Showing 241 - 250 of 257 Items
Investigating the Role of Heterochromatin Protein 1 (HP1) in the Formation of Chromosome Compartments Access to this record is restricted to members of the Bowdoin community. Log in here to view.
- Restriction End Date: 2025-06-01
Date: 2020-01-01
Creator: Diego Andres Villamarin
Access: Access restricted to the Bowdoin Community
Comparing enhancer action in cis and in trans
Date: 2012-08-01
Creator: Jack R. Bateman, Justine E. Johnson, Melissa N. Locke
Access: Open access
- Studies from diverse systems have shown that distinct interchromosomal interactions are a central component of nuclear organization. In some cases, these interactions allow an enhancer to act in trans, modulating the expression of a gene encoded on a separate chromosome held in close proximity. Despite recent advances in uncovering such phenomena, our understanding of how a regulatory element acts on another chromosome remains incomplete. Here, we describe a transgenic approach to better understand enhancer action in trans in Drosophila melanogaster. Using phiC31-based recombinase-mediated cassette exchange (RMCE), we placed transgenes carrying combinations of the simple enhancer GMR, a minimal promoter, and different fluorescent reporters at equivalent positions on homologous chromosomes so that they would pair via the endogenous somatic pairing machinery of Drosophila. Our data demonstrate that the enhancer GMR is capable of activating a promoter in trans and does so in a variegated pattern, suggesting stochastic interactions between the enhancer and the promoter when they are carried on separate chromosomes. Furthermore, we quantitatively assessed the impact of two concurrent promoter targets in cis and in trans to GMR, demonstrating that each promoter is capable of competing for the enhancer's activity, with the presence of one negatively affecting expression from the other. Finally, the single-cell resolution afforded by our approach allowed us to show that promoters in cis and in trans to GMR can both be activated in the same nucleus, implying that a single enhancer can share its activity between multiple promoter targets carried on separate chromosomes. © 2012 by the Genetics Society of America.
Direct selection for sequences encoding proteases of known specificity
Date: 1991-06-15
Creator: Tracy A. Smith, Bruce D. Kohorn
Access: Open access
- We have developed a simple genetic selection that could be used to isolate eukaryotic cDNAs encoding proteases that cleave within a defined amino acid sequence. The selection was developed by using the transcription factor GAL4 from Saccharomyces cerevisiae as a selectable marker, a cloned protease from tobacco etch virus (TEV), and an 18-amino acid TEV protease target sequence. In yeast, TEV protease cleaves its target even when the target is fused to internal regions of the GAL4 protein. This cleavage separates the DNA binding domain from the transcription activation domain of GAL4, rendering it transcriptionally inactive. The proteolytic cleavage can be detected phenotypically by the inability of cells to metabolize galactose. Cells expressing the TEV protease can also be selected on the suicide substrate 2-deoxygalactose. DNA binding studies show that the TEV protease decreases the activity of the GAL4/target fusion protein. Because another protease target sequence of 55 amino acids can be inserted into GAL4 without any loss of transcriptional activity, this assay offers the opportunity to use high-efficiency cDNA cloning and expression vectors to select coding sequences of other proteases from various species. The assay could also be used to help define both target specificities and functional domains of proteases.
Mutations in a signal sequence for the thylakoid membrane identify multiple protein transport pathways and nuclear suppressors
Date: 1994-07-01
Creator: Tracy A. Smith, Bruce D. Kohorn
Access: Open access
- The apparatus that permits protein translocation across the internal thylakoid membranes of chloroplasts is completely unknown, even though these membranes have been the subject of extensive biochemical analysis. We have used a genetic approach to characterize the translocation of Chlamydomonas cytochrome f, a chloroplast-encoded protein that spans the thylakoid once. Mutations in the hydrophobic core of the cytochrome f signal sequence inhibit the accumulation of cytochrome f, lead to an accumulation of precursor, and impair the ability of Chlamydomonas cells to grow photosynthetically. One hydrophobic core mutant also reduces the accumulation of other thylakoid membrane proteins, but not those that translocate completely across the membrane. These results suggest that the signal sequence of cytochrome f is required and is involved in one of multiple insertion pathways. Suppressors of two signal peptide mutations describe at least two nuclear genes whose products likely describe the translocation apparatus, and selected second- site chloroplast suppressors further define regions of the cytochrome f signal peptide.
