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991.
Fernando Pistón Santiago Marín Alberto Hernando Francisco Barro 《Molecular breeding : new strategies in plant improvement》2009,23(4):655-667
The wheat grain is the most important organ for human food and therefore is the target for much research focused on modifying
its composition to improve nutritional and functional components. Genetic transformation provides a precise tool to alter
the composition of wheat grain by expressing new genes or by down-regulating groups of proteins encoded by multigene families
such as gliadins, which contain clusters of epitopes that are active groups in triggering celiac disease. For such work, specific
promoters are required to express such constructs in the wheat endosperm. In the present study we report the isolation and
characterization of a γ-gliadin promoter from transgenic wheat, and the analysis of gliadin synthesis during grain development
in bread wheat by Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight Mass Spectrometry (MALDI/TOF MS). The γ-gliadin
promoter fragment was isolated from bread wheat by genome walking and was re-introduced, driving the expression of the gusA gene, by particle bombardment, giving fifteen independent transgenic lines. Detailed analysis of the sequence of the 885 bp
promoter fragment showed that it contains three prolamin boxes but only one is conserved according to the consensus sequence
reported. The AACA/TA motif is present twice in published γ-gliadin promoter sequences. The RY element i.e., CATGCAT or CATGCAC,
is also present twice in the published promoter. Transgenic lines were classified as high, medium, and low expressers. The
expression of the gusA gene was found only in the seeds of the transgenic lines. GUS staining was first detected in the outer endosperm of the lobes,
and then it extended to the whole outer endosperm. GUS staining was not found in the aleurone layer nor in the embryo. The
qRT-PCR data confirmed the data obtained by GUS staining. The expression of the gusA gene determined by qRT-PCR for the high expresser line (B281) was 4 and 8 times higher than that of medium (B282) and low
(B286) expresser lines, respectively. MALDI/TOF-MS showed that gliadins exhibited different patterns of synthesis during the
course of seed maturation. Thus, gliadins with masses higher than 36,000 Da were synthesised within the first 12 days post
anthesis while those with masses lower than 36,000 Da were synthesised later. Results of GUS staining, qRT-PCR and MALDI/TOF-MS
showed that the γ-gliadin promoter reported in this work could be a good candidate to downregulate wheat gliadins. 相似文献
992.
993.
994.
Vera Pingoud Wolfgang Wende Monika Reuter Albert Jeltsch Monika Fuxreiter 《Journal of molecular biology》2009,393(1):140-94
Restriction endonucleases of the PD…D/EXK family need Mg2+ for DNA cleavage. Whereas Mg2+ (or Mn2+) promotes catalysis, Ca2+ (without Mg2+) only supports DNA binding. The role of Mg2+ in DNA cleavage by restriction endonucleases has elicited many hypotheses, differing mainly in the number of Mg2+ involved in catalysis. To address this problem, we measured the Mg2+ and Mn2+ concentration dependence of DNA cleavage by BamHI, BglII, Cfr10I, EcoRI, EcoRII (catalytic domain), MboI, NgoMIV, PspGI, and SsoII, which were reported in co-crystal structure analyses to bind one (BglII and EcoRI) or two (BamHI and NgoMIV) Me2+ per active site. DNA cleavage experiments were carried out at various Mg2+ and Mn2+ concentrations at constant ionic strength. All enzymes show a qualitatively similar Mg2+ and Mn2+ concentration dependence. In general, the Mg2+ concentration optimum (between ∼ 1 and 10 mM) is higher than the Mn2+ concentration optimum (between ∼ 0.1 and 1 mM). At still higher Mg2+ or Mn2+ concentrations, the activities of all enzymes tested are reduced but can be reactivated by Ca2+. Based on these results, we propose that one Mg2+ or Mn2+ is critical for restriction enzyme activation, and binding of a second Me2+ plays a role in modulating the activity. Steady-state kinetics carried out with EcoRI and BamHI suggest that binding of a second Mg2+ or Mn2+ mainly leads to an increase in Km, such that the inhibitory effect of excess Mg2+ or Mn2+ can be overcome by increasing the substrate concentration. Our conclusions are supported by molecular dynamics simulations and are consistent with the structural observations of both one and two Me2+ binding to these enzymes. 相似文献
995.
