Literatura académica sobre el tema "Protease encoding genes"
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Artículos de revistas sobre el tema "Protease encoding genes"
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Xavier-Filho, J. y F. A. Paiva Campos. "Genes encoding protease inhibitors". Plant Molecular Biology Reporter 12, n.º 2 (junio de 1994): S58—S59. http://dx.doi.org/10.1007/bf02671572.
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Reid, Vernita J., Louwrens W. Theron, Maret du Toit y Benoit Divol. "Identification and Partial Characterization of Extracellular Aspartic Protease Genes from Metschnikowia pulcherrima IWBT Y1123 and Candida apicola IWBT Y1384". Applied and Environmental Microbiology 78, n.º 19 (20 de julio de 2012): 6838–49. http://dx.doi.org/10.1128/aem.00505-12.
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ABSTRACTThe extracellular acid proteases of non-Saccharomyceswine yeasts may fulfill a number of roles in winemaking, which include increasing the available nitrogen sources for the growth of fermentative microbes, affecting the aroma profile of the wine, and potentially reducing protein haze formation. These proteases, however, remain poorly characterized, especially at genetic level. In this study, two extracellular aspartic protease-encoding genes were identified and sequenced, from two yeast species of enological origin: one gene fromMetschnikowia pulcherrimaIWBT Y1123, namedMpAPr1, and the other gene fromCandida apicolaIWBT Y1384, namedCaAPr1.In silicoanalysis of these two genes revealed a number of features peculiar to aspartic protease genes, and both the MpAPr1 and CaAPr1 putative proteins showed homology to proteases of yeast genera. Heterologous expression ofMpAPr1inSaccharomyces cerevisiaeYHUM272 confirmed that it encodes an aspartic protease. MpAPr1 production, which was shown to be constitutive, and secretion were confirmed in the presence of bovine serum albumin (BSA), casein, and grape juice proteins. TheMpAPr1gene was found to be present in 12 otherM. pulcherrimastrains; however, plate assays revealed that the intensity of protease activity was strain dependent and unrelated to the gene sequence.
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Akula, Srinivas, Zhirong Fu, Sara Wernersson y Lars Hellman. "The Evolutionary History of the Chymase Locus -a Locus Encoding Several of the Major Hematopoietic Serine Proteases". International Journal of Molecular Sciences 22, n.º 20 (11 de octubre de 2021): 10975. http://dx.doi.org/10.3390/ijms222010975.
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Several hematopoietic cells of the immune system store large amounts of proteases in cytoplasmic granules. The absolute majority of these proteases belong to the large family of chymotrypsin-related serine proteases. The chymase locus is one of four loci encoding these granule-associated serine proteases in mammals. The chymase locus encodes only four genes in primates, (1) the gene for a mast-cell-specific chymotryptic enzyme, the chymase; (2) a T-cell-expressed asp-ase, granzyme B; (3) a neutrophil-expressed chymotryptic enzyme, cathepsin G; and (4) a T-cell-expressed chymotryptic enzyme named granzyme H. Interestingly, this locus has experienced a number of quite dramatic expansions during mammalian evolution. This is illustrated by the very large number of functional protease genes found in the chymase locus of mice (15 genes) and rats (18 genes). A separate expansion has also occurred in ruminants, where we find a new class of protease genes, the duodenases, which are expressed in the intestinal region. In contrast, the opossum has only two functional genes in this locus, the mast cell (MC) chymase and granzyme B. This low number of genes may be the result of an inversion, which may have hindered unequal crossing over, a mechanism which may have been a major factor in the expansion within the rodent lineage. The chymase locus can be traced back to early tetrapods as genes that cluster with the mammalian genes in phylogenetic trees can be found in frogs, alligators and turtles, but appear to have been lost in birds. We here present the collected data concerning the evolution of this rapidly evolving locus, and how these changes in gene numbers and specificities may have affected the immune functions in the various tetrapod species.
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Lewis, Janina P., Divya Iyer y Cecilia Anaya-Bergman. "Adaptation of Porphyromonas gingivalis to microaerophilic conditions involves increased consumption of formate and reduced utilization of lactate". Microbiology 155, n.º 11 (1 de noviembre de 2009): 3758–74. http://dx.doi.org/10.1099/mic.0.027953-0.
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Porphyromonas gingivalis, previously classified as a strict anaerobe, can grow in the presence of low concentrations of oxygen. Microarray analysis revealed alteration in gene expression in the presence of 6 % oxygen. During the exponential growth phase, 96 genes were upregulated and 79 genes were downregulated 1.4-fold. Genes encoding proteins that play a role in oxidative stress protection were upregulated, including alkyl hydroperoxide reductase (ahpCF), superoxide dismutase (sod) and thiol peroxidase (tpx). Significant changes in gene expression of proteins that mediate oxidative metabolism, such as cytochrome d ubiquinol oxidase-encoding genes, cydA and cydB, were detected. The expression of genes encoding formate uptake transporter (PG0209) and formate tetrahydrofolate ligase (fhs) was drastically elevated, which indicates that formate metabolism plays a major role under aerobic conditions. The concomitant reduction of expression of a gene encoding the lactate transporter PG1340 suggests decreased utilization of this nutrient. The concentrations of both formate and lactate were assessed in culture supernatants and cells, and they were in agreement with the results obtained at the transcriptional level. Also, genes encoding gingipain protease secretion/maturation regulator (porR) and protease transporter (porT) had reduced expression in the presence of oxygen, which also correlated with reduced protease activities under aerobic conditions. In addition, metal transport was affected, and while iron-uptake genes such as the genes encoding the haemin uptake locus (hmu) were downregulated, expression of manganese transporter genes, such as feoB2, was elevated in the presence of oxygen. Finally, genes encoding putative regulatory proteins such as extracellular function (ECF) sigma factors as well as small proteins had elevated expression levels in the presence of oxygen. As P. gingivalis is distantly related to the well-studied model organism Escherichia coli, results from our work may provide further understanding of oxygen metabolism and protection in other related bacteria belonging to the phylum Bacteroidetes.
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Lundwall, Åke y Adam Clauss. "Genes encoding WFDC- and Kunitz-type protease inhibitor domains: are they related?" Biochemical Society Transactions 39, n.º 5 (21 de septiembre de 2011): 1398–402. http://dx.doi.org/10.1042/bst0391398.
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We have previously demonstrated that the genes of SCPs (semen coagulum proteins) and the WFDC (whey acidic protein four-disulfide core)-type protease inhibitor elafin are homologous in spite of lacking similarity between their protein products. This led to the discovery of a locus on human chromosome 20, encompassing genes of the SCPs, SEMG1 (semenogelin I) and SEMG2, and 14 genes containing the sequence motif that is characteristic of WFDC-type protease inhibitors. We have now identified additional genes at the locus that are similarly organized, but which give rise to proteins containing the motif of Kunitz-type protease inhibitors. Here, we discuss the evolution of genes encoding SCPs and describe mechanisms by which they and genes with Kunitz motifs might have evolved from genes with WFDC motifs. We can also demonstrate an expansion of the WFDC locus with 0.6 Mb in the cow. The region, which seems to be specific to ruminants, contains several genes and pseudogenes with Kunitz motifs, one of which is the much-studied BPTI (bovine pancreatic trypsin inhibitor).
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Basta, David W., David Angeles-Albores, Melanie A. Spero, John A. Ciemniecki y Dianne K. Newman. "Heat-shock proteases promote survival of Pseudomonas aeruginosa during growth arrest". Proceedings of the National Academy of Sciences 117, n.º 8 (6 de febrero de 2020): 4358–67. http://dx.doi.org/10.1073/pnas.1912082117.
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When nutrients in their environment are exhausted, bacterial cells become arrested for growth. During these periods, a primary challenge is maintaining cellular integrity with a reduced capacity for renewal or repair. Here, we show that the heat-shock protease FtsH is generally required for growth arrest survival of Pseudomonas aeruginosa, and that this requirement is independent of a role in regulating lipopolysaccharide synthesis, as has been suggested for Escherichia coli. We find that ftsH interacts with diverse genes during growth and overlaps functionally with the other heat-shock protease-encoding genes hslVU, lon, and clpXP to promote survival during growth arrest. Systematic deletion of the heat-shock protease-encoding genes reveals that the proteases function hierarchically during growth arrest, with FtsH and ClpXP having primary, nonredundant roles, and HslVU and Lon deploying a secondary response to aging stress. This hierarchy is partially conserved during growth at high temperature and alkaline pH, suggesting that heat, pH, and growth arrest effectively impose a similar type of proteostatic stress at the cellular level. In support of this inference, heat and growth arrest act synergistically to kill cells, and protein aggregation appears to occur more rapidly in protease mutants during growth arrest and correlates with the onset of cell death. Our findings suggest that protein aggregation is a major driver of aging and cell death during growth arrest, and that coordinated activity of the heat-shock response is required to ensure ongoing protein quality control in the absence of growth.
