Academic literature on the topic 'DnaG primase'
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Journal articles on the topic "DnaG primase"
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Sharma, Dhakaram Pangeni, Ramachandran Vijayan, Syed Arif Abdul Rehman, and Samudrala Gourinath. "Structural insights into the interaction of helicase and primase in Mycobacterium tuberculosis." Biochemical Journal 475, no. 21 (November 15, 2018): 3493–509. http://dx.doi.org/10.1042/bcj20180673.
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The helicase–primase interaction is an essential event in DNA replication and is mediated by the highly variable C-terminal domain of primase (DnaG) and N-terminal domain of helicase (DnaB). To understand the functional conservation despite the low sequence homology of the DnaB-binding domains of DnaGs of eubacteria, we determined the crystal structure of the helicase-binding domain of DnaG from Mycobacterium tuberculosis (MtDnaG-CTD) and did so to a resolution of 1.58 Å. We observed the overall structure of MtDnaG-CTD to consist of two subdomains, the N-terminal globular region (GR) and the C-terminal helical hairpin region (HHR), connected by a small loop. Despite differences in some of its helices, the globular region was found to have broadly similar arrangements across the species, whereas the helical hairpins showed different orientations. To gain insights into the crucial helicase–primase interaction in M. tuberculosis, a complex was modeled using the MtDnaG-CTD and MtDnaB-NTD crystal structures. Two nonconserved hydrophobic residues (Ile605 and Phe615) of MtDnaG were identified as potential key residues interacting with MtDnaB. Biosensor-binding studies showed a significant decrease in the binding affinity of MtDnaB-NTD with the Ile605Ala mutant of MtDnaG-CTD compared with native MtDnaG-CTD. The loop, connecting the two helices of the HHR, was concluded to be largely responsible for the stability of the DnaB–DnaG complex. Also, MtDnaB-NTD showed micromolar affinity with DnaG-CTDs from Escherichia coli and Helicobacter pylori and unstable binding with DnaG-CTD from Vibrio cholerae. The interacting domains of both DnaG and DnaB demonstrate the species-specific evolution of the replication initiation system.
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Huang, Yen-Hua, and Cheng-Yang Huang. "Structural Insight into the DNA-Binding Mode of the Primosomal Proteins PriA, PriB, and DnaT." BioMed Research International 2014 (2014): 1–14. http://dx.doi.org/10.1155/2014/195162.
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Replication restart primosome is a complex dynamic system that is essential for bacterial survival. This system uses various proteins to reinitiate chromosomal DNA replication to maintain genetic integrity after DNA damage. The replication restart primosome inEscherichia coliis composed of PriA helicase, PriB, PriC, DnaT, DnaC, DnaB helicase, and DnaG primase. The assembly of the protein complexes within the forked DNA responsible for reloading the replicative DnaB helicase anywhere on the chromosome for genome duplication requires the coordination of transient biomolecular interactions. Over the last decade, investigations on the structure and mechanism of these nucleoproteins have provided considerable insight into primosome assembly. In this review, we summarize and discuss our current knowledge and recent advances on the DNA-binding mode of the primosomal proteins PriA, PriB, and DnaT.
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Hayashi, Chihiro, Erika Miyazaki, Shogo Ozaki, Yoshito Abe, and Tsutomu Katayama. "DnaB helicase is recruited to the replication initiation complex via binding of DnaA domain I to the lateral surface of the DnaB N-terminal domain." Journal of Biological Chemistry 295, no. 32 (June 15, 2020): 11131–43. http://dx.doi.org/10.1074/jbc.ra120.014235.
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The DNA replication protein DnaA in Escherichia coli constructs higher-order complexes on the origin, oriC, to unwind this region. DnaB helicase is loaded onto unwound oriC via interactions with the DnaC loader and the DnaA complex. The DnaB–DnaC complex is recruited to the DnaA complex via stable binding of DnaB to DnaA domain I. The DnaB–DnaC complex is then directed to unwound oriC via a weak interaction between DnaB and DnaA domain III. Previously, we showed that Phe46 in DnaA domain I binds to DnaB. Here, we searched for the DnaA domain I–binding site in DnaB. The DnaB L160A variant was impaired in binding to DnaA complex on oriC but retained its DnaC-binding and helicase activities. DnaC binding moderately stimulated DnaA binding of DnaB L160A, and loading of DnaB L160A onto oriC was consistently and moderately inhibited. In a helicase assay with partly single-stranded DNA bearing a DnaA-binding site, DnaA stimulated DnaB loading, which was strongly inhibited in DnaB L160A even in the presence of DnaC. DnaB L160A was functionally impaired in vivo. On the basis of these findings, we propose that DnaB Leu160 interacts with DnaA domain I Phe46. DnaB Leu160 is exposed on the lateral surface of the N-terminal domain, which can explain unobstructed interactions of DnaA domain I–bound DnaB with DnaC, DnaG primase, and DnaA domain III. We propose a probable structure for the DnaA–DnaB–DnaC complex, which could be relevant to the process of DnaB loading onto oriC.
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Thirlway, Jenny, and Panos Soultanas. "In the Bacillus stearothermophilus DnaB-DnaG Complex, the Activities of the Two Proteins Are Modulated by Distinct but Overlapping Networks of Residues." Journal of Bacteriology 188, no. 4 (February 15, 2006): 1534–39. http://dx.doi.org/10.1128/jb.188.4.1534-1539.2006.
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ABSTRACTWe demonstrate the primase activity ofBacillus stearothermophilusDnaG and show that it initiates at 3′-ATC-5′ and 3′-ATT-5′ sites synthesizing primers that are 22 or 23 nucleotides long. In the presence of the helicase DnaB the size distribution of primers is different, and a range of additional smaller primers are also synthesized. Nine residues from the N- and C-terminal domains of DnaB, as well as its linker region, have been reported previously to affect this interaction. InBacillus stearothermophilusonly three residues from the linker region (I119 and I125) and the N-terminal domain (Y88) of DnaB have been shown previously to have direct structural importance, and I119 and I125 mediate DnaG-induced effects on DnaB activity. The functions of the other residues (L138, T191, E192, R195, and M196) are still a mystery. Here we show that the E15A, Y88A, and E15A Y88A mutants bind DnaG but are not able to modulate primer size, whereas the R195A M196A mutant inhibited the primase activity. Therefore, four of these residues, E15 and Y88 (N-terminal domain) and R195 and M196 (C-terminal domain), mediate DnaB-induced effects on DnaG activity. Overall, the data suggest that the effects of DnaB on DnaG activity and vice versa are mediated by distinct but overlapping networks of residues.
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Ilic, Stefan, Shira Cohen, Meenakshi Singh, Benjamin Tam, Adi Dayan, and Barak Akabayov. "DnaG Primase—A Target for the Development of Novel Antibacterial Agents." Antibiotics 7, no. 3 (August 13, 2018): 72. http://dx.doi.org/10.3390/antibiotics7030072.
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The bacterial primase—an essential component in the replisome—is a promising but underexploited target for novel antibiotic drugs. Bacterial primases have a markedly different structure than the human primase. Inhibition of primase activity is expected to selectively halt bacterial DNA replication. Evidence is growing that halting DNA replication has a bacteriocidal effect. Therefore, inhibitors of DNA primase could provide antibiotic agents. Compounds that inhibit bacterial DnaG primase have been developed using different approaches. In this paper, we provide an overview of the current literature on DNA primases as novel drug targets and the methods used to find their inhibitors. Although few inhibitors have been identified, there are still challenges to develop inhibitors that can efficiently halt DNA replication and may be applied in a clinical setting.
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Koepsell, Scott A., Marilynn A. Larson, Mark A. Griep, and Steven H. Hinrichs. "Staphylococcus aureus Helicase but Not Escherichia coli Helicase Stimulates S. aureus Primase Activity and Maintains Initiation Specificity." Journal of Bacteriology 188, no. 13 (July 1, 2006): 4673–80. http://dx.doi.org/10.1128/jb.00316-06.
