De novo mammalian prion synthesis

Prions are responsible for a heterogeneous group of fatal neurodegenerative diseases. They can manifest as infectious, sporadic or genetic disorders involving posttranslational modifications of the cellular prion protein (PrP^C). Prions (PrP^Sc) are characterized by their infectious property and intrinsic ability to convert the physiological PrP^C into the pathological form, acting as a template. The “protein-only” hypothesis, postulated by Stanley B. Prusiner, implies the possibility to generate de novo prions in vivo and in vitro. Here we will describe major milestones towards proving this hypothesis, taking into account physiological environment/s, biochemical properties and interactors of the PrP^C.     

De novo generation of prion strains

Prions are self-replicating proteins that can cause neurodegenerative disorders such as bovine spongiform encephalopathy (also known as mad cow disease). Aberrant conformations of prion proteins accumulate in the central nervous system, causing spongiform changes in the brain and eventually death. Since the inception of the prion hypothesis - which states that misfolded proteins are the infectious agents that cause these diseases - researchers have sought to generate infectious proteins from defined components in the laboratory with varying degrees of success. Here, we discuss several recent studies that have produced an array of novel prion strains in vitro that exhibit increasingly high titres of infectivity. These advances promise unprecedented insight into the structure of prions and the mechanisms by which they originate and propagate.     

The reconstitution of mammalian prion infectivity de novo

The discovery of prion disease transmission in mammals, as well as a non-Mendelian type of inheritance in yeast, has led to the establishment of a new concept in biology, the prion hypothesis. The prion hypothesis postulates that an abnormal protein conformation propagates itself in an autocatalytic manner using the normal isoform of the same protein as a substrate and thereby acts either as a transmissible agent of disease (in mammals), or as a heritable determinant of phenotype (in yeast and fungus). While the prion biology of yeast and fungus supports this idea strongly, the direct proof of the prion hypothesis in mammals, specifically the reconstitution of the disease-associated isoform of the prion protein (PrP(Sc)) in vitro de novo from noninfectious prion protein, has been difficult to achieve despite many years of effort. The present review summarizes our current knowledge about the biochemical nature of the prion infectious agent and structure of PrP(Sc), describes potential strategies for generating prion infectivity de novo and provides some insight on why the reconstitution of infectivity has been difficult to achieve in vitro. Several hypotheses are proposed to explain the apparently low infectivity of the first generation of recently reported synthetic mammalian prions.     

De novo purine synthesis in skeletal muscle

Myoblast and primary muscle cultures from rat were found to contain the complete pathway of de novo purine nucleotide synthesis. Quantitative assessment of the pathway in skeletal muscle in mice in vivo, revealed a more intensive purine production in muscle than in liver. Skeletal muscle is thus a major site of de novo purine production in the mammalian body.     

De novo identification of mammalian ciliary motility proteins using cryo-EM

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De novo synthesis of DNA in human platelets

Platelets, incubated with radiolabeled thymidine and purified free of contaminating nucleated cells, were analyzed for their ability to synthesize DNA. The only DNA species isolated from these purified platelets was mitochondrial DNA. The CsCl gradient-purified platelet DNA was treated with the restriction endonucleases EcoRI, HindIII and HpaI yielding the expected pattern for human mitochondrial DNA. Nitrocellulose blots of the electrophoresed, restriction endonuclease-treated DNA were fluorographed. All of the DNA fragments generated by the restriction enzymes were labeled, indicating de novo synthesis. This was further substantiated by inhibition of DNA synthesis by ethidium bromide and 2',3'-dideoxythymidine. Platelet DNA appeared to become greatly fragmented after 4 to 7 days storage while all of the thymidine incorporated was observed in intact mitochondrial DNA. These results indicate a continuous degradation of platelet mitochondrial DNA with no apparent repair mechanism. The ability of platelets to synthesize DNA may be associated with the protein synthetic capacity of platelets previously described.     