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Bidochka, Michael J. y Michael J. Melzer. "Genetic polymorphisms in three subtilisin-like protease isoforms (Pr1A, Pr1B, and Pr1C) from Metarhizium strains". Canadian Journal of Microbiology 46, n.º 12 (1 de diciembre de 2000): 1138–44. http://dx.doi.org/10.1139/w00-112.
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Restriction fragment length polymorphisms (RFLP) were examined in three isoforms of a gene family encoding subtilisin-like proteases (Pr1A, Pr1B, and Pr1C) in several isolates of the entomopathogenic fungus Metarhizium anisopliae. RFLP variation was not observed in any of the Pr1 genes from isolates within the same genetically related group. Between genetically related groups and between isolates from disparate geographical areas, the greatest variation in RFLP patterns was observed for Pr1A. When variation does occur at Pr1B and Pr1C, it was generally observed at an EcoRI site. Metarhizium anisopliae var. majus strain 473 and a M. flavoviride isolate were most dissimilar in RFLP patterns at all Pr1 genes when compared to the M. anisopliae strains. We suggest that Pr1 genes represent a gene family of subtilisin-like proteases and that the Pr1A gene encodes for the ancestral subtilisin-like protease which has subsequently duplicated and rearranged within the genome.Key words: Metarhizium anisopliae, protease, RFLP, entomopathogen.
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Lewis, Janina P. y Francis L. Macrina. "IS195, an Insertion Sequence-Like Element Associated with Protease Genes in Porphyromonas gingivalis". Infection and Immunity 66, n.º 7 (1 de julio de 1998): 3035–42. http://dx.doi.org/10.1128/iai.66.7.3035-3042.1998.
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ABSTRACT Porphyromonas gingivalis is recognized as an important etiologic agent in adult and early-onset periodontal disease. Proteases produced by this organism contribute to its virulence in mice. Protease-encoding genes have been shown to contain multiple copies of repeated nucleotide sequences. These conserved sequences have also been found in hemagglutinin genes. In the process of studying the genetic loci containing the conserved repeated sequences, we have characterized a prtP gene homolog from P. gingivalis W83 encoding a cysteine protease with Lys-X specificity. However, thisprtP gene was interrupted by an insertion sequence-like element which we designated IS195. Furthermore, IS195 and another element, IS1126, were present downstream of prtP gene homologs (kgp) found inP. gingivalis H66 and 381. IS195, a 1,068-bp insertion sequence-like element, contained 11-bp inverted repeats at its termini and was bordered by 9-bp direct repeats presumed to be a transposition-mediated target site duplication. Its central region contained one large open reading frame encoding a predicted 300-amino-acid protein which appeared to be a transposase. We isolated two naturally occurring variants of P. gingivalis W83, one carrying IS195 within the coding region of theprtP gene and another containing an intact prtPgene. Biochemical characterization revealed a lack of trypsin-like Lys-X specific proteolytic activity in the P. gingivalisW83 variant carrying the disrupted prtP gene. Studies using a mouse model revealed a reduction of virulence resulting from insertion of IS195 into the coding region of theprtP gene. An allelic-exchange mutant defective in theprtP gene also was constructed and tested in vivo. It displayed intermediate virulence compared to that of the wild-type andprtP::IS195 mutant strains. We conclude that the Lys-X cysteine protease contributes to virulence in soft tissue infections.
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GARNIER, Gérard, Antonella CIRCOLO, Yuanyuan XU y John E. VOLANAKIS. "Complement C1r and C1s genes are duplicated in the mouse: differential expression generates alternative isomorphs in the liver and in the male reproductive system". Biochemical Journal 371, n.º 2 (15 de abril de 2003): 631–40. http://dx.doi.org/10.1042/bj20021555.
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C1r and C1s are the serine proteases that form the catalytic unit of the C1 complex, the first component of complement. In the present study, we found that the genes encoding murine C1r and C1s are duplicated. One set of these genes, referred to as c1rA and c1sA, are primarily expressed in the liver and are therefore the homologues of the human C1r and C1s genes. The other two genes, termed c1rB and c1sB, are expressed exclusively in male reproductive tissues, specifically the coagulating gland and the prostate. The predicted C1rB and C1sB proteins share 96 and 93% amino acid identity with C1rA and C1sA respectively. Most of the substitutions are clustered in the serine protease domains, suggesting differences in catalytic efficiencies and/or substrate specificities or alternatively adaptation to different physiological environments. The high homology of C1rB and C1sB with C1rA and C1sA in the non-catalytic regions indicates that they are probably capable of assembling the C1 complex. The expression of alternative genes encoding isomorphs of activating components of complement in male reproductive tissues raises the possibility of new mechanisms of complement activation in the male genital tract or of novel functions for complement proteases in reproduction.
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Heusel, JW, EM Scarpati, NA Jenkins, DJ Gilbert, NG Copeland, SD Shapiro y TJ Ley. "Molecular cloning, chromosomal location, and tissue-specific expression of the murine cathepsin G gene". Blood 81, n.º 6 (15 de marzo de 1993): 1614–23. http://dx.doi.org/10.1182/blood.v81.6.1614.1614.
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Abstract We previously have characterized a cluster of genes encoding cathepsin G (CG) and two other CG-like hematopoietic serine proteases, CGL-1 and CGL-2, on human chromosome 14. In this report, we clone and characterize a novel, related murine hematopoietic serine protease gene using human CG (hCG) cDNA as the probe. This murine gene spans approximately 2.5 kb of genomic DNA, is organized into five exons and four introns, and bears a high degree of homology to hCG at both nucleic acid (73%) and deduced amino acid (66%) levels. The predicted cDNA contains an open reading frame of 783 nucleotides that encodes a nascent protein of 261 amino acids. Processing of a putative signal (pre) peptide of 18 residues and an activation (pro) dipeptide would generate a mature enzyme of approximately 27 Kd that has an estimated pI of 12.0. Conserved residues at His44, Asp88, and Ser181 form the characteristic catalytic triad of the serine protease superfamily. The gene is tightly linked to the CTLA-1 locus on murine chromosome 14, where the serine protease genes mCCP1–4 are clustered. Expression of this gene is detected only in the bone marrow and is restricted to a small population of early myeloid cells. These findings are consistent with the identification of the gene encoding murine CG.
Tesis sobre el tema "Protease encoding genes"
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Camilleri, Raymond Stephen. "Molecular genetic and biochemical studies of the D1-processing protease of Arabidopsis Thaliana". Thesis, Royal Holloway, University of London, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.322223.
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Matsebula, Aaron Mfanuzile. "Cloning and expression of a chimeric protease inhibitor encoding gene in Escherichia coli and Pichia pastoris". Master's thesis, University of Cape Town, 1996. http://hdl.handle.net/11427/9649.
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Includes bibliographical references.Squash family protease inhibitors are small peptides of 27-32 residues, hence they are ideal subjects for structure-function studies. Their small size is within the reach of peptide chemical synthesis, which enables one to produce enough peptide material for experimental purposes within a reasonable time frame.
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Rawat, Reetika. "Characterization of the promoter of SmCP, the gene encoding Solanum melongena cysteine proteinase". Thesis, Click to view the E-thesis via HKUTO, 2004. http://sunzi.lib.hku.hk/hkuto/record/B34740156.
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Lugli, Elena. "Analysis of a multi-gene family, PRT1. encoding subtilism-like serine proteases in Pneumocystis carinii". Thesis, University of Oxford, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.302531.
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Ma, Pei-yin. "Chemical synthesis, cloning and expression of a gene encoding systemin, a proteinase inhibitor-inducing factor". Master's thesis, University of Cape Town, 1996. http://hdl.handle.net/11427/9241.
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Bibliography: leaves 116-120.Wound-inducible proteinase inhibitors in plants elicit a defence mechanism by inactivating the proteinases of insects. This triggers a feedback mechanism causing overproduction of digestive enzymes together with a decrease in appetite, leading to starvation. System in, a polypeptide proteinase inhibitor-inducing factor, when applied to cut stems of young tomato plants induces the accumulation of inhibitors in a manner similar to the normal wounding response. We designed and synthesised the minus strand oligonucleotide template complementary to the system in DNA sequence using Escherichia coli codon preferences. The double stranded fragment encoding the 18 amino acid residue systemin was cloned into pUCJ 8 for amplification and subcloning into pMAL-pk for expression as a maltose binding-fusion protein. The recombinant systemin was released by enterokinase and isolated by HPLC. After further purification, the physical characteristics including amino acid composition, peptide sequence and molecular weight of r-systemin were determined. When the recombinant peptide was applied to young tomato plants, it induced the accumulation of proteinase inhibitor I messenger RNA.