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ABSTRACT Bacterial primases are essential for DNA replication due to their role in polymerizing the formation of short RNA primers repeatedly on the lagging-strand template and at least once on the leading-strand template. The ability of recombinant Staphylococcus aureus DnaG primase to utilize different single-stranded DNA templates was tested using oligonucleotides of the sequence 5′-CAGA (CA)5 XYZ (CA)3-3′, where XYZ represented the variable trinucleotide. These experiments demonstrated that S. aureus primase synthesized RNA primers predominately on templates containing 5′-d(CTA)-3′ or TTA and to a much lesser degree on GTA-containing templates, in contrast to results seen with the Escherichia coli DnaG primase recognition sequence 5′-d(CTG)-3′. Primer synthesis was initiated complementarily to the middle nucleotide of the recognition sequence, while the third nucleotide, an adenosine, was required to support primer synthesis but was not copied into the RNA primer. The replicative helicases from both S. aureus and E. coli were tested for their ability to stimulate either S. aureus or E. coli primase. Results showed that each bacterial helicase could only stimulate the cognate bacterial primase. In addition, S. aureus helicase stimulated the production of full-length primers, whereas E. coli helicase increased the synthesis of only short RNA polymers. These studies identified important differences between E. coli and S. aureus related to DNA replication and suggest that each bacterial primase and helicase may have adapted unique properties optimized for replication.
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Kuron, Aneta, Malgorzata Korycka-Machala, Anna Brzostek, Marcin Nowosielski, Aidan Doherty, Bozena Dziadek, and Jaroslaw Dziadek. "Evaluation of DNA Primase DnaG as a Potential Target for Antibiotics." Antimicrobial Agents and Chemotherapy 58, no. 3 (December 30, 2013): 1699–706. http://dx.doi.org/10.1128/aac.01721-13.
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ABSTRACTMycobacteria contain genes for several DNA-dependent RNA primases, includingdnaG, which encodes an essential replication enzyme that has been proposed as a target for antituberculosis compounds. Anin silicoanalysis revealed that mycobacteria also possess archaeo-eukaryotic superfamily primases (AEPs) of unknown function. Using a homologous recombination system, we obtained direct evidence that wild-typednaGcannot be deleted from the chromosome ofMycobacterium smegmatiswithout disrupting viability, even in backgrounds in which mycobacterial AEPs are overexpressed. In contrast, single-deletion AEP mutants or mutants defective for all four identifiedM. smegmatisAEP genes did not exhibit growth defects under standard laboratory conditions. Deletion of nativednaGinM. smegmatiswas tolerated only after the integration of an extra intact copy of theM. smegmatisorMycobacterium tuberculosisdnaGgene, under the control of chemically inducible promoters, into theattBsite of the chromosome.M. tuberculosisandM. smegmatisDnaG proteins were overproduced and purified, and their primase activities were confirmed using radioactive RNA synthesis assays. The enzymes appeared to be sensitive to known inhibitors (suramin and doxorubicin) of DnaG. Notably,M. smegmatisbacilli appeared to be sensitive to doxorubicin and resistant to suramin. The growth and survival of conditional mutant mycobacterial strains in which DnaG was significantly depleted were only slightly affected under standard laboratory conditions. Thus, although DnaG is essential for mycobacterial viability, only low levels of protein are required for growth. This suggests that very efficient inhibition of enzyme activity would be required for mycobacterial DnaG to be useful as an antibiotic target.
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Britton, Robert A., and James R. Lupski. "Isolation and Characterization of Suppressors of Two Escherichia coli dnaG Mutations, dnaG2903 and parB." Genetics 145, no. 4 (April 1, 1997): 867–75. http://dx.doi.org/10.1093/genetics/145.4.867.
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The dnaG gene of Escherichia coli encodes the primase protein, which synthesizes a short pRNA that is essential for the initiation of both leading and lagging strand DNA synthesis. Two temperature-sensitive mutations in the 3′ end of the dnaG gene, dnaG2903 and parB, cause a defect in chromosome partitioning at the nonpermissive temperature 42°. We have characterized 24 cold-sensitive suppressor mutations of these two dnaG alleles. By genetic mapping and complementation, five different classes of suppressors have been assigned: sdgC, sdgD, sdgE, sdgG and sdgH. The genes responsible for suppression in four of the five classes have been determined. Four of the sdgC suppressor alleles are complemented by the dnaE gene, which encodes the enzymatic subunit of DNA polymerase III. The sdgE class are mutations in era, an essential GTPase of unknown function. The sdgG suppressor is likely a mutation in one of three genes: ubiC, ubiA or yjbI. The sdgH class affects rpsF, which encodes the ribosomal protein S6. Possible mechanisms of suppression by these different classes are discussed.
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Paschalis, Vasileios, Emmanuelle Le Chatelier, Matthew Green, François Képès, Panos Soultanas, and Laurent Janniere. "Interactions of the Bacillus subtilis DnaE polymerase with replisomal proteins modulate its activity and fidelity." Open Biology 7, no. 9 (September 2017): 170146. http://dx.doi.org/10.1098/rsob.170146.
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During Bacillus subtilis replication two replicative polymerases function at the replisome to collectively carry out genome replication. In a reconstituted in vitro replication assay, PolC is the main polymerase while the lagging strand DnaE polymerase briefly extends RNA primers synthesized by the primase DnaG prior to handing-off DNA synthesis to PolC. Here, we show in vivo that (i) the polymerase activity of DnaE is essential for both the initiation and elongation stages of DNA replication, (ii) its error rate varies inversely with PolC concentration, and (iii) its misincorporations are corrected by the mismatch repair system post-replication. We also found that the error rates in cells encoding mutator forms of both PolC and DnaE are significantly higher (up to 15-fold) than in PolC mutants. In vitro , we showed that (i) the polymerase activity of DnaE is considerably stimulated by DnaN, SSB and PolC, (ii) its error-prone activity is strongly inhibited by DnaN, and (iii) its errors are proofread by the 3′ > 5′ exonuclease activity of PolC in a stable template-DnaE–PolC complex. Collectively our data show that protein–protein interactions within the replisome modulate the activity and fidelity of DnaE, and confirm the prominent role of DnaE during B. subtilis replication.
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Li, Jie, Jingfang Liu, Ligang Zhou, Huadong Pei, Jian Zhou, and Hua Xiang. "Two Distantly Homologous DnaG Primases from Thermoanaerobacter tengcongensis Exhibit Distinct Initiation Specificities and Priming Activities." Journal of Bacteriology 192, no. 11 (March 26, 2010): 2670–81. http://dx.doi.org/10.1128/jb.01511-09.
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ABSTRACT Primase, encoded by dnaG in bacteria, is a specialized DNA-dependent RNA polymerase that synthesizes RNA primers de novo for elongation by DNA polymerase. Genome sequence analysis has revealed two distantly related dnaG genes, TtdnaG and TtdnaG 2, in the thermophilic bacterium Thermoanaerobacter tengcongensis. Both TtDnaG (600 amino acids) and TtDnaG2 (358 amino acids) exhibit primase activities in vitro at a wide range of temperatures. Interestingly, the template recognition specificities of these two primases are quite distinctive. When trinucleotide-specific templates were tested, TtDnaG initiated RNA primer synthesis efficiently only on templates containing the trinucleotide 5′-CCC-3′, not on the other 63 possible trinucleotides. When the 5′-CCC-3′ sequence was flanked by additional cytosines or guanines, the initiation efficiency of TtDnaG increased remarkably. Significantly, TtDnaG could specifically and efficiently initiate RNA primer synthesis on a limited set of tetranucleotides composed entirely of cytosines and guanines, indicating that TtDnaG initiated RNA primer synthesis more preferably on GC-containing tetranucleotides. In contrast, it seemed that TtDnaG2 had no specific initiation nucleotides, as it could efficiently initiate RNA primer synthesis on all templates tested. The DNA binding affinity of TtDnaG2 was usually 10-fold higher than that of TtDnaG, which might correlate with its high activity but low template specificity. These distinct priming activities and specificities of TtDnaG and TtDnaG2 might shed new light on the diversity in the structure and function of the primases.
Dissertations / Theses on the topic "DnaG primase"
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Wolter, Gwen Annette. "Charakterisierung der Strahlen-induzierten Komplexbildung von DNA-Polymerase [alpha]-Primase [Alpha-Primase] mit dem Tumorsuppressorprotein p53." [S.l.] : [s.n.], 2001. http://deposit.ddb.de/cgi-bin/dokserv?idn=967492394.
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Pan, Hu. "Structural and biochemical studies of DNA primase from Bacillus stearothermophilus." Thesis, University of Oxford, 1999. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.302122.
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Borazjani, Gholami Farimah. "Role of replicative primase in lesion bypass during DNA replication." Thesis, University of Sussex, 2017. http://sro.sussex.ac.uk/id/eprint/68762/.
