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Krishnan, A, Zhang S, Liu Y, Tadmori KA, Bryant DA, Dismukes GC.  2016.  Consequences of ccmR deletion on respiration, fermentation and H2 metabolism in cyanobacterium Synechococcus sp. PCC 7002. Biotechnol Bioeng. Abstract
CcmR, a LysR-type transcriptional regulator, represses the genes encoding components of the high-affinity carbon concentration mechanism in cyanobacteria. Unexpectedly, deletion of the ccmR gene was found to alter the expression of the terminal oxidase and fermentative genes, especially the hydrogenase operon in the cyanobacterium Synechococcus sp. PCC 7002. Consistent with the transcriptomic data, the deletion strain exhibits flux increases (30-50%) in both aerobic O2 respiration and anaerobic H2 evolution. To understand how CcmR influences anaerobic metabolism, the kinetics of autofermentation were investigated following photoautotrophic growth. The autofermentative H2 yield increased by 50% in the CcmR deletion strain compared to the wild-type strain, and increased to 160% (within 20 h) upon continuous removal of H2 from the medium ("milking") to suppress uptake. Consistent with this greater reductant flux to H2 , the mutant excreted less lactate during autofermentation (NAD(P)H consuming pathway). To enhance the rate of NADH production during anaerobic metabolism, the ccmR mutant was engineered to introduce GAPDH overexpression (more NADH production) and LDH deletion (less NADH consumption). The triple mutant (ccmR deletion + GAPDH overexpression + LDH deletion) showed 6-8-fold greater H2 yield than the WT strain, achieving conversion rates of 17 nmol 108 cells-1 h-1 and yield of 0.87 H2 per glucose equivalent (8.9% theoretical maximum). Simultaneous monitoring of the intracellular NAD(P)H concentration and H2 production rate by these mutants reveals an inverse correspondence between these variables indicating hydrogenase-dependent H2 production as a major sink for consuming NAD(P)H in preference to excretion of reduced carbon as lactate during fermentation.
Druzhinin, SY, Tran NT, Skalenko KS, Goldman SR, Knoblauch JG, Dove SL, Nickels BE.  2015.  A Conserved Pattern of Primer-Dependent Transcription Initiation in Escherichia coli and Vibrio cholerae Revealed by 5' RNA-seq. PLoS Genet. 11(7):e1005348.
Norrander, J, Kempe T, Messing J.  1983.  Construction of improved M13 vectors using oligodeoxynucleotide-directed mutagenesis. Gene. 26:101-6. AbstractWebsite
The restriction endonuclease cleavage sites for SphI and KpnI have been added to the lac cloning region of the phage vectors M13mp10 and M13mp11, using oligodeoxynucleotide-directed in vitro mutagenesis. Complementary deoxy 16-, 21- or 18-mers with the desired base changes were annealed to the M13mp DNA strand and extended with the Klenow fragment of DNA polymerase I. In adding these sites we have shown that this technique can be used as a general method for inserting sequences of DNA as well as introducing deletions and base pair changes.
Song, R, Messing J.  2002.  Contiguous genomic DNA sequence comprising the 19-kD zein gene family from maize. Plant physiology. 130:1626-35. AbstractWebsite
A new approach has been undertaken to analyze the sequences and linear organization of the 19-kD zein genes in maize (Zea mays). A high-coverage, large-insert genomic library of the inbred line B73 based on bacterial artificial chromosomes was used to isolate a redundant set of clones containing members of the 19-kD zein gene family, which previously had been estimated to consist of 50 members. The redundant set of clones was used to create bins of overlapping clones that represented five distinct genomic regions. Representative clones containing the entire set of 19-kD zein genes were chosen from each region and sequenced. Seven bacterial artificial chromosome clones yielded 1,160 kb of genomic DNA. Three of them formed a contiguous sequence of 478 kb, the longest contiguous sequenced region of the maize genome. Altogether, these DNA sequences provide the linear organization of 25 19-kD zein genes, one-half the number previously estimated. It is suggested that the difference is because of haplotypes exhibiting different degrees of gene amplification in the zein multigene family. About one-half the genes present in B73 appear to be expressed. Because some active genes have only been duplicated recently, they are so conserved in their sequence that previous cDNA sequence analysis resulted in "unigenes" that were actually derived from different gene copies. This analysis also shows that the 22- and 19-kD zein gene families shared a common ancestor. Although both ancestral genes had the same incremental gene amplification, the 19-kD zein branch exhibited a greater degree of far-distance gene translocations than the 22-kD zein gene family.