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BARANIYA, DIVYASHRI. "Microbial protease encoding genes in soil: diversity, abundance and enzymatic activity". Doctoral thesis, 2016. http://hdl.handle.net/2158/1040253.
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The objective of this research was to identify potential factors that govern the abundance, diversity and expression of genes encoding proteases in soil. I studied the response of protease encoding genes and proteolytic community structure of soil under different conditions. Three different approaches have been used for this thesis :
1. Using a rhizobox approach effect of root exudates from plants differing in Nitrogen utilizing efficiencies (NUE), on protease enzyme activity, molecular diversity and abundance of proteolytic genes was investigated. We observed a higher molecular diversity, abundance and higher enzyme activity associated with higher NUE cultivar. Furthermore, effect of root exudates can’t be ignored and rhizosphere soil exhibited significantly higher results.
2. Effect of elevated atmospheric CO2 was studied on rhizosphere and bulk soil from a Free Air Carbon dioxide Enrichment (FACE) field that was exposed to 550 ppm of CO2. We observed a reduction in abundance of apr protease genes. This decrease has been observed in both rhizosphere and bulk soil. We concluded that in eCO2 conditions, the action on soil microbes is not limited via root exudates but also mediated through fixation of CO2 directly by mircobes under high partial pressure.
3. Changes in expression of proteases during day and night has been studied in a green-house experiment where Barley rhizosphere soil was subjected to metatranscriptome analysis. Samples were collected during dark and light exposed period and mRNA was isolated which was later sequenced on an Illumina sequencer.
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Su, Xun Zheng y 蘇訓正. "Cloning and sequence analysis of the gene encoding the protease of vibrio parahaemolyticus". Thesis, 1994. http://ndltd.ncl.edu.tw/handle/85027532088389443602.
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Yu, Mei Xuan y 余美萱. "Expression and characterization of the gene encoding the extracellular protease of vibrio parahemolyticus". Thesis, 1995. http://ndltd.ncl.edu.tw/handle/41066787887294982987.
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Lin, Tzu-Jay y 林子傑. "Characterization and sequences analysis on the gene prtS1 encoding serine protease of Aeromonas hydrophila". Thesis, 2000. http://ndltd.ncl.edu.tw/handle/78571215317033041633.
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碩士國立臺灣大學
農業化學研究所
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Aeromonas hydrophila and related aeromonads are gram negative, facultatively anaerobic fresh water bacteria, many strains of which are pathogens of humans and animals. The pathogenicity may involve with several extracellular enzymes including haemolysins, lipase, and protease. It has been suggested that proteolytic enzymes produced by A. hydrophila may play an important role in invasiveness and in establishment of infection by such as overcoming initial host defense and providing nutrients for cell proliferation. During further study, we have cloned and sequenced a proteolytic gene prtS1 from clinical strain A. hydrophila CKH29. In this study we characterized the gene product of prtS1. prtS1 can be translated into a 453 a.a. protein which displays high homology with E. coli HtrA/DegQ/DegS family. PrtS1 was overexpressed in E.coli with a multicopy plasmid pUC18, after expression, it can be translocated into periplasma by postulated recognition of E. coli leader peptidase, but most gene product remained in cytosol after translation. Purification of PrtS1 was proceeded by ammonium sulfate precipitation, following of ion-exchge and gel filtration fractionation. The molecular weight of PrtS1 was estimated as 45 kD and 41 kD by SDS PAGE and 378 kD by CL-6B elution analysis, according to these results, it is predicted that PrtS1 can form an octomer by oligomerization after protein synthesized and achieved most proteolytic activity. The PrtS1 protein was shown to be a specific endopeptidase could be inhibited by PMSF, it showed high temperature stability, suggesting that PrtS1 is a thermostable serine protease. The optimal reaction pH and temperature with casein as substrate was pH 8.0 and 45℃. Inorganic ion Zn2+ showed inhibition to proteolytic activity of PrtS1, in contrast, the activity of PrtS1 was increased by Mg2+and Ca2+. Towards the N-termial near middle of PrtS1 amino sequences shows serine protease catalytic triad and GNSGGAL motif, PrtS1 may involve putative PDZ domains behind the catalytic domain, which may in response to its multi-function.
Capítulos de libros sobre el tema "Protease encoding genes"
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Mulks, M. H., D. A. Simpson y R. J. Shoberg. "Restriction site polymorphism in genes encoding type 2 but not type 1 gonococcal IgAl proteases". En Gonococci and Meningococci, 419–26. Dordrecht: Springer Netherlands, 1988. http://dx.doi.org/10.1007/978-94-009-1383-7_67.
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Hershberger, R. J., C. Mueller, H. K. Gershenfeld y I. L. Weissman. "A Serine Protease-Encoding Gene That Marks Activated Cytotoxic T Cells In Vivo and In Vitro". En Current Topics in Microbiology and Immunology, 81–92. Berlin, Heidelberg: Springer Berlin Heidelberg, 1988. http://dx.doi.org/10.1007/978-3-642-73911-8_7.
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Sama, Babru, Tom Boone, Barbara Karan, Kenneth Chen, Raj Sachdev y Tsutomo Arakawa. "Cloning and Expression of the Gene Encoding a Novel Proteinase from TritiraChium Album Limber". En Advances in Experimental Medicine and Biology, 95–104. Boston, MA: Springer US, 1996. http://dx.doi.org/10.1007/978-1-4613-0319-0_10.
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Takeuchi, M., K. Ogura, T. Hamamoto y Y. Kobayashi. "Molecular Cloning and Sequence Analysis of a Gene Encoding an Aspartic Proteinase from Aspergillus Oryzae". En Aspartic Proteinases, 577–80. Boston, MA: Springer US, 1995. http://dx.doi.org/10.1007/978-1-4615-1871-6_77.
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Jayagoudar, Santoshkumar, Ch Ramesh, Pradeep Kumar, Aditya Banyal, Bhagwan Rekadwad, Ramchander Merugu, Tiina Nõges y Rahul Kumar. "Thermophilic Fungi and their Applications in Biotechnology". En Mycology: Current and Future Developments, 359–83. BENTHAM SCIENCE PUBLISHERS, 2022. http://dx.doi.org/10.2174/9789815040340122040023.
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Thermophilic fungi survive at a growth temperature above 45°C. Although widely spread in the terrestrial region, they have remained underexplored, diverging from other thermophiles of eubacteria and archaea. Thermophilic fungi elicited a lot of interest due to their ability to produce thermostable and thermotolerant compounds with practical applications in the field of biotechnology. Studies on growth kinetics, physiology, supplement uptake, and protein breakdown rate in thermophilic fungi have provided important information on these fungi. Thermophilic fungi can degrade biomass such as polysaccharides into monomers. The characteristics of their enzymes show contrasts among species, just as among strains of comparative species. Some extracellular compounds separated from thermophilic fungi are commercially produced, and a couple of others have the potential due to financial advantages.<br>Thermophilic fungal genes encoding lipase, protease, and various other vital enzymes have been cloned and overexpressed in heterologous fungi for understanding the mechanisms of their natural thermostability and catalysis.
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A. Elkonin, Lev, Valery M. Panin, Odissey A. Kenzhegulov y Saule Kh. Sarsenova. "RNAi-Mutants of Sorghum bicolor (L.) Moench with Improved Digestibility of Seed Storage Proteins". En Grain and Seed Proteins Functionality [Working Title]. IntechOpen, 2021. http://dx.doi.org/10.5772/intechopen.96204.