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Maintenance of genome integrity and stability is fundamental for any form of life. This is complicated as DNA is highly reactive and always under attack from a wide range of endogenous and exogenous sources which can lead to different damages in the DNA. To preserve the integrity of DNA replication, cells hav evolved a variety of DNA repair pathways. DNA damage tolerance mechanisms serve as the last line of defence to rescue the stalled replications forks. TLS, error-prone type of DNA damage tolerance, acts to bypass DNA lesions and allows continuation of DNA replication. Surprisingly majority of archaeal species lack canonical TLS polymerases. This poses a question as to how archaea restart stalled replication in the absence of TLS or lesion repair pathways. This thesis establishes that archaeal replicative primase (PriS/L), a member of the archaeo-eukaryotic primase (AEP) superfamily, possessing both primase and polymerase activities, is able to bypass the most common oxidative damages and highly distorting lesions caused by UV radiation. It has been postulated that archaeal replicative polymerases (Pol B and Pol D family Pols) can bind tightly to the deaminated bases uracil and cause replication fork stalling four bases prior to dU. A specific mechanism for resuming replication of uracil containing DNA by PriS/L is suggested in this thesis. In this thesis, we also reported how the enzymatic activities of archaeal PriS/L are regulated. Here, it is demonstrated that in contrast to archaeal replicative polymerases, single-strand binding proteins (RPA) significantly limit the polymerase activity of PriS/L. The remaining results chapter interrogates the possible interactions between PriS/L and RPA. Finally, the attempts to reconstitute an archaeal CMG complex in vitro, with the aim of shedding light on the role of archaeal replicative primase in replication-specific TLS are described.
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Desogus, Gianluigi. "Structural studies of lysyl-tRNA synthetases and DNA primases." Thesis, Imperial College London, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.369258.
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Liku, Muluye E. "CDK regulation of replication proteins: Mcm2-7 and DNA polymerase alpha primase." Diss., Search in ProQuest Dissertations & Theses. UC Only, 2008. http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3324598.
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Ligat, Gaëtan. "Cytomégalovirus humain, mutations de résistance et nouvelles cibles thérapeutiques." Thesis, Limoges, 2017. http://www.theses.fr/2017LIMO0046/document.
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Le cytomégalovirus humain (CMVH) est un pathogène opportuniste majeur en cas d’immunodépression et représente la principale cause d’infection congénitale d’origine virale. Bien qu’efficaces, l’utilisation des molécules conventionnelles est limitée par l’émergence de résistance et leur toxicité. Il devient alors nécessaire de développer de nouveaux traitements.L’étude des nouvelles mutations émergeant sous traitement antiviral demeure donc essentielle. L’introduction de ces nouvelles mutations, par mutagénèse « en passant », dans un chromosome bactérien artificiel contenant le génome viral nous permet, après transfection en cellules humaines, de tester la sensibilité de la souche recombinantes aux antiviraux.Différentes mutations de résistances ont ainsi été caractérisées. Afin de mettre en évidence de nouvelles cibles antivirales, des analyses bio-informatiques et la production de virus recombinants ont permis d’identifier de potentiels motifs fonctionnels essentiels à la réplication au sein du complexe terminase et hélicase-primase. Ainsi, nous avons montré quela sous-unité pUL56 du complexe terminase appartient à la famille des LAGLIDADG Homing Endonuclease. En effet, pUL56 contient un motif LATLNDIERFL et un motif de liaison à l’ADN. La technologie Alpha utilisant des protéines purifiées a permis de valider le caractère essentiel du fragment WMVVKYMGFF de pUL56 pour l’interaction avec pUL89. Enfin, nous avons mis en évidence les résidus impliqués dans la fixation de l’ATP au sein de l’hélicase et dans la stabilisation du zinc de la primase. Ainsi, la compréhension de la structure de ces protéines pourrait permettent de mieux appréhender leur fonctionnement au sein du processus de réplication du CMVH et le développement de nouvelles thérapies ciblant ces domainesHuman cytomegalovirus (HCMV) is an important opportunistic pathogen for immunecompromised patients and is the leading cause of congenital viral infection. Although they are effective, using of conventional molecules is limited by the emergence of resistance and their toxicity. Then it becomes necessary to develop new treatments. Study of new mutationsemerging under antiviral treatment is therefore essential. Introduction of these new mutations, by « en passant » mutagenesis, into an artificial bacterial chromosome containing the viral genome allows us, after transfection into human cells, testing antivirals sensitivity of the recombinant. Different mutations of resistances have been characterized. In order tohighlight new antiviral targets, bioinformatics and recombinant viruses production allowed to identify potential functional patterns essential for viral replication within terminase and helicase-primase complex. Thus, we have shown that pUL56 subunit of the terminase complex belongs to the LAGLIDADG Homing Endonuclease family. Indeed, pUL56 contains aLATLNDIERFL motif and a DNA binding motif. Alpha technology using purified proteins allowed to validate the essential character of the WMVVKYMGFF fragment of pUL56 for the interaction with pUL89. Finally, we highlighted the residues involved in ATP binding within the helicase and in the stabilization of zinc within the primase. Thus, understanding of these proteins structure could allow us to better understand their role within the viral replication process and the development of new therapies targeting these domains
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Morley, Stewart Anthony. "Interactions Between the Organellar Pol1A, Pol1B, and Twinkle DNA Replication Proteins and Their Role in Plant Organelle DNA Replication." BYU ScholarsArchive, 2019. https://scholarsarchive.byu.edu/etd/8128.
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Plants maintain organelle genomes that are descended from ancient microbes. Ages ago, these ancient microbes were engulfed by larger cells, beginning a process of co-evolution we now call the endo-symbiotic theory. Over time, DNA from the engulfed microbe was transferred to the genome of the larger engulfing cell, eventually losing the ability to be free-living, and establishing a permanent residency in the larger cell. Similarly, the larger cell came to rely so much on the microbe it had engulfed, that it too lost its ability to survive without it. Thus, mitochondria and plastids were born. Nearly all multicellular eukaryotes possess mitochondria; however, different evolutionary pressures have created drastically different genomes in plants versus animals. For one, animals have very compact, efficient mitochondrial genomes, with about 97% of the DNA coding for genes. These genomes are very consistent in size across different animal species. Plants, on the other hand, have mitochondrial genomes 10 to more than 100 times as large as animal mitochondrial genomes. Plants also use a variety of mechanisms to replicate and maintain their DNA. Central to these mechanisms are nuclear-encoded, organelle targeted replication proteins. To date, there are two DNA polymerases that have been identified in plant mitochondria and chloroplasts, Pol1A and Pol1B. There is also a DNA helicase-primase that localizes to mitochondria and chloroplasts called Twinkle, which has similarities to the gp4 protein from T7 phage. In this dissertation, we discuss the roles of the polymerases and the effects of mutating the Pol1A and Pol1B genes respectively. We show that organelle genome copy number decreases slightly and over time but with little effect on plant development. We also detail the interactions between Twinkle and Pol1A or Pol1B. Plants possess the same organellar proteins found in animal mitochondria, which are homologs to T7 phage DNA replication proteins. We show that similar to animals and some phage, plants utilize the same proteins in similar interactions to form the basis of a DNA replisome. However, we also show that plants mutated for Twinkle protein show no discernable growth defects, suggesting there are alternative replication mechanisms available to plant mitochondria that are not accessible in animals.
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Merryweather, Andrew. "The role of DNA primases specified by plasmids RP4 and ColIb-P9." Thesis, University of Leicester, 1986. http://hdl.handle.net/2381/34448.
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Bayes, Michelle. "A molecular phylogenetic study of the galagos, strepsirrhine primates and archontan mammals." Thesis, Oxford Brookes University, 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.266454.
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Rudd, Sean G. "Cellular and biochemical characterisation of PrimPol, a novel eukaryotic primase-polymerase involved in DNA damage tolerance." Thesis, University of Sussex, 2013. http://sro.sussex.ac.uk/id/eprint/45543/.
