Bibliografías temáticas / In vitro biosynthesis

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Literatura académica sobre el tema "In vitro biosynthesis"

Autor: Grafiati
Publicado: 25 de mayo de 2024

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Artículos de revistas sobre el tema "In vitro biosynthesis"

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Cheng, Fang, Timo M. Takala y Per E. J. Saris. "Nisin Biosynthesis in vitro". Journal of Molecular Microbiology and Biotechnology 13, n.º 4 (2007): 248–54. http://dx.doi.org/10.1159/000104754.

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Araki, Yasuko, Takayoshi Awakawa, Motomichi Matsuzaki, Rihe Cho, Yudai Matsuda, Shotaro Hoshino, Yasutomo Shinohara et al. "Complete biosynthetic pathways of ascofuranone and ascochlorin inAcremonium egyptiacum". Proceedings of the National Academy of Sciences 116, n.º 17 (5 de abril de 2019): 8269–74. http://dx.doi.org/10.1073/pnas.1819254116.

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Ascofuranone (AF) and ascochlorin (AC) are meroterpenoids produced by various filamentous fungi, includingAcremonium egyptiacum(synonym:Acremonium sclerotigenum), and exhibit diverse physiological activities. In particular, AF is a promising drug candidate against African trypanosomiasis and a potential anticancer lead compound. These compounds are supposedly biosynthesized through farnesylation of orsellinic acid, but the details have not been established. In this study, we present all of the reactions and responsible genes for AF and AC biosyntheses inA. egyptiacum, identified by heterologous expression, in vitro reconstruction, and gene deletion experiments with the aid of a genome-wide differential expression analysis. Both pathways share the common precursor, ilicicolin A epoxide, which is processed by the membrane-bound terpene cyclase (TPC) AscF in AC biosynthesis. AF biosynthesis branches from the precursor by hydroxylation at C-16 by the P450 monooxygenase AscH, followed by cyclization by a membrane-bound TPC AscI. All genes required for AC biosynthesis (ascABCDEFG) and a transcriptional factor (ascR) form a functional gene cluster, whereas those involved in the late steps of AF biosynthesis (ascHIJ) are present in another distantly located cluster. AF is therefore a rare example of fungal secondary metabolites requiring multilocus biosynthetic clusters, which are likely to be controlled by the single regulator, AscR. Finally, we achieved the selective production of AF inA. egyptiacumby genetically blocking the AC biosynthetic pathway; further manipulation of the strain will lead to the cost-effective mass production required for the clinical use of AF.
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Preie, Gianfranco Del, Enrico Maggi, Sergio Romagnani y Mario Ricci. "Human IgE Biosynthesis In Vitro". Clinical Reviews in Allergy 7, n.º 2 (junio de 1989): 193–216. http://dx.doi.org/10.1007/bf02914466.

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Wiltshire, Michael D. y Simon J. Foster. "Identification and Analysis of Staphylococcus aureus Components Expressed by a Model System of Growth in Serum". Infection and Immunity 69, n.º 8 (1 de agosto de 2001): 5198–202. http://dx.doi.org/10.1128/iai.69.8.5198-5202.2001.

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ABSTRACT A model system mimicking Staphylococcus aureusbacteremia was developed by growth in serum under microaerobic conditions. Eight genes induced by growth in serum were identified, including an antimicrobial peptide biosynthesis locus, amino acid biosynthetic loci, and genes encoding putative surface proteins. Nine independent insertions were found in the major lysine biosynthesis operon, which encodes eight genes, is repressed by lysine in vitro, and is expressed in vivo.
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Li, Li, Jun Wu, Zixin Deng, T. Mark Zabriskie y Xinyi He. "Streptomyces lividans Blasticidin S Deaminase and Its Application in Engineering a Blasticidin S-Producing Strain for Ease of Genetic Manipulation". Applied and Environmental Microbiology 79, n.º 7 (1 de febrero de 2013): 2349–57. http://dx.doi.org/10.1128/aem.03254-12.

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ABSTRACTBlasticidin S is a peptidyl nucleoside antibiotic produced byStreptomyces griseochromogenesthat exhibits strong fungicidal activity. To circumvent an effective DNA uptake barrier system in the native producer and investigate its biosynthesisin vivo, the blasticidin S biosynthetic gene cluster (bls) was engrafted to the chromosome ofStreptomyces lividans. However, the resulting mutant, LL2, produced the inactive deaminohydroxyblasticidin S instead of blasticidin S. Subsequently, a blasticidin S deaminase (SLBSD, forS. lividansblasticidin S deaminase) was identified inS. lividansand shown to govern thisin vivoconversion. Purified SLBSD was found to be capable of transforming blasticidin S to deaminohydroxyblasticidin Sin vitro. It also catalyzed deamination of the cytosine moiety of cytosylglucuronic acid, an intermediate in blasticidin S biosynthesis. Disruption of the SLBSD gene inS. lividansLL2 led to successful production of active blasticidin S in the resultant mutant,S. lividansWJ2. To demonstrate the easy manipulation of the blasticidin S biosynthetic gene cluster,blsE,blsF, andblsL, encoding a predicted radicalS-adenosylmethionine (SAM) protein, an unknown protein, and a guanidino methyltransferase, were individually inactivated to access their role in blasticidin S biosynthesis.
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Mohammed, Yousef, Ding Ye, Mudan He, Houpeng Wang, Zuoyan Zhu y Yonghua Sun. "Production of Astaxanthin by Animal Cells via Introduction of an Entire Astaxanthin Biosynthetic Pathway". Bioengineering 10, n.º 9 (11 de septiembre de 2023): 1073. http://dx.doi.org/10.3390/bioengineering10091073.

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Astaxanthin is a fascinating molecule with powerful antioxidant activity, synthesized exclusively by specific microorganisms and higher plants. To expand astaxanthin production, numerous studies have employed metabolic engineering to introduce and optimize astaxanthin biosynthetic pathways in microorganisms and plant hosts. Here, we report the metabolic engineering of animal cells in vitro to biosynthesize astaxanthin. This was accomplished through a two-step study to introduce the entire astaxanthin pathway into human embryonic kidney cells (HEK293T). First, we introduced the astaxanthin biosynthesis sub-pathway (Ast subp) using several genes encoding β-carotene ketolase and β-carotene hydroxylase enzymes to synthesize astaxanthin directly from β-carotene. Next, we introduced a β-carotene biosynthesis sub-pathway (β-Car subp) with selected genes involved in Ast subp to synthesize astaxanthin from geranylgeranyl diphosphate (GGPP). As a result, we unprecedentedly enabled HEK293T cells to biosynthesize free astaxanthin from GGPP with a concentration of 41.86 µg/g dry weight (DW), which represented 66.19% of the total ketocarotenoids (63.24 µg/g DW). Through optimization steps using critical factors in the astaxanthin biosynthetic process, a remarkable 4.14-fold increase in total ketocarotenoids (262.10 µg/g DW) was achieved, with astaxanthin constituting over 88.82%. This pioneering study holds significant implications for transgenic animals, potentially revolutionizing the global demand for astaxanthin, particularly within the aquaculture sector.
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Pan, Guohui, Zhengren Xu, Zhikai Guo, Hindra, Ming Ma, Dong Yang, Hao Zhou et al. "Discovery of the leinamycin family of natural products by mining actinobacterial genomes". Proceedings of the National Academy of Sciences 114, n.º 52 (11 de diciembre de 2017): E11131—E11140. http://dx.doi.org/10.1073/pnas.1716245115.

