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Traller, JC, Cokus SJ, Lopez DA, Gaidarenko O, Smith SR, McCrow JP, Gallaher SD, Podell S, Thompson M, Cook O, Morselli M, Jaroszewicz A, Allen EE, Allen AE, Merchant SS, Pellegrini M, Hildebrand M.  2016.  Genome and methylome of the oleaginous diatom Cyclotella cryptica reveal genetic flexibility toward a high lipid phenotype. Biotechnology for Biofuels. 9:258.   10.1186/s13068-016-0670-3   Abstract

Improvement in the performance of eukaryotic microalgae for biofuel and bioproduct production is largely dependent on characterization of metabolic mechanisms within the cell. The marine diatom Cyclotella cryptica, which was originally identified in the Aquatic Species Program, is a promising strain of microalgae for large-scale production of biofuel and bioproducts, such as omega-3 fatty acids.

Cook, O, Hildebrand M.  2016.  Enhancing LC-PUFA production in Thalassiosira pseudonana by overexpressing the endogenous fatty acid elongase genes. Journal of Applied Phycology. 28:897-905.   10.1007/s10811-015-0617-2   AbstractWebsite

The health beneficial omega-3 long-chain polyunsaturated fatty acids (LC-PUFAs), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) are naturally synthesized by diatoms through consecutive steps of fatty acid elongase and desaturase enzymes. In Thalassiosira pseudonana, these fatty acids constitute about 10-20 % of the total fatty acids, with EPA accumulation being five to ten times higher than DHA. In order to identify the subcellular localization of enzymes in the pathway of LC-PUFA biosynthesis in T. pseudonana and to manipulate the production of EPA and DHA, we generated constructs for overexpressing each of the T. pseudonana long-chain fatty acid elongase genes. Full-length proteins were fused to GFP, and transgenic lines were generated. In addition, overexpressed native proteins with no GFP fusion were tested. The subcellular localization of each elongase protein was determined. We then examined the total amount of lipids and analyzed the fatty acid profile in each of the transgenic lines compared to wild type. Lines with overexpressed elongases showed an increase of up to 1.4-fold in EPA and up to 4.5-fold in DHA, and the type of fatty acid that was increased (EPA vs. DHA) depended on the type of elongase that was overexpressed. This data informs future metabolic engineering approaches to further improve EPA and DHA content in diatoms.

Hildebrand, M, Davis A, Abbriano R, Pugsley HR, Traller JC, Smith SR, Shrestha RP, Cook O, Sanchez E, Manabe S, Manandhar-Shrestha K, Alderete B.  2016.  Applications of Imaging Flow Cytometry for Microalgae. Imaging flow cytometry: Methods and protocols. ( Barteneva NS, Vorobjev IA, Eds.).:xiii,295pages.: Humana Press   10.1007/978-1-4939-3302-0   Abstract

The ability to image large numbers of cells at high resolution enhances flow cytometric analysis of cells and cell populations. In particular, the ability to image intracellular features adds a unique aspect to analyses, and can enable correlation between molecular phenomena resulting in alterations in cellular phenotype. Unicellular microalgae are amenable to high-throughput analysis to capture the diversity of cell types in natural samples, or diverse cellular responses in clonal populations, especially using imaging cytometry. Using examples from our laboratory, we review applications of imaging cytometry, specifically using an Amnis(®) ImageStream(®)X instrument, to characterize photosynthetic microalgae. Some of these examples highlight advantages of imaging flow cytometry for certain research objectives, but we also include examples that would not necessarily require imaging and could be performed on a conventional cytometer to demonstrate other concepts in cytometric evaluation of microalgae. We demonstrate the value of these approaches for (1) analysis of populations, (2) documentation of cellular features, and (3) analysis of gene expression.