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Genome stability is of upmost importance to life. DNA polymerases are essential for the duplication and maintenance of the genome but they cannot themselves begin synthesis of a DNA chain, and require the activity of specialised RNA polymerases called primases. In eukaryotic cells distinct enzymes catalyse these two essential processes. This thesis contains the characterisation of coiled-coil domain containing protein (CCDC)111, a previously uncharacterised protein conserved in a broad range of unicellular and multicellular eukaryotes including humans. CCDC111 is a member of the archaeaoeukaroytic primase (AEP) superfamily and uniquely for a eukaryotic enzyme possesses both primase and polymerase activities, and was thus renamed PrimPol. The work in this thesis implicates PrimPol in the process of DNA damage tolerance, a universal mechanism by which cells complete genome duplication in spite of potentially lethal DNA damage. The first results chapters detail the essential role of a PrimPol homologue (TbPrimPol2) in the important protozoan pathogen Trypanosoma brucei. A combination of molecular, cell biology, and biochemical analyses indicate a role for TbPrimPol2 in the post-replication tolerance of endogenously occurring DNA damage using its trans-lesion DNA synthesis activity. The remaining results chapters characterise PrimPol in human cultured cells, and demonstrate that this enzyme is present in both the nucleus and mitochondria. In the nucleus PrimPol functions in the cellular tolerance of ultraviolet (UV)- induced DNA damage, and is required to protect xeroderma pigmentosum variant (XP-V) cells, deficient in the UV lesion bypass polymerase Pol !, from the cytotoxic affects of UV radiation. Together, this thesis establishes the involvement of PrimPol in DNA damage tolerance from one of the earliest diverging eukaryotic organisms to man.
Books on the topic "DnaG primase"
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Schultz-Biegert Symposium (2nd 1991 Kartause Ittingen). Paternity in primates: Genetic tests and theories : implications of human DNA fingerprinting : 2nd Schultz-Biegert Symposium, Kartause Ittingen, Switzerland, September 16-20, 1991. Edited by Martin R. D. 1942-, Dixson A. F, and Wickings E. J. Basel: Karger, 1992.
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Marx, Christy. Watson And Crick And Dna (Primary Sources of Revolutionary Scientific Discoveries and Theories). Rosen Publishing Group, 2005.
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Taberlet, Pierre, Aurélie Bonin, Lucie Zinger, and Eric Coissac. DNA metabarcoding data analysis. Oxford University Press, 2018. http://dx.doi.org/10.1093/oso/9780198767220.003.0008.
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DNA metabarcoding generates huge amounts of data containing noise introduced by molecular methods. Chapter 8 “DNA metabarcoding data analysis” discusses the analytic steps and available software to curate and evaluate DNA metabarcoding data prior to final ecological analyses. It provides command lines to perform primary analyses of Illumina sequencing data with the OBITools, ranging from read assignment to samples to the formation of molecular operational taxonomic units (MOTUs) and their assignment to a taxon through comparison against reference databases. Chapter 8 also develops several methods to further curate sequencing data from contaminants or dysfunctional PCRs by using DNA extraction, PCR, and sequencing blank controls as well as PCR/biological replicates. It also presents several classical analyses to ensure that the diversity of the sample or the study site is appropriately covered. Finally, this chapter considers what conclusions on biodiversity and ecological processes can be really drawn from metabarcoding data.
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Martin, R. D., and Alan F. Dixson. Paternity in Primates: Genetic Tests and Theories : Implications of Human DNA Fingerprinting. S Karger Pub, 1992.
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Harley, Ross. The detection of in vitro DNA damage in primary rat hepatocytes using the alkaline comet assay. 1995.
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Lucchesi, John C. Epigenetics, Nuclear Organization & Gene Function. Oxford University Press, 2019. http://dx.doi.org/10.1093/oso/9780198831204.001.0001.
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Epigenetics is the study of heritable changes in gene function that do not involve changes in the DNA sequence. Epigenetic changes, consisting principally of DNA methylation, histone modifications and non-coding RNAs, maintain and modulate the initial impact of regulatory factors that recognize and associate with particular genomic sequences. This book’s primary goal is to establish a framework that can be used to understand the basis of epigenetic regulation and to appreciate both its derivation from genetics and its interdependence with genetic mechanisms. A further aim is to highlight the role played by the three-dimensional organization of the genetic material itself (the complex of DNA, histones and non-histone proteins referred to as chromatin) and its distribution within a functionally compartmentalized nucleus. Dysfunctions at any level of genetic regulation have the potential to result in an increased susceptibility to disease or actually give rise to overt pathologies. As illustrated in this book, research is continuously uncovering the role of epigenetics in a variety of human disorders, providing new avenues for therapeutic interventions and advances in regenerative medicine.
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Fidelity of primate cell repair of a double-strand break in a (CTG)·(CAG) tract: Effect of slipped DNA structures. Ottawa: National Library of Canada, 2003.
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(Editor), Bryan D. Ness, and Christina J. Moose (Editor), eds. Magills Encyclopedia of Science Plant Life: DNA Replication-Metabolites : Primary Vs. Secondary (Magills Encyclopedia of Science, Vol. 2). Salem Press, 2003.
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Lachmann, Robin H., and Nigel Manning. Trimethylaminuria. Oxford University Press, 2016. http://dx.doi.org/10.1093/med/9780199972135.003.0064.
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Trimethylaminuria (TMAU) or “Fish Odor Syndrome” is a disorder caused by increased concentrations of the volatile amine trimethylamine (TMA) in body fluids resulting in an unpleasant odor. The excess TMA may occur either due to deficient hepatic oxidation (primary) or increased bacterial generation (secondary). Testing urine for TMA concentration is the first line of investigation, preferably following a dietary load of a TMA precursor such as choline. Measurement of TMA and TMA-oxide are used as a guide to determine a primary or secondary cause, which can be confirmed by DNA analysis. FMO3 deficiency may have further clinical consequences due to the wide range of substrates oxidized by the enzyme including many drugs. Treatment of both primary and secondary TMAU relies on restriction of dietary precursors of TMA, antibiotic-based reduction of gut flora, and odor chelators. Riboflavin may also benefit some patients.
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Gapstur, Susan M., and Philip John Brooks. Alcohol and Cancer Risk. Oxford University Press, 2017. http://dx.doi.org/10.1093/oso/9780190238667.003.0012.
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In 2010, alcoholic beverage consumption caused an estimated 3.3 million deaths worldwide, and contributed to injuries, violence, liver cirrhosis, social disruption and at least seven different types of cancer. The International Agency for Research on Cancer (IARC) classifies exposure to both ethanol in alcoholic beverages and acetaldehyde, the primary metabolite of ethanol, as carcinogenic to humans (Group 1) based on “sufficient” evidence that alcoholic beverage consumption is causally related to cancers of the oral cavity, pharynx, larynx, esophagus, liver, colorectum and female breast. The biologic mechanisms by which alcohol and its primary metabolite acetaldehyde affect cancer risk appear to vary across anatomic sites. Broadly, these mechanisms involve DNA and protein damage from acetaldehyde and oxidative stress, nutritional malabsorption and metabolic effects, and for breast cancer, increased estrogen levels. The World Health Organization has increased global surveillance of alcohol consumption and encourages national efforts to apply evidence-based policies to reduce consumption.
Book chapters on the topic "DnaG primase"
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Messaoudi, Ilhem, Blossom Damania, and Scott W. Wong. "Primate Models for Gammaherpesvirus-Associated Malignancies." In DNA Tumor Viruses, 703–33. New York, NY: Springer US, 2008. http://dx.doi.org/10.1007/978-0-387-68945-6_27.
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Kemp, Chris, and David Kuninger. "Nonintegrating DNA Virus." In Primary and Stem Cells, 85–102. Hoboken, NJ, USA: John Wiley & Sons, Inc., 2011. http://dx.doi.org/10.1002/9781118147177.ch5.
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Soultanas, Panos, and Edward Bolt. "Replicative DNA Helicases and Primases." In Molecular Life Sciences, 1–9. New York, NY: Springer New York, 2014. http://dx.doi.org/10.1007/978-1-4614-6436-5_57-6.
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Soultanas, Panos, and Edward Bolt. "Replicative DNA Helicases and Primases." In Molecular Life Sciences, 1062–69. New York, NY: Springer New York, 2018. http://dx.doi.org/10.1007/978-1-4614-1531-2_57.
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Piette, Jacques. "Drug-DNA Interaction." In Primary Photo-Processes in Biology and Medicine, 229–40. Boston, MA: Springer US, 1985. http://dx.doi.org/10.1007/978-1-4684-1224-6_13.
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Tsvetanova, Billyana, Lansha Peng, Xiquan Liang, Ke Li, Jian-Ping Yang, Tony Ho, Josh Shirley, et al. "DNA Assembly Technologies Based on Homologous Recombination." In Primary and Stem Cells, 1–17. Hoboken, NJ, USA: John Wiley & Sons, Inc., 2011. http://dx.doi.org/10.1002/9781118147177.ch1.