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Nature’s ability to generate diverse natural products from simple building blocks has inspired combinatorial biosynthesis. The knowledge-based approach to combinatorial biosynthesis has allowed the production of designer analogs by rational metabolic pathway engineering. While successful, structural alterations are limited, with designer analogs often produced in compromised titers. The discovery-based approach to combinatorial biosynthesis complements the knowledge-based approach by exploring the vast combinatorial biosynthesis repertoire found in Nature. Here we showcase the discovery-based approach to combinatorial biosynthesis by targeting the domain of unknown function and cysteine lyase domain (DUF–SH) didomain, specific for sulfur incorporation from the leinamycin (LNM) biosynthetic machinery, to discover the LNM family of natural products. By mining bacterial genomes from public databases and the actinomycetes strain collection at The Scripps Research Institute, we discovered 49 potential producers that could be grouped into 18 distinct clades based on phylogenetic analysis of the DUF–SH didomains. Further analysis of the representative genomes from each of the clades identified 28 lnm-type gene clusters. Structural diversities encoded by the LNM-type biosynthetic machineries were predicted based on bioinformatics and confirmed by in vitro characterization of selected adenylation proteins and isolation and structural elucidation of the guangnanmycins and weishanmycins. These findings demonstrate the power of the discovery-based approach to combinatorial biosynthesis for natural product discovery and structural diversity and highlight Nature’s rich biosynthetic repertoire. Comparative analysis of the LNM-type biosynthetic machineries provides outstanding opportunities to dissect Nature’s biosynthetic strategies and apply these findings to combinatorial biosynthesis for natural product discovery and structural diversity.
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O'Hanlon, Karen A., Lorna Gallagher, Markus Schrettl, Christoph Jöchl, Kevin Kavanagh, Thomas O. Larsen y Sean Doyle. "Nonribosomal Peptide Synthetase GenespesLandpes1Are Essential for Fumigaclavine C Production in Aspergillus fumigatus". Applied and Environmental Microbiology 78, n.º 9 (17 de febrero de 2012): 3166–76. http://dx.doi.org/10.1128/aem.07249-11.

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ABSTRACTThe identity of metabolites encoded by the majority of nonribosomal peptide synthetases in the opportunistic pathogen,Aspergillus fumigatus, remains outstanding. We found that the nonribosomal peptide (NRP) synthetases PesL and Pes1 were essential for fumigaclavine C biosynthesis, the end product of the complex ergot alkaloid (EA) pathway inA. fumigatus. Deletion of eitherpesL(ΔpesL) orpes1(Δpes1) resulted in complete loss of fumigaclavine C biosynthesis, relatively increased production of fumitremorgins such as TR-2, fumitremorgin C and verruculogen, increased sensitivity to H2O2, and increased sensitivity to the antifungals, voriconazole, and amphotericin B. Deletion ofpesLresulted in severely reduced virulence in an invertebrate infection model (P< 0.001). These findings indicate that NRP synthesis plays an essential role in mediating the final prenylation step of the EA pathway, despite the apparent absence of NRP synthetases in the proposed EA biosynthetic cluster forA. fumigatus. Liquid chromatography/diode array detection/mass spectrometry analysis also revealed the presence of fumiquinazolines A to F in bothA. fumigatuswild-type and ΔpesLstrains. This observation suggests that alternative NRP synthetases can also function in fumiquinazoline biosynthesis, since PesL has been shown to mediate fumiquinazoline biosynthesisin vitro. Furthermore, we provide here the first direct link between EA biosynthesis and virulence, in agreement with the observed toxicity associated with EA exposure. Finally, we demonstrate a possible cluster cross-talk phenomenon, a theme which is beginning to emerge in the literature.
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Fecik, Robert A. "Natural product biosynthesis moves in vitro". Nature Chemical Biology 3, n.º 9 (septiembre de 2007): 531–32. http://dx.doi.org/10.1038/nchembio0907-531.

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Johnson, M. E. y K. V. Rajagopalan. "In vitro system for molybdopterin biosynthesis." Journal of Bacteriology 169, n.º 1 (1987): 110–16. http://dx.doi.org/10.1128/jb.169.1.110-116.1987.

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Tesis sobre el tema "In vitro biosynthesis"

1

Smith, Kristina J. "In vitro biosynthesis of pectic polysaccharides". Thesis, University of Stirling, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.322091.

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Gillespie, Charles Stewart. "Myelin membrane protein biosynthesis : an in vitro study". Thesis, University of Stirling, 1988. http://hdl.handle.net/1893/22868.

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The sites of biosynthesis and incorporation of the abundant CNS myelin proteins 2' , 3' -cyclic nucleotide-3'-phosphodiesterase (CNPase) and P2 protein into the growing myelin membrane were investigated. Cell-free translation systems programmed with mRNA from rat brain, rabbit spinal cord, free and bound polysomes and purified myelin demonstrated conclusively that both CNPase and P2 are synthesized on free polysomes like the myelin basic proteins (MBPs) but unlike the proteolipid protein (PLP), the major intrinsic membrane protein of CNS myelin, which is known to be synthesized at the oligodendrocyte endoplasmic reticulum on bound polysomes (Colman et al., 1982) . These observations were supported by labelling studies on rats in vivo during the period of maximal myelin deposition. Newly synthesized CNPase associated with the myelin membrane very rapidly after labelling (~2 minutes) and this is consistent with the view that there is only a brief delay between synthesis and incorporation into their target membrane for extrinsic-type plasma membrane proteins. An RNA fraction isolated from purified CNS myelin was not enriched in mRNAs coding for CNPase and P2 but a considerable enrichment of mRNAs coding for MBPs was observed. This phenomenon has important implications for the cell biology of myelination since it suggests that although MBPs, CNPase and P2 are all basic extrinsic membrane proteins, and synthesized on free polysomes, different mechanisms for their transport to the myelin membrane exist. The addition of dog pancreatic microsomes (DPM) during translation showed no membrane association for CNPase however, at least 50% of MBPs were observed to non-specifically associate with these membranes. When newly synthesized MBP and P2 were incubated post-translationally with DPM or rabbit spinal cord myelin P2 only associated with myelin whereas MBP showed an equal affinity for both types of membranes. The segregation of MBP free polysomes at the myelin membrane during synthesis ensures that the nascent MBP polypeptides associate with the correct membrane. Recent evidence has shown that the free polysome-mRNA complex is bound to the cytoskeleton during protein synthesis. After extensive characterization of the purified rat brain oligodendrocyte and myelin-associated cytoskeletons it was shown that the synthesis of MBPs and CNPase only occurs from mRNA that is associated with the cytoskeleton and not when it is part of the cytoplasmic mRNA pool. Lipid analysis of the purified rat brain myelin-associated cytoskeleton revealed the presence of tightly bound lipid with a considerable enrichment of cerebroside and sphingomyelin (the latter at the expense of phosphatidylethanolamine). These studies on the cytoskeletal involvement in myelinogenesis suggest that extrinsic CNS myelin proteins are synthesized on the cytoskeleton and that post-translational cytoskeletal transport of these proteins to the growing myelin membrane may take place.
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Smith, Susan Janette. "In vitro biosynthesis of steroids in equine testicular tissue". Thesis, University of Liverpool, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.328222.

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Nordman, Tomas. "In vitro studies on the biosynthesis and reduction of ubiquinone /". Stockholm, 2003. http://diss.kib.ki.se/2003/91-7349-475-5/.

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Lucas, Richard James. "In vitro peptidoglycan biosynthesis on silicon-supported tethered lipid bilayers". Thesis, University of Warwick, 2005. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.429714.

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Coxon, M. M. "Studies of pantothenate biosynthesis in Arabidopsis in vivo and in vitro". Thesis, University of Cambridge, 2007. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.598113.

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With a view to developing inhibitors, two enzymes of the Arabidopsis pantothenate pathway (KPHMT2 and PS) were expressed in E. coli, as MBP fusion proteins purified and used to perform kinetic studies to identify their characteristics. KPHMT2 showed an apparent Km, Vmax and kcat of 0.534 ± 0.089 mM, 0.802 ± 0.032 nmoles/min and 2.79 min/1 respectively for α-KIVA whilst the enzyme appeared to be inhibited by its co-factor, 5,10-methylene tetrahydrofolate, at levels above approximately 25μM. In addition to its potential as a target for herbicides and anti-microbials the pathway is also of commercial interest for the production of pantothenate for use as a supplement in animal feed and for addition to cosmetics. By constitutive expression of the genes of the E. coli pathway, Arabidopsis lines with higher levels of pantothenate than WT were obtained. Expression of E. coli panC resulted in increased levels of the vitamin in some lines, as did the expression of panD, however, expression of panB showed no increase. The β-alanine levels of the transgenic lines ere also assayed and showed hugely elevated levels in the lines expressing panD.  To determine if expression of the E. coli genes or the accumulation of pantothenate effected the expression of the endogenous genes, RT-PCR was performed and showed no significant difference in the expression of endogenous panB2 or panC in lines expressing E. coli panB, panC or panD. Expression of the genes of the pathway was also studied using promoter:GUS fusions, RT-PCR, western blot analysis and enzyme assays. The results showed expression of the genes throughout the plant suggesting de novo pantothenate biosynthesis with panB1 and panB2 promoter activity showing up-regulation in flowers and siliques, tissues known to require large amounts of pantothenate.
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Jacobson, Annica. "Regulation of hyaluronan biosynthesis : Expression in vitro and importance for tumor progression". Doctoral thesis, Uppsala University, Department of Medical Biochemistry and Microbiology, 2002. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-2004.