Carrieri, D, Ananyev GM, Lenz O, Bryant DA, Dismukes CG.  2011.  Contribution of a sodium ion gradient to energy conservation during fermentation in the cyanobacterium Arthrospira (Spirulina) maxima CS-328.. Applied and environmental microbiology. 77(20):7185-94. Abstract
Sodium gradients in cyanobacteria play an important role in energy storage under photoautotrophic conditions but have not been well studied during autofermentative metabolism under the dark, anoxic conditions widely used to produce precursors to fuels. Here we demonstrate significant stress-induced acceleration of autofermentation of photosynthetically generated carbohydrates (glycogen and sugars) to form excreted organic acids, alcohols, and hydrogen gas by the halophilic, alkalophilic cyanobacterium Arthrospira (Spirulina) maxima CS-328. When suspended in potassium versus sodium phosphate buffers at the start of autofermentation to remove the sodium ion gradient, photoautotrophically grown cells catabolized more intracellular carbohydrates while producing 67% higher yields of hydrogen, acetate, and ethanol (and significant amounts of lactate) as fermentative products. A comparable acceleration of fermentative carbohydrate catabolism occurred upon dissipating the sodium gradient via addition of the sodium-channel blocker quinidine or the sodium-ionophore monensin but not upon dissipating the proton gradient with the proton-ionophore dinitrophenol (DNP). The data demonstrate that intracellular energy is stored via a sodium gradient during autofermentative metabolism and that, when this gradient is blocked, the blockage is compensated by increased energy conversion via carbohydrate catabolism.
Okajima, T, Reddy BVVG, Matsuda T, Irvine KD.  2008.  Contributions of chaperone and glycosyltransferase activities of O-fucosyltransferase 1 to Notch signaling. BMC Biology. 6:1. AbstractWebsite
BACKGROUND: O-fucosyltransferase1 (OFUT1) is a conserved ER protein essential for Notch signaling. OFUT1 glycosylates EGF domains, which can then be further modified by the N-acetylglucosaminyltransferase Fringe. OFUT1 also possesses a chaperone activity that promotes the folding and secretion of Notch. Here, we investigate the respective contributions of these activities to Notch signaling in Drosophila. RESULTS: We show that expression of an isoform lacking fucosyltransferase activity, Ofut1R245A, rescues the requirement for Ofut1 in embryonic neurogenesis. Lack of requirement for O-fucosylation is further supported by the absence of embryonic phenotypes in Gmd mutants, which lack all forms of fucosylation. Requirements for O-fucose during imaginal development were evaluated by characterizing clones of cells expressing only Ofut1R245A. These clones phenocopy fringe mutant clones, indicating that the absence of O-fucose is functionally equivalent to the absence of elongated O-fucose. CONCLUSION: Our results establish that Notch does not need to be O-fucosylated for fringe-independent Notch signaling in Drosophila; the chaperone activity of OFUT1 is sufficient for the generation of functional Notch.
Gallavotti, A, Long JA, Stanfield S, Yang X, Jackson D, Vollbrecht E, Schmidt RJ.  2010.  The Control of Axillary Meristem fate in the Maize Ramosa Pathway. Development. 137:2849-2856. AbstractWebsite
Plant axillary meristems are composed of highly organized, self-renewing stem cells that produce indeterminate branches or terminate in differentiated structures, such as the flowers. These opposite fates, dictated by both genetic and environmental factors, determine interspecific differences in the architecture of plants. The Cys(2)-His(2) zinc-finger transcription factor RAMOSA1 (RA1) regulates the fate of most axillary meristems during the early development of maize inflorescences, the tassel and the ear, and has been implicated in the evolution of grass architecture. Mutations in RA1 or any other known members of the ramosa pathway, RAMOSA2 and RAMOSA3, generate highly branched inflorescences. Here, we report a genetic screen for the enhancement of maize inflorescence branching and the discovery of a new regulator of meristem fate: the RAMOSA1 ENHANCER LOCUS2 (REL2) gene. rel2 mutants dramatically increase the formation of long branches in ears of both ra1 and ra2 mutants. REL2 encodes a transcriptional co-repressor similar to the TOPLESS protein of Arabidopsis, which is known to maintain apical-basal polarity during embryogenesis. REL2 is capable of rescuing the embryonic defects of the Arabidopsis topless-1 mutant, suggesting that REL2 also functions as a transcriptional co-repressor throughout development. We show by genetic and molecular analyses that REL2 physically interacts with RA1, indicating that the REL2/RA1 transcriptional repressor complex antagonizes the formation of indeterminate branches during maize inflorescence development. Our results reveal a novel mechanism for the control of meristem fate and the architecture of plants.