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Moore, Jennifer C., and Ronald P. Hart. "Strategies for the Delivery of Naked DNA." In Primary and Stem Cells, 37–47. Hoboken, NJ, USA: John Wiley & Sons, Inc., 2011. http://dx.doi.org/10.1002/9781118147177.ch3.
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Plevani, P., M. Foiani, S. Francesconi, A. Pizzagalli, C. Santocanale, M. Falconi Muzi, S. Piatti, et al. "Genetic Control of the DNA Polymerase α-Primase complex in the Yeast Saccharomyces cerevisiae." In DNA Replication: The Regulatory Mechanisms, 285–94. Berlin, Heidelberg: Springer Berlin Heidelberg, 1992. http://dx.doi.org/10.1007/978-3-642-76988-7_26.
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Seymour, C. B., and C. Mothersill. "Radiation Induced Transformation in Primary Differentiated Thyroid Cultures." In Radiation Carcinogenesis and DNA Alterations, 209–16. Boston, MA: Springer US, 1986. http://dx.doi.org/10.1007/978-1-4684-5269-3_12.
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Kleihues, P., A. Aguzzi, T. Shibata, and O. D. Wiestler. "Immunohistochemical Assessment of Differentiation and DNA Replication in Human Brain Tumors." In Primary Brain Tumors, 123–32. New York, NY: Springer New York, 1989. http://dx.doi.org/10.1007/978-1-4612-3676-4_9.
Full textConference papers on the topic "DnaG primase"
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Boldinova, E. O., A. G. Baranovskiy, Yu V. Filina, R. R. Miftakhova, A. A. Manukyan, T. Tagirov, and A. V. Makarova. "THE ROLE OF C-TERMINAL DOMAIN IN ACTIVITY OF HUMAN PRIMASE-POLYMERASE PRIMPOL." In X Международная конференция молодых ученых: биоинформатиков, биотехнологов, биофизиков, вирусологов и молекулярных биологов — 2023. Novosibirsk State University, 2023. http://dx.doi.org/10.25205/978-5-4437-1526-1-300.
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Primase-polymerase PrimPol plays an important role in maintaining the stability of the genome by participating in the mechanism of DNA synthesis restart. The work shows that the C-terminal domain of PrimPol plays a key role in the regulation of enzyme activity, modulating the affinity of PrimPol for DNA and providing stabilization of the incoming initiating nucleotide. The activity of clinically significant PrimPol variants with substitutions in the C-terminal domain was also studied in the work.
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Adami, Andrea, Fausto Borghetti, Nicola Massari, Massimiliano Decarli, Cristian Collini, Leandro Lorenzelli, and David Stoppa. "Design of a cantilever-based system for DNA detection." In 2011 7th Conference on Ph.D. Research in Microelectronics and Electronics (PRIME). IEEE, 2011. http://dx.doi.org/10.1109/prime.2011.5966217.
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Caboni, Alessandra, Massimo Barbaro, Alexandra Homsy, Peter van der Wal, Vincent Linder, and Nico de Rooij. "Integration of a microfluidic flow cell on a CMOS biosensor for DNA detection." In 2008 Ph.D. Research in Microelectronics and Electronics (PRIME). IEEE, 2008. http://dx.doi.org/10.1109/rme.2008.4595740.
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Caruntu, Dumitru I., and Reynaldo Oyervides. "Primary Resonance of MEMS/NEMS Circular Plate Biosensors." In ASME 2014 International Design Engineering Technical Conferences and Computers and Information in Engineering Conference. American Society of Mechanical Engineers, 2014. http://dx.doi.org/10.1115/detc2014-35496.
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This investigation deals with M/NEMS circular plates under electrostatic actuation. Such structures can be used as resonator sensors for medicine and biology applications such as virus, bacteria or DNA detection. The system consists of a clamped circular plate over a ground. The actuation of the plate is done through an AC voltage whose frequency is near half natural frequency of the plate. This produces a primary resonance to be used afterwards for sensing purposes. It is showed that a saddle-node bifurcation occurs. The effects of damping, voltage, Casimir, and van der Waals forces are predicted.
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Son, Joo-Hiuk. "Active Demethylation of Cancer Cells using Terahertz Radiation for Potential Cancer Treatment." In Conference on Lasers and Electro-Optics/Pacific Rim. Washington, D.C.: Optica Publishing Group, 2022. http://dx.doi.org/10.1364/cleopr.2022.cmp3a_02.
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Carcinogenesis involves DNA methylation which is a primary alteration in DNA in the development of cancer before genetic mutation. Because the abnormal DNA methylation is found in most cancer cells, the assessment of DNA methylation using terahertz radiation can be a novel optical method to detect and control cancer. The methylation has been directly observed by terahertz time-domain spectroscopy and this epigenetic chemical change could be manipulated to the state of demethylation using resonant terahertz radiation. Demethylation of cancer cells is a key issue in epigenetic cancer therapy and our results may lead to the treatment of cancer using electromagnetic waves.
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Yang, Yueqian, Jimei Zhang, Dai Zhao, Guo Zheng, Bo Sun, Shuqing Sun, Hainan Wang, et al. "Primary Designation and Characterization of a Molecular Beacon DNA Sensing Probe." In 2011 International Conference on Control, Automation and Systems Engineering (CASE). IEEE, 2011. http://dx.doi.org/10.1109/iccase.2011.5997529.
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Kindhi, Berlian Al, Muhammad Afif Hendrawan, Diana Purwitasari, Tri Arief Sardjono, and Mauridhi Hery Purnomo. "Distance-based pattern matching of DNA sequences for evaluating primary mutation." In 2017 2nd International Conferences on Information Technology, Information Systems and Electrical Engineering (ICITISEE). IEEE, 2017. http://dx.doi.org/10.1109/icitisee.2017.8285518.
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Esfandyarpour, Hesaam, and Ronald W. Davis. "Gate-Controlled Microfluidic Chamber With Magnetic Bead for DNA Sequencing-by-Synthesis Technology." In ASME 2007 5th International Conference on Nanochannels, Microchannels, and Minichannels. ASMEDC, 2007. http://dx.doi.org/10.1115/icnmm2007-30119.
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In this paper we present a novel microfluidic platform for DNA sequencing-by-synthesis methods (e.g. pyrosequencing). The proposed platform is based on the valve-controllable PDMS channel technology with DNA-coated magnetic beads. The encapsulation of the reaction of DNA polymerization in picoliter-sized wells provides for excellent isolation and control for detection. This separation prevents cross-talk amongst neighbor reactors which is one of the most limitations for higher integration of the current technologies. Through application of an external magnetic field the beads can be allocated with better accuracy. In addition this property can help mixing for the reaction. The proposed system is useful for a number of other bio-species detection and sorting templates. This paper illustrates the design and experimental results of a primary template as well as different advantages and potential applications of the Gate-Controlled Magnetic Bead (GCMB) platform in the world of DNA sequencing and genetics.
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Sabedot, Thais S., Tathiane M. Malta, James Snyder, Tobias Walbert, Ian Lee, Steven Kalkanis, Ana Valeria Castro, and Houtan Noushmehr. "Abstract A12: DNA methylation-based liquid biopsy detects primary and recurrent meningioma." In Abstracts: AACR Special Conference on Advances in Liquid Biopsies; January 13-16, 2020; Miami, FL. American Association for Cancer Research, 2020. http://dx.doi.org/10.1158/1557-3265.liqbiop20-a12.
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Ma, Xin, and Lefu Hu. "Identification of DNA-binding Residues of a Protein from Its Primary Sequence." In 2012 5th International Symposium on Computational Intelligence and Design (ISCID). IEEE, 2012. http://dx.doi.org/10.1109/iscid.2012.80.
Full textReports on the topic "DnaG primase"
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DeMartini, James C., Abraham Yaniv, Jonathan O. Carlson, Arnona Gazit, Leonard E. Pearson, Kalman Perk, J. K. Young, Noam Safran, and A. Friedman. Evaluation of Naked Proviral DNA as a Vaccine for Ovine Lentivirus Infection. United States Department of Agriculture, September 1994. http://dx.doi.org/10.32747/1994.7570553.bard.