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Hyaluronan, a component of the extracellular matrix, is synthesized by either of three hyaluronan-synthesizing enzymes termed Has1, Has2 and Has3. The expression level of each Has gene varies between cell types of mesenchymal origin and is differentially regulated in response to external stimuli. For example, stimulation of mesothelial cells with PDGF-BB induced an up-regulation of the Has2 gene, whereas the Has1 and Has3 genes remained unaffected. The induction of Has2 gene expression correlated well with increased Has2 protein levels and accumulation of hyaluronan. Moreover, treatment of mesothelial cells with hydrocortisone suppressed hyaluronan synthesis in cell culture primarily through down-regulation of the Has2 gene. Thus, among the Has isoforms, Has2 seems to be most markedly regulated in response to external stimuli.

In an attempt to investigate the importance of hyaluronan in tumor progression, the hyaluronan synthesizing enzyme Has2 and the hyaluronan degrading enzyme Hyal1 were over-expressed in a rat colon adenocarcinoma cell line, PROb. We found that Has2 gene over-expression in colon carcinoma cells promoted cell growth in vitro and progression of transplantable tumors. In contrast, over-expression of Hyal1 lead to a considerable reduction of growth rates both in vivo and in vitro. A linear correlation between tumor growth rate and hyaluronan amount in tumor tissue was observed. In another tumor model, experimental anaplastic thyroid carcinoma, the effects of TGF-β inhibition on hyaluronan and collagen contents in tumor xenografts were investigated. We found that inhibition of TGF-β, a stimulator of hyaluronan and collagen synthesis, lead to reduced collagen deposition whereas the hyaluronan levels in stromal tissue only marginally differed. Our results indicate that a high ratio of collagen to hyaluronan may be characteristic of a pathogenic mechanism that leads to elevated interstitual tumor pressure.

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Champattanachai, Voraratt. "Effects of hexosamine biosynthesis on an in vitro model of cardiac ischemia". Thesis, Birmingham, Ala. : University of Alabama at Birmingham, 2008. https://www.mhsl.uab.edu/dt/2008d/champattanachai.pdf.

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Chiang, Cheng Ching Kurt. "Natural Rubber Biosynthesis: Perspectives from Polymer Chemistry". University of Akron / OhioLINK, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=akron1386367354.

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Cross, Stuart. "In vitro studies of the enzymes involved in fluorometabolite biosynthesis in Streptomyces cattleya /". St Andrews, 2009. http://hdl.handle.net/10023/720.

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Libros sobre el tema "In vitro biosynthesis"

1

J, Tymms Martin, ed. In vitro transcription and translation protocols. Totowa, N.J: Humana Press, 1995.

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International Symposium on In Vitro Immunization in Hybridoma Technology (1987 Tylösand, Sweden). In vitro immunization in hybridoma technology: Proceedings of the International Symposium on In Vitro Immunization in Hybridoma Technology, Tylösand, Sweden, September 7-8, 1987. Amsterdam: Elsevier, 1988.

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Recombinant And In Vitro Rna Synthesis Methods And Protocols. Humana Press, 2012.

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In vitro transcription and translation protocols. 2a ed. Totowa, NJ: Humana Press, 2005.

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International Symposium on In Vitro Immunization in Hybridoma Technology (1987 Tylösand, Sweden). In vitro immunization in hybridoma technology. Elsevier, 1988.

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Grandi, Guido. In Vitro Transcription and Translation Protocols. Humana Press, 2010.

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In Vitro Transcription and Translation Protocols. 2a ed. Humana Press, 2007.

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Jacobson, Annica. Regulation of Hyaluronan Biosynthesis: Expression in Vitro & Importance for Tumor Progression (Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, 1148). Uppsala Universitet, 2002.

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Capítulos de libros sobre el tema "In vitro biosynthesis"

1

Kakegawa, Koichi y Atsushi Komamine. "Anthocyanin biosynthesis in vitro". En Plant Tissue Culture Manual, 935–57. Dordrecht: Springer Netherlands, 1995. http://dx.doi.org/10.1007/978-94-009-0103-2_52.

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Kobayashi, Shiro y Hiroshi Uyama. "In Vitro Biosynthesis of Polyesters". En Biopolyesters, 241–62. Berlin, Heidelberg: Springer Berlin Heidelberg, 2001. http://dx.doi.org/10.1007/3-540-40021-4_8.

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Wilson, D. Wright. "The Biosynthesis of Pyrimidines in vitro". En Ciba Foundation Symposium - Isotopes in Biochemistry, 152–63. Chichester, UK: John Wiley & Sons, Ltd., 2008. http://dx.doi.org/10.1002/9780470715161.ch14.

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Kurz, Wolfgang G. W., Friedrich Constabel y Robert T. Tyler. "Biosynthesis of Monoterpene Indole Alkaloids in vitro". En Plant Tissue Culture Manual, 37–59. Dordrecht: Springer Netherlands, 1995. http://dx.doi.org/10.1007/978-94-011-0303-9_3.

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Ardao, Inés, Ee Taek Hwang y An-Ping Zeng. "In Vitro Multienzymatic Reaction Systems for Biosynthesis". En Fundamentals and Application of New Bioproduction Systems, 153–84. Berlin, Heidelberg: Springer Berlin Heidelberg, 2013. http://dx.doi.org/10.1007/10_2013_232.

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Kurz, Wolfgang G. W., Friedrich Constabel y Robert T. Tyler. "Biosynthesis of Monoterpene Indole Alkaloids in vitro". En Plant Tissue Culture Manual, 959–81. Dordrecht: Springer Netherlands, 1995. http://dx.doi.org/10.1007/978-94-009-0103-2_53.

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Pichersky, Eran, Natalia Dudareva, Jihong Wang, Leland Cseke y Efraim Lewinsohn. "Biosynthesis of Scent and Flavor Compounds". En Plant Biotechnology and In Vitro Biology in the 21st Century, 601–4. Dordrecht: Springer Netherlands, 1999. http://dx.doi.org/10.1007/978-94-011-4661-6_135.

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Caughey, B. "In Vitro Expression and Biosynthesis of Prion Protein". En Current Topics in Microbiology and Immunology, 93–107. Berlin, Heidelberg: Springer Berlin Heidelberg, 1991. http://dx.doi.org/10.1007/978-3-642-76540-7_6.

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Thomas, Lebin, Zeeshan ur Rahman, Kuldeep Sharma, Devendra Nagar, Akanksha Vashishtha, Gaurav Kumar y Siva P. K. Chetri. "In Vitro Biosynthesis of Natural Products in Plant Roots". En Rhizobiology: Molecular Physiology of Plant Roots, 475–95. Cham: Springer International Publishing, 2021. http://dx.doi.org/10.1007/978-3-030-84985-6_24.

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Marsh, J. P., L. Jean y B. T. Mossman. "Asbestos and Fibrous Glass Induce Biosynthesis of Polyamines in Tracheobronchial Epithelial Cells In Vitro". En In Vitro Effects of Mineral Dusts, 305–11. Berlin, Heidelberg: Springer Berlin Heidelberg, 1985. http://dx.doi.org/10.1007/978-3-642-70630-1_39.

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Actas de conferencias sobre el tema "In vitro biosynthesis"

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Fang, Wen-Feng, Ya-Ting Chang, Chia-Cheng Tseng, Hung-Chen Chen, Ya-Chun Chang, Shih-Feng Liu, Chin-Chou Wang y Meng-Chih Lin. "Leukotriene Biosynthesis Inhibitor Attenuates In Vitro Lipid A Induced Acute Lung Injury (ALI)". En American Thoracic Society 2012 International Conference, May 18-23, 2012 • San Francisco, California. American Thoracic Society, 2012. http://dx.doi.org/10.1164/ajrccm-conference.2012.185.1_meetingabstracts.a1421.

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Ramm, Michael, Silke Steinbach y Volker Schroeckh. "IN VITRO BIOSYNTHESIS OF AN AI-2 LIKE BACTERIAL AUTOINDUCER BY RECOMBINANT ESCHERICHIA COLI ENZYMES". En XXIst International Carbohydrate Symposium 2002. TheScientificWorld Ltd, 2002. http://dx.doi.org/10.1100/tsw.2002.700.