Goldstein, RE, Cook O, Dinur T, Pisante A, Karandikar UC, Bidwai A, Paroush Z.  2005.  An eh1-like motif in odd-skipped mediates recruitment of Groucho and repression in vivo. Molecular and Cellular Biology. 25:10711-10720.   10.1128/Mcb.25.24.10711-10720.2005   AbstractWebsite

Drosophila Groucho, like its vertebrate Transducin-like Enhancer-of-split homologues, is a corepressor that silences gene expression in numerous developmental settings. Groucho itself does not bind DNA but is recruited to target promoters by associating with a large number of DNA-binding negative transcriptional regulators. These repressors tether Groucho via short conserved polypeptide sequences, of which two have been defined. First, WRPW and related tetrapeptide motifs have been well characterized in several repressors. Second, a motif termed Engrailed homology 1 (eh1) has been found predominantly in homeodomain-containing transcription factors. Here we describe a yeast two-hybrid screen that uncovered physical interactions between Groucho and transcription factors, containing eh1 motifs, with different types of DNA-binding domains. We show that one of these, the zinc finger protein Odd-skipped, requires its eh1-like sequence for repressing specific target genes in segmentation. Comparison between diverse eh1 motifs reveals a bias for the phosphoacceptor amino acids serine and threonine at a fixed position, and a mutational analysis of Odd-skipped indicates that these residues are critical for efficient interactions with Groucho and for repression in vivo. Our data suggest that phosphorylation of these phosphomeric residues, if it occurs, will down-regulate Groucho binding and therefore repression, providing a mechanism for posttranslational control of Groucho-mediated repression.

Cook, O, Biehs B, Bier E.  2004.  Brinker and optomotor-blind act coordinately to initiate development of the L5 wing vein primordium in Drosophila. Development. 131:2113-2124.   10.1242/dev.01100   AbstractWebsite

The stereotyped pattern of Drosophila wing veins is determined by the action of two morphogens, Hedgehog (Hh) and Decapentaplegic (Dpp), which act sequentially to organize growth and patterning along the anterior-posterior axis of the wing primordium. An important unresolved question is how positional information established by these morphogen gradients is translated into localized development of morphological structures such as wing veins in precise locations. In the current study, we examine the mechanism by which two broadly expressed Dpp signaling target genes, optomotor-blind (omb) and brinker (brk), collaborate to initiate formation of the fifth longitudinal (L5) wing vein. omb is broadly expressed at the center of the wing disc in a pattern complementary to that of brk, which is expressed in the lateral regions of the disc and represses omb expression. We show that a border between omb and brk expression domains is necessary and sufficient for inducing L5 development in the posterior regions. Mosaic analysis indicates that brk-expressing cells produce a short-range signal that can induce vein formation in adjacent omb-expressing cells. This induction of the L5 primordium is mediated by abrupt, which is expressed in a narrow stripe of cells along the brklomb border and plays a key role in organizing gene expression in the L5 primordium. Similarly, in the anterior region of the wing, brk helps define the position of the L2 vein in combination with another Dpp target gene, spalt. The similar mechanisms responsible for the induction of L5 and L2 development reveal how boundaries set by dosage-sensitive responses to a long-range morphogen specify distinct vein fates at precise locations.

Goldstein, RE, Jimenez G, Cook O, Gur D, Paroush Z.  1999.  Huckebein repressor activity in Drosophila terminal patterning is mediated by Groucho. Development. 126:3747-3755. AbstractWebsite

The Groucho corepressor mediates negative transcriptional regulation in association with various DNA-binding proteins in diverse developmental contexts. We have previously implicated Groucho in Drosophila embryonic terminal patterning, showing that it is required to confine tailless and huckebein terminal gap gene expression to the pole regions of the embryo. Here we reveal an additional requirement for Groucho in this developmental process by establishing that Groucho mediates repressor activity of the Huckebein protein. Putative Huckebein target genes are derepressed in embryos lacking maternal groucho activity and biochemical experiments demonstrate that Huckebein physically interacts with Groucho. Using an in vivo repression assay, we identify a functional repressor domain in Huckebein that contains an FRPW tetrapeptide, similar to the WRPW Groucho-recruitment domain found in Hairy-related repressor proteins. Mutations in Huckebein's FRPW motif abolish Groucho binding and in vivo repression activity, indicating that binding of Groucho through the FRPW motif is required for the repressor function of Huckebein. Taken together with our earlier results, these findings show that Groucho-repression regulates sequential aspects of terminal patterning in Drosophila.

