SEA-PHAGES CURE and can help students gain a meaningful understanding of Vision and Change competencies. Journal of Microbiology & Biology Education
A higher-throughput microfluidic in vitro bioreactor coupled with fluorescence microscopy has been used to study bacterial biofilm growth and morphology, including Pseudomonas aeruginosa (P. aeruginosa). Here, we will describe how the system can be used to study the growth kinetics and the morphological properties such as the surface roughness and textural entropy of P. aeruginosa strain PA01 that expresses an enhanced green fluorescent protein (PA01-EGFP). A detailed protocol will describe how to grow and seed PA01-EGFP cultures, how to set up the microscope and autorun, and conduct the image analysis to determine growth rate and morphological properties using a variety of shear forces that are controlled by the microfluidic device. This article will provide a detailed description of a technique to improve the study of PA01-EGFP biofilms which eventually can be applied towards other strains of bacteria, fungi, or algae biofilms using the microfluidic platform.
A higher-throughput microfluidic in vitro bioreactor coupled with fluorescence microscopy has been used to study bacterial biofilm growth and morphology, including Pseudomonas aeruginosa (P. aeruginosa). Here, we will describe how the system can be used to study the growth kinetics and the morphological properties such as the surface roughness and textural entropy of P. aeruginosa strain PA01 that expresses an enhanced green fluorescent protein (PA01-EGFP). A detailed protocol will describe how to grow and seed PA01-EGFP cultures, how to set up the microscope and autorun, and conduct the image analysis to determine growth rate and morphological properties using a variety of shear forces that are controlled by the microfluidic device. This article will provide a detailed description of a technique to improve the study of PA01-EGFP biofilms which eventually can be applied towards other strains of bacteria, fungi, or algae biofilms using the microfluidic platform. Video LinkThe video component of this article can be found at https://www.jove.com/video/58926/ Biofilms are communities of microorganisms, such as bacteria, organized by an extracellular polymeric substance that are attached to a support, and are typically found at the interface between a liquid and a solid surface 1 . These biofilm communities can be beneficial to the environment, such as improving water quality in water supply lines and in bioremediation of recalcitrant compounds 2,3 . However, biofilms can also be highly harmful to human health with undesirable consequences. For example, medical devices, such as hip and knee implants, are one type of surface where biofilm accumulation has been a challenge and causes severe medical complications 4,5. Biofilms can also enter natural water systems, such as rivers and lakes, and infiltrate water supply pipes leading to bacteria contamination in drinking water resulting in infections 6,7,8. Biofilms formed in marine environments adhere to ships and other man-made substrates and present a major economic and environmental problem as increased friction leads to increase fuel consumption 9,10. Antimicrobial coatings, such as Tributyltin, have been developed to prevent these problems but are toxic to marine life 11 . P. aeruginosa is a Gram-negative bacterium with high thriving capabilities in a variety of environmental and nutrimental conditions 12 . P. aeruginosa is a common cause of community-and hospital-acquired infections and found to be closely associated to injuries, such as severe burns, and immunocompromised hosts, such as in cystic fibrosis (CF) 5,12,13 , AIDS, and cancer patients 5,13. The formation of P. aeruginosa biofilms has been most seriously connected to CF, where chronic lung infections are the leading cause of death for this disease 5
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