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Msdcalf, R. L., E. K. O. Kruithof y W.-D. Schleuning. "TRANSIENT INDUCTION OF PLASMINOGEN ACTIVATOR INHIBITOR 2 (PAI-2) GENE TRANSCRIPTION BY THE TUMOUR PROMOTING PHORBOL ESTER PMA IN THE HUMAN MACROPHAGE-LIKE CELT, LINE U-937". En XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1642857.

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The human hematopoietic cell line U-937 differentiates to a macrophage/monocyte phenotype after exposure to the tumour promoter PMA. We have recently shown that PMA concomitantly induces PAI-2 biosynthesis in these cells. Now, we have employed an 1880 base pair cDNA to study PAI-2 biosynthesis on the level of transcription and mRNA stability. By in vitro elongation of initiated PAI-2 transcripts in isolated nuclei in the presence of 32p-labelled UTP followed by hybridization to cloned DNA (“run-on” transcription assay), we have demonstrated that PAI-2 gene transcription rates are induced 50-fold after exposure of the cells to PMA for 4 h in the presence of 5% fetal bovine serum. Transcription rates subsequently declined to base-line levels within 48 h. This transient increase of PAI-2 transcription rate was reflected by a transient induction of PAI-2 mRNA with maximal levels (60-fold) occuring after 16 h and subsiding to near base-levels after 48 h. Similar experiments performed in the absence of fetal bovine serum revealed that maximal levels of PAI-2 mRNA occured after only 4 hours exposure to PMA and were virtually absent after 16 h. Our results indicate that PMA induction of PAI-2 biosynthesis occurs at the level of gene transcription and is influenced by the presence of serum. Since PAI-2 is a major protein of U-937 cells, these date also suggest a role for hormonally modulated PAI-2 biosynthesis in macrophage physiology.
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Yamauchi, Kevin A., Christopher B. Raub, Albert C. Chen, Robert L. Sah, Scott J. Hazelwood y Stephen M. Klisch. "Glycosaminoglycan and Collagen Remodeling During In Vitro Dynamic Compression of Articular Cartilage: Experiments and Finite Element Modeling". En ASME 2013 Summer Bioengineering Conference. American Society of Mechanical Engineers, 2013. http://dx.doi.org/10.1115/sbc2013-14286.

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The biomechanical properties of articular cartilage (AC) can be altered by chemical and mechanical stimuli. Dynamic unconfined compression (UCC) has been shown to increase biosynthesis at moderate strain amplitudes (1–4%) and frequencies from 0.01Hz. to 0.1Hz [1]. Furthermore, interstitial fluid velocity and maximum principle strain have been proposed as candidates for controlling glycosaminoglycan (GAG) and collagen (COL) remodeling, respectively [2,3]. The goal of this study was to integrate in vitro growth data, including biochemical and microstructural properties, into a computational continuum mixture model to elucidate potential mechanical triggers for AC tissue remodeling.
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5

Paye, M., T. Krieg y Ch M. Lapière. "FACTOR XIII (FXIIIa) OF BLOOD COAGULATION NORMALIZES COLLAGEN SYNTHESIS OF SCLERODERMA FIBROBLASTS". En XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644650.

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Progressive systemic scleroderma (PSS) fibroblasts display, in some cases, an excessive collagen production which leads to fibrosis of the skin and internal organs. Administration of FXIIIa has been reported to be beneficial to some of the PSS patients. The effect of FXIIIa on collagen synthesis by PSS fibroblasts was studied in vitro in two different culture conditions: in a confluent monolayer on plastic and in a three-dimensional collagen lattice.Proteins and collagen synthesis was measurfed by metabolic labeling for 24 h with 3H-proline in absence or presence of FXIIIa (1 U/ml) in dermal fibroblasts from an active lesion (PSS forearm), from an uninvolved area of the skin of the same patient (control abdomen) and from the skin of a normal subject.Proteins synthesis was similar for the three strains under both culture conditions while collagen synthesis was strongly increased in PSS forearm fibroblasts as compared to the uninvolved and to the normal skin fibroblasts. The addition of FXIIIa repressed collagen synthesis of PSS forearm synthesis to the level observed in the abdominal skin fibroblasts of the patient and the normal cells in absence of FXIIIa. When cultured in a collagen lattice collagen synthesis was repressed in a similar proportion in all fibroblasts. Addition of FXIIIa further reduced collagen biosynthesis in the active PSS fibroblasts. The addition of FXIIIa in the lattice largely increased the degradation of newly synthesized collagen in all the strains of fibroblasts.The action of FXIIIa on collagen biosynthesis was also tested in monolayer in 3 other strains of PSS fibroblasts and 3 controls. In all fibroblasts, collagen biosynthesis was reduced by 75 % after addition of FXIIIa.Our results suggest that the beneficial effect of FXIIIa administration in PSS patient might be related to a reduction o1 excessive collagen production and perhaps to an increased degradation of newly synthesized molecules.
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6

Medcalf, R. L., E. van den Berg y W.-D. Schleuning. "THE INFLUENCE OF GLUCOCORTICOID HORMONES ON THE GENE TRANSCRIPTION OF POUR COMPONENTS OF THE FIBRINOLYTIC SYSTEM". En XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644611.

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The hormonal regulation of plasminogen activator (urokinase type (u-PA) and tissue-type (t-PA)) biosynthesis plays an important role in fibrinolysis and extracellular matrix turnover during invasive growth and cell migration. Recently, two genetically distinct inhibitors of both PA's (PA inhibitor 1 (PAI-1) and PA inhibitor 2 (PAI-2)) have been described which may contribute to the modulation of matrix stability. We have employed cloned cDNA probes to study the regulation of biosynthesis of these proteins in the human fibrosarcoma line HT1080. These cells constitutively express high levels of pro-u-PA. PAI-1, PAI-2 and t-PA are also present at relatively low levels. Treatment of the cells with the glucocorticoid dexamethasone (Dex; 10−7 M), almost completely suppresses u-PA gene transcription, as determined by measurement of in vitro elongation of initiated u-PA transcripts in isolated nuclei ("run-on" transcription assay). Concomitantly, Dex also induces PAI-1 and t-PA gene transcription, whereas PAI-2 gene transcription appeared to remain ' unaffected. These changes in transcription rates are also reflected at the level of mRNA: u-PA mRNA is decreased, whereas PAI-1 and t-PA mRNA are simultaneously induced. PAI-2 mRNA is apparently unchanged. These results demonstrate that glucocorticoid hormones reprogramne the expression of components of the fibrinolytic system, and are of possible relevance in the context of inflammatory disease and malignancy.
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7

Kieffer, N., M. Titeux, A. Henri, J. Breton-Gorius y W. Vainchenker. "MEGAKARYOCYTIC ORIGIN OF PLATELET HLA CLASS I ANTIGEN". En XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643546.

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The existence of HLA class I antigens on human platelets is well established. However, several authors have suggested that platelet HLA antigens are not integral membrane components but are acquired from soluble plasma sources and adsorbed to the platelet surface.In the present study, we used the monoclonal antibody W6/32, directed against a monomorphic epitope of the HLA class I antigen for the immunochemical characterization of platelet HLA. Immunoprecipitation experiments, performed after in vitro metabolic radiolabeling of human platelets revealed a band of molecular weight 44,000 identical to that precipitated from metabolic labeled U937 or HEL cells. When the same antibody was tested by indirected immunofluorescence in a double labeling technique on in vitro cultures of human megakaryocytes, performed in the absence of human serum in the culture medium, megakaryocytes identified by an anti-vWF MoAb revealed a membrane staining with W6/32 identical to that observed on other bone marrow cells, e.g. macrophages. Our results provide evidence that platelet HLA has a megaka-ryocytic origin and that residual biosynthesis of HLA antigen does still occur in circulating platelets. However, our results do not exclude the ability of human platelets to adsord circulating HLA class I antigen from plasma.
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8

Rampart, M., H. Bult, A. G. Herman, P. J. Jose y T. J. Williams. "PROSTACYCLIN (PGI2) FORMATION IN RELATION TO ANAPHYLATOXIN C5a GENERATION DURING RABBIT ENDOTOXIN SHOCK". En XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1642837.