Hepatitis C virus NS5A protein modulates template selection by the RNA polymerase in in vitro system
Alexander V. Ivanov Vera L. Tunitskaya Vladimir A. Mitkevich Vladimir S. Prassolov Marina K. Kukhanova 《FEBS letters》2009,583(2):277-1996
Hepatitis C virus (HCV) NS5A phosphoprotein is a component of virus replicase. Here we demonstrate that in vitro unphosphorylated NS5A protein inhibits HCV RNA-dependent RNA polymerase (RdRp) activity in polyA-oligoU system but has little effect on synthesis of viral RNA. The phosphorylated casein kinase (CK) II NS5A protein causes the opposite effect on RdRp in each of these systems. The phosphorylation of NS5A protein with CKII does not affect its affinity to the HCV RdRp and RNA. The NS5A phosphorylation with CKI does not change the RdRp activity. Herein we report evidence that the NS5A prevents template binding to the RdRp.
Structured summary
MINT-6803697: CKI (uniprotkb:P97633) phosphorylates (MI:0217) NS5A (uniprotkb:P26662) by protein kinase assay (MI:0424)MINT-6803713: CKII (uniprotkb:P67870) phosphorylates (MI:0217) NS5A (uniprotkb:P26662) by protein kinase assay (MI:0424) 相似文献996.
997.
Peter Lazar Dean Konjević Uwe Kierdorf Vera Njemirovskij Jan Čurlík Marijan Grubešić 《European Journal of Wildlife Research》2009,55(1):85-89
The paper presents the skull of a male gray wolf shot in the region of Snina, eastern Slovakia. Age at death of the wolf was
estimated at 6 years. The skull was characterized by the almost-complete absence of the alveolar processes of the incisive
bones and multiple dental abnormalities that were attributed to a severe trauma. All maxillary incisors and both maxillary
first premolars were missing. In addition, both maxillary canines were fractured with only tooth fragments being left. The
pulp exposure associated with the crown fractures of the maxillary canines had caused pulp necrosis and periapical lesions,
as evidenced on radiographs. The right P2, P3, and P4 were fractured, with only the remaining tooth structure of the second and third premolars being left. In the right P4 and the left M1, periapical bone resorption was diagnosed radiologically. In the mandibular dentition, the right I2 and left P1 had been lost in life. The mandibular canines exhibited distinct wear facets that were caused by contact with the maxillary
third incisors, which were subsequently lost. The fact that all fractured teeth showed signs of wear indicates that the trauma
had occurred some time before the wolf was shot. 相似文献
998.
999.
Vera Tai Ian T. Paulsen Katherine Phillippy D. Aaron Johnson Brian Palenik 《Environmental microbiology》2009,11(10):2698-2709
Microbes live in diverse communities yet their physiologies are typically studied in axenic culture. To begin to address this dichotomy, whole-genome microarray analyses were used and revealed that several major metabolic pathways were affected in Synechococcus sp. WH8102, a model phototroph, when grown with Vibrio parahaemolyticus , a model heterotroph. In co-cultures with V. parahaemolyticus , although phosphate was not depleted, Synechococcus sp. WH8102 may have experienced phosphate stress since the expression of phosphate acquisition genes increased and alkaline phosphatase activity was higher than in monocultures. Expression of cell wall synthesis genes and the components of a zinc transporter were also upregulated. In contrast, a ferric uptake regulation (Fur) family gene was downregulated as were genes that encode proteins rich in iron or involved in detoxifying oxygen radicals. Nitrogen use may also have been affected in co-cultures as the gene expression changes share similarities with ammonia-grown Synechococcus . This study demonstrates the multiple impacts that interspecific microbial interactions can have on the physiology of a major primary producer and the importance of investigating microbial physiology from a community perspective. 相似文献
1000.
Gianna Moscato Olivier Vandenplas Roy Gerth Van Wijk Jean-Luc Malo Luca Perfetti Santiago Quirce Jolanta Walusiak Roberto Castano Gianni Pala Denyse Gautrin Hans De Groot Ilenia Folletti Mona Rita Yacoub Andrea Siracusa 《Respiratory research》2009,10(1):1-20
The present document is the result of a consensus reached by a panel of experts from European and non-European countries on Occupational Rhinitis (OR), a disease of emerging relevance which has received little attention in comparison to occupational asthma. The document covers the main items of OR including epidemiology, diagnosis, management, socio-economic impact, preventive strategies and medicolegal issues. An operational definition and classification of OR tailored on that of occupational asthma, as well as a diagnostic algorithm based on steps allowing for different levels of diagnostic evidence are proposed. The needs for future research are pointed out. Key messages are issued for each item. 相似文献