De novo DNA synthesis using single molecule PCR

The throughput of DNA reading (sequencing) has dramatically increased recently due to the incorporation of in vitro clonal amplification. The throughput of DNA writing (synthesis) is trailing behind, with cloning and sequencing constituting the main bottleneck. To overcome this bottleneck, an in vitro alternative for in vivo DNA cloning must be integrated into DNA synthesis methods. Here we show how a new single molecule PCR (smPCR)-based procedure can be employed as a general substitute to in vivo cloning thereby allowing for the first time in vitro DNA synthesis. We integrated this rapid and high fidelity in vitro procedure into our earlier recursive DNA synthesis and error correction procedure and used it to efficiently construct and error-correct a 1.8-kb DNA molecule from synthetic unpurified oligos completely in vitro. Although we demonstrate incorporating smPCR in a particular method, the approach is general and can be used in principle in conjunction with other DNA synthesis methods as well.     

De novo synthesis of phytochrome in pumpkin hooks

Phytochrome becomes density labeled in the hook of pumpkin (Cucurbita pepo L.) seedlings grown in the dark on D₂O, indicating that the protein moiety of the pigment is synthesized de novo during development. Red light causes a rapid decline of the total phytochrome level in the hook of etiolated seedlings but upon return to the dark, phytochrome again accumulates. These newly appearing molecules are also synthesized de novo. Newly synthesized phytochrome in both dark-grown and red-irradiated seedlings is in the red-absorbing form. Turnover of the red-absorbing form is indicated by the density labeling of phytochrome during a period when the total phytochrome level in the hook of dark-grown seedlings remains constant. However, it was not possible to determine whether this results from intracellular turnover or turnover of the whole cell population during hook growth.     

De novo synthesis of pyrimidine nucleotides by Helicobacter pylori

The incorporation of pyrimidine nucleotide precursors into Helicobacter pylori and the activities of enzymes involved in their synthetic pathways were investigated by radioactive tracer analysis and ³¹P nuclear magnetic resonance spectroscopy. The bacterium was found to take up aspartate and bicarbonate and to incorporate carbon atoms from these precursors into its genomic DNA. Orotate, an intermediate of de novo pyrimidine biosynthesis, and uracil and uridine, precursors for pyrimidine pathways, were also incorporated by the micro-organism. Radiolabelled substrates were used to assess the activities of aspartate transcarbamoylase, orotate phosphoribosyltransferase, orotidylate decarboxylase, CTP synthetase, uracil phosphoribosyltransferase, thymidine kinase and deoxycytidine kinase in bacterial lysates. The study provided evidence for the presence in H. pylori of an operational de novo pathway, and a less active salvage pathway for the biosynthesis of pyrimidine nucleotides.     

De novo fatty acid synthesis in developing rat lung

The rate of de novo fatty acid synthesis in developing rat lung was measured by the rate of incorporation of 3H from 3H2O into fatty acids in lung slices and by the activity of acetyl-CoA carboxylase in fetal, neonatal and adult lung. Both tritium incorporation and acetyl-CoA carboxylase activity increased sharply during late gestation, peaked on the last fetal day, and declined by 50% 1 day after birth. In the adult, values were only one-half the peak fetal rates. In vitro regulation of acetyl-CoA carboxylase activity in fetal lung was similar to that described in adult non-pulmonary tissues: activation by citrate and inhibition by palmitoyl-CoA. Similarly, incubation conditions that favored enzyme phosphorylation inhibited acetyl-CoA carboxylase activity in lung while dephosphorylating conditions stimulated activity. Incorporation of [U-14 C]glucose into lung lipids during development was influenced heavily by incorporation into fatty acids, which generally paralleled the rate of tritium incorporation into fatty acids. The relative utilization of acetyl units from exogenous glucose for overall fatty acid synthesis was greater in adult lung than in fetal or neonatal lung, suggesting that other substrates may be important for fatty acid synthesis in developing lung. In fetal lung explants, de novo fatty acid synthesis was inhibited by exogenous palmitate. Taken together, these data suggest that de novo synthesis may be an important source of saturated fatty acids in fetal lung but of lesser importance in the neonatal period. Furthermore, the regulation of acetyl-CoA carboxylase activity and fatty acid synthesis in lung may be similar to non-pulmonary tissues.     