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Injection of endotoxin (LPS) in animals, a model for gram-negative septic shock, leads to intravascular activation of the complement system, and is one of the few conditions in which 6-oxo-PGF]CX and thromboxane (TX) B2 (non-enzymic metabolites of PGI2 and TXA2) can be detected in arterial blood. Previously we reported associations between complement activation, PGI2 biosynthesis and LPS-induced hypotension in rabbits. As C5a and C5adesArg trigger endothelial PGI2 formation in vitro, we have now measured the plasma levels of immunoreactive (ir) C5a in relation to generation of PGI2 and changes in arterial blood pressure in LPS shock. Pentobarbitone anaesthethized rabbits received LPS (E. coli 0111:B4, 0.5 mg/kg) or saline via the marginal ear vein. A catheter in the left carotid artery was used to collect blood and to monitor mean arterial blood pressure (MABP). Platelet and leukocyte numbers, haemolytic complement titre (CH50), and plasma ir6-oxo-PGFioc , irTXB2 and irC5a were measured 15 min before and at different times after saline or LPS injection. LPS caused a dose- and time-dependent formation of irC5a in rabbit serum in vitro, predominantly via the classical pathway. LPS also activated complement in vivo, as indicated by about 20 % reduction of CH50 titre (measured after 3h) and a marked increase of arterial irC5a (20-120 ng/ml) in the first 2 to 5 min. After 30 min, irC5a had returned to baseline levels (< 2-5 ng/ml) and remained so up to 3h after injection of LPS. This irC5a peak correlated with a shortlasting initiation of PGI2 release (from < 20 pg/ml up to 550 pg/ml) and a drop in MABP (from about 95 mmHg to 50 mmHg) 2-5 min after LPS. None of these changes occurred after saline injection.In conclusion, LPS activates complement in vivo with concomitant formation of C5a. This peptide may trigger -either directly or after phagocyte activation - endothelial PGI2 biosynthesis, leading to arterial hypotension. This is supported by the suppression of the initial rise of arterial ir6-oxo-PGF1α and hypotension in complement-depleted rabbits. Inhibition of C5a formation or activity may prove to be a meaningful approach to the treatment of septic circulatory shock.
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9

Vernon, Lauren L., David G. Wilensky, Chong Wang, Lee D. Kaplan y Chun-Yuh C. Huang. "Mechanical Loading Reduces Chondrocyte Death After Single Impact Trauma: Porcine Knee Model". En ASME 2012 Summer Bioengineering Conference. American Society of Mechanical Engineers, 2012. http://dx.doi.org/10.1115/sbc2012-80833.

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Osteoarthritis often results from degenerative changes induced by trauma such as joint impact injuries sustained during athletics, combat, or motor vehicle accidents. Articular cartilage, avascular in nature, relies of synovial nutrition [1] and lacks sufficient regenerative capabilities [2]. Acute cartilage injuries have been shown to induce cell death [3, 4, 5], leading to reduced chondrocyte density and degenerative changes to the cartilage matrix composition; over time the tissue becomes compromised and loses its ability to maintain and restore itself. It has been demonstrated, that mechanical loading can affect local perfusion and diffusion through the matrix thereby altering the flow of nutrients and metabolites [2, 6]. Furthermore, mechanical loading modulates the chondrocyte biosynthesis of extracellular matrix that is required in the cartilage repair process. In this study, a two part in-vitro porcine knee model was utilized to investigate articular cartilage response immediately following a single impact injury under cyclic mechanical loading conditions.
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10

Jovanović-Šanta, Suzana S., Aleksandar M. Oklješa, Antos B. Sachanka, Yaraslau U. Dzichenka y Sergei A. Usanov. "17-SUBSTITUTED STEROIDAL TETRAZOLES – NOVEL LIGANDS FOR HUMAN STEROID-CONVERTING CYP ENZYMES". En 1st INTERNATIONAL Conference on Chemo and BioInformatics. Institute for Information Technologies, University of Kragujevac, 2021. http://dx.doi.org/10.46793/iccbi21.336js.

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In animal and human organisms, there are many enzymes, members of the family of heme- containing proteins, cytochromes P450 (CYPs), included in the biosynthesis and metabolism of many biomolecules, as cholesterol, bile acids, sex, and corticosteroid hormones, as well as in metabolism of drugs and xenobiotics. It is also well-known that different imidazole and triazole derivatives are efficient inhibitors of CYPs activity. In this study, we present in vitro screening of binding of novel androstane derivatives with tetrazole- containing substituents in position 17 to human recombinant steroid-converting CYP enzymes: CYP7A1, CYP7B1, CYP17A1, CYP19, and CYP21. Initial screening was performed using a high throughput screening approach, while the affinity of the ligands was analyzed using spectrophotometric titration. For some among tested compounds type I spectral response (substrate-like binding) for CYP7A1 selectively, while for one compound type II spectral response (inhibitor-like binding) for CYP21 were detected, with micromolar values of Kds. Interestingly, one compound with mixed spectral response was found to bind for CYP7B1, which means that there are two optimal positions of the ligand inside the protein active site. Such results could be useful in CYP-inhibiting drug development, during a fast, high-throughput screening of pharmacological potential of novel compounds, as well as in side- effects recognizing.
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Informes sobre el tema "In vitro biosynthesis"

1

Applebaum, Shalom W., Lawrence I. Gilbert y Daniel Segal. Biochemical and Molecular Analysis of Juvenile Hormone Synthesis and its Regulation in the Mediterranean Fruit Fly (Ceratitis capitata). United States Department of Agriculture, 1995. http://dx.doi.org/10.32747/1995.7570564.bard.

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Original Objectives and revisions: (1) "To determine the biosynthetic pathway of JHB3 in the adult C. capitata CA in order to establish parameters for the future choice and synthesis of suitable inhibitors". Modified: to determine the pattern of FR-7 biosynthesis during normal reproductive maturation, and identify enzymes potentially involved in its synthesis. (2) "To correlate allatal epoxidase activity to the biosynthesis of JHB3 at different stages of reproductive maturation/vitellogenesis and evaluate the hypothesis that a specific JH-epoxidase may be rate limiting". Modified: to study the effects of epoxidase inhibitors on the pattern of allatal JH biosynthesis in vitro and on female reproduction in vive. (3) "To probe and clone the gene homologous to ap from C. capitata, determine its exon-intron organization, sequence it and demonstrate its spatial and temporal expression in larvae, pupae and adults." The "Medfly" (Ceratitis capitata) is a serious polyphagous fruit pest, widely distributed in subtropical regions. Damage is caused by oviposition and subsequent development of larvae. JH's are dominant gonadotropic factors in insects. In the higher Diptera, to which the Medfly belongs, JHB3 is a major homolog. It comprises 95% of the total JH produced in vitro in D. melanogaster, with JH-III found as a minor component. The biosynthesis of both JH-III and JHB3 is dependent on epoxidation of double bonds in the JH molecule. The specificity of such epoxidases is unknown. The male accessory gland D. melanogaster produces a Sex Peptide, transferred to the female during copulation. SP reduces female receptivity while activating specific JH biosynthesis in vitro and inducing oviposition in vive. It also reduces pheromone production and activates CA of the moth Helicoverpa armigera. In a previous study, mutants of the apterous (ap) gene of D. melanogaster were analyzed. This gene induces previteilogenic arrest which can be rescued by external application of JH. Considerable progress has been made in recombinant DNA technology of the Medfly. When fully operative, it might be possible to effectively transfer D. melanogaster endocrine gene-lesions into the Medfly as a strategy for their genetic control. A marked heterogeneity in the pattern of JH homologs produced by Medfly CA was observed. Contrary to the anticipated biosynthesis of JHB;, significant amounts of an unknown JH-like compound, of unknown structure and provisionally termed FR-7, were produced, in addition to significant amounts of JH-III and JHB3. Inhibitors of monooxygenases, devised for their effects on ecdysteroid biosynthesis, affect Medfly JH biosynthesis but do not reduce egg deposition. FR-7 was isolated from incubation media of Medfly CA and examined by various MS procedures, but its structure is not yet resolved. MS analysis is being done in collaboration with Professor R.R.W. Rickards of the Australian National University in Canberra, Australia. A homologue of the ap gene of D. melanogaster exists in the Medfly. LIM domains and the homeo-domain, important for the function of the D. melanogaster ap gene, are conserved here too. Attempts to clone the complete gene were unsuccessful. Due to the complexity of JH homologs, presence of related FR-7 in the biosynthetic products of Medfly CA and lack of reduction in eggs deposited in the presence of monooxygenase inhibitors, inhibition of epoxidases is not a feasible alternative to control Medfly reproduction, and raises questions which cannot be resolved within the current dogma of hormonal control of reproduction in Diptera. The Medfly ap gene has similar domains to the D. melanogaster ap gene. Although mutant ap genes are involved in JH deficiency, ap is a questionable candidate for an endocrine lesion, especially since the D. melanogoster gene functions is a transcription factor.
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2

O`Brien, David Allen. Carotenoid biosynthesis in bacteria: In vitro studies of a crt/bch transcription factor from Rhodobacter capsulatus and carotenoid enzymes from Erwinia herbicola. Office of Scientific and Technical Information (OSTI), noviembre de 1992. http://dx.doi.org/10.2172/10140835.