Sun, G, Irvine KD.  2014.  Control of growth during regeneration.. Current topics in developmental biology. 108:95-120. AbstractWebsite
Regeneration is a process by which organisms replace damaged or amputated organs to restore normal body parts. Regeneration of many tissues or organs requires proliferation of stem cells or stem cell-like blastema cells. This regenerative growth is often initiated by cell death pathways induced by damage. The executors of regenerative growth are a group of growth-promoting signaling pathways, including JAK/STAT, EGFR, Hippo/YAP, and Wnt/β-catenin. These pathways are also essential to developmental growth, but in regeneration, they are activated in distinct ways and often at higher strengths, under the regulation by certain stress-responsive signaling pathways, including JNK signaling. Growth suppressors are important in termination of regeneration to prevent unlimited growth and also contribute to the loss of regenerative capacity in nonregenerative organs. Here, we review cellular and molecular growth regulation mechanisms induced by organ damage in several models with different regenerative capacities.
Irvine, KD, Harvey KF.  2015.  Control of organ growth by patterning and hippo signaling in Drosophila.. Cold Spring Harbor perspectives in biology. 7 AbstractWebsite
Control of organ size is of fundamental importance and is controlled by genetic, environmental, and mechanical factors. Studies in many species have pointed to the existence of both organ-extrinsic and -intrinsic size-control mechanisms, which ultimately must coordinate to regulate organ size. Here, we discuss organ size control by organ patterning and the Hippo pathway, which both act in an organ-intrinsic fashion. The influence of morphogens and other patterning molecules couples growth and patterning, whereas emerging evidence suggests that the Hippo pathway controls growth in response to mechanical stimuli and signals emanating from cell-cell interactions. Several points of cross talk have been reported between signaling pathways that control organ patterning and the Hippo pathway, both at the level of membrane receptors and transcriptional regulators. However, despite substantial progress in the past decade, key questions in the growth-control field remain, including precisely how and when organ patterning and the Hippo pathway communicate to control size, and whether these communication mechanisms are organ specific or general. In addition, elucidating mechanisms by which organ-intrinsic cues, such as patterning factors and the Hippo pathway, interface with extrinsic cues, such as hormones to control organ size, remain unresolved.
Ebright, RH, Ebright YW, Pendergrast PS, Gunasekera A.  1990.  Conversion of a helix-turn-helix motif sequence-specific DNA binding protein into a site-specific DNA cleavage agent.. Proceedings of the National Academy of Sciences of the United States of America. 87(8):2882-6. Abstract
Escherichia coli catabolite gene activator protein (CAP) is a helix-turn-helix motif sequence-specific DNA binding protein [de Crombrugghe, B., Busby, S. & Buc, H. (1984) Science 224, 831-838; and Pabo, C. & Sauer, R. (1984) Annu. Rev. Biochem. 53, 293-321]. In this work, CAP has been converted into a site-specific DNA cleavage agent by incorporation of the chelator 1,10-phenanthroline at amino acid 10 of the helix-turn-helix motif. [(N-Acetyl-5-amino-1,10-phenanthroline)-Cys178]CAP binds to a 22-base-pair DNA recognition site with Kobs = 1 x 10(8) M-1. In the presence of Cu(II) and reducing agent, [(N-acetyl-5-amino-1,10-phenanthroline)-Cys178]CAP cleaves DNA at four adjacent nucleotides on each DNA strand within the DNA recognition site. The DNA cleavage reaction has been demonstrated using 40-base-pair and 7164-base-pair DNA substrates. The DNA cleavage reaction is not inhibited by dam methylation of the DNA substrate. Such semisynthetic site-specific DNA cleavage agents have potential applications in chromosome mapping, cloning, and sequencing.
