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Edgell, MH, Hardies SC, Loeb DD, Shehee WR, Padgett RW, Burton FH, Comer MB, Casavant NC, Funk FD, Hutchison CA.  1987.  The L1 family in mice. Progress in clinical and biological research. 251:107-29.Website
Irvine, KD, Helfand SL, Hogness DS.  1991.  The large upstream control region of the Drosophila homeotic gene Ultrabithorax. Development. 111:407-24. AbstractWebsite
Ultrabithorax (Ubx) is a Drosophila homeotic gene that determines the segmental identities of parts of the thorax and abdomen. Appropriate Ubx transcription requires a long upstream control region (UCR) that is defined genetically by the bithoraxoid (bxd) and postbithorax (pbx) subfunction mutations. We have directly analyzed UCR functions by the examination of beta-galactosidase expression in flies containing Ubx-lacZ fusion genes. 35 kb of UCR DNA confers upon beta-galactosidase an expression pattern that closely parallels normal Ubx expression throughout development. In contrast, 22 kb of UCR DNA confers fewer features of normal Ubx expression, and with 5 kb of UCR DNA the expression pattern has no resemblance to Ubx expression except in the visceral mesoderm. We have also shown that bxd chromosome breakpoint mutants form a comparable 5' deletion series in which the severity of the effect on Ubx expression correlates with the amount of upstream DNA remaining in the mutant. In Ubx-lacZ fusions containing 22 kb of UCR DNA, and in comparable bxd mutants, there is a persistent pair-rule pattern of metameric expression in early development, demonstrating that there are distinct mechanisms with different sequence requirements for the initial activation of Ubx in different metameres. The correction of this pair-rule pattern later in embryogenesis shows that there are also distinct mechanisms for the activation of Ubx at different times during development.
Radford, SJ, Hoang TL, Gluszek AA, Ohkura H, McKim KS.  2015.  Lateral and End-On Kinetochore Attachments Are Coordinated to Achieve Bi-orientation in Drosophila Oocytes. PLoS Genetics. 11(10):e1005605.Website
Dismukes, CG, McNeely K, Eng JF.  2011.  An LC-MS-based chemical and analytical method for targeted metabolite quantification in the model cyanobacterium Synechococcus sp. PCC 7002.. Analytical chemistry. 83(10):3808-16. Abstract
Herein, we detail the development of a method for the chemical isolation and tandem LC-MS/MS quantification of a targeted subset of internal metabolites from cyanobacteria. We illustrate the selection of target compounds; requirements for and optimization of mass spectral detection channels, screening, and optimization of chromatography; and development of a sampling protocol that seeks to achieve complete, representative, and stable metabolite extraction on the seconds time scale. Several key factors influencing the separation by reversed-phase ion pairing chromatography, specifically the hydrophobicity of the sample matrix and sensitivity to mobile phase acidity, are identified and resolved. We illustrate this methodology with an example from the autofermentative metabolism in the model cyanobacterium Synechococcus sp. PCC 7002, for which intracellular levels of 25 metabolites were monitored over 48 h, including intermediates in central carbon metabolism together with those involved in the cellular energy charge and redox poise. Upon removal of alternative reductant sinks (nitrate), anoxia induces autofermentation of carbohydrates with a parallel rise in the intracellular pyridine nucleotide redox poise that is specific to NAD(H) and alongside a gradual decline in the adenylate energy charge. This LC-MS/MS-based method provides for accurate time-resolved quantification of multiple metabolites in parallel, thus enabling experimental verification of the active metabolic pathways.
Rongo, C, Whitfield CW, Rodal A, Kim SK, Kaplan JM.  1998.  LIN-10 is a shared component of the polarized protein localization pathways in neurons and epithelia. Cell. 94:751-9. AbstractWebsite
We tested the model that neurons and epithelial cells use a shared mechanism for polarized protein sorting by comparing the pathways for localizing basolateral and postsynaptic proteins in C. elegans. GLR-1 glutamate receptors are localized to postsynaptic elements of central synapses and, when ectopically expressed, to basolateral membranes of epithelial cells. Proper localization of GLR-1 in both neurons and epithelia requires the PDZ protein LIN-10, defining LIN-10 as a shared component of the basolateral and postsynaptic localization pathways. Changing the GLR-1 carboxy-terminal sequence from a group I PDZ-binding consensus (-TAV) to a group II consensus (-FYV) restores GLR-1 synaptic localization in lin-10 mutants. Thus, these interneurons utilize at least two separate postsynaptic localization pathways.