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3

O'Brien, D. A. Carotenoid biosynthesis in bacteria: In vitro studies of a crt/bch transcription factor from Rhodobacter capsulatus and carotenoid enzymes from Erwinia herbicola. Office of Scientific and Technical Information (OSTI), noviembre de 1992. http://dx.doi.org/10.2172/6880113.

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4

Fridman, Eyal y Eran Pichersky. Tomato Natural Insecticides: Elucidation of the Complex Pathway of Methylketone Biosynthesis. United States Department of Agriculture, diciembre de 2009. http://dx.doi.org/10.32747/2009.7696543.bard.

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Plant species synthesize a multitude of specialized compounds 10 help ward off pests. and these in turn may well serve as an alternative to synthetic pesticides to reduce environmental damage and health risks to humans. The general goal of this research was to perform a genetic and biochemical dissection of the natural-insecticides methylketone pathway that is specific to the glandular trichomes of the wild species of tomato, Solanumhabrochaites f. glabratum (accession PI126449). Previous study conducted by us have demonstrated that these compounds are synthesized de novo as a derivate pathway of the fatty acid biosynthesis, and that a key enzyme. designated MethylketoneSynthase 1 (MKS 1). catalyzes conversion of the intermediate B-ketoacyl- ACPs to the corresponding Cn-1 methylketones. The approach taken in this proposed project was to use an interspecific F2 population. derived from the cross between the cultivated lV182 and the wild species PIl26449. for three objectives: (i) Analyze the association between allelic status of candidate genes from the fatty acid biosynthesis pathway with the methylketone content in the leaves (ii) Perform bulk segregant analysis of genetic markers along the tomato genome for identifying genomic regions that harbor QTLs for 2TD content (iii) Apply differential gene expression analysis using the isolated glands of bulk segregant for identifying new genes that are involved in the pathway. The genetic mapping in the interspecific F2 population included app. 60 genetic markers, including the candidate genes from the FAS pathway and SSR markers spread evenly across the genome. This initial; screening identified 5 loci associated with MK content including the candidate genes MKS1, ACC and MaCoA:ACP trans. Interesting observation in this genetic analysis was the connection between shape and content of the glands, i.e. the globularity of the four cells, typical to the wild species. was associated with increased MK in the segregating population. In the next step of the research transcriptomic analysis of trichomes from high- and 10w-MK plants was conducted. This analysis identified a new gene, Methy1ketone synthase 2 (MKS2), whose protein product share sequence similarity to the thioesterase super family of hot-dog enzymes. Genetic analysis in the segregating population confirmed its association with MK content, as well as its overexpression in E. coli that led to formation of MK in the media. There are several conclusions drawn from this research project: (i) the genetic control of MK accumulation in the trichomes is composed of biochemical components in the FAS pathway and its vicinity (MKS 1 and MKS2). as well as genetic factors that mediate the morphology of these specialized cells. (ii) the biochemical pathway is now realized different from what was hypothesized before with MKS2 working upstream to I\1KS 1 and serves as the interface between primary (fatty acids) and secondary (MK) metabolism. We are currently testing the possible physical interactions between these two proteins in vitro after the genetic analysis showed clear epistatic interactions. (iii) the regulation of the pathway that lead to specialized metabolism in the wild species is largely mediated by transcription and one of the achievements of this project is that we were able to isolate and verify the specificity of the MKS1 promoter to the trichomes which allows manipulation of the pathways in these cells (currently in progress). The scientific implications of this research project is the advancement in our knowledge of hitherto unknown biochemical pathway in plants and new leads for studying a new family in plants (hot dog thioesterase). The agricultural and biotechnological implication are : (i) generation of new genetic markers that could assist in importing this pathway to cultivated tomato hence enhancing its natural resistance to insecticides, (ii) the discovery of MKS2 adds a new gene for genetic engineering of plants for making new fatty acid derived compounds. This could be assisted with the use of the isolated and verified MKS1 promoter. The results of this research were summarized to a manuscript that was published in Plant Physiology (cover paper). to a chapter in a proceeding book. and one patent was submitted in the US.
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5

Rafaeli, Ada, Russell Jurenka y Daniel Segal. Isolation, Purification and Sequence Determination of Pheromonotropic-Receptors. United States Department of Agriculture, julio de 2003. http://dx.doi.org/10.32747/2003.7695850.bard.

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Moths constitute a major group of pest insects in agriculture. Pheromone blends are utilised by a variety of moth species to attract conspecific mates, which is under circadian control by the neurohormone, PBAN (pheromone-biosynthesis-activating neuropeptide). Our working hypothesis was that, since the emission of sex-pheromone is necessary to attract a mate, then failure to produce and emit pheromone is a potential strategy for manipulating adult moth behavior. The project aimed at identifying, characterising and determining the sequence of specific receptors responsible for the interaction with pheromonotropic neuropeptide/s using two related moth species: Helicoverpa armigera and H. lea as model insects. We established specific binding to a membrane protein estimated at 50 kDa in mature adult females using a photoaffinity-biotin probe for PBAN. We showed that JH is required for the up-regulation of this putative receptor protein. In vitro studies established that the binding initiates a cascade of second messengers including channel opening for calcium ions and intracellular cAMP production. Pharmacological studies (using sodium fluoride) established that the receptor is coupled to a G-protein, that is, the pheromone-biosynthesis-activating neuropeptide receptor (PBAN-R) belongs to the family of G protein-coupled receptor (GPCR)'s. We showed that PBAN-like peptides are present in Drosophila melanogaster based on bioassay and immunocytochemical data. Using the annotated genome of D. melanogaster to search for a GPCR, we found that some were similar to neuromedin U- receptors of vertebrates, which contain a similar C-terminal ending as PBAN. We established that neuromedin U does indeed induce pheromone biosynthesis and cAMP production. Using a PCR based cloning strategy and mRNA isolated from pheromone glands of H. zea, we successfully identified a gene encoding a GPCR from pheromone glands. The full-length PBAN-R was subsequently cloned and expressed in Sf9 insect cells and was shown to mobilize calcium in response to PBAN in a dose-dependent manner. The successful progress in the identification of a gene, encoding a GPCR for the neurohormone, PBAN, provides a basis for the design of a novel battery of compounds that will specifically antagonize pheromone production. Furthermore, since PBAN belongs to a family of insect neuropeptides with more than one function in different life stages, this rationale may be extended to other physiological key-regulatory processes in different insects.
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6

Cohen, Jerry D. y Ephraim Epstein. Metabolism of Auxins during Fruit Development and Ripening. United States Department of Agriculture, agosto de 1995. http://dx.doi.org/10.32747/1995.7573064.bard.