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Modification of the composition of grain storage proteins is an intensively developing area of plant biotechnology, which is of particular importance for sorghum – high-yielding drought tolerant crop. Compared to other cereals, the majority of sorghum cultivars and hybrids are characterized by reduced nutritional value that is caused by a low content of essential amino acids in the seed storage proteins (kafirins), and resistance of kafirins to protease digestion. RNA interference (RNAi) by suppressing synthesis of individual kafirin subclasses may be an effective approach to solve this problem. In this chapter, we review published reports on RNAi silencing of the kafirin-encoding genes. In addition, we present new experimental data on phenotypic effects of RNAi-silencing of γ-KAFIRIN-1 gene in sorghum cv. Avans. To obtain RNAi mutants with γ-KAFIRIN-1 gene silencing we used Agrobacterium-mediated genetic transformation. Transgenic kernels had modified endosperm type with reduced vitreous layer and significantly improved in vitro protein digestibility (93% vs. 57%, according to the densitometry of SDS-PAGE patterns). SDS-PAGE of transgenic kernels showed lowered level of kafirins and appearance of globulin proteins, which were not observed in the original cultivar. For the first time, the cases of instability of inserted genetic construct were identified: elimination of ubi1-intron that is a constituent part of the genetic construct for RNAi silencing, or nos-promotor governing expression of the marker gene (bar) (in the RNAi mutants of cv. Zheltozernoe 10). The research findings presented in this chapter provide strong evidence that RNA interference can be used for improvement of the nutritional properties of sorghum grain.
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Grant, Seth G. N. "Synaptic Disease in Psychiatry". En Neurobiology of Mental Illness, editado por Pamela Sklar, 311–21. Oxford University Press, 2013. http://dx.doi.org/10.1093/med/9780199934959.003.0024.
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Human synapses contain over 1000 proteins forming the synapse proteome and diseases that disrupt these proteins cause synaptopathy. Hundreds of gene mutations in synapse proteins cause over 130 brain diseases including major psychiatric, neurological and childhood developmental disorders. The synapse proteome is organized into the presynaptic and postsynaptic proteome and each is further organized into macromolecular multiprotein complexes that perform key functions. Disruption of these complexes underlies a common etiology of diseases with related clinical features such as Schizophrenia, Autism and Intellectual Disability. Mutations in the different genes encoding the proteins comprising these complexes also underlies the multigenic nature of diseases. Synaptic disease is emerging as a major cause of brain disease and understanding the organization and function of the synapse proteome opens important new avenues for disease classification and therapeutic intervention.
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Primrose, Sandy B. "Tracking Horizontal Gene Transfer: Staphylococcus aureus". En Microbiology of Infectious Disease, 124–32. Oxford University Press, 2022. http://dx.doi.org/10.1093/oso/9780192863843.003.0016.
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Staphylococcus aureus is a much-feared pathogen that can cause a number of different diseases depending on the strains involved and the site of entry to the body. Staphylococcus has many different virulence factors. Staphyloxanthin (pigment), coagulase, staphylokinase, and the capsule interfere with host defences. Other virulence factors are toxins that cause food poisoning (enterotoxins) or tissue damage (proteases). Ninety genes encoding virulence factors have been identified and these are split between the core genome and the accessory genome. Of these genes, thirty-five have been found in all strains. Strains that are resistant to methicillin (MRSA) have the mecA gene. This is encoded by a mobile genetic element that also harbours other antibiotic-resistance genes. Using ribotyping it has been shown that certain clonal complexes of MRSA predominate worldwide. Community-acquired MRSA are more virulent than hospital-acquired MRSA because they produce the Panton–Valentine leucocidin. Pigs have been shown to be a source of MRSA. Vancomycin is one of a small number of antibiotics that can be used to treat MRSA. Resistance to this antibiotic only occurs sporadically. Mupirocin can be used to eliminate nasal carriage of MRSA in patients being admitted to hospital for cold surgery.
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I. Kane, Emma y Donald E. Spratt. "New Discoveries on the Roles of “Other” HECT E3 Ubiquitin Ligases in Disease Development". En Ubiquitin - Proteasome Pathway. IntechOpen, 2020. http://dx.doi.org/10.5772/intechopen.91770.
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HECT E3 ubiquitin ligases selectively recognize, bind, and ubiquitylate their substrate proteins to target them for 26S proteasomal degradation. There is increasing evidence that HECT E3 ubiquitin ligase dysfunction due to misfolding and/or the gene encoding the protein being mutated is responsible for the development of different diseases. Apart from the more prominent and well-characterized E6AP and members of the NEDD4 family, new studies have begun to reveal how other members of the HECT E3 ubiquitin ligase family function as well as their links to disease and developmental disorders. This chapter provides a comprehensive discussion on the more mysterious members of the HECT E3 ubiquitin ligase family and how they control intracellular processes. Specifically, AREL1, HACE1, HECTD1, HECTD4, G2E3, and TRIP12 will be examined as these enzymes have recently been identified as contributors to disease development.
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Ravichandiran, Nerethika, Muneesh Kumar Barman, Sai Tejaswi Lavuri, Manjita Srivastava, Shalini Sakthivel, Meenakshi Singh, Kailash Chand, Subash C. Sonkar y Prudhvilal Bhukya. "Precision Medicine in Cancer". En Handbook of Research on Advancements in Cancer Therapeutics, 433–66. IGI Global, 2021. http://dx.doi.org/10.4018/978-1-7998-6530-8.ch015.
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Cancer is the one of the deadliest diseases and takes the lives of millions of people every year across the world. Due to disease heterogeneity and multi-factorial reasons, traditional treatment such as radiation therapy, immunotherapy, or chemotherapy are effective only among a small population of the patients. Tumors can have different fundamental genetic causes and protein expressions that differ from one patient to another. This variability among individual lends itself to the field of precision and personalized medicine. Following the completion of human genome sequencing, significant progress has been observed in the characterization of human epigenome, proteome, and metabolome. Pharmacogenetics and pharmacogenomics use this sequence to study the genetic causes of individual variations in drug response and the simultaneous impact of change in genome that decide the patient's response to drug respectively. On summation, identify the subpopulation of patient and provide them tailored therapy thus increasing the effectiveness of treatment. All these evolved the field of precision or personalized medicine that plays a crucial role in cancer prevention, prognosis, diagnosis, and therapeutics. These tailored therapies are characterized by increased efficiency and reduced toxicity. Not all cancers have genetic variability; some are also influenced by polymorphism of gene encoding enzymes that play an important role in pharmacokinetics of drug. The discoveries of cancer predisposition genes allow diagnosis of a patient at risk of cancer development and let them make the decision on précised individual risk modification characteristic. The use of CYP2D6 genotyping for breast cancer, mutation in KRAS in colorectal cancer, genomic variation in EGFR in small lung cancer, melanoma are some of the examples of importance of cancer predisposition genes. In recent times, distinct molecular subtypes of cancers have been identified with requirement of different treatment for each subtype. Precision medicine shifts the trend from reaction to prevention and forestalls disease progression.
Actas de conferencias sobre el tema "Protease encoding genes"
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Pannekoek, H., M. Linders, J. Keijer, H. Veerman, H. Van Heerikhuizen y D. J. Loskutoff. "THE STRUCTURE OF THE HUMAN ENDOTHELIAL PLASMINOGEN ACTIVATOR INHIBITOR (PAI-1) GENE: NON-RANDOM POSITIONING OF INTRONS". En XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1644767.
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The endothelium plays a crucial role in the regulation of the fibrinolytic process, since it synthesizes and secretes tissue-type plasminogen activator (t-PA) as well as the fast-acting plasminogen activator inhibitor (PAI-1). Molecular cloning of full-length PAI-1 cDNA, employing a human endothelial cDNA expression library, and a subsequent determination of the complete nucleotide sequence, allowed a prediction of the amino-acid sequence of the PAI-1 glycoprotein. It was observed that the amino-acid sequence is significantly homologous to those of members of the serine protease inhibitor ("Serpin") family, e.g. αl-antitrypsin and antithrombin III. Serpins are regulators of various processes, such as coagulation, inflammatory reactions, complement activation and share a common functional principle and a similar structure, indicative for a common primordial gene. The intron-exon arrangement of Serpin genes may provide a record for the structure of a primordial gene. A comparison of the location of introns among members of the Serpin family reveals that some introns are indeed present at identical or almost identical positions, however in many other cases there is no correspondence between the intron positions among different Serpin genes.Obviously, more data on the chromosomal gene structure of members of this family are required to formulate a scheme for the evolutionary creation of the Serpins. To that end, we have established the number and the precise location of the introns in the PAI-1 gene and have compared these data with those reported on other Serpin genes. For that purpose a human genomic cosmid DNA library of about 340.000 independent colonies was screened with radiolabelled full-length PAI-1 cDNA as probe. Two clones were found which contain the entire PAI-1 gene. Restriction site mapping, electron microscopic inspection of heteroduplexes and nucleotide sequence analysis demonstrate that the PAI-1 gene comprises about 12.2kilo basepairs and consists of nine exons and eight introns. Intron-exon boundaries are all in accord with the "GT-AG" rule, including a cryptic acceptor splice site found in intron 7. Furthermore, it is observed that intron 3 of the PAI-1 gene occupies an identical position as intron E of chicken ovalbumin and intron E of the ovalbumin-related gene Y. The location of the other seven introns is unrelated to the known location of introns in the genes encoding the Serpins, rat angiotensin, chicken ovalbumin (and gene Y), human antithrombin III and human al-antitrypsin. The 3' untranslated region of the PAI-1 gene is devoid of introns, indicating that the two mRNA species detected in cultured endothelial cells which share an identical 5' untranslated segment and codogenic region, but differ in the length of the 3' untranslated region, arise by alternative polyadenylation. An extrapolation of the position of the introns to the amino-acid sequence of PAI-1, and adaption of the view that the subdomain structure of the Serpins is analogous, shows that the introns of PAI-1 are non-randomly distributed. Except for intron 7, the position of the other seven introns corresponds with randon-coil regions of the protein or with the borders of β-sheets and a-helices. Extrapolation of the position of introns in the genes of other Serpins to their respective amino-acid sequences and subdomain structures also reveals a preference for random-coil regions and borders of subdomains. These observations are reminiscent of an evolutionary model, called "intron sliding", that accounts for variations in surface loops of the same protein in different species by aberrant splicing (Craik et al., Science 220 (1983) 1125). The preferential presence of introns in gene segments, encoding these variable regions, and absence in regions determining the general folding of these proteins would explain conservation of the structure during the evolution of those genes.