Xanthophyll cycle activity in two prominent arctic shrub species
Date: 2017-01-01
Creator: T.S. Magney, B.A. Logan, J.S. Reblin, N.T. Boelman, J.U.H., Eitel, H.E. Greaves, K.L. Griffin, C.M. Prager, L.A. Vierling
Access: Open access
Human protein Sam68 relocalization and interaction with poliovirus RNA polymerase in infected cells
Date: 1996-03-19
Creator: A. E. Mcbride, A. Schlegel, K. Kirkegaard
Access: Open access
- A HeLa cDNA expression library was screened for human polypeptides that interacted with the poliovirus RNA-dependent RNA polymerase, 3D, using the two-hybrid system in the yeast Saccharomyces cerevisiae. Sam68 (Src- associated in mitosis, 68 kDa) emerged as the human cDNA that, when fused to a transcriptional activation domain, gave the strongest 3D interaction signal with a LexA-3D hybrid protein. 3D polymerase and Sam68 coimmunoprecipitated from infected human cell lysates with antibodies that recognized either protein. Upon poliovirus infection. Sam68 relocalized from the nucleus to the cytoplasm, where poliovirus replication occurs. Sam68 was isolated from infected cell lysates with an antibody that recognizes poliovirus protein 2C, suggesting that it is found on poliovirus-induced membranes upon which viral RNA synthesis occurs. These data, in combination with the known RNA- and protein-binding properties of Sam68, make Sam68 a strong candidate for a host protein with a functional role in poliovirus replication.
Identification of methylated proteins in the yeast small ribosomal subunit: A role for SPOUT methyltransferases in protein arginine methylation
Date: 2012-06-26
Creator: Brian D. Young, David I. Weiss, Cecilia I. Zurita-Lopez, Kristofor J. Webb, Steven G., Clarke, Anne E. McBride
Access: Open access
- We have characterized the posttranslational methylation of Rps2, Rps3, and Rps27a, three small ribosomal subunit proteins in the yeast Saccharomyces cerevisiae, using mass spectrometry and amino acid analysis. We found that Rps2 is substoichiometrically modified at arginine-10 by the Rmt1 methyltransferase. We demonstrated that Rps3 is stoichiometrically modified by ω- monomethylation at arginine-146 by mass spectrometric and site-directed mutagenic analyses. Substitution of alanine for arginine at position 146 is associated with slow cell growth, suggesting that the amino acid identity at this site may influence ribosomal function and/or biogenesis. Analysis of the three-dimensional structure of Rps3 in S. cerevisiae shows that arginine-146 makes contacts with the small subunit rRNA. Screening of deletion mutants encoding potential yeast methyltransferases revealed that the loss of the YOR021C gene results in the absence of methylation of Rps3. We demonstrated that recombinant Yor021c catalyzes ω-monomethylarginine formation when incubated with S-adenosylmethionine and hypomethylated ribosomes prepared from a YOR021C deletion strain. Interestingly, Yor021c belongs to the family of SPOUT methyltransferases that, to date, have only been shown to modify RNA substrates. Our findings suggest a wider role for SPOUT methyltransferases in nature. Finally, we have demonstrated the presence of a stoichiometrically methylated cysteine residue at position 39 of Rps27a in a zinc-cysteine cluster. The discovery of these three novel sites of protein modification within the small ribosomal subunit will now allow for an analysis of their functional roles in translation and possibly other cellular processes. © 2012 American Chemical Society.
Protein arginine methylation in Candida albicans: Role in nuclear transport
Date: 2007-07-01
Creator: Anne E. McBride, Cecilia Zurita-Lopez, Anthony Regis, Emily Blum, Ana, Conboy, Shannon Elf, Steven Clarke
Access: Open access
- Protein arginine methylation plays a key role in numerous eukaryotic processes, such as protein transport and signal transduction. In Candida albicans, two candidate protein arginine methyltransferases (PRMTs) have been identified from the genome sequencing project. Based on sequence comparison, C. albicans candidate PRMTs display similarity to Saccharomyces cerevisiae Hmt1 and Rmt2. Here we demonstrate functional homology of Hmt1 between C. albicans and S. cerevisiae: CaHmt1 supports growth of S. cerevisiae strains that require Hmt1, and CaHmt1 methylates Npl3, a major Hmt1 substrate, in S. cerevisiae. In C. albicans strains lacking CaHmt1, asymmetric dimethylarginine and ω-monomethylarginine levels are significantly decreased, indicating that Hmt1 is the major C. albicans type I PRMT1. Given the known effects of type I PRMTs on nuclear transport of RNA-binding proteins, we tested whether Hmt1 affects nuclear transport of a putative Npl3 ortholog in C. albicans. CaNpl3 allows partial growth of S. cerevisiae npl3Δ strains, but its arginine-glycine-rich C terminus can fully substitute for that of ScNpl3 and also directs methylation-sensitive association with ScNpl3. Expression of green fluorescent protein-tagged CaNpl3 proteins in C. albicans strains with and without CaHmt1 provides evidence for CaHmt1 facilitating export of CaNpl3 in this fungus. We have also identified the C. albicans Rmt2, a type IV fungus- and plant-specific PRMT, by amino acid analysis of an rmt2Δ/rmt2Δ strain, as well as biochemical evidence for additional cryptic PRMTs. Copyright © 2007, American Society for Microbiology. All Rights Reserved.