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We had proposed to look at several aspects of auxin metabolism in fruit tissues: 1) IAA biosynthesis from tryptophan and IAA biosynthesis via the non-tryptophan pathway; 2) changes in the capacity to form conjugates and catabolites of auxin at different times during fruit development and; 3) the effects of modifying auxin metabolism in fruit tissues. The latter work focused primarily on the maize iaglu gene, with initial studies also using a bacterial gene for hydrolysis of IAA-aspartate. These metabolic and molecular studies were necessary to define potential benefits of auxin metabolism modification and will direct future efforts for crop improvement by genetic methods. An in vitro system was developed for the production of tomato fruit in culture starting from immature flowers in order to ascertain the effect of auxin modification on fruit ripening. IAA supplied to the fruit culture media prior to breaker stage resulted in an increase in the time period between breaker and red-ripe stages from 7 days without additional IAA to 12 days when 10-5 M IAA was added. These results suggest that significant changes in the ripening period could be obtained by alteration of auxin relationships in tomato fruit. We generated transgenic tomato plants that express either the maize iaglu gene or reduced levels of the gene that encodes the enzyme IAA-glucose synthetase. A modified shuttle vector pBI 121 expressing the maize iaglu gene in both sense and antisense orientations under a 35S promoter was used for the study. The sense plants showed total lack of root initiation and development. The antisense transgenic plants, on the other hand, had unusually well developed root systems at early stages in development. Analysis showed that the amount and activity of the endogenous 75 kDa IAGLU protein was reduced in these plants and consequently these plants had reduced levels of IAA-glucose and lower overall esterified IAA.
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7

Dudareva, Natalia, Alexander Vainstein, Eran Pichersky y David Weiss. Integrating biochemical and genomic approaches to elucidate C6-C2 volatile production: improvement of floral scent and fruit aroma. United States Department of Agriculture, septiembre de 2007. http://dx.doi.org/10.32747/2007.7696514.bard.

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The specific objectives of approved proposal include to: 1. Elucidate the C6-C2 biochemical pathways leading to the biosynthesis of phenylacetaldehyde, phenylethyl alcohol and phenylethyl acetate in floral tissues of ornamentally important plants, pefunia and roses. 2. Isolate and characterrze genes responsible for the production of these C6-C2 compounds and those involved in the regulation of the pathway using genomic and transcriptomic tools. 3. Determine whether altering the expression of key genes of this pathway can result in changing the aroma characteristics of flowers. Aldehydes are intermediates in a variety of biochemical pathways including those involved in the metabolism of carbohydrates, vitamins, steroids, amino acids, benzylisoquinoline alkaloids, hormones, and lipids. In plants they are also synthesized in response to environmental stresses such as salinity, cold, and heat shock or as flavors and aromas in fruits and flowers. Phenylacetaldehyde along with 2-phenylethanol and its acetate ester, are important scent compounds in numerous flowers, including petunias and roses. However, little is known about the biosynthesis of these volatile compounds in plants. We have shown that the formation PHA and 2-phenylethanol from Phe does not occur via trans-cinnamic acid and instead competes with the key enzyme of phenypropanoid metabolism Pheammonia-lyase (PAL) for Phe utilization. Using functional genomic approach and comparative gene expression profiling, we have isolated and characterized a novel enzyme from petunia and rose flowers that catalyzes the formation of the Ca-Czcompound phenylacetaldehyde (PHA) from L-phenylalanine (Phe) by the removal of both the carboxyl and amino groups. This enzyme, designated as phenylacetaldehyde synthases (PAAS), is a bifunctional enzyme that catalyzes the unprecedented efficient coupling of phenylalanine decarboxylation to oxidation, generating phenylacetaldehyde, CO2, ammonia, and hydrogen peroxide in stoichiometric amounts. Down-regulation of PAAS expression via RNA interference-based (RNAi) technology in petunia resulted in no PHA emission when compared with controls. These plants also produced no 2-phenylethanol, supporting our conclusion that PHA is a precursor of 2-phenylethanol. To understand the regulation of scent formation in plants we have also generated transgenic petunia and tobacco plants expressing the rose alcohol acetyltransferase (RhAAT) gene under the control of a CaMV-35S promoter. Although the preferred substrate of RhAAT in vitro is geraniol, in transgenic petunia flowers, it used phenylethyl alcohol and benzyl alcohol to produce the corresponding acetate esters, not generated by control flowers. These results strongly point to the dependence of volatile production on substrate availability. Analysis of the diurnal regulation of scent production in rose flowers revealed that although the daily emission of most scent compounds is synchronized, various independently evolved mechanisms control the production, accumulation and release of different volatiles. This research resulted in a fundamental discovery of biochemical pathway, enzymes and genes involved in biosynthesis of C6-C2s compounds, and provided the knowledge for future engineering plants for improved scent quality.
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8

Meidan, Rina y Robert Milvae. Regulation of Bovine Corpus Luteum Function. United States Department of Agriculture, marzo de 1995. http://dx.doi.org/10.32747/1995.7604935.bard.

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The main goal of this research plan was to elucidate regulatory mechanisms controlling the development, function of the bovine corpus luteum (CL). The CL contains two different sterodigenic cell types and therefore it was necessary to obtain pure cell population. A system was developed in which granulosa and theca interna cells, isolated from a preovulatory follicle, acquired characteristics typical of large (LL) and small (SL) luteal cells, respectively, as judged by several biochemical and morphological criteria. Experiments were conducted to determine the effects of granulosa cells removal on subsequent CL function, the results obtained support the concept that granulosa cells make a substaintial contribution to the output of progesterone by the cyclic CL but may have a limited role in determining the functional lifespan of the CL. This experimental model was also used to better understand the contribution of follicular granulosa cells to subsequent luteal SCC mRNA expression. The mitochondrial cytochrome side-chain cleavage enzyme (SCC), which converts cholesterol to pregnenolone, is the first and rate-limiting enzyme of the steroidogenic pathway. Experiments were conducted to characterize the gene expression of P450scc in bovine CL. Levels of P450scc mRNA were higher during mid-luteal phase than in either the early or late luteal phases. PGF 2a injection decreased luteal P450scc mRNA in a time-dependent manner; levels were significantly reduced by 2h after treatment. CLs obtained from heifers on day 8 of the estrous cycle which had granulosa cells removed had a 45% reduction in the levels of mRNA for SCC enzymes as well as a 78% reduction in the numbers of LL cells. To characterize SCC expression in each steroidogenic cell type we utilized pure cell populations. Upon luteinization, LL expressed 2-3 fold higher amounts of both SCC enzymes mRNAs than SL. Moreover, eight days after stimulant removal, LL retained their P4 production capacity, expressed P450scc mRNA and contained this protein. In our attempts to establish the in vitro luteinization model, we had to select the prevulatory and pre-gonadotropin surge follicles. The ratio of estradiol:P4 which is often used was unreliable since P4 levels are high in atretic follicles and also in preovulatory post-gonadotropin follicles. We have therefore examined whether oxytocin (OT) levels in follicular fluids could enhance our ability to correctly and easily define follicular status. Based on E2 and OT concentrations in follicular fluids we could more accurately identify follicles that are preovulatory and post gonadotropin surge. Next we studied OT biosynthesis in granulosa cells, cells which were incubated with forskolin contained stores of the precursor indicating that forskolin (which mimics gonadotropin action) is an effective stimulator of OT biosynthesis and release. While studying in vitro luteinization, we noticed that IGF-I induced effects were not identical to those induced by insulin despite the fact that megadoses of insulin were used. This was the first indication that the cells may secrete IGF binding protein(s) which regonize IGFs and not insulin. In a detailed study involving several techniques, we characterized the species of IGF binding proteins secreted by luteal cells. The effects of exogenous polyunsaturated fatty acids and arachidonic acid on the production of P4 and prostanoids by dispersed bovine luteal cells was examined. The addition of eicosapentaenoic acid and arachidonic acid resulted in a dose-dependent reduction in basal and LH-stimulated biosynthesis of P4 and PGI2 and an increase in production of PGF 2a and 5-HETE production. Indomethacin, an inhibitor of arachidonic acid metabolism via the production of 5-HETE was unaffected. Results of these experiments suggest that the inhibitory effect of arachidonic acid on the biosynthesis of luteal P4 is due to either a direct action of arachidonic acid, or its conversion to 5-HETE via the lipoxgenase pathway of metabolism. The detailed and important information gained by the two labs elucidated the mode of action of factors crucially important to the function of the bovine CL. The data indicate that follicular granulosa cells make a major contribution to numbers of large luteal cells, OT and basal P4 production, as well as the content of cytochrome P450 scc. Granulosa-derived large luteal cells have distinct features: when luteinized, the cell no longer possesses LH receptors, its cAMP response is diminished yet P4 synthesis is sustained. This may imply that maintenance of P4 (even in the absence of a Luteotropic signal) during critical periods such as pregnancy recognition, is dependent on the proper luteinization and function of the large luteal cell.
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9

Sionov, Edward, Nancy Keller y Shiri Barad-Kotler. Mechanisms governing the global regulation of mycotoxin production and pathogenicity by Penicillium expansum in postharvest fruits. United States Department of Agriculture, enero de 2017. http://dx.doi.org/10.32747/2017.7604292.bard.