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O'hara, Patrick J., Frank A. Grant, A. Betty, J. Haldmen y Mark J. Murray. "Structure of the Human Factor VII Gene". En XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643786.
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Factor VII is a member of a family of vitamin K-dependent, gamma-carboxylated plasma protein which includes factor IX, factor X, protein C, protein S and prothrombin. Activated factor VII (factor Vila) is a plasma serine protease which participates in a cascade of reactions leading to the coagulation of blood. Two overlapping genomic clones containing sequences encoding human factor VII were isolated and characterized. The complete sequence of the gene was determined and found to span 12.8 kilobases. The mRNA for factor VII as demonstrated by cDNA cloning is polyadenylated at multiple sites but contains only one AAUAAA poly-A signal sequence. The mRNA can undergo alternative splicing forming one transcript containing eight segments as exons and another with an additional exon which encodes a larger pre-pro leader sequence. The portion of the pre-pro leader coded for by the additional exon has no known counterpart in the other vitamin K-dependent proteins. The positions of the introns with respect to the amino acid sequence encoded by the eight essential exons of factor VII are the same as those present in factor IX, factor X, protein C and the first three exons of prothrombin. These exons code for domains generally conserved among members of this gene family, including a pre-pro leader (the essential exon la and alternative exon lb), a gamma-carboxylated domain (exons 2 and 3) a growth factor domain (exons 4 and 5) an activation region (exon 6) and a serine protease (exon 8). The corresponding introns in these genes are dissimilar with respect to size and sequence, with the exception of the third intron in factor VII and protein C. Four introns and a portion of exon 8 in factor VII contain regions made up of tandem repeats of oligonucleotide monomer elements. More than a quarter of the intron sequences and more than a third of the 3' untranslated portion of the mRNA transcript consist of these minisatellite tandem repeats. This type of structure is responsible for polymorphisms due to allelic variation in repeat copy number in other areas of the human genome. Tandem repeats can evolve as a result of random crossover in DNA whose sequence is not maintained by selection. This suggests that much of the sequence information present in the introns and untranslated portion of the message is dispensable.
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Pannekok, H., A. J. Van Zonneveid, C. J. M. de vries, M. E. MacDonald, H. Veerman y F. Blasi. "FUNCTIONAL PROPERTIES OF DELETION-MUTANTS OF TISSUE-TYPE PLASMINOGEN ACTIVATOR". En XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643724.
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Over the past twenty-five years, genetic methods have generated a wealth of information on the regulation and the structure-function relationship of bacterial genes.These methods are based on the introduction of random mutations in a gene to alter its function. Subsequently, genetic techniques cure applied to localize the mutation, while the nature of the impairedfunction could be determined using biochemical methods. Classic examples of this approach is now considered to be the elucidation of the structure and function of genes, constituting the Escherichia coli lactose (lac) and tryptophan (trp) operons,and the detailed establishment of the structure and function of the repressor (lacl) of the lac operon. Recombinant DNA techniques and the development of appropriate expression systems have provided the means both to study structure and functionof eukaryotic (glyco-) proteins and to create defined mutations with a predestinedposition. The rationale for the construction of mutant genes should preferentiallyrely on detailed knowledge of the three-dimensional structure of the gene product.Elegant examples are the application of in vitro mutagenesis techniques to substitute amino-acid residues near the catalytic centre of subtilisin, a serine proteasefrom Bacillus species and to substituteanamino acid in the reactive site (i.e. Pi residue; methionine) of α-antitrypsin, a serine protease inhibitor. Such substitutions have resulted into mutant proteins which are less susceptible to oxidation and, in some cases, into mutant proteins with a higher specific activity than the wild-type protein.If no data are available on the ternary structure of a protein, other strategies have to be developed to construct intelligent mutants to study the relation between the structure and the function of a eukaryotic protein. At least for a number of gene families, the gene structure is thought to be created by "exon shuffling", an evolutionary recombinational process to insert an exon or a set of exons which specify an additional structural and/or functional domain into a pre-existing gene. Both the structure of the tissue-type plasminogen activator protein(t-PA) and the t-PA gene suggest that this gene has evolved as a result of exon shuffling. As put forward by Gilbert (Science 228 (1985) 823), the "acid test"to prove the validity of the exon shuffling theory is either to delete, insert or to substitute exon(s) (i.e. in the corresponding cDNA) and toassay the properties of the mutant proteins to demonstrate that an exon or a set of adjacent exons encode (s) an autonomousfunction. Indeed, by the construction of specific deletions in full-length t-PA cDNA and expression of mutant proteins intissue-culture cells, we have shown by this approach that exon 2 of thet-PA gene encodes the function required forsecretion, exon 4 encodes the "finger" domain involved in fibrin binding(presumably on undegraded fibrin) and the set of exons 8 and 9 specifies kringle 2, containing a lysine-binding sit(LBS) which interacts with carboxy-terminal lysines, generated in fibrin after plasmic digestion. Exons 10 through 14 encode the carboxy-ter-minal light chain of t-PA and harbor the catalytic centre of the molecule and represents the predominant "target site" for the fast-acting endothelial plasminogen activator inhibitor (PAI-1).As a follow-up of this genetic approach to construct deletion mutants of t-PA, we also created substitution mutants of t-PA. Different mutants were constructed to substitute cDNA encoding thelight chain of t-PA by cDNA encoding the B-chain of urokinase (u-PA), in order to demonstrate that autonomous structural and functional domains of eitherone of the separate molecules are able toexert their intrinsic properties in a different context (C.J.M. de Vries et al., this volume). The possibilities and the limitations of this approach to study the structure and the function of t-PA and of other components of the fibrinolytic process will be outlined.
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de Vries, C. J. M., N. K. Veerman y H. Pannekoek. "ARTIFICIAL EXON SHUFFLING: CONSTRUCTION OF HYBRID cDNAS CONTAINING DOMAINS OF TISSUE-TYPE PLASMINOGEN ACTIVATOR (T-PA) AND UROKINASE (u-PA)". En XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643940.
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The intriguing finding that functions of t-PA coincide with structural domains and that these domains occur in related proteins, has been the basis to construct hybrid proteins by artificial exon shuffling to prove the conservation of functions in the shuffled domains. The heavy chain (Hch) of t-PA mediates both binding to fibrin and stimulation of plasminogen activator activity via its Finger- and Kringle-2 domain, whereas the light chain (Lch) contains the serine protease moiety of the protein. The Hch of u-PA is very homologous to the Lch of t-PA, but exhibits a higher plasminogen activator activity. This activity of u-PA is not stimulated by fibrin. We employed the ‘M13 in vitro outlooping’ technique to fuse the Hch of t-PA cDNA and the Hch of u-PA cDNA, to create two different hybrid cDNAs. On one hybrid cDNA, the t-PA and the u-PA sequences are coupled precisely at the exon-intron boundaries of the corresponding genes, while the other hybrid cDNA lacks a u-PA segment at the junction, encoding 13 amino acids of u-PA. The hybrid cDNAs were transiently expressed in mouse Ltk- cells and the recombinant proteins were characterized. The plasminogen activator activity of these proteins was determined in an indirect amidolytic assay, using plasminogen and the chromogenic substrate S2251. As anticipated, the activity of both t-PA/u-PA hybrid proteins is stimulated by fibrin, however, not to the same extent as t-PA. Remarkably, we found a decreased inhibition of the hybrid proteins by the endothelial plasminogen activator inhibitor (PAI-1) as compared to t-PA and u-PA, although stable complexes between the hybrid proteins and the inhibitor are formed. We conclude that functions of structural domains are maintained during exon shuffling
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Geddes, V. A., G. V. Louie, G. D. Brayer y R. T. A. MacGillivray. "MOLECULAR BASIS OF HEMOPHILIA B: IDENTIFICATION OF THE DEFECT IN FACTOR IX VANCOUVER". En XIth International Congress on Thrombosis and Haemostasis. Schattauer GmbH, 1987. http://dx.doi.org/10.1055/s-0038-1643872.