Radford, SJ, Go A MM, McKim KS.  2016.  Cooperation Between Kinesin Motors Promotes Spindle Symmetry and Chromosome Organization in Oocytes.. Genetics. Abstract
The oocyte spindle in most animal species is assembled in the absence of the microtubule-organizing centers called centrosomes. Without the organization provided by centrosomes, acentrosomal meiotic spindle organization may rely heavily on the bundling of microtubules by kinesin motor proteins. Indeed, the minus-end directed kinesin-14 NCD and the plus-end directed kinesin-6 Subito are known to be required for oocyte spindle organization in Drosophila melanogaster How multiple microtubule-bundling kinesins interact to produce a functional acentrosomal spindle is not known. In addition, there have been few studies on the meiotic function of one of the most important microtubule-bundlers in mitotic cells, the kinesin-5 KLP61F. We have found that the kinesin-5 KLP61F is required for spindle and centromere symmetry in oocytes. The asymmetry observed in the absence of KLP61F depends on NCD, the kinesin-12 KLP54D, and the microcephaly protein ASP. In contrast, KLP61F and Subito work together in maintaining a bipolar spindle. We propose that the prominent central spindle, stabilized by Subito, provides the framework for the coordination of multiple microtubule-bundling activities. The activities of several proteins, including NCD, KLP54D, and ASP, generate asymmetries within the acentrosomal spindle, while KLP61F and Subito balance these forces resulting in the capacity to accurately segregate chromosomes.
Koppen, M, Simske JS, Sims PA, Firestein BL, Hall DH, Radice AD, Rongo C, Hardin JD.  2001.  Cooperative regulation of AJM-1 controls junctional integrity in Caenorhabditis elegans epithelia. Nat Cell Biol. 3:983-91. AbstractWebsite
The function of epithelial cell sheets depends on the integrity of specialized cell-cell junctions that connect neighbouring cells. We have characterized the novel coiled-coil protein AJM-1, which localizes to an apical junctional domain of Caenorhabditis elegans epithelia basal to the HMR-HMP (cadherin-catenin) complex. In the absence of AJM-1, the integrity of this domain is compromised. Proper AJM-1 localization requires LET-413 and DLG-1, homologues of the Drosophila tumour suppressors Scribble and Discs large, respectively. DLG-1 physically interacts with AJM-1 and is required for its normal apical distribution, and LET-413 mediates the rapid accumulation of both DLG-1 and AJM-1 in the apical domain. In the absence of both dlg-1 and let-413 function AJM-1 is almost completely lost from apical junctions in embryos, whereas HMP-1 (alpha-catenin) localization is only mildly affected. We conclude that LET-413 and DLG-1 cooperatively control AJM-1 localization and that AJM-1 controls the integrity of a distinct apical junctional domain in C. elegans.
Oh, H, Irvine KD.  2011.  Cooperative Regulation of Growth by Yorkie and Mad through bantam. Developmental Cell. 20:109-22. AbstractWebsite
The Dpp and Fat-Hippo signaling pathways both regulate growth in Drosophila. Dpp is a BMP family ligand and acts via a Smad family DNA-binding transcription factor, Mad. Fat-Hippo signaling acts via a non-DNA-binding transcriptional coactivator protein, Yorkie. Here, we show that these pathways are directly interlinked. They act synergistically to promote growth, in part via regulation of the microRNA gene bantam, and their ability to promote growth is mutually dependent. Yorkie and Mad physically bind each other, and we identify a 410 bp minimal enhancer of bantam that responds to Yorkie:Mad in vivo and in cultured cells, and show that both Yorkie and Mad associate with this enhancer in vivo. Our results indicate that in promoting the growth of Drosophila tissues, Fat-Hippo and Dpp signaling contribute distinct subunits of a shared transcriptional activation complex, Yorkie:Mad.