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Ovine lentivirus (OvLV) infection is widespread in sheep of the United States and Israel and is responsible for substantial economic losses. The primary goal of this project was to evaluate naked proviral DNA as a vaccine to induce protective immunity in sheep in endemic areas. Contrary to expectations, inoculation of sheep with proviral DNA derived from the full length OvLV molecular clone pkv72 did not result in detectable OvLV infection, but infectious virus was recovered from transfected ovine cells. Kv72 virus produced by these cells infected sheep and induced antibody responses, and was used as a viral challenge in subsequent experiments. To improve in vivo transfection efficiency and compare the viral LTR with other romoters, expression of reporter genes was studied in sheep transfected in vivo by injection of cationic liposome-DNA complexes; one formulation produced gene expression in a sheep for 4 months following a single intravenous injection. Since the pol-deleted OvLV construct was not stable in vivo, twelve lambs were injected with plasmids containing the Kv72 gag region (pCMVgag) or env region (pCMVenv), or saline. Prior to challenge, no detectable anti-OvLV immune responses were detected. Following homologous challenge with OvLV. Although the naked DNA approach to vaccination holds promise for control of ovine lentivirus-induced disease, further work needs to be done to develop more effective methods of transfecting sheep with DNA.
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Tzfira, Tzvi, Michael Elbaum, and Sharon Wolf. DNA transfer by Agrobacterium: a cooperative interaction of ssDNA, virulence proteins, and plant host factors. United States Department of Agriculture, December 2005. http://dx.doi.org/10.32747/2005.7695881.bard.
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Agrobacteriumtumefaciensmediates genetic transformation of plants. The possibility of exchanging the natural genes for other DNA has led to Agrobacterium’s emergence as the primary vector for genetic modification of plants. The similarity among eukaryotic mechanisms of nuclear import also suggests use of its active elements as media for non-viral genetic therapy in animals. These considerations motivate the present study of the process that carries DNA of bacterial origin into the host nucleus. The infective pathway of Agrobacterium involves excision of a single-stranded DNA molecule (T-strand) from the bacterial tumor-inducing plasmid. This transferred DNA (T-DNA) travels to the host cell cytoplasm along with two virulence proteins, VirD2 and VirE2, through a specific bacteriumplant channel(s). Little is known about the precise structure and composition of the resulting complex within the host cell and even less is known about the mechanism of its nuclear import and integration into the host cell genome. In the present proposal we combined the expertise of the US and Israeli labs and revealed many of the biophysical and biological properties of the genetic transformation process, thus enhancing our understanding of the processes leading to nuclear import and integration of the Agrobacterium T-DNA. Specifically, we sought to: I. Elucidate the interaction of the T-strand with its chaperones. II. Analyzing the three-dimensional structure of the T-complex and its chaperones in vitro. III. Analyze kinetics of T-complex formation and T-complex nuclear import. During the past three years we accomplished our goals and made the following major discoveries: (1) Resolved the VirE2-ssDNA three-dimensional structure. (2) Characterized VirE2-ssDNA assembly and aggregation, along with regulation by VirE1. (3) Studied VirE2-ssDNA nuclear import by electron tomography. (4) Showed that T-DNA integrates via double-stranded (ds) intermediates. (5) Identified that Arabidopsis Ku80 interacts with dsT-DNA intermediates and is essential for T-DNA integration. (6) Found a role of targeted proteolysis in T-DNA uncoating. Our research provide significant physical, molecular, and structural insights into the Tcomplex structure and composition, the effect of host receptors on its nuclear import, the mechanism of T-DNA nuclear import, proteolysis and integration in host cells. Understanding the mechanical and molecular basis for T-DNA nuclear import and integration is an essential key for the development of new strategies for genetic transformation of recalcitrant plant species. Thus, the knowledge gained in this study can potentially be applied to enhance the transformation process by interfering with key steps of the transformation process (i.e. nuclear import, proteolysis and integration). Finally, in addition to the study of Agrobacterium-host interaction, our research also revealed some fundamental insights into basic cellular mechanisms of nuclear import, targeted proteolysis, protein-DNA interactions and DNA repair.
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Wright, Adam, Marija Milacic, Karen Rothfels, Joel Weiser, Quang Trinh, Bijay Jassal, Robin Haw, and Lincoln Stein. Evaluating the Predictive Accuracy of Reactome's Curated Biological Pathways. Reactome, November 2022. http://dx.doi.org/10.3180/poster/20221109wright.
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Reactome is a database of human biological pathways manually curated from the primary literature and peer-reviewed by experts. To evaluate the utility of Reactome pathways for predicting functional consequences of genetic perturbations, we compared predictions of perturbation effects based on Reactome pathways against published empirical observations. Ten cancer-relevant Reactome pathways, representing diverse biological processes such as signal transduction, cell division, DNA repair, and transcriptional regulation, were selected for testing. For each pathway, root input nodes and key pathway outputs were defined. We then used pathway-diagram-derived logic graphs to predict, either by inspection by biocurators or using a novel algorithm MP-BioPath, the effects of bidirectional perturbations (upregulation/activation or downregulation/inhibition) of single root inputs on the status of key outputs. These predictions were then compared to published empirical tests.
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Heifetz, Yael, and Michael Bender. Success and failure in insect fertilization and reproduction - the role of the female accessory glands. United States Department of Agriculture, December 2006. http://dx.doi.org/10.32747/2006.7695586.bard.
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The research problem. Understanding of insect reproduction has been critical to the design of insect pest control strategies including disruptions of mate-finding, courtship and sperm transfer by male insects. It is well known that males transfer proteins to females during mating that profoundly affect female reproductive physiology, but little is known about the molecular basis of female mating response and no attempts have yet been made to interfere with female post-mating responses that directly bear on the efficacy of fertilization. The female reproductive tract provides a crucial environment for the events of fertilization yet thus far those events and the role of the female tract in influencing them are poorly understood. For this project, we have chosen to focus on the lower reproductive tract because it is the site of two processes critical to reproduction: sperm management (storage, maintenance, and release from storage) and fertilization. E,fforts during this project period centered on the elucidation of mating responses in the female lower reproductive tract The central goals of this project were: 1. To identify mating-responsive genes in the female lower reproductive tract using DNA microarray technology. 2. In parallel, to identify mating-responsive genes in these tissues using proteomic assays (2D gels and LC-MS/MS techniques). 3. To integrate proteomic and genomic analyses of reproductive tract gene expression to identify significant genes for functional analysis. Our main achievements were: 1. Identification of mating-responsive genes in the female lower reproductive tract. We identified 539 mating-responsive genes using genomic and proteomic approaches. This analysis revealed a shift from gene silencing to gene activation soon after mating and a peak in differential gene expression at 6 hours post-mating. In addition, comparison of the two datasets revealed an expression pattern consistent with the model that important reproductive proteins are pre-programmed for synthesis prior to mating. This work was published in Mack et al. (2006). Validation experiments using real-time PCR techniques suggest that microarray assays provide a conservativestimate of the true transcriptional activity in reproductive tissues. 2.lntegration of proteomics and genomics data sets. We compared the expression profiles from DNA microarray data with the proteins identified in our proteomic experiments. Although comparing the two data sets poses analyical challenges, it provides a more complete view of gene expression as well as insights into how specific genes may be regulated. This work was published in Mack et al. (2006). 3. Development of primary reproductive tract cell cultures. We developed primary cell cultures of dispersed reproductive tract cell types and determined conditions for organ culture of the entire reproductive tract. This work will allow us to rapidly screen mating-responsive genes for a variety of reproductive-tract specifi c functions. Scientific and agricultural significance. Together, these studies have defined the genetic response to mating in a part of the female reproductive tract that is critical for successful fertllization and have identified alarge set of mating-responsive genes. This work is the first to combine both genomic and proteomic approaches in determining female mating response in these tissues and has provided important insights into insect reproductive behavior.
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Palmer, Guy, Varda Shkap, Wendy Brown, and Thea Molad. Control of bovine anaplasmosis: cytokine enhancement of vaccine efficacy. United States Department of Agriculture, March 2007. http://dx.doi.org/10.32747/2007.7695879.bard.