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Factor IX Vancouver (fIX-V) is the cause of a moderate form of hemophilia B. An individual presenting with this disorder had 2.6% of normal procoagulant activity in his plasma but had 62% of the normal factor IX antigen level. Specific antibodies showed that fIX-V contains epitopes for both the heavy and light chains of factor IXa. To identify the defect involved, DNA was isolated from the lymphocytes of the male hemophiliac. Southern blot analysis using a full-length factor IX cDNA as a hybridization probe showed no gross differences between the fIX-V gene and the normal factor IX gene. The DNA from the hemophiliac was then partially digested with Sau3A and the resulting fragments (10-20kbp in size) were ligated into the BamHI site of λEMBL3. The DNA was then packaged into phage particles in vitro, and the recombinant phage were screened with the factor IX cDNA as a probe. Eight phage were isolated that contained overlapping DNA covering the complete gene for fIX-V. DNA sequence analysis of the protein-encoding regions, the intron/exon junctions and 5'-and 3'-flanking sequences revealed a single nucleotide change from the normal factor IX gene. The codon for amino acid 397 was changed from ATA (lie) to ACA (Thr). This mutation is in the catalytic domain of factor IXa and is novel amongst those hemophilia B mutations reported to date. Based on the known three dimensional structures of the pancreatic serine proteases, trypsin, elastase and chymotrypsin, models have been constructed for the structures of the catalytic domains of both the normal and Thr-397 mutant of factor IXa. These results suggest that the Thr-397 mutation may alter the conformation of the substrate binding region in the active site of factor IXa Vancouver through the formation of a hydrogen bond between the hydroxyl group of the Thr-397 side chain and the main chain carbonyl group of Trp-385. The postulated conformational change would lead to reduced binding affinity for the factor IXa substrate resulting in a reduction in the catalytic activity of fIXa-Vancouver.Supported in part by grants from the Medical Research Council of Canada (to GDB and RTAM).
Informes sobre el tema "Protease encoding genes"
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Barkan, Alice y Zach Adam. The Role of Proteases in Regulating Gene Expression and Assembly Processes in the Chloroplast. United States Department of Agriculture, enero de 2003. http://dx.doi.org/10.32747/2003.7695852.bard.
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Chloroplasts house many biochemical processes that are essential for plant viability. Foremost, among these is photosynthesis, which requires the protein-rich thylakoid membrane system. The activation of chloroplast genes encoding thylakoid membrane proteins and the targeting and assembly of these proteins together with their nuclear-encoded partners are essential for the elaboration of the thylakoid membrane. Several nuclear-encoded proteins that regulate chloroplast gene expression and that mediate the targeting of proteins to the thylakoid membrane have been identified in recent years, and many more remain to be discovered. The abundance of such proteins is critical and is likely to be determined to a significant extent by their stability, which in turn, is influenced by chloroplast protease activities. The primary goal of this project was to link specific proteases to specific substrates, and in particular, to specific regulatory and assembly proteins. We proposed a two-pronged approach, involving genetic analysis of the consequences of the mutational loss of chloroplast proteases, and biochemical analysis of the degradation pathways of specific proteins that have been shown to control chloroplast gene expression. Our initial bioinformatic analysis of chloroplast proteases allowed us to identify the set of pro teases that is targeted to the chloroplast. We used that information to recover three Arabidopsis mutants with T - DNA insertions in specific chloroplast protease genes. We carried out the first analysis of the stability of a regulator of chloroplast gene expression (CRS2), and found that the protein is much less stable than are typical components of the photosynthetic apparatus. Genetic reagents and analytical methods were developed that have set the stage for a rapid advancement of our understanding of chloroplast proteolysis. The results obtained may be useful for manipulating the expression of transgenes in the chloroplast and for engineering plants whose photosynthetic activity is optimized under harsh environmental conditions.
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Ohad, Itzhak y Himadri Pakrasi. Role of Cytochrome B559 in Photoinhibition. United States Department of Agriculture, diciembre de 1995. http://dx.doi.org/10.32747/1995.7613031.bard.
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The aim of this research project was to obtain information on the role of the cytochrome b559 in the function of Photosystem-II (PSII) with special emphasis on the light induced photo inactivation of PSII and turnover of the photochemical reaction center II protein subunit RCII-D1. The major goals of this project were: 1) Isolation and sequencing of the Chlamydomonas chloroplast psbE and psbF genes encoding the cytochrome b559 a and b subunits respectively; 2) Generation of site directed mutants and testing the effect of such mutation on the function of PSII under various light conditions; 3) To obtain further information on the mechanism of the light induced degradation and replacement of the PSII core proteins. This information shall serve as a basis for the understanding of the role of the cytochrome b559 in the process of photoinhibition and recovery of photosynthetic activity as well as during low light induced turnover of the D1 protein. Unlike in other organisms in which the psbE and psbF genes encoding the a and b subunits of cytochrome b559, are part of an operon which also includes the psbL and psbJ genes, in Chlamydomonas these genes are transcribed from different regions of the chloroplast chromosome. The charge distribution of the derived amino-acid sequences of psbE and psbF gene products differs from that of the corresponding genes in other organisms as far as the rule of "positive charge in" is concerned relative to the process of the polypeptide insertion in the thylakoid membrane. However, the sum of the charges of both subunits corresponds to the above rule possibly indicating co-insertion of both subunits in the process of cytochrome b559 assembly. A plasmid designed for the introduction of site-specific mutations into the psbF gene of C. reinhardtii. was constructed. The vector consists of a DNA fragment from the chromosome of C. reinhardtii which spans the region of the psbF gene, upstream of which the spectinomycin-resistance-conferring aadA cassette was inserted. This vector was successfully used to transform wild type C. reinhardtii cells. The spectinomycin resistant strain thus obtained can grow autotrophically and does not show significant changes as compared to the wild-type strain in PSII activity. The following mutations have been introduced in the psbF gene: H23M; H23Y; W19L and W19. The replacement of H23 involved in the heme binding to M and Y was meant to permit heme binding but eventually alter some or all of the electron transport properties of the mutated cytochrome. Tryptophane W19, a strictly conserved residue, is proximal to the heme and may interact with the tetrapyrole ring. Therefore its replacement may effect the heme properties. A change to tyrosine may have a lesser affect on the potential or electron transfer rate while a replacement of W19 by leucine is meant to introduce a more prominent disturbance in these parameters. Two of the mutants, FW19L and FH23M have segregated already and are homoplasmic. The rest are still grown under selection conditions until complete segregation will be obtained. All mutants contain assembled and functional PSII exhibiting an increased sensitivity of PSII to the light. Work is still in progress for the detailed characterization of the mutants PSII properties. A tobacco mutant, S6, obtained by Maliga and coworkers harboring the F26S mutation in the b subunit was made available to us and was characterized. Measurements of PSII charge separation and recombination, polypeptide content and electron flow indicates that this mutation indeed results in light sensitivity. Presently further work is in progress in the detailed characterization of the properties of all the above mutants. Information was obtained demonstrating that photoinactivation of PSII in vivo initiates a series of progressive changes in the properties of RCII which result in an irreversible modification of the RCII-D1 protein leading to its degradation and replacement. The cleavage process of the modified RCII-D1 protein is regulated by the occupancy of the QB site of RCII by plastoquinone. Newly synthesized D1 protein is not accumulated in a stable form unless integrated in reassembled RCII. Thus the degradation of the irreversibly modified RCII-D1 protein is essential for the recovery process. The light induced degradation of the RCII-D1 protein is rapid in mutants lacking the pD1 processing protease such as in the LF-1 mutant of the unicellular alga Scenedesmus obliquus. In this case the Mn binding site of PSII is abolished, the water oxidation process is inhibited and harmful cation radicals are formed following light induced electron flow in PSII. In such mutants photo-inactivation of PSII is rapid, it is not protected by ligands binding at the QB site and the degradation of the inactivated RCII-D1 occurs rapidly also in the dark. Furthermore the degraded D1 protein can be replaced in the dark in absence of light driven redox controlled reactions. The replacement of the RCII-D1 protein involves the de novo synthesis of the precursor protein, pD1, and its processing at the C-terminus end by an unknown processing protease. In the frame of this work, a gene previously isolated and sequenced by Dr. Pakrasi's group has been identified as encoding the RCII-pD1 C-terminus processing protease in the cyanobacterium Synechocystis sp. PCC 6803. The deduced sequence of the ctpA protein shows significant similarity to the bovine, human and insect interphotoreceptor retinoid-binding proteins. Results obtained using C. reinhardtii cells exposes to low light or series of single turnover light flashes have been also obtained indicating that the process of RCII-D1 protein turnover under non-photoinactivating conditions (low light) may be related to charge recombination in RCII due to back electron flow from the semiquinone QB- to the oxidised S2,3 states of the Mn cluster involved in the water oxidation process.