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The original objectives of the study, as defined in the approved proposal, are: To characterize the relationship of CreA and LaeA in regulation of P T production To understand how PacC modulates P. expansumpathogenicity on apples To examine if other secondary metabolites are involved in virulence or P. expansumfitness To identify the signaling pathways leading to PAT synthesis Penicilliumexpansum, the causal agent of blue mould rot, is a critical health concern because of the production of the mycotoxinpatulin (PAT) in colonized apple fruit tissue. Although PAT is produced by many Penicilliumspecies, the factors activating its biosynthesis were not clear. This research focused on host and fungal mechanisms of activation of LaeA (the global regulator of secondary metabolism), PacC (the global pH modulator) and CreA (the global carbon catabolite regulator) on PAT synthesis with intention to establish P. expansumas the model system for understanding mycotoxin synthesis in fruits. The overall goal of this proposal is to identify critical host and pathogen factors that mechanistically modulate P. expansumgenes and pathways to control activation of PAT production and virulence in host. Several fungal factors have been correlated with disease development in apples, including the production of PAT, acidification of apple tissue by the fungus, sugar content and the global regulator of secondary metabolism and development, LaeA. An increase in sucrose molarity in the culture medium from 15 to 175 mM negatively regulated laeAexpression and PAT accumulation, but, conversely, increased creAexpression, leading to the hypothesis that CreA could be involved in P. expansumPAT biosynthesis and virulence, possibly through the negative regulation of LaeA. We found evidence for CreAtranscriptional regulation of laeA, but this was not correlated with PAT production either in vitro or in vivo, thus suggesting that CreA regulation of PAT is independent of LaeA. Our finding that sucrose, a key ingredient of apple fruit, regulates PAT synthesis, probably through suppression of laeAexpression, suggests a potential interaction between CreA and LaeA, which may offer control therapies for future study. We have also identified that in addition to PAT gene cluster, CreA regulates other secondary metabolite clusters, including citrinin, andrastin, roquefortine and communesins, during pathogenesis or during normal fungal growth. Following creation of P. expansumpacCknockout strain, we investigated the involvement of the global pH regulator PacC in fungal pathogenicity. We demonstrated that disruption of the pH signaling transcription factor PacC significantly decreased the virulence of P. expansumon deciduous fruits. This phenotype is associated with an impairment in fungal growth, decreased accumulation of gluconic acid and reduced synthesis of pectolytic enzymes. We showed that glucose oxidase- encoding gene, which is essential for gluconic acid production and acidification during fruit colonization, was significantly down regulated in the ΔPepacCmutant, suggesting that gox is PacC- responsive gene. We have provided evidence that deletion of goxgene in P. expansumled to a reduction in virulence toward apple fruits, further indicating that GOX is a virulence factor of P. expansum, and its expression is regulated by PacC. It is also clear from the present data that PacC in P. expansumis a key factor for the biosynthesis of secondary metabolites, such as PAT. On the basis of RNA-sequencing (RNA-seq) analysis and physiological experimentation, the P. expansumΔlaeA, ΔcreAand ΔpacCmutants were unable to successfully colonize apples for a multitude of potential mechanisms including, on the pathogen side, a decreased ability to produce proteolytic enzymes and to acidify the environment and impaired carbon/nitrogen metabolism and, on the host side, an increase in the oxidative defence pathways. Our study defines these global regulatory factors and their downstream signalling pathways as promising targets for the development of strategies to fight against this post-harvest pathogen.
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Prusky, Dov, Nancy P. Keller y Amir Sherman. global regulation of mycotoxin accumulation during pathogenicity of Penicillium expansum in postharvest fruits. United States Department of Agriculture, enero de 2014. http://dx.doi.org/10.32747/2014.7600012.bard.

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Background to the topic- Penicilliumas a postharvest pathogen and producer of the mycotoxin PAT. Penicilliumspp. are destructive phytopathogens, capable of causing decay in many deciduous fruits, during postharvest handling and storage; and the resulting losses can amount to 10% of the stored produce and the accumulation of large amounts of the mycotoxinpatulin. The overall goal of this proposal is to identify critical host and pathogen factors that modulate P. expansummycotoxin genes and pathways which are required for PAT production and virulence. Our preliminary results indicated that gluconic acid are strongly affecting patulin accumulation during colonization. P. expansumacidifies apple fruit tissue during colonization in part through secretion of gluconic acid (GLA). Several publications suggested that GLA accumulation is an essential factor in P. expansumpathogenicity. Furthermore, down regulation of GOX2 significantly reduced PAT accumulation and pathogenicity. PAT is a polyketide and its biosynthesis pathway includes a 15-gene cluster. LaeA is a global regulator of mycotoxin synthesis. It is now known that patulin synthesis might be subjected to LaeA and sometimes by environmental sensing global regulatory factors including the carbon catabolite repressor CreA as well as the pH regulator factor PacC and nitrogen regulator AreA. The mechanisms by which LaeA regulates patulin synthesis was not fully known and was part of our work. Furthermore, the regulatory system that controls gene expression in accordance with ambient pH was also included in our work. PacC protein is in an inactive conformation and is unable to bind to the promoter sites of the target genes; however, under alkaline growth conditions activated PacC acts as both an activator of alkaline-expressed genes and a repressor of acid-expressed genes. The aims of the project- This project aims to provide new insights on the roles of LaeA and PacC and their signaling pathways that lead to GLA and PAT biosynthesis and pathogenicity on the host. Specifically, our specific aims were: i) To elucidate the mechanism of pH-controlled regulation of GLA and PAT, and their contribution to pathogenesis of P. expansum. We are interested to understanding how pH and/or GLA impact/s under PacC regulation affect PAT production and pathogenesis. ii) To characterize the role of LaeA, the global regulator of mycotoxin production, and its effect on PAT and PacC activity. iii) To identify the signaling pathways leading to GLA and PAT synthesis. Using state- of-the-art RNAseq technologies, we will interrogate the transcriptomes of laeAand pacCmutants, to identify the common signaling pathways regulating synthesis of both GLA and PAT. Major conclusions, solutions, achievements- In our first Aim our results demonstrated that ammonia secreted at the leading edge of the fungal colony induced transcript activation of the global pH modulator PacC and PAT accumulation in the presence of GLA. We assessed these parameters by: (i) direct exogenous treatment of P. expansumgrowing on solid medium; (ii) direct exogenous treatment on colonized apple tissue; (iii) growth under self-ammonia production conditions with limited carbon; and (iv) analysis of the transcriptional response to ammonia of the PAT biosynthesis cluster. Ammonia induced PAT accumulation concurrently with the transcript activation of pacCand PAT biosynthesis cluster genes, indicating the regulatory effect of ammonia on pacCtranscript expression under acidic conditions. Transcriptomic analysis of pH regulated processes showed that important genes and BARD Report - Project 4773 Page 2 of 10 functionalities of P. expansumwere controlled by environmental pH. The differential expression patterns of genes belonging to the same gene family suggest that genes were selectively activated according to their optimal environmental conditions to enable the fungus to cope with varying conditions and to make optimal use of available enzymes. Concerning the second and third Aims, we demonstrated that LaeA regulates several secondary metabolite genes, including the PAT gene cluster and concomitant PAT synthesis invitro. Virulence studies of ΔlaeAmutants of two geographically distant P. expansumisolates (Pe-21 from Israel and Pe-T01 from China) showed differential reduction in disease severity in freshly harvested fruit ranging from no reduction for Ch-Pe-T01 strains in immature fruit to 15–25% reduction for both strains in mature fruit, with the ΔlaeAstrains of Is-Pe-21 always showing a greater loss in virulence. Results suggest the importance of LaeA regulation of PAT and other secondary metabolites on pathogenicity. Our work also characterized for the first time the role of sucrose, a key nutritional factor present in apple fruit, as a negative regulator of laeAexpression and consequent PAT production in vitro. This is the first report of sugar regulation of laeAexpression, suggesting that its expression may be subject to catabolite repression by CreA. Some, but not all of the 54 secondary metabolite backbone genes in the P. expansumgenome, including the PAT polyketide backbone gene, were found to be regulated by LaeA. Together, these findings enable for the first time a straight analysis of a host factor that potentially activates laeAand subsequent PAT synthesis.
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