高GC含量DNA模板的PCR扩增

目的：探索高GC含量DNA的PCR扩增条件,为扩增达托霉素生物合成基因簇及拼接奠定基础。方法：在PCR扩增体系中,使用高保真的聚合酶及添加不同浓度的DMSO、7-deaza-dGTP等增强剂,并选择合适的PCR循环程序,优化富含GC的DNA的PCR扩增条件。结果：向反应体系中额外添加1%~4%的DMSO可以显著提高富含GC的DNA的PCR扩增产物量,但会降低其特异性;7-deaza-dGTP可以提高扩增产物的特异性及保真度,但产量会有所下降。应用touch down PCR并在体系中添加7-deaza-dGTP能够提高扩增产物的特异性和产率,增加扩增的保真度。结论：应用优化的PCR扩增条件将所有达托霉素生物合成基因簇分段扩增出来,并可扩增出长达6 kb的片段,且序列完全正确,可以进行后续拼接。     

De Novo Synthesis of Steroids and Oxysterols in Adipocytes

Local production and action of cholesterol metabolites such as steroids or oxysterols within endocrine tissues are currently recognized as an important principle in the cell type- and tissue-specific regulation of hormone effects. In adipocytes, one of the most abundant endocrine cells in the human body, the de novo production of steroids or oxysterols from cholesterol has not been examined. Here, we demonstrate that essential components of cholesterol transport and metabolism machinery in the initial steps of steroid and/or oxysterol biosynthesis pathways are present and active in adipocytes. The ability of adipocyte CYP11A1 in producing pregnenolone is demonstrated for the first time, rendering adipocyte a steroidogenic cell. The oxysterol 27-hydroxycholesterol (27HC), synthesized by the mitochondrial enzyme CYP27A1, was identified as one of the major de novo adipocyte products from cholesterol and its precursor mevalonate. Inhibition of CYP27A1 activity or knockdown and deletion of the Cyp27a1 gene induced adipocyte differentiation, suggesting a paracrine or autocrine biological significance for the adipocyte-derived 27HC. These findings suggest that the presence of the 27HC biosynthesis pathway in adipocytes may represent a defense mechanism to prevent the formation of new fat cells upon overfeeding with dietary cholesterol.     

Enhancement of PCR Amplification of Moderate GC-Containing and Highly GC-Rich DNA Sequences

PCR is a commonly used and highly efficient technique in biomolecular laboratories for specific amplification of DNA. However, successful DNA amplification can be very time consuming and troublesome because many factors influence PCR efficiency. Especially GC-rich DNA complicates amplification because of generation of secondary structures that hinder denaturation and primer annealing. We investigated the impact of previously recommended additives such as dimethylsulfoxide (DMSO), magnesium chloride (MgCl₂), bovine serum albumin (BSA), or formamide. Furthermore, we tested company-specific substances as Q-Solution, High GC Enhancer, and Hi-Spec; various actively promoted polymerases as well as different PCR conditions for their positive effects on DNA amplification of templates with moderate and extremely high CG-content. We found considerable differences of specificity and quantity of product between different terms. In this article, we introduce conditions for optimized PCR to help resolve problems amplifying moderate to high GC-rich templates.     

De Novo Synthesis of Isocitritase in Peanut (Arachis hypogaea L.) Cotyledons

Germination of peanut seed is accompanied by a rapid increase in isocitritase (isocitrate lyase, EC 4.1.3.1) during the first 4 days. The presence of cycloheximide (50 μg/ml) during water imbibition inhibited the increase in isocitritase activity. Actinomycin D conversely did not inhibit isocitritase activity until the second day of imbibition while RNA synthesis was inhibited. Germination of peanut seed in ¹⁴C-reconstituted amino acids followed by fractionation of a 20 to 35% ammonium sulfate preparation on a Sephadex G-200 column (57-fold purification) showed that the active enzymic fraction coincided with a large peak of radioactivity. Germination of peanut seed in 45% D₂O followed by enzyme purification and CsCl equilibrium centrifugation revealed that all the enzyme from D₂O seed had a higher density than normal isocitritase. These data indicate that isocitritase in peanut seed is synthesized de novo.     

Content and De Novo Synthesis of Cocaine in Embryos and Endosperms from Fruit of Erythroxylum coca Lam

There is controversy as to whether the mature fruit of Erythroxylumcoca var. coca Lam. contains the cocaine alkaloid (benzoylmethylecgonine).In the present study, cocaine was monitored to determine ifit was present in embryos and endosperms of mature fruit ofE. coca var. coca Lam., and if present, the time required forde novo synthesis in imbibing seed. Seeds from mature fruitof E. coca were dissected to separate the embryos from the endosperms.The separated embryos and endosperms were analysed for cocaine.Subsequently, endosperms and embryos from seed imbibed. undera light and dark treatment were separated on days 3, 6, 9, 12and 15 and analysed for cocaine. Cocaine was present in embryos(0.005% of d. wt) and endosperms (0–001% of d. wt) ofmature fruit of E. coca. De novo synthesis of cocaine occurredonly in embryos of seed imbibed under light after day 9 of imbibition. Erythroxylum coca, alkaloid, benzoylmethylecgonine, cocaine, embryo, endosperm, seed imbibition     

De Novo Messenger RNA and Protein Synthesis Are Required for Phytoalexin-mediated Disease Resistance in Soybean Hypocotyls