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Adam, Zach y Eran Pichersky. Degradation of Abnormal Proteins in Chloroplasts of Higher Plants. United States Department of Agriculture, agosto de 1994. http://dx.doi.org/10.32747/1994.7568768.bard.
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In this study we attempted to get a better understanding of processes involved in the degradation of abnormal proteins i chloroplasts. To achieve this goal, we used a number of complementary approaches. We first characterized the expression of the two subunits of Clp protease. We demonstrated that both of them were expressed in chloroplasts in a constitutive fashion, but the expression of the regulatory subunit ClpC was enhanced by light. We generated a mutant the lumenal protein OEE33 which was targeted to the stroma in in vitro experiments. In the wrong compartment it was found unstable, and characterization of its degradation revealed that it was degraded by a soluble, ATP-dependent serine protease, which are also the characteristics of Clp protease. In search of other homologues of bacterial proteases, we found that chloroplasts contain a homologue of the FtsH protease. It is an ATP-dependent metallo-protease, bound to the stromal side of the thylakoid membrane, whose expression is dependent on light. The gene encodig this protease was cloned and characterized. In attempt to generate Arabidopsis mutant plants impaired in their capability to degrade abnormal chloroplast proteins, we fused the gene for mistargeted OEE33 to the streptomycin-detoxifying gene. This chimeric gene was introduced into Arabodipsis plants, to generate transformed plants. This transformants plants were sensitive to streptomycin due to the rapid turn-over of the chimeric protein. Seeds from these plants were then chemically mutagenised, and seedlings were selected for their capability to grow on streptomycin. The ability of these mutant transformants to grow on streptomycin is presumably due to stabilization of the chimeric protein. These plants will allow us in the future to identify the effected genes, which are likely to be involved in the protein degradation process.
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Manulis-Sasson, Shulamit, Christine D. Smart, Isaac Barash, Laura Chalupowicz, Guido Sessa y Thomas J. Burr. Clavibacter michiganensis subsp. michiganensis-tomato interactions: expression and function of virulence factors, plant defense responses and pathogen movement. United States Department of Agriculture, febrero de 2015. http://dx.doi.org/10.32747/2015.7594405.bard.
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Clavibactermichiganensissubsp. michiganensis(Cmm), the causal agent of bacterial wilt and canker of tomato, is the most destructive bacterial disease of tomato causing substantial economic losses in Israel, the U.S.A. and worldwide. The goal of the project was to unravel the molecular strategies that allow Cmm, a Gram-positive bacterium, to develop a successful infection in tomato. The genome of Cmm contains numerous genes encoding for extracellular serine proteases and cell wall degrading enzymes. The first objective was to elucidate the role of secreted serine proteases in Cmm virulence. Mutants of nine genes encoding serine proteases of 3 different families were tested for their ability to induce wilting, when tomato stems were puncture-inoculated, as compared to blisters formation on leaves, when plants were spray-inoculated. All the mutants showed reduction in wilting and blister formation as compared to the wild type. The chpCmutant displayed the highest reduction, implicating its major role in symptom development. Five mutants of cell wall degrading enzymes and additional genes (i.e. perforin and sortase) caused wilting but were impaired in their ability to form blisters on leaves. These results suggest that Cmm differentially expressed virulence genes according to the site of penetration. Furthermore, we isolated and characterized two Cmmtranscriptional activators, Vatr1 and Vatr2 that regulate the expression of virulence factors, membrane and secreted proteins. The second objective was to determine the effect of bacterial virulence genes on movement of Cmm in tomato plants and identify the routes by which the pathogen contaminates seeds. Using a GFP-labeledCmm we could demonstrate that Cmm extensively colonizes the lumen of xylem vessels and preferentially attaches to spiral secondary wall thickening of the protoxylem and formed biofilm-like structures composed of large bacterial aggregates. Our findings suggest that virulence factors located on the chp/tomAPAI or the plasmids are required for effective movement of the pathogen in tomato and for the formation of cellular aggregates. We constructed a transposon plasmid that can be stably integrated into Cmm chromosome and express GFP, in order to follow movement to the seeds. Field strains from New York that were stably transformed with this construct, could not only access seeds systemically through the xylem, but also externally through tomato fruit lesions, which harbored high intra-and intercellular populations. Active movement and expansion of bacteria into the fruit mesocarp and nearby xylem vessels followed, once the fruit began to ripen. These results highlight the ability of Cmm to invade tomato fruit and seed through multiple entry routes. The third objective was to assess correlation between disease severity and expression levels of Cmm virulence genes and tomato defense genes. The effect of plant age on expression of tomato defense related proteins during Cmm infection was analyzed by qRT-PCR. Five genes out of eleven showed high induction at early stages of infection of plants with 19/20 leaves compared to young plants bearing 7/8 leaves. Previous results showed that Cmm virulence genes were expressed at early stages of infection in young plants compared to older plants. Results of this study suggest that Cmm virulence genes may suppress expression of tomato defense-related genes in young plants allowing effective disease development. The possibility that chpCis involved in suppression of tomato defense genes is currently under investigation by measuring the transcript level of several PR proteins, detected previously in our proteomics study. The fourth objective was to define genome location and stability of virulence genes in Cmm strains. New York isolates were compared to Israeli, Serbian, and NCPPB382 strains. The plasmid profiles of New York isolates were diverse and differed from both Israeli and Serbian strains. PCR analysis indicated that the presence of putative pathogenicity genes varied between isolates and highlighted the ephemeral nature of pathogenicity genes in field populations of Cmm. Results of this project significantly contributed to the understanding of Cmm virulence, its movement within tomato xylem or externally into the seeds, the role of serine proteases in disease development and initiated research on global regulation of Cmm virulence. These results form a basis for developing new strategies to combat wilt and canker disease of tomato.
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Lers, Amnon, E. Lomaniec, S. Burd, A. Khalchitski, L. Canetti y Pamela J. Green. Analysis of Senescence Inducible Ribonuclease in Tomato: Gene Regulation and Function. United States Department of Agriculture, febrero de 2000. http://dx.doi.org/10.32747/2000.7570563.bard.