Ambegaonkar, AA, Irvine KD.  2015.  Coordination of planar cell polarity pathways through Spiny legs. eLife. 4:pii:e09946.
Ebright, R, Dong Q, Messing J.  1992.  Corrected nucleotide sequence of M13mp18 gene III.. Gene. 114(1):81-3. Abstract
There are seven differences between the actual nucleotide (nt) sequence of bacteriophage M13mp18 gene III and the previously reported nt sequence (which had been compiled based on the nt sequence of wild-type bacteriophage M13 gene III).
Westra, ER, van Erp PB, Wong SP, Kunne T, Staals R, Seegers CL, Bollen S, Jore MM, de Vos WM, Dame RT et al..  2012.  CRISPR immunity relies on the conservative binding and degradation of negatively supercoiled invader DNA by Cascade and Cas3. Mol. Cell. 46:595-605.
Djordjevic, M, Djordjevic M, Severinov K.  2012.  CRISPR transcript processing: an unusual mechanism for strong amplification of small RNAs. Biol. Direct. 7:24.
Hursh, DA, Padgett RW, Gelbart WM.  1993.  Cross regulation of decapentaplegic and Ultrabithorax transcription in the embryonic visceral mesoderm of Drosophila. Development (Cambridge, England). 117:1211-22. AbstractWebsite
The Drosophila decapentaplegic gene (dpp) encodes a TGF-beta family member involved in signal transduction during embryonic midgut formation. The shortvein (shv) class of cis-regulatory dpp mutants disrupt expression in parasegments 4 and 7 (ps4 and ps7) of the embryonic visceral mesoderm (VM) surrounding the gut and cause abnormalities in gut morphogenesis. We demonstrate that cis-regulatory elements directing expression in ps4 and ps7 are separable and identify DNA fragments that generate ps4 and ps7 expression patterns using reporter gene constructs. dpp reporter gene expression in both ps4 and ps7 is autoregulated as it requires endogenous dpp+ activity. Reporter gene ps7 expression requires the wild-type action of Ultra-bithorax (Ubx), and abdominal-A. Furthermore, the expression of certain Ubx reporter genes is coincident with dpp in the VM. Both the mis-expression of Ubx reporter genes in the developing gastric caecae at ps4 and its normal expression in ps7 are dependent upon endogenous dpp+ activity. We conclude that dpp both responds to and regulates Ubx in ps7 of the visceral mesoderm and that Ubx autoregulation within this tissue may be indirect as it requires more components than have previously been thought.
Montano, SP, Cot'e ML, Fingerman I, Pierce M, Vershon AK, Georgiadis MM.  2002.  Crystal Structure of the DNA-binding Domain from Ndt80, a Transcriptional Activator Required for Meiosis in Yeast. Proc Natl Acad Sci U S A. 99:14041-14046. Abstract
Ndt80 is a transcriptional activator required for meiosis in the yeast Saccharomyces cerevisiae. Here, we report the crystal structure at 2.3 A resolution of the DNA-binding domain of Ndt80 experimentally phased by using the anomalous and isomorphous signal from a single ordered Se atom per molecule of 272-aa residues. The structure reveals a single approximately 32-kDa domain with a distinct fold comprising a beta-sandwich core elaborated with seven additional beta-sheets and three short alpha-helices. Inspired by the structure, we have performed a mutational analysis and defined a DNA-binding motif in this domain. The DNA-binding domain of Ndt80 is homologous to a number of proteins from higher eukaryotes, and the residues that we have shown are required for DNA binding by Ndt80 are highly conserved among this group of proteins. These results suggest that Ndt80 is the defining member of a previously uncharacterized family of transcription factors, including the human protein (C11orf9), which has been shown to be highly expressed in invasive or metastatic tumor cells.