De novo reconstitution of a functional mammalian urinary bladder by tissue engineering

Human organ replacement is limited by a donor shortage, problems with tissue compatibility, and rejection. Creation of an organ with autologous tissue would be advantageous. In this study, transplantable urinary bladder neo-organs were reproducibly created in vitro from urothelial and smooth muscle cells grown in culture from canine native bladder biopsies and seeded onto preformed bladder-shaped polymers. The native bladders were subsequently excised from canine donors and replaced with the tissue-engineered neo-organs. In functional evaluations for up to 11 months, the bladder neo-organs demonstrated a normal capacity to retain urine, normal elastic properties, and histologic architecture. This study demonstrates, for the first time, that successful reconstitution of an autonomous hollow organ is possible using tissue-engineering methods.     

哺乳动物抑制型转录因子及启动子对的全新设计与构建

转录因子及启动子是基因回路的基础。相较于原核启动子,真核启动子作用机制复杂,增加了全新设计与改造的难度。目前有限数量的真核转录因子及启动子成为在哺乳动物细胞中设计并实现复杂基因回路以满足各类临床或工业应用需求的瓶颈。文中介绍了基于能够结合特定DNA序列的DNA结合结构域,通过柔性连接肽连接到转录抑制模块KRAB,构建抑制型转录因子以及通过在SV40启动子下游插入结合序列构建对应启动子的方法。而后,在哺乳动物细胞系中通过流式细胞术对其抑制转录的强度、不同转录因子及启动子对之间的正交性进行了测定。文中提供了一套标准化的、可调节的转录因子及启动子的全新设计与构建方案。基于该方案所构建的5对抑制型转录因子及启动子对能够在哺乳动物细胞中起到不同程度的抑制效果且相互正交。文中构建的哺乳动物转录因子及启动子对扩充了哺乳动物生物元件库,为构建复杂真核基因回路打下了基础;运用该设计方法能够根据需求构建更多正交的人工转录因子及启动子对。     

De novo pyrimidine nucleotide synthesis in the preimplantation mouse embryo

Utilizing phosphonacetyl- -aspartate (PALA), the transition state analog which specifically inhibits aspartate carbamyl transferase, we have shown that the preimplantation mouse embryo in culture has a functioning de novo pyridmidine biosynthetic pathway. This pathway accounts for some of the carbon dioxide fixation into nucleic acids previously described. Inhibition of de novo pyrimidine nucleotide synthesis during 2-cell to 8-cell development does not prevent morula development, but does prevent blastocyst development in nearly all embryos. Inhibition of the morula to blastocyst transition is most likely caused by a diminished pyrimidine nucleotide pool. Both de novo and salvage pathways appear active from the 2-cell embryo through blastocyst formation.     

De novo synthesis of peroxidase in cotton ovule culture medium

The biosynthesis of cotton ( Gossypium hirsutum L. 'Stoneville 208') peroxidase (EC 1.11.1.7) has been investigated in an organ culture system, since this enzyme may play a role in cell wall biogenesis or host defense mechanisms. Electrophoretic analysis of proteins from cotton ovule cultures indicated relatively few proteins being released into the surrounding medium. De novo synthesis of released peroxidase and other medium proteins was determined by in vivo labeling of ovule cultures with [³⁵S]-methionine. Analysis of labeled culture medium by denatured gel electrophoresis followed by fluorography showed incorporation of isotope into 2 major proteins with molecular weights of 30 kD and 56 kD, as well as a limited number of minor proteins. Similar analysis of native isoelectric focusing gels coupled with autoradiography demonstrated [³⁵S]-methionine incorporation into 2 major proteins with pI values of 4.3 and 5.0. The pI 5.0 protein was shown to have a molecular weight of 30 kD. The pI 4.3 protein had a molecular weight of 56 kD and was shown to be peroxidase by activity staining. Minor radiolabeled proteins were observed in the cationic region of the isoelectric focusing gels.     