Texto completoResumen
Natural leaf senescence has a negative influence on yield. Postharvest induced senescence contributes to the losses of quality in flowers, foliage, and vegetables. Strategies designed to control the senescence process in crop plants could therefore have great applied significance. Senescence is regulated by differential gene expression yet, functional characterization of the genes specifically induced and study of their expression control, is still in its infancy. Study of senescence-specific genes is required to allow identification of regulatory elements participating in senescence-induced expression and thus provide insights into the genetic regulation of senescence. A main feature of senescence is the hydrolysis of macromolecules by hydrolases of various types such as RNases and proteases. This study was aimed a analysis of senescence-inducible RNases in tomato with the following objectives: Isolation of senescence-inducible RNase cDNA clones; Expression analyses of RNase genes during senescence; Identification of sequences required for senescence-induced gene expression; Functional analyses of senescence-inducible RNases. We narrowed our aims somewhat to focus on the first three objectives because the budget we were awarded was reduced from that requested. We have expanded our research for identification senescence-related RNase/nuclease activities as we thought it will direct us to new RNase/nuclease genes. We have also carried out research in Arabidopsis and parsley, which enabled us to draw mire general conclusions. We completed the first and second objectives and have made considerable progress on the remaining two. We have defined growth conditions suitable for this research and defined the physiological and biochemical parameters characteristic to the advance of leaf senescence. In tomato and arabidopsis we have focused on natural leaf senescence. Parsley was used mainly for study of postharvest senescence in detached leaves. We have identified a 41-kD a tomato nuclease, LeNUCI, specifically induced during senescence which can degrade both RNA and DNA. This activity could be induced by ethylene in young leaves and was subjected to detailed analysis, which enabled its classification as Nuclease I enzyme. LeNUCI may be involved in nucleic acid metabolism during tomato leaf senescence. In parsley senescing leaves we identified 2 main senescence-related nuclease activities of 41 and 39-kDa. These activities were induced in both naturally or artificially senescing leaves, could degrade both DNA and RNA and were very similar in their characteristics to the LeNUCI. Two senescence-induced RNase cDNAs were cloned from tomato. One RNase cDNA was identical to the tomato LX RNase while the second corresponded to the LE RNase. Both were demonstrated before to be induced following phosphate starvation of tomato cell culture but nothing was known about their expression or function in plants. LX gene expression was much more senescence specific and ethylene could activate it in detached young leaves. LE gene expression, which could be transiently induced by wounding, appeared to be activated by abscisic acid. We suggest that the LX RNase has a role in RNA catabolism in the final stage of senescence, and LE may be a defense-related protein. Transgenic plants were generated for altering LX gene expression. No major visible alterations in the phenotype were observed so far. Detailed analysis of senescence in these plants is performed currently. The LX promoter was cloned and its analysis is performed currently for identification of senescence-specific regulatory elements. In Arabidopsis we have identified and characterized a senescence-associated nuclease 1 gene, BFN1, which is highly expressed during leaf and stem senescence. BFN1, is the first example of a senescence- associated gene encoding a nuclease I enzyme as well as the first nuclease I cloned and characterized from Arabidopsis. Our progress should provide excellent tools for the continued analysis of regulation and function of senescence-inducible ribonucleases and nucleases in plants. The cloned genes can be used in reverse genetic approaches, already initiated, which can yield a more direct evidence for the function of these enzymes. Another contribution of this research will be in respect to the molecular mechanism, which controls senescence. We had already initiated in this project and will continue to identify and characterize regulatory elements involved in senescence-specific expression of the genes isolated in this work.
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Lers, Amnon y Pamela J. Green. LX Senescence-Induced Ribonuclease in Tomato: Function and Regulation. United States Department of Agriculture, septiembre de 2003. http://dx.doi.org/10.32747/2003.7586455.bard.
Texto completoResumen
Natural leaf senescence, which occurs even when growth conditions are near optimal, has a negative influence on yield. Postharvest induced senescence contributes to the losses of quality in flowers, foliage, and vegetables. Strategies designed to control the senescence process in crop plants could therefore have great applied significance. However, the successful design of such strategies requires a better insight into the senescence machinery and control in higher plants. A main feature of senescence is the hydrolysis of macromolecules by hydrolases of various types such as ribonucleases (RNases) and proteases. Previously we had identified and characterized the tomato LX RNase gene demonstrating its transcript to be highly and specifically induced during senescence. This reported study was focused on LX but also had broadened our research to other senescence-associated nucleic acids degrading enzymes to learn about their function and the regulation of their encoding genes. Beside tomato we used parsley and Arabidopsis for the study of: the bi-functional nuclease which has a role in senescence. The study of different senescence- associated nucleases in few plant systems will allow a more general view on function and regulation of these enzymes in senescence. The specific original proposed objectives included: 1. Study the consequences of alterations in LX RNase level on tomato leaf senescence and general development; 2. Analyze stimuli which may participate in senescence-specific activation of the LX gene; 3. Clone the senescence-associated BFNI nuclease gene homologue from tomato. 4. Further characterize the sequences required for senescence-specific gene expression. Homozygous transgenic plants in which LX gene was either inhibited or over-expressed were generated. In both of these LX mutated plants no major phenotypic consequences were observed, which may suggests that LX is not essential for plant growth under optimal growth conditions. Lack of any abnormalities in the LX over-expressing lines suggests that special system exist to allow function of the RNase only when needed. Detailed analyses of growth under stress and consequences to RNA metabolism are underway. We have analyzed LX expression on the protein level demonstrating that it is involved also in petal senescing. Our results suggest that LX is responding to complex regulation involving developmental, organ dependent factors and responds differently to hormonal or environmental stimuli in the different plant organs. The cloned 1.4 kb promoter was cloned and its analysis revealed that probably not all required elements for senescence induction are included. Biochemical analysis of senescence-associated be-functional nucleases in the different plants, tomato, parsley and Arabidopsis, suggests they belong to a sub-class within the type I plant nucleases. The parsley PcNUC1/2 nuclease protein was purified from senescing leaves its and activity was studied in vitro revealing endo-, double strand, nucleolytic activity and exo-nucleolytic activity. Its encoding gene was cloned and found to be induced on the mRNA level. The promoter of the related Arabidopsis BFNI nuclease was shown in both tomato and Arabidopsis to be able and direct senescence-specific expression suggesting that, at least part, the gene is regulated on the transcriptional level and that the mechanism for this senescence-specific regulation is conserved between different plants. Few plants in which the BFNI gene is mutated were identified which are subjected now to detailed analysis. Our results suggest that the senescence-related nucleic acid degrading enzymes share similarities in both function and regulation between different plants and possibly have important functions in processes un-related to senescence. Still, the function of these enzymes, at least in some cases is not essential to plant development under optimal growth conditions. We are now at the stage which permits in depth investigation of the specific functions and mode of molecular regulation of senescence-associated nucleases with the aid of the research tools developed. The isolated senescence-specific promoter, shown to be active in heterologous plant system, could be utilized in agricultural-related biotechnological applications for retardation of senescence.
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Harman, Gary E. y Ilan Chet. Enhancement of plant disease resistance and productivity through use of root symbiotic fungi. United States Department of Agriculture, julio de 2008. http://dx.doi.org/10.32747/2008.7695588.bard.
Texto completoResumen
The objectives of the project were to (a) compare effects ofT22 and T-203 on growth promotion and induced resistance of maize inbred line Mol7; (b) follow induced resistance of pathogenesis-related proteins through changes in gene expression with a root and foliar pathogen in the presence or absence of T22 or T-203 and (c) to follow changes in the proteome of Mol? over time in roots and leaves in the presence or absence of T22 or T-203. The research built changes in our concepts regarding the effects of Trichoderma on plants; we hypothesized that there would be major changes in the physiology of plants and these would be reflected in changes in the plant proteome as a consequence of root infection by Trichoderma spp. Further, Trichoderma spp. differ in their effects on plants and these changes are largely a consequence of the production of different elicitors of elicitor mixtures that are produced in the zone of communication that is established by root infection by Trichoderma spp. In this work, we demonstrated that both T22 and T-203 increase growth and induce resistance to pathogens in maize. In Israel, it was shown that a hydrophobin is critical for root colonization by Trichoderma strains, and that peptaibols and an expansin-like protein from Ttrichoderma probably act as elicitors of induced resistance in plants. Further, this fungus induces the jasmonate/ethylene pathway of disease resistance and a specific cucumber MAPK is required for transduction of the resistance signal. This is the first such gene known to be induced by fungal systems. In the USA, extensive proteomic analyses of maize demonstrated a number of proteins are differentially regulated by T. harzianum strain T22. The pattern of up-regulation strongly supports the contention that this fungus induces increases in plant disease resistance, respiratory rates and photosynthesis. These are all very consistent with the observations of effects of the fungus on plants in the greenhouse and field. In addition, the chitinolytic complex of maize was examined. The numbers of maize genes encoding these enzymes was increased about 3-fold and their locations on maize chromosomes determined by sequence identification in specific BAC libraries on the web. One of the chitinolytic enzymes was determined to be a heterodimer between a specific exochitinase and different endochitinases dependent upon tissue differences (shoot or root) and the presence or absence of T. harzianum. These heterodimers, which were discovered in this work, are very strongly antifungal, especially the one from shoots in the presence of the biocontrol fungus. Finally, RNA was isolated from plants at Cornell and sent to Israel for transcriptome assessment using Affymetrix chips (the chips became available for maize at the end of the project). The data was sent back to Cornell for bioinformatic analyses and found, in large sense, to be consistent with the proteomic data. The final assessment of this data is just now possible since the full annotation of the sequences in the maize Affy chips is just now available. This work is already being used to discover more effective strains of Trichoderma. It also is expected to elucidate how we may be able to manipulate and breed plants for greater disease resistance, enhanced growth and yield and similar goals. This will be possible since the changes in gene and protein expression that lead to better plant performance can be elucidated by following changes induced by Trichoderma strains. The work was in, some parts, collaborative but in others, most specifically transcriptome analyses, fully synergistic.