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Anaplasmosis an arthropod-born disease of cattle caused by the rickettsia Anaplasma marginale and is an impediment to efficient production of healthy livestock in both Israel and the United States. Currently the only effective vaccines are derived from the blood of infected cattle. The risk of widespread transmission of both known and newly emergent pathogens has prevented licensure of live blood-based vaccines in the U.S. and is a major concern for their continued use in Israel. Consequently development of a safe, effective vaccine is a high priority. In this collaborative project we focused on two approaches to vaccine development. The first focused o n improving antigen delivery to livestock and specifically examined how DNA vaccines could be improved to enhance priming and expansion of the immune response. This research resulted in development and testing of two novel vaccine delivery systems--one that targeted antigen spread among dendritic cells (the key cell in priming immune responses and a follow-on construct that also specifically targeted antigen to the endosomal-lysosomal compartment the processing organelle within the dendritic cell that directs vaccine antigen to the MHC class ll-CD4* T cell priming pathway). The optimized construct targeting vaccine antigen to the dendritic cell MHC class II pathway was tested for ability to prime A. marginale specific immune responses in outbred cattle. The results demonstrated both statistically significant effects of priming with a single immunization, continued expansion of the primary immune response including development of high affinity lgG antibodies and rapid recall of the memory response following antigen challenge. This portion of the study represented a significant advance in vaccine delivery for livestock. Importantly the impact of these studies is not limited to A. marginale a s the targeting motifs are optimized for cattle and can be adapted to other cattle vaccinations by inserting a relevant pathogen-specific antigen. The second approach (which represented an addition to the project for which approval was requested as part of the first annual report) was a comparative approach between A . marginale and the Israel A . centrale vaccines train. This addition was requested as studies on Major Surface Protein( MSP)- 2 have shown that this antigen is highly antigenically variable and presented solely as a "static vaccine" antigen does not give cross-strain immunity. In contrast A. . centrale is an effective vaccine which Kimron Veterinary institute has used in the field in Israel for over 50 years. Taking advantage of this expertise, a broad comparison of wild type A. marginale and vaccine strain was initiated. These studies revealed three primary findings: i) use of the vaccine is associated with superinfection, but absence of clinical disease upon superinfection with A. marginale; ii) the A. centrale vaccine strain is not only less virulent but transmission in competent in Dermacentor spp. ticks; and iii) some but not all MSPs are conserved in basic orthologous structure but there are significant polymorphisms among the strains. These studies clearly indicated that there are statistically significant differences in biology (virulence and transmission) and provide a clear path for mapping of biology with the genomes. Based on these findings, we initiated complete genome sequencing of the Israel vaccine strain (although not currently funded by BARD) and plant to proceed with a comparative genomics approach using already sequenced wild-type A. marginale. These findings and ongoing collaborative research tie together filed vaccine experience with new genomic data, providing a new approach to vaccine development against a complex pathogen.
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Barkan, Alice, and Zach Adam. The Role of Proteases in Regulating Gene Expression and Assembly Processes in the Chloroplast. United States Department of Agriculture, January 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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Freeman, Stanley, and Daniel Legard. Epidemiology and Etiology of Colletotrichum Species Causing Strawberry Diseases. United States Department of Agriculture, September 2001. http://dx.doi.org/10.32747/2001.7695845.bard.
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Diseases caused by Colletotrichum spp. are one of the most important limitations on international strawberry production, affecting all vegetative and fruiting parts of the plant. From 1995 to 1997, C. acutatum infections reached epidemic levels in Israeli strawberry nurseries, causing extensive loss of transplants in fruit-bearing fields and additional reductions in yield. Although C. acutatum also occurs on strawberry in Florida, recent crown rot epidemics have been primarily caused by C. gloeosporioides. Little is known about the basic epidemiology of these important diseases on strawberry. The source of initial inoculum for epidemics in Israel, Florida (other US states including California) and the rest of the world is not well understood. Subspecies relationships between Colletotrichum isolates that cause the different diseases on strawberry (i.e. attack different tissues) are also not well understood. Objectives of this proposal were to detennine the potential of infested soil, strawberry debris and other hosts as sources of primary inoculum for strawberry diseases caused by Colletotrichum spp. in Israel and Florida. In addition, traditional (ie. morphological characteristics, benomyl sensitivity, vegetative compatibility grouping) and DNA based methods were used to investigate the etiology of these diseases in order to resolve epidemiologically important subspecies variation. In Israel it was found that C. gloeosporioides and C. acutatum infecting strawberry could remain viable in sterilized soil for up to one year and in methyl-bromide fumigated soil for up to 4 months; inoculum in mummified fruit remained viable for at least 5 months under field conditions whereas that in infected crowns was not recovered. Therefore, the contribution of these inocula to disease epidemics should be considered. The host range and specificity of C. acutatum from strawberry was examined on pepper, eggplant, tomato, bean and strawberry under greenhouse conditions. The fungus was recovered from all plant species over a three-month period but caused disease symptoms only on strawberry. C. acutatum was also isolated from healthy looking, asymptomatic plants of the weed species, Vicia and Conyza, growing in infected strawberry fruiting fields. Isolates of C. acutatum originating from strawberry and anemone infected both plant species in artificial inoculations. The habitation of a large number of plant species including weeds by C. acutatum suggests that although it causes disease only on strawberry and anemone in Israel, these plants may serve as a potential inoculum source for strawberry infection and pennit survival of the pathogen between seasons. In Florida, isolates of Colletotrichum spp. from diseased strawberry fruit and crowns were evaluated to detennine their etiology and the genetic diversity of the pathogens. Only C. acutatum was recovered from fruit and C. gloeosporioides were the main species recovered from crowns. These isolates were evaluated at 40 putative genetic loci using random amplified polymorphic DNA (RAPD). Genetic analysis of RAPD markers revealed that the level of linkage disequilibrium among polymorphic loci in C. gloeosporioides suggested that they were a sexually reproducing population. Under field conditions in Florida, it was detennined that C. gloeosporioides in buried crowns survived
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Sherman, A., D. N. Kuhn, Y. Cohen, R. Ophir, and R. Goenaga. Exploring the polyembryonic seed trait in mango as a basis for a biotechnology platform for fruit tree crops. Israel: United States-Israel Binational Agricultural Research and Development Fund, 2021. http://dx.doi.org/10.32747/2021.8134176.bard.
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Mango is one of the most important fruit crops. However, the biology of this fruit tree is under studied. The lack of genetic and genomic resources has limited progress in mango research and breeding. Several research groups have recently started developing genomic tools for mango by creating transcriptome and genomic data. Sexual reproduction in plants is the main pathway for the creation of new genetic combinations. In modern agriculture, breeders exploit the genetic diversity generated through sexual reproduction to develop elite cultivars; however, these cultivars require genetic stabilization before they are suitable for mass propagation for uniform crop production. In heterozygous plants such as fruit trees, vegetative propagation (cloning) is the primary path for the propagation of genetically uniform plants. Another natural plant mechanism that can create genetically uniform plants (clones) is apomixes. Apomixis is defined as asexual reproduction through seeds that lead to the production of clonal progeny whose genotype is identical to that of the mother plant. In fruit crops like citrus and mango, sporophytic apomixes result in polyembryony, where seeds contain multiple embryos, one of which is sexually originated, and the others are clones of the mother tree. As part of this research, the reference genome of mango was established as a basic platform for mango breeding and research. It was used to map two important mango traits fruit size and polyembryony. The draft genome 'Tommy Atkins' sequence was generated using NRGene de-novo Magic on high molecular weight DNA of 'Tommy Atkins,' supplemented by 10X Genomics long read sequencing to improve the initial assembly. The final 'Tommy Atkins' genome assembly was a consensus sequence that included 20 pseudomolecules representing the 20 chromosomes of mango. The availability of a genome enables the genetic dissection of important traits. We demonstrated the utility of the genome assembly and the 'Tommy Atkins' x 'Kensington Pride' map by analyzing fruit weight phenotypic data and identifying two QTLs for this trait.
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Minz, Dror, Stefan J. Green, Noa Sela, Yitzhak Hadar, Janet Jansson, and Steven Lindow. Soil and rhizosphere microbiome response to treated waste water irrigation. United States Department of Agriculture, January 2013. http://dx.doi.org/10.32747/2013.7598153.bard.