Cook, O, Low W, Rahamimoff H.  1998.  Membrane topology of the rat brain Na+-Ca2+ exchanger. Biochimica Et Biophysica Acta-Biomembranes. 1371:40-52.   Doi 10.1016/S0005-2736(97)00272-1   AbstractWebsite

To provide experimental evidence for the topology of the Na+-Ca2+ exchanger protein NCX1 in the membrane, indirect immunofluorescence studies using site specific anti-peptide antibodies and Flag-epitope insertion into chosen locations of the protein were carried out. Anti-peptide antibodies AbO-6 mid AbO-8 were raised against peptide segments present in a large hydrophilic loop of about 500 amino acids, which separates the hydrophobic amino terminal part of the protein from the hydrophobic carboxy terminal. AbO-10 was raised against the C-terminal tail or the protein. All three antibodies hound to the exchanger protein expressed in transfected cells, in rat brain synaptic plasma membrane and in dog sarcolemmal preparations. The antibodies bound only to those NCX1 isoforms that contained the epitope against which they were raised. Detection of the exchanger protein in transfected cells in situ required the addition of permeabilizing agents suggesting an intracellular location of the epitopes to which AbO-6. AbO-8 and AbO-10 bind. The Flag epitope was inserted into ten putative extramembraneous segments along the exchanger protein. For topology studies, only the Flag-mutants that retained Na+-Ca2+ exchange activity in whole HeLa cells, were used. Immunofluorescence studies indicated, that the N-terminnl of the protein is extracellular, the first hydrophilic loop separating transmembrane helices 1 and 2 as well as the C-terminal, are intracellular. (C) 1998 Elsevier Science B.V.

Rahamimoff, H, Low W, Cook O, Furman I, Kasir J, Vatashski R.  1996.  The structural basis of Na+-Ca2+ exchange activity. Sodium-Calcium Exchange. 779:29-36.   Doi 10.1111/J.1749-6632.1996.Tb44767.X   AbstractWebsite
Furman, I, Cook O, Kasir J, Low W, Rahamimoff H.  1995.  The Putative Amino-Terminal Signal Peptide of the Cloned Rat-Brain Na+-Ca2+ Exchanger Gene (Rbe-1) Is Not Mandatory for Functional Expression. Journal of Biological Chemistry. 270:19120-19127. AbstractWebsite

The rat brain Na+-Ca2+ exchanger (RBE) gene, as well as other isoforms of this protein family, can be organized into 12 transmembrane alpha helices, the first of which was proposed by Durkin et al. (14) to constitute a cleavable signal peptide. We have prepared three amino-terminal mutants, in which 21, 26, and 31 amino acids beyond the initiating methionine were deleted. The deletions include the hydrophobic core of the putative signal peptide (N21), the entire putative signal peptide and parts of the putative signal peptidase cleavage site (N26), and the entire putative signal peptide and putative signal peptidase cleavage site (N31). Ah three mutant clones were transiently expressed in HeLa cells. The average Na+ gradient-dependent Ca2+ transport activity of the mutant exchangers was 108% (N21), 37.2% (N26), and 60.06% (N31) of the wild-type clone. Mutation of the putative cleavage site by an exchange of Ala-32 --> Asp, resulted in a decrease in Na+-Ca2+ exchange activity to 7.7%, relative to the wild-type exchanger. Functional reconstitution of the proteins that were expressed in the transfected cells, resulted in transport activities of: 60.1% (N21), 26.75% (N26), 85.36% (N31), and 31% (Ala-32 --> Asp) relative to the wild-type exchanger. Western blot analysis of the protein profile of RBE-I, N21, N26, N31 and Ala-32 --> Asp-transfected HeLa cells was carried out by using an antipeptide antibody directed against a pentadecapeptide segment derived from the large putative cytoplasmic loop of the cloned rat exchanger gene. In the total cell extract and in the plasma membrane-enriched fraction, in addition to a major protein band of about 125 kDa, which corresponds to the molecular mass of the mature fully processed Na+-Ca2+ exchanger, an additional protein of about 135 kDa is revealed in the profile of N21- and N26-transfected cells. This band is not detected in the protein profile of RBE-1, N31, or Ala-32 --> Asp. The amino-terminal truncated mutants of the cloned Na+-Ca2+ exchanger could be expressed and processed also in a reticulocyte lysate supplemented with dog microsomes. Our results suggest that the putative signal peptide of the cloned Na+-Ca2+ exchanger gene does not play a mandatory role in functional expression of the protein in HeLa cells.

