Stem cells divide both to produce new stem cells and to generate daughter cells that can differentiate. The underlying mechanisms are not well understood, but conceptually are of two kinds. Intrinsic mechanisms may control the unequal partitioning of determinants leading to asymmetric cell divisions that yield one stem cell and one differentiated daughter cell. Alternatively, extrinsic mechanisms, involving stromal cell signals, could cause daughter cells that remain in their proper niche to stay stem cells, whereas daughter cells that leave this niche differentiate. Here we use Drosophila spermatogenesis as a model stem cell system to show that there are excess stem cells and gonialblasts in testes that are deficient for Raf activity. In addition, the germline stem cell population remains active for a longer fraction of lifespan than in wild type. Finally, raf is required in somatic cells that surround germ cells. We conclude that a cell-extrinsic mechanism regulates germline stem cell behaviour.

Acetylcholine (ACh) induces repetitive, propagating intracellular Ca2+ concentration ([Ca2+]i) oscillations in porcine tracheal smooth muscle (TSM) cells. Using real-time confocal microscopy, we examined the role of sarcoplasmic reticulum (SR) Ca2+ release through inositol 1,4,5-trisphosphate (IP3) receptor and ryanodine receptor (RyR) channels in ACh-induced [Ca2+]i oscillations. In beta-escin permeabilized TSM cells, exposure to ACh in the presence of GTP also resulted in [Ca2+]i oscillations. [Ca2+]i oscillations could not be initiated by IP3 alone; however, an elevation of [Ca2+]i was observed. During ongoing [Ca2+]i oscillations, exposure to heparin, an IP3 receptor antagonist, caused a slowing of oscillation frequency but not complete inhibition. In contrast, ruthenium red, a RyR antagonist, completely abolished ACh-induced [Ca2+]i oscillations. Reverse transcriptase-polymerase chain reaction of TSM mRNA demonstrated the expression of RyR-2 and RyR-3 isoforms of the RyR. These results indicate that SR Ca2+ release through RyR channels is critical for ACh-induced [Ca2+]i oscillations in porcine TSM cells.

Snu114 is the only GTPase required for mRNA splicing. As a homolog of elongation factor G, it contains three domains (III-V) predicted to undergo a large rearrangement following GTP hydrolysis. To assess the functional importance of the domains of Snu114, we used random mutagenesis to create conditionally lethal alleles. We identified three main classes: (1) mutations that are predicted to affect GTP binding and hydrolysis, (2) mutations that are clustered in 10-to 20-amino-acid stretches in each of domains III-V, and (3) mutations that result in deletion of up to 70 amino acids from the C terminus. Representative mutations from each of these classes blocked the first step of splicing in vivo and in vitro. The growth defects caused by most alleles were synthetically exacerbated by mutations in PRP8, a U5 snRNP protein that physically interacts with Snu114, as well as in genes involved in snRNP biogenesis, including SAD1 and BRR1. The allele snu114-60, which truncates the C terminus, was synthetically lethal with factors required for activation of the spliceosome, including the DExD/H-box ATPases BRR2 and PRP28. We propose that GTP hydrolysis results in a rearrangement between Prp8 and the C terminus of Snu114 that leads to release of U1 and U4, thus activating the spliceosome for catalysis. P RE-mRNA splicing is catalyzed by the spliceosome, of GTP hydrolysis to drive rearrangements of the spliceosome (Fabrizio et al. 1997). a large dynamic complex composed of five small nuclear RNAs (snRNAs) and Ͼ80 proteins (Burge et Snu114 is packaged with other proteins and the U5 snRNA to form the U5 small ribonucleoprotein particle al. 1998; Jurica and Moore 2003). The chemistry of (snRNP). Prior to formation of the spliceosome, U5 splicing comprises two sequential transesterification resnRNP interacts with the U4/U6 di-snRNP, in which actions (Moore et al. 1993). In the first reaction, the 5Ј U4 and U6 snRNAs are extensively base paired, thus splice site is cleaved and a branched lariat structure is forming U4/U6·U5 tri-snRNP (reviewed in Burge et al. formed within the intron. In the second reaction, the 1998). According to the canonical model of spliceosome 3Ј splice site is cleaved and the two exons are joined assembly, the tri-snRNP is then recruited to the pretogether. During the splicing cycle, the RNA and prospliceosome, in which U1 snRNA is base paired with tein components of the spliceosome undergo numerous the 5Ј splice site and U2 snRNA is base paired with the rearrangements, which must be highly coordinated to branchpoint sequence, an intronic consensus sequence ensure fidelity of the process (Staley and Guthrie near the 3Ј splice site. Although the addition of tri-1998). Most of these rearrangements appear to be ensnRNP forms the complete spliceosome, this complex ergy dependent and are correlated with the activity of is catalytically inert. Activation requires that the U1/5Ј individual ATPases of the DExD/H-box family. Eight splice site interaction and the base pairing between U4 known DExD/H-box prot...