Major, RJ, Irvine KD.  2006.  Localization and requirement for Myosin II at the dorsal-ventral compartment boundary of the Drosophila wing. Developmental dynamics : an official publication of the American Association of Anatomists. 235:3051-8. AbstractWebsite
As organisms develop, their tissues can become separated into distinct cell populations through the establishment of compartment boundaries. Compartment boundaries have been discovered in a wide variety of tissues, but in many cases the molecular mechanisms that separate cells remain poorly understood. In the Drosophila wing, a stripe of Notch activation maintains the dorsal-ventral compartment boundary, through a process that depends on the actin cytoskeleton. Here, we show that the dorsal-ventral boundary exhibits a distinct accumulation of Myosin II, and that this accumulation is regulated downstream of Notch signaling. Conversely, the dorsal-ventral boundary is depleted for the Par-3 homologue Bazooka. We further show that mutations in the Myosin heavy chain subunit encoded by zipper can impair dorsal-ventral compartmentalization without affecting anterior-posterior compartmentalization. These observations identify a distinct accumulation and requirement for Myosin activity in dorsal-ventral compartmentalization, and suggest a novel mechanism in which contractile tension along an F-actin cable at the compartment boundary contributes to compartmentalization.
Kim, J, Bortz E, Zhong H, Leeuw T, Leberer E, Vershon AK, Hirsch JP.  2000.  Localization and Signaling of G(beta) Subunit Ste4p are Controlled by A-factor Receptor and the A-specific Protein Asg7p. Mol Cell Biol. 20:8826-8835. Abstract
Haploid yeast cells initiate pheromone signaling upon the binding of pheromone to its receptor and activation of the coupled G protein. A regulatory process termed receptor inhibition blocks pheromone signaling when the a-factor receptor is inappropriately expressed in MATa cells. Receptor inhibition blocks signaling by inhibiting the activity of the G protein beta subunit, Ste4p. To investigate how Ste4p activity is inhibited, its subcellular location was examined. In wild-type cells, alpha-factor treatment resulted in localization of Ste4p to the plasma membrane of mating projections. In cells expressing the a-factor receptor, alpha-factor treatment resulted in localization of Ste4p away from the plasma membrane to an internal compartment. An altered version of Ste4p that is largely insensitive to receptor inhibition retained its association with the membrane in cells expressing the a-factor receptor. The inhibitory function of the a-factor receptor required ASG7, an a-specific gene of previously unknown function. ASG7 RNA was induced by pheromone, consistent with increased inhibition as the pheromone response progresses. The a-factor receptor inhibited signaling in its liganded state, demonstrating that the receptor can block the signal that it initiates. ASG7 was required for the altered localization of Ste4p that occurs during receptor inhibition, and the subcellular location of Asg7p was consistent with its having a direct effect on Ste4p localization. These results demonstrate that Asg7p mediates a regulatory process that blocks signaling from a G protein beta subunit and causes its relocalization within the cell.
Sun, S, Reddy BVVG, Irvine KD.  2015.  Localization of Hippo Signaling complexes and Warts activation in vivo. Nature Communications. 6:8402.
Rongo, C, Gavis ER, Lehmann R.  1995.  Localization of oskar RNA regulates oskar translation and requires Oskar protein. Development. 121:2737-46. AbstractWebsite
The site of oskar RNA and protein localization within the oocyte determines where in the embryo primordial germ cells form and where the abdomen develops. Initiation of oskar RNA localization requires the activity of several genes. We show that ovaries mutant for any of these genes lack Oskar protein. Using various transgenic constructs we have determined that sequences required for oskar RNA localization and translational repression map to the oskar 3'UTR, while sequences involved in the correct temporal activation of translation reside outside the oskar 3'UTR. Upon localization of oskar RNA and protein at the posterior pole, Oskar protein is required to maintain localization of oskar RNA throughout oogenesis. Stable anchoring of a transgenic reporter RNA at the posterior pole is disrupted by oskar nonsense mutations. We propose that initially localization of oskar RNA permits translation into Oskar protein and that subsequently Oskar protein regulates its own RNA localization through a positive feedback mechanism.
Tang, H, Severinov K, Goldfarb A, Fenyo D, Chait B, Ebright RH.  1994.  Location, structure, and function of the target of a transcriptional activator protein.. Genes & development. 8(24):3058-67. Abstract
We have isolated and characterized single-amino-acid substitution mutants of RNA polymerase alpha subunit defective in CAP-dependent transcription at the lac promoter but not defective in CAP-independent transcription. Our results establish that (1) amino acids 258-265 of alpha constitute an "activation target" essential for CAP-dependent transcription at the lac promoter but not essential for CAP-independent transcription, (2) amino acid 261 is the most critical amino acid of the activation target, (3) amino acid 261 is distinct from the determinants for alpha-DNA interaction, and (4) the activation target may fold as a surface amphipathic alpha-helix. We propose a model for transcriptional activation at the lac promoter that integrates these and other recent results regarding transcriptional activation and RNA polymerase structure and function.