De novo synthesis of monoterpenes by Saccharomyces cerevisiae wine yeasts

This paper reports the production of monoterpenes, which elicit a floral aroma in wine, by strains of the yeast Saccharomyces cerevisiae. Terpenes, which are typical components of the essential oils of flowers and fruits, are also present as free and glycosylated conjugates amongst the secondary metabolites of certain wine grape varieties of Vitis vinifera. Hence, when these compounds are present in wine they are considered to originate from grape and not fermentation. However, the biosynthesis of monoterpenes by S. cerevisiae in the absence of grape derived precursors is shown here to be of de novo origin in wine yeast strains. Higher concentration of assimilable nitrogen increased accumulation of linalool and citronellol. Microaerobic compared with anaerobic conditions favored terpene accumulation in the ferment. The amount of linalool produced by some strains of S. cerevisiae could be of sensory importance in wine production. These unexpected results are discussed in relation to the known sterol biosynthetic pathway and to an alternative pathway for terpene biosynthesis not previously described in yeast.     

De novo design and synthesis of HIV-1 integrase inhibitors

Existing AIDS therapies are out of reach for most HIV-infected people in developing countries and, where available, they are limited by their toxicity and their cost. New anti-HIV agents are needed urgently to combat emerging viral resistance and reduce the side effects associated with currently available drugs. Toward this end, LeapFrog, a de novo drug design program was used to design novel, potent, and selective inhibitors of HIV-1 integrase. The designed compounds were synthesized and tested for in vitro inhibition of HIV-1 integrase. Out of the 25 compounds that were designed, and synthesized, four molecules (compounds 23, 26, 43, and 59) showed moderate to low inhibition of HIV-1 integrase for 3'-processing and 3'-strand transfer activities. Nonetheless, these compounds possess structural features not seen in known HIV-1 integrase inhibitors and thus can serve as excellent leads for further optimization of anti-HIV-1 integrase activity.     

De novo synthesis of 3'-nucleotidase in germinating wheat embryo

The enzyme 3′-AMP nucleotidase was purified 2,500- to 5,000-fold from extracts of an acetone powder of wheat (Triticum aestivum) embryonic axes germinated for 40 hours. Sodium dodecyl sulfate acrylamide gel electrophoresis and chromatography on Biogel-P100 indicate that the enzyme is monomeric with a molecular weight of 39,000. Extracts of embryos germinated up to 6 hours have only 1% of the 40-hour level of enzyme activity. To see if the increase to 40 hours represents de novo synthesis, extracts were compared for their ability to react with a rabbit antibody prepared against the enzyme. In immunodiffusion tests, 40-hour extracts showed a strong precipitin line coincident with that of the purified enzyme, whereas no precipitation was observed with 1-hour extracts. When the enzyme present in 40-hour extracts was partially inactivated by EDTA, it still blocked the ability of the antibody to inhibit enzyme activity. Extracts of 1-hour embryos, in contrast, were not able to block the inhibitory activity of the antibody. Embryos allowed to take up ³⁵SO₄ between 40 and 46 hours of germination synthesized ³⁵S-labeled 3′-nucleotidase. In contrast, no radioactive protein synthesized by embryos during the first 6 hours of germination coincided on gel electrophoresis with the enzyme. These results indicate that the increase in 3′-nucleotidase activity is a consequence of de novo synthesis of the enzyme.     

De novo RNA synthesis catalyzed by HCV RNA-dependent RNA polymerase

The 65 kDa RNA-dependent RNA polymerase (NS5B), encoded by the hepatitis C virus (HCV) genome, is a key component involved in viral replication. Here we provide the direct evidence that purified HCV polymerase catalyzed de novo RNA synthesis in a primer-independent manner using homopolymers and HCV RNA as templates. The enzyme could utilize both polyC and polyU as templates for de novo RNA synthesis, suggesting that NS5B specifically recognized pyrimidine bases for initiation. More importantly, NS5B also catalyzed de novo RNA synthesis with an HCV RNA template; the resulting nascent RNA products, smaller than the template used, contained ATP as the first nucleotide. These results indicate that the newly synthesized RNAs did not result from template self-priming and suggest that a replication initiation site in the HCV RNA genome is a uridylate.     

De novo synthesis of peroxidase isozymes in sweet potato slices

The peroxidase content of sweet potato slices (Ipomoea batatas Lam.) increased nearly 100-fold following 84 hours incubation in an air atmosphere containing ethylene, 1 microliter per liter. The object of experiments reported here is to determine if this increase in peroxidase activity results from synthesis de novo of the enzyme or from activation of a preexisting inactive form of the enzyme.