Actinomycin D inhibited the synthesis of poly(A)-containing messenger RNA in healthy soybean (Glycine max [L.] Merr. cv. Harosoy 63) hypocotyls and in hypocotyls inoculated with the pathogenic fungus Phytophthora megasperma var. sojae A. A. Hildb., but had little effect on protein synthesis within 6 hours. Blasticidin S, conversely, inhibited protein synthesis in the hypocotyls without exhibiting significant effects on messenger RNA synthesis. The normal cultivar-specific resistance of the Harosoy 63 soybean hypocotyls to the fungus was completely diminished by actinomycin D or blasticidin S. The fungus grew as well in hypocotyls treated with either inhibitor as it did in the near isogenic susceptible cultivar Harosoy, and production of the phytoalexin glyceollin was concomitantly reduced. The effects of actinomcyin D and blasticidin S were pronounced when the treatments were made at the time of fungus inoculation or within 2 to 4 hours after inoculation, but not after longer times. These results indicated that the normal expression of resistance to the fungus and production of glyceollin both required de novo messenger RNA and protein synthesis early after infection. Furthermore, actinomycin D and blasticidin S also were effective in suppressing resistance expression and glyceollin production in soybean hypocotyls when inoculated with various Phytophthora species that were normally nonpathogenic to the plants. This indicated that the mechanism of general resistance to these normally nonpathogenic fungi also involves de novo messenger RNA and protein synthesis and production of glyceollin.     

De Novo Methylation of Repeated Sequences in Coprinus Cinereus

We have examined the stability of duplicated DNA sequences in the sexual phase of the life cycle of the basidiomycete fungus, Coprinus cinereus. We observed premeiotic de novo methylation in haploid nuclei containing either a triplication, a tandem duplication, or an ectopic duplication. Methylation changes were not observed in unique sequences. Repeated sequences underwent methylation changes during the dikaryotic stage. In one cross, 27% of the segregants exhibited methylation-directed gene inactivation. However, all auxotrophs eventually reverted to prototrophy. C to T transition mutations were not observed in this study. Our studies also revealed one inversion that occurred in 50% of the segregants in a single triplication cross, and a single pop-out event that occurred during vegetative growth. These alterations were similar to changes reported in experiments with duplicated sequences in Neurospora crassa and Ascobolus immersus. However, significant differences were also noted. First, the extent of methylation was much less in C. cinereus than in the other two fungi. Second, CpG sequences appeared to be the preferred targets of methylation.     

De Novo Synthesis of 4,5-Dimethoxycatechol and 2,5-Dimethoxyhydroquinone by the Brown Rot Fungus Gloeophyllum trabeum

The new dimethoxycatechol 4,5-dimethoxy-1,2-benzenediol (DMC) and the new dimethoxyhydroquinone 2,5-dimethoxy-1,4-benzenediol (DMH) were isolated from stationary cultures of the brown rot fungus Gloeophyllum trabeum growing on a glucose mineral medium protected from light. The structure was elucidated by gas chromatography-mass spectrometry through comparison to a synthetic standard. Further confirmation was obtained by forming a dimethoxyoxazole derivative by condensation of DMC with methylene chloride and through examination of methylated derivatives. DMC and DMH may serve as ferric chelators, oxygen-reducing agents, and redox-cycling molecules, which would include functioning as electron transport carriers to Fenton’s reactions. Thus, they appear to be important components of the brown rot decay system of the fungus.     

De Novo Structural Pattern Mining in Cellular Electron Cryotomograms

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Small-Molecule Screen Identifies De Novo Nucleotide Synthesis as a Vulnerability of Cells Lacking SIRT3

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Quantification of Local De Novo Synthesis Versus Blood Contributions to Quinolinic Acid Concentrations in Brain and Systemic Tissues

Abstract: The source of the neurotoxin quinolinic acid (QUIN) in brain and systemic tissues under normal and pathologic circumstances reflects either de novo synthesis from l -tryptophan and other precursors, or entry of QUIN itself from the blood. To quantify the relative contributions of blood- versus tissue-derived QUIN, [¹³C₇]QUIN was infused subcutaneously via osmotic pumps (0.55 µl/h, 30 mM) in gerbils, and the fraction of QUIN in tissue (Ti; measured in tissue homogenates) derived from blood (BI; measured in serum) was calculated by the formula ([¹³C₇]QUIN_Ti/QUIN_Ti)/([¹³C₇]QUIN_Bl/QUIN_Bl). In controls, blood QUIN contributed 38–49% of QUIN in brain, 70% in CSF, between 40 and 70% in kidney, heart, and skeletal muscle, but <5% in spleen, lung, liver, and intestine. Systemic endotoxin (450 µg/kg) increased blood, brain, CSF, and systemic tissue QUIN levels. Notably, the relative proportion of QUIN derived from blood in brain, spleen, lung, and intestine was unchanged by endotoxin, but increased in kidney, heart, and skeletal muscle. In contrast, cerebral ischemic injury (10 min of bilateral carotid artery occlusion) increased regional brain QUIN concentrations at 4 days post ischemia, with a proportional increase in the amount of QUIN derived from de novo synthesis by brain tissue. In the blood and systemic tissues of postischemic gerbils, there were no changes in systemic tissue or blood QUIN levels, or changes in the relative proportions of blood- versus systemic tissue-derived QUIN. These results establish that the brain normally synthesizes QUIN, that the blood is a significant source of QUIN in controls and during acute systemic immune activation, and that the rate of QUIN formation by brain tissue increases in conditions of brain and systemic immune activation.