Furman, I, Cook O, Kasir J, Rahamimoff H.  1993.  Cloning of 2 Isoforms of the Rat-Brain Na+-Ca-2+ Exchanger Gene and Their Functional Expression in Hela-Cells. Febs Letters. 319:105-109.   Doi 10.1016/0014-5793(93)80046-W   AbstractWebsite

Two functional isoforms of the rat brain Na+ Ca2+ exchanger were isolated from a lambdaZAP hippocampus cDNA library. The open reading frame of clone RBE-1 codes for a protein 935 amino acids long, and that of clone RBE-2 codes for a protein 958 amino acids long. Expression in HeLa cells of Na+ gradient dependent Ca2+ transport activity was determined following transfection of the cells with either RBE-1 or RBE-2. Both clones expressed proteins that exchange Na+ with Ca2+ in an electrogenic manner and none of them exhibited a dependency of the antiport on K+, since they transported Ca2+ in an Na+ gradient dependent manner in external choline chloride as well.

C, S, O W, Z B, DM C.  1993.  Subunit association in acetohydroxy acid synthase isozyme III. J Bacteriol. 1993 Sep;175(17):5339-43.. AbstractWebsite

Acetohydroxy acid synthase isozyme III (AHAS III) from Escherichia coli is composed of large and small subunits (encoded by the genes ilvI and ilvH) in an alpha 2 beta 2 structure. The large (61-kDa) subunit apparently contains the catalytic machinery of the enzyme, while the small (17-kDa) subunit is required for specific stabilization of the active conformation of the large subunit as well as for valine sensitivity. The interaction between subunits has been studied by using purified enzyme and extracts containing subcloned subunits. The association between large and small subunits is reversible, with a dissociation constant sufficiently high to have important experimental consequences: the activity of the enzyme shows a concentration dependence curve which is concave upward, and this dependence becomes linear upon the addition of excess large or small subunits. We estimate that at a concentration of 10(-7) M for each subunit (7 micrograms of enzyme ml-1), the large subunits are only half associated as the I2H2 active holoenzyme. This dissociation constant is high enough to cause underestimation of the activity of AHAS III in bacterial extracts. The true activity of this isozyme in extracts is observed in the presence of excess small subunits, which maintain the enzyme in its associated form. Reexamination of an E. coli K-12 ilvBN+ ilvIH+ strain grown in glucose indicates that AHAS III is the major isozyme expressed. As an excess of small subunits does not influence the apparent Ki for valine inhibition of the purified enzyme, it is likely that valine binds to and inhibits I2H2 rather than inducing dissociation. AHAS I and II seem to show a much lower tendency to dissociate than does AHAS III.

O, W, C S, DM C, Z B.  1992.  Properties of subcloned subunits of bacterial acetohydroxy acid synthases. J Bacteriol. AbstractWebsite

The acetohydroxy acid synthase (AHAS) isozymes from enterobacteria are each composed of a large and small subunit in an alpha 2 beta 2 structure. It has been generally accepted that the large (ca. 60-kDa) subunits are catalytic, while the small ones are regulatory. In order to further characterize the roles of the subunits as well as the nature and the specificities of their interactions, we have constructed plasmids encoding the large or small subunits of isozymes AHAS I and AHAS III, each with limited remnants of the other peptide. The catalytic properties of the large subunits have been characterized and compared with those of extracts containing the intact enzyme or of purified enzymes. Antisera to the isolated subunits have been used in Western blot (immunoblot) analyses for qualitative and semiquantitative determinations of the presence of the polypeptides in extracts. The large subunits of AHAS isozymes I and III have lower activities than the intact enzymes: Vmax/Km is 20 to 50 times lower in both cases. However, for AHAS I, most of this difference is due to the raised Km of the large subunit alone, while for AHAS III, it is due to a lowered Vmax. The substrate specificities, R, of large subunits are close to those of the intact enzymes. The catalytic activity of the large subunits of AHAS I is dependent on flavin adenine dinucleotide (FAD), as is that of the intact enzyme, although the apparent affinities of the large subunits alone for FAD are 10-fold lower. Isolated subunits are insensitive to valine inhibition. Nearly all of the properties of the intact AHAS isozyme I or III can be reconstituted by mixing extracts containing the respective large and small subunits. The mixing of subunits from different enzymes does not lead to activation of the large subunits. It is concluded that the catalytic machinery of these AHAS isozymes is entirely contained within the large subunits. The small subunits are required, however, for specific stabilization of an active conformation of the large subunits as well as for value sensitivity.