Snu114 is aU5snRNP protein essential for pre-mRNA splicing. Based on its homology with the ribosomal translocase EF-G, it is thought that GTP hydrolysis by Snu114 induces conformational rearrangements in the spliceosome. We recently identified allele-specific genetic interactions between SNU114 and genes encoding three other U5 snRNP components, Prp8 and two RNA-dependent ATPases, Prp28 and Brr2, required for destabilization of U1 and U4 snRNPs prior to catalysis. To shed more light onto the function of Snu114, we have now directly analyzed snRNP and spliceosome assembly in SNU114 mutant extracts. The Snu114-60 C-terminal truncation mutant, which is synthetically lethal with the ATPase mutants prp28-1 and brr2-1, assembles spliceosomes but subsequently blocks U4 snRNP release. Conversely, mutants in the GTPase domain fail to assemble U5 snRNPs. These mutations prevent the interaction of Snu114 with Prp8 as well as with U5 snRNA. Since Prp8 is thought to regulate the activity of the DEAD-box ATPases, this strategy of snRNP assembly could ensure that Prp8 activity is itself regulated by aG TP-dependent mechanism.

BackgroundPlant biotechnology can be leveraged to produce food, fuel, medicine, and materials. Standardized methods advocated by the synthetic biology community can accelerate the plant design cycle, ultimately making plant engineering more widely accessible to bioengineers who can contribute diverse creative input to the design process.ResultsThis paper presents work done largely by undergraduate students participating in the 2010 International Genetically Engineered Machines (iGEM) competition. Described here is a framework for engineering the model plant Arabidopsis thaliana with standardized, BioBrick compatible vectors and parts available through the Registry of Standard Biological Parts (http://www.partsregistry.org). This system was used to engineer a proof-of-concept plant that exogenously expresses the taste-inverting protein miraculin.ConclusionsOur work is intended to encourage future iGEM teams and other synthetic biologists to use plants as a genetic chassis. Our workflow simplifies the use of standardized parts in plant systems, allowing the construction and expression of heterologous genes in plants within the timeframe allotted for typical iGEM projects.

In the past ten years, increasing evidence has demonstrated that scientific teaching and active learning improve student retention and learning gains in the sciences. Graduate teaching assistants (GTAs), who play an important role in undergraduate education at many universities, require training in these methods to encourage implementation, long-term adoption, and advocacy. Here, we describe the design and evaluation of a two-day training workshop for first-year GTAs in the life sciences. This workshop combines instruction in current research and theory supporting teaching science through active learning as well as opportunities for participants to practice teaching and receive feedback from peers and mentors. Postworkshop assessments indicated that GTA participants' knowledge of key topics increased during the workshop. In follow-up evaluations, participants reported that the workshop helped them prepare for teaching. This workshop design can easily be adapted to a wide range of science disciplines. Overall, the workshop prepares graduate students to engage, include, and support undergraduates from a variety of backgrounds when teaching in the sciences. © 2018 by The International Union of Biochemistry and Molecular Biology, 46:318-326, 2018.

No abstract