Xu, J-H, Wang R, Li X, Miclaus M, Messing J.  2016.  Locus- and Site-Specific DNA Methylation of 19 kDa Zein Genes in Maize.. PloS one. 11(1):e0146416. Abstract
An interesting question in maize development is why only a single zein gene is highly expressed in each of the 19-kDa zein gene clusters (A and B types), z1A2-1 and z1B4, in the immature endosperm. For instance, epigenetic marks could provide a structural difference. Therefore, we investigated the DNA methylation of the arrays of gene copies in both promoter and gene body regions of leaf (non-expressing tissue as a control), normal endosperm, and cultured endosperm. Although we could show that expressed genes have much lower methylation levels in promoter regions than silent ones in both leaf and normal endosperm, there was surprisingly also a difference in the pattern of the z1A and z1B gene clusters. The expression of z1B gene is suppressed by increased DNA methylation and activated with reduced DNA methylation, whereas z1A gene expression is not. DNA methylation in gene coding regions is higher in leaf than in endosperm, whereas no significant difference is observed in gene bodies between expressed and non-expressed gene copies. A median CHG methylation (25-30%) appears to be optimal for gene expression. Moreover, tissue-cultured endosperm can reset the DNA methylation pattern and tissue-specific gene expression. These results reveal that DNA methylation changes of the 19-kDa zein genes is subject to plant development and tissue culture treatment, but varies in different chromosomal locations, indicating that DNA methylation changes do not apply to gene expression in a uniform fashion. Because tissue culture is used to produce transgenic plants, these studies provide new insights into variation of gene expression of integrated sequences.
Maduzia, L, Gumienny T, Zimmerman C, Wang H, Shetgiri P, Krishna S, Roberts A, Padgett R.  2002.  Lon-1 regulates Caenorhabditis elegans body size downstream of the Dbl-1 TGFβ signaling pathway. Dev Biol. 246:418-428. AbstractWebsite
In Caenorhabditis elegans, two well-characterized TGF beta signaling cascades have been identified: the Small/Male tail abnormal (Sma/Mab) and Dauer formation (Daf) pathways. The Sma/Mab pathway regulates body size morphogenesis and male tail development. The ligand of the pathway, dbl-1, transmits its signal through two receptor serine threonine kinases, daf-4 and sma-6, which in turn regulate the activity of the Smads, sma-2, sma-3, and sma-4. In general, Smads have been shown to both positively and negatively regulate the transcriptional activity of downstream target genes in various organisms. In C. elegans, however, target genes have remained elusive. We have cloned and characterized lon-1, a gene with homology to the cysteine-rich secretory protein (CRISP) family of proteins. lon-1 regulates body size morphogenesis, but does not affect male tail development. lon-1 is expressed in hypodermal tissues, which is the focus of body size determination, similar to sma-2, sma-4, and sma-6. Using genetic methods, we show that lon-1 lies downstream of the Sma/Mab signaling cascade and demonstrate that lon-1 mRNA levels are up-regulated in sma-6-null mutant animals. This provides evidence that lon-1 is negatively regulated by Sma/Mab pathway signaling. Taken together, these data identify lon-1 as a novel downstream target gene of the dbl-1 TGF beta-like signaling pathway.
Ebright, RH, Gunasekera A, Zhang XP, Kunkel TA, Krakow JS.  1990.  Lysine 188 of the catabolite gene activator protein (CAP) plays no role in specificity at base pair 7 of the DNA half site.. Nucleic acids research. 18(6):1457-64. Abstract
Two similar, but not identical, models have been proposed for the amino acid-base pair contacts in the CAP-DNA complex ('model I,' Weber, I. and Steitz, T., Proc. Natl. Acad. Sci. USA, 81, 3973-3977, 1984; 'model II,' Ebright, et al., Proc. Natl. Acad. Sci. USA, 81, 7274-7278, 1984). The most important difference between the two models involves Lys188 of CAP. Model I predicts that Lys188 of CAP makes a specificity determining contact with base pair 7 of the DNA half site. In contrast, model II predicts that Lys188 makes no contact with base pair 7 of the DNA half site. In the present work, we have used site-directed mutagenesis to replace Lys188 of CAP by Asn, an amino acid unable to make the putative contact. We have assessed the specificities of the following proteins, both in vitro and in vivo: wild-type CAP, [Asn188]CAP, [Val181]CAP, and [Val181;Asn188]CAP. The results indicate that Lys188 makes no contribution to specificity at base pair 7 of the DNA half site. We propose, contrary to model I, that Lys188 makes no contact with base pair 7 of the DNA half site.