Specific sequences within arginine-glycine-rich domains affect mRNA-binding protein function
Date: 2009-08-05
Creator: Anne E. McBride, Ana K. Conboy, Shanique P. Brown, Chaiyaboot Ariyachet, Kate L., Rutledge
Access: Open access
- The discovery of roles for arginine methylation in intracellular transport and mRNA splicing has focused attention on the methylated arginine-glycine (RG)-rich domains found in many eukaryotic RNA-binding proteins. Sequence similarity among these highly repetitive RG domains, combined with interactions between RG-rich proteins, raises the question of whether these regions are general interaction motifs or whether there is specificity within these domains. Using the essential Saccharomyces cerevisiae mRNA-binding protein Npl3 (ScNpl3) as a model system, we first tested the importance of the RG domain for protein function. While Npl3 lacking the RG domain could not support growth of cells lacking Npl3, surprisingly, expression of the RG domain alone supported partial growth of these cells. To address the specificity of this domain, we created chimeric forms of ScNpl3 with RG-rich domains of S. cerevisiae nucleolar proteins, Gar1 and Nop1 (ScGar1, ScNop1), or of the Candida albicans Npl3 ortholog (CaNpl3). Whereas the CaNpl3 RG chimeric protein retained nearly wild-type function in S. cerevisiae, the ScGar1 and ScNop1 RG domains significantly reduced Npl3 function and self-association, indicating RG domain specificity. Nuclear localization of Npl3 also requires specific RG sequences, yet heterologous RG domains allow similar modulation of Npl3 transport by arginine methylation.
Long-term maintenance of channel distribution in a central pattern generator neuron by neuromodulatory inputs revealed by decentralization in organ culture
Date: 2001-09-15
Creator: Adi Mizrahi, Patsy S. Dickinson, Peter Kloppenburg, Valerie Fénelon, Deborah J., Baro, Ronald M. Harris-Warrick, Pierre Meyrand, John Simmers
Access: Open access
- Organotypic cultures of the lobster (Homarus gammarus) stomatogastric nervous system (STNS) were used to assess changes in membrane properties of neurons of the pyloric motor pattern-generating network in the long-term absence of neuromodulatory inputs to the stomatogastric ganglion (STG). Specifically, we investigated decentralization-induced changes in the distribution and density of the transient outward current, IA, which is encoded within the STG by the shal gene and plays an important role in shaping rhythmic bursting of pyloric neurons. Using an antibody against lobster shal K+ channels, we found shal immunoreactivity in the membranes of neuritic processes, but not somata, of STG neurons in 5 d cultured STNS with intact modulatory inputs. However, in 5 d decentralized STG, shal immunoreactivity was still seen in primary neurites but was likewise present in a subset of STG somata. Among the neurons displaying this altered shal localization was the pyloric dilator (PD) neuron, which remained rhythmically active in 5 d decentralized STG. Two-electrode voltage clamp was used to compare IA in synaptically isolated PD neurons in long-term decentralized STG and nondecentralized controls. Although the voltage dependence and kinetics of IA changed little with decentralization, the maximal conductance of IA in PD neurons increased by 43.4%. This increase was consistent with the decentralization-induced increase in shal protein expression, indicating an alteration in the density and distribution of functional A-channels. Our results suggest that, in addition to the short-term regulation of network function, modulatory inputs may also play a role, either directly or indirectly, in controlling channel number and distribution, thereby maintaining the biophysical character of neuronal targets on a long-term basis.