Li, T, Jin Y, Vershon AK, Wolberger C.  1998.  Crystal Structure of the MATa1/MATalpha2 Homeodomain Heterodimer in Complex with DNa Containing an A-tract. Nucleic Acids Res. 26:5707-5718. Abstract
The crystal structure of the heterodimer formed by the DNA binding domains of the yeast mating type transcription factors, MATa1 and MATalpha2, bound to a 21 bp DNA fragment has been determined at 2.5 A resolution. The DNA fragment in the present study differs at four central base pairs from the DNA sequence used in the previously studied ternary complex. These base pair changes give rise to a (dA5).(dT5) tract without changing the overall base composition of the DNA. The resulting A-tract occurs near the center of the overall 60 degrees bend in the DNA. Comparison of the two structures shows that the structural details of the DNA bend are maintained despite the DNA sequence changes. Analysis of the A5-tract DNA subfragment shows that it contains a bend toward the minor groove centered at one end of the A-tract. The observed bend is larger than that observed in the crystal structures of A-tracts embedded in uncomplexed DNA, which are straight and have been presumed to be quite rigid. Variation of the central DNA base sequence reverses the two AT base pairs contacted in the minor groove by Arg7 of the alpha2 N-terminal arm without significantly altering the DNA binding affinity of the a1/alpha2 heterodimer. The Arg7 side chain accommodates the sequence change by forming alternate H bond interactions, in agreement with the proposal that minor groove base pair recognition is insensitive to base pair reversal. Furthermore, the minor groove spine of hydration, which stabilizes the narrowed minor groove caused by DNA bending, is conserved in both structures. We also find that many of the water-mediated hydrogen bonds between the a1 and alpha2 homeodomains and the DNA are highly conserved, indicating an important role for water in stabilization of the a1/alpha2-DNA complex.
Montano, SP, Pierce M, Cot'e ML, Vershon AK, Georgiadis MM.  2002.  Crystallographic Studies of a Novel DNA-binding Domain from the Yeast Transcriptional Activator Ndt80. Acta Crystallogr D Biol Crystallogr. 58:2127-2130. Abstract
The Ndt80 protein is a transcriptional activator that plays a key role in the progression of the meiotic divisions in the yeast Saccharomyces cerevisiae. Ndt80 is strongly induced during the middle stages of the sporulation pathway and binds specifically to a promoter element called the MSE to activate transcription of genes required for the meiotic divisions. Here, the preliminary structural and functional studies to characterize the DNA-binding activity of this protein are reported. Through deletion analysis and limited proteolysis studies of Ndt80, a novel 32 kDa DNA-binding domain that is sufficient for DNA-binding in vitro has been defined. Crystals of the DNA-binding domain of Ndt80 in two distinct lattices have been obtained, for which diffraction data extend to 2.3 A resolution.
McKim, KS, Joyce EF, Jang JK.  2009.  Cytological analysis of meiosis in fixed Drosophila ovaries. Methods Mol Biol. 558:197-216. AbstractWebsite
Methods are described to analyze two different parts of the Drosophila ovary, which correspond to early stages (pachytene) and late stages (metaphase I and beyond) of meiosis. In addition to taking into account morphology, the techniques differ by fixation conditions and the method to isolate the tissue. Most of these methods are whole mounts, which preserve the three-dimensional structure.
Manheim, EA, Jang JK, Dominic D, McKim KS.  2002.  Cytoplasmic localization and evolutionary conservation of MEI-218, a protein required for meiotic crossing over in Drosophila. Mol. Biol. Cell. 13:84-95.
Rauskolb, C, Sun S, Sun G, Pan Y, Irvine KD.  2014.  Cytoskeletal Tension Inhibits Hippo Signaling through an Ajuba-Warts Complex.. Cell. 158:143-156. AbstractWebsite
Mechanical forces have been proposed to modulate organ growth, but a molecular mechanism that links them to growth regulation in vivo has been lacking. We report that increasing tension within the cytoskeleton increases Drosophila wing growth, whereas decreasing cytoskeletal tension decreases wing growth. These changes in growth can be accounted for by changes in the activity of Yorkie, a transcription factor regulated by the Hippo pathway. The influence of myosin activity on Yorkie depends genetically on the Ajuba LIM protein Jub, a negative regulator of Warts within the Hippo pathway. We further show that Jub associates with α-catenin and that its localization to adherens junctions and association with α-catenin are promoted by cytoskeletal tension. Jub recruits Warts to junctions in a tension-dependent manner. Our observations delineate a mechanism that links cytoskeletal tension to regulation of Hippo pathway activity, providing a molecular understanding of how mechanical forces can modulate organ growth.