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Research objectives : Identify genetic potential and community structure of soil and rhizosphere microbial community structure as affected by treated wastewater (TWW) irrigation. This objective was achieved through the examination soil and rhizosphere microbial communities of plants irrigated with fresh water (FW) and TWW. Genomic DNA extracted from soil and rhizosphere samples (Minz laboratory) was processed for DNA-based shotgun metagenome sequencing (Green laboratory). High-throughput bioinformatics was performed to compare both taxonomic and functional gene (and pathway) differences between sample types (treatment and location). Identify metabolic pathways induced or repressed by TWW irrigation. To accomplish this objective, shotgun metatranscriptome (RNA-based) sequencing was performed. Expressed genes and pathways were compared to identify significantly differentially expressed features between rhizosphere communities of plants irrigated with FW and TWW. Identify microbial gene functions and pathways affected by TWW irrigation*. To accomplish this objective, we will perform a metaproteome comparison between rhizosphere communities of plants irrigated with FW and TWW and selected soil microbial activities. Integration and evaluation of microbial community function in relation to its structure and genetic potential, and to infer the in situ physiology and function of microbial communities in soil and rhizospere under FW and TWW irrigation regimes. This objective is ongoing due to the need for extensive bioinformatics analysis. As a result of the capabilities of the new PI, we have also been characterizing the transcriptome of the plant roots as affected by the TWW irrigation and comparing the function of the plants to that of the microbiome. *This original objective was not achieved in the course of this study due to technical issues, especially the need to replace the American PIs during the project. However, the fact we were able to analyze more than one plant system as a result of the abilities of the new American PI strengthened the power of the conclusions derived from studies for the 1ˢᵗ and 2ⁿᵈ objectives. Background: As the world population grows, more urban waste is discharged to the environment, and fresh water sources are being polluted. Developing and industrial countries are increasing the use of wastewater and treated wastewater (TWW) for agriculture practice, thus turning the waste product into a valuable resource. Wastewater supplies a year- round reliable source of nutrient-rich water. Despite continuing enhancements in TWW quality, TWW irrigation can still result in unexplained and undesirable effects on crops. In part, these undesirable effects may be attributed to, among other factors, to the effects of TWW on the plant microbiome. Previous studies, including our own, have presented the TWW effect on soil microbial activity and community composition. To the best of our knowledge, however, no comprehensive study yet has been conducted on the microbial population associated BARD Report - Project 4662 Page 2 of 16 BARD Report - Project 4662 Page 3 of 16 with plant roots irrigated with TWW – a critical information gap. In this work, we characterize the effect of TWW irrigation on root-associated microbial community structure and function by using the most innovative tools available in analyzing bacterial community- a combination of microbial marker gene amplicon sequencing, microbial shotunmetagenomics (DNA-based total community and gene content characterization), microbial metatranscriptomics (RNA-based total community and gene content characterization), and plant host transcriptome response. At the core of this research, a mesocosm experiment was conducted to study and characterize the effect of TWW irrigation on tomato and lettuce plants. A focus of this study was on the plant roots, their associated microbial communities, and on the functional activities of plant root-associated microbial communities. We have found that TWW irrigation changes both the soil and root microbial community composition, and that the shift in the plant root microbiome associated with different irrigation was as significant as the changes caused by the plant host or soil type. The change in microbial community structure was accompanied by changes in the microbial community-wide functional potential (i.e., gene content of the entire microbial community, as determined through shotgun metagenome sequencing). The relative abundance of many genes was significantly different in TWW irrigated root microbiome relative to FW-irrigated root microbial communities. For example, the relative abundance of genes encoding for transporters increased in TWW-irrigated roots increased relative to FW-irrigated roots. Similarly, the relative abundance of genes linked to potassium efflux, respiratory systems and nitrogen metabolism were elevated in TWW irrigated roots when compared to FW-irrigated roots. The increased relative abundance of denitrifying genes in TWW systems relative FW systems, suggests that TWW-irrigated roots are more anaerobic compare to FW irrigated root. These gene functional data are consistent with geochemical measurements made from these systems. Specifically, the TWW irrigated soils had higher pH, total organic compound (TOC), sodium, potassium and electric conductivity values in comparison to FW soils. Thus, the root microbiome genetic functional potential can be correlated with pH, TOC and EC values and these factors must take part in the shaping the root microbiome. The expressed functions, as found by the metatranscriptome analysis, revealed many genes that increase in TWW-irrigated plant root microbial population relative to those in the FW-irrigated plants. The most substantial (and significant) were sodium-proton antiporters and Na(+)-translocatingNADH-quinoneoxidoreductase (NQR). The latter protein uses the cell respiratory machinery to harness redox force and convert the energy for efflux of sodium. As the roots and their microbiomes are exposed to the same environmental conditions, it was previously hypothesized that understanding the soil and rhizospheremicrobiome response will shed light on natural processes in these niches. This study demonstrate how newly available tools can better define complex processes and their downstream consequences, such as irrigation with water from different qualities, and to identify primary cues sensed by the plant host irrigated with TWW. From an agricultural perspective, many common practices are complicated processes with many ‘moving parts’, and are hard to characterize and predict. Multiple edaphic and microbial factors are involved, and these can react to many environmental cues. These complex systems are in turn affected by plant growth and exudation, and associated features such as irrigation, fertilization and use of pesticides. However, the combination of shotgun metagenomics, microbial shotgun metatranscriptomics, plant transcriptomics, and physical measurement of soil characteristics provides a mechanism for integrating data from highly complex agricultural systems to eventually provide for plant physiological response prediction and monitoring. BARD Report
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Evans, Donald L., Avigdor Eldar, Liliana Jaso-Friedmann, and Herve Bercovier. Streptococcus Iniae Infection in Trout and Tilapia: Host-Pathogen Interactions, the Immune Response Towards the Pathogen and Vaccine Formulation. United States Department of Agriculture, February 2005. http://dx.doi.org/10.32747/2005.7586538.bard.
Full textAbstract:
The objectives of the BARD proposal were to determine the mechanisms of nonspecific cytotoxic cells (NCC) that are necessary to provide heightened innate resistance to infection and to identify the antigenic determinants in Streptococcus iniae that are best suited for vaccine development. Our central hypothesis was that anti-bacterial immunity in trout and tilapia can only be acquired by combining "innate" NCC responses with antibody responses to polysaccharide antigens. These Objectives were accomplished by experiments delineated by the following Specific Aims: Specific aim (SA) #1 (USA) "Clone and Identify the Apoptosis Regulatory Genes in NCC"; Specific aim #2 (USA)"Identify Regulatory Factors that Control NCC Responses to S. iniae"; Specific aim #3 (Israel) "Characterize the Biological Properties of the S. iniae Capsular Polysaccharide"; and Specific aim #4 (Israel) "Development of an Acellular Vaccine". Our model of S. iniae pathogenesis encompassed two approaches, identify apoptosis regulatory genes and proteins in tilapia that affected NCC activities (USA group) and determine the participation of S.iniae capsular polysaccharides as potential immunogens for the development of an acellular vaccine (Israel group). We previously established that it was possible to immunize tilapia and trout against experimental S. difficile/iniaeinfections. However these studies indicated that antibody responses in protected fish were short lived (3-4 months). Thus available vaccines were useful for short-term protection only. To address the issues of regulation of pathogenesis and immunogens of S. iniae, we have emphasized the role of the innate immune response regarding activation of NCC and mechanisms of invasiveness. Considerable progress was made toward accomplishing SA #1. We have cloned the cDNA of the following tilapia genes: cellular apoptosis susceptibility (CAS/AF547173»; tumor necrosis factor alpha (TNF / A Y 428948); and nascent polypeptide-associated complex alpha polypeptide (NACA/ A Y168640). Similar attempts were made to sequence the tilapia FasLgene/cDNA, however these experiments were not successful. Aim #2 was to "Identify Regulatory Factors that Control NCC Responses to S. iniae." To accomplish this, a new membrane receptor has been identified that may control innate responses (including apoptosis) of NCC to S. iniae. The receptor is a membrane protein on teleost NCC. This protein (NCC cationic antimicrobial protein-1/ncamp-1/AAQ99138) has been sequenced and the cDNA cloned (A Y324398). In recombinant form, ncamp-l kills S. iniae in vitro. Specific aim 3 ("Characterize the Biological Properties of the S.iniae Capsular Polysaccharide") utilized an in- vitro model using rainbow trout primary skin epithelial cell mono layers. These experiments demonstrated colonization into epithelial cells followed by a rapid decline of viable intracellular bacteria and translocation out of the cell. This pathogenesis model suggested that the bacterium escapes the endosome and translocates through the rainbow trout skin barrier to further invade and infect the host. Specific aim #4 ("Development of an Acellular Vaccine") was not specifically addressed. These studies demonstrated that several different apoptotic regulatory genes/proteins are expressed by tilapia NCC. These are the first studies demonstrating that such factors exist in tilapia. Because tilapia NCC bind to and are activated by S. iniae bacterial DNA, we predict that the apoptotic regulatory activity of S. iniae previously demonstrated by our group may be associated with innate antibacterial responses in tilapia.