Novel Interaction of the Z-DNA Binding Domain of Human ADAR1 with the Oncogenic c-Myc Promoter G-Quadruplex

Both G-quadruplex and Z-DNA can be formed in G-rich and repetitive sequences on genome, and their formation and biological functions are controlled by specific proteins. Z-DNA binding proteins, such as human ADAR1, have a highly conserved Z-DNA binding domain having selective affinity to Z-DNA. Here, our study identifies the Z-DNA binding domain of human ADAR1 (hZα_ADAR1) as a novel G-quadruplex binding protein that recognizes c-myc promoter G-quadruplex formed in NHEIII₁ region and represses the gene expression. An electrophoretic migration shift assay shows the binding of hZα_ADAR1 to the intramolecular c-myc promoter G-quadruplex-forming DNA oligomer. To corroborate the binding of hZα_ADAR1 to the G-quadruplex, we conducted CD and NMR chemical shift perturbation analyses. CD results indicate that hZα_ADAR1 stabilizes the parallel-stranded conformation of the c-myc G-quadruplex. The NMR chemical shift perturbation data reveal that the G-quadruplex binding region in hZα_ADAR1 was almost identical with the Z-DNA binding region. Finally, promoter assay and Western blot analysis show that hZα_ADAR1 suppresses the c-myc expression promoted by NHEIII₁ region containing the G-quadruplex-forming sequence. This finding suggests a novel function of Z-DNA binding protein as a regulator of G-quadruplex-mediated gene expression.     

Effect of externally added carnitine on the synthesis of acetylcholine in rat cerebral cortex cells

Acetylcholine synthesis from radiolabelled glucose was monitored in cerebral cortex cells isolated from brains of suckling and adult rats. Acetylcholine synthesis was found much higher in suckling animals, both in the absence and presence of acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) inhibitor, paraoxon. Together with choline (20 μM), carnitine was found to stimulate acetylcholine synthesis in a synergistic way in cortex cells from adult rats (18%). Choline, however, was incapable of reversing an inhibitory effect exerted by carnitine on acetylcholine synthesis in cortex cells from suckling animals. Distribution of carnitine derivatives was found significantly different in the cells from young and old animals, the content of acetylcarnitine decreased with age with a corresponding increase of free carnitine. The observed differences in carnitine effect on acetylcholine synthesis suggested that high acetylcarnitine in cells capable of β-oxidation might be correlated with the lower level of acetylcholine synthesis.     

Genetic analysis of replicating instabilities in yeast

Strains showing ethyl methanesulfonate (EMS)-induced replicating instability were genetically analysed to test whether within a given line, mosaics from different plating generations carry a mutation at the same site within the locus. A forward mutation system involving five loci controlling adenine biosynthesis in Schizosaccharomyces pombe was used. Genetic analysis was carried out by interallelic complementation and intragenic recombination tests. The data showed that EMS-induced instabilities are site-specific in being confined to the same recombination unit. This finding is discussed in relation to the possible mechanism(s) of replicating instabilities after different mutagenic treatments in a variety of biological systems.     

Induction of unscheduled DNA synthesis by the carcinogen 7-bromomethylbenz(a)anthracene and its removal from the DNA of normal and xeroderma pigmentosum lymphocytes

The carcinogen 7-bromomethylbenz(a)anthracene (BBA), which can bind strongly to DNA, induces unscheduled DNA synthesis (DNA repair) in normal lymphocytes but almost none in lymphocytes from patients with Xeroderma pigmentosum (XP), and inherited disease known to be defective in excision repair of ultraviolet-damaged DNA. We studied [³H]BBA's ability to bind to DNA of normal and XP lymphocytes, its influence on unscheduled DNA synthesis, and its removal from the DNA of both cell types. We found that 20–30% of the BBA is bound to macromolecules other than DNA and that its binding to DNA is essentially complete after 30 min. The induction of unscheduled DNA synthesis by the carcinogen in XP lymphocytes was approximately 10% of that induced in normal lymphocytes. While 15–20% of the BBA was removed from the DNA of normal cells 6 h after treatment, only 1–2% was removed from the DNA of XP cells. Thus, XP cells not only are defective in repairing ultraviolet-damaged DNA and excising thymine dimers but also fail to repair DNA damaged by certain carcinogens, and, most importantly, fail to remove the DNA-bound carcinogen, BBA.     

Studies of Blastocystis hominis

Blastocystis hominis, grown in Boeck-Drbohlav culture medium, modified by the omission of rice starch and the addition of 20% human serum and mineral oil cover to the Locke's solution overlay, can assume 3 morphologic forms. In the absence of human serum the vacuolated form, which divides by binary fission predominates. In medium with high serum content the granular form appears, with 3 types of granules. Spheroid or more elongate cytoplasmic granules predominate. In older organisms, lipid granules are found either in the peripheral cytoplasm or in the central vacuolar space. In occasional cells, variable numbers of reproductive granules develop in the central vacuolar space. These latter granules are released from the organism and give rise to typical B. hominis cells. The 3rd form, the ameba form, appears in small numbers in older cultures and in those treated with antibiotics. Ameba forms feed on bacteria and have slow pseudopodial activity. Exposure to oxygen causes rapid damage to cell membrane, with resulting leakage and collapse.     

Effects of proximate cholesterol precursors and steroid hormones on mouse myeloma growth in serum-free medium

Summary The proximate cholesterol precursors lathosterol, 7-dehydrocholesterol and desmosterol supported the growth of NS-1 and X63 mouse myeloma cells. These cells and X63.653 cells are cholesterol auxotrophs, yet each was able to convert [³H]lathosterol to [³H]cholesterol. These results are consistent with the conclusion that cholesterol auxotrophy in these myeloma cells is due to a deficiency in 3-ketosteroid reductase activity. The steroid hormones testosterone, progesserone and hydrocortisone could not replace cholesterol as a medium supplement. These results provide a greater understanding of the cholesterol auxotrophy characteristic of cell lines clonally-derived from the MOPC 21 myeloma tumor, and they provide a rational basis for the use of sterols in defined culture medium for mouse myeloma cells. This work was supported by National Institute of Health grants CA40294 and CA37589 to G. H. Sato and by a grant from RJR nabisco Inc. Editor's Statement These results help identify the defect in myeloma cells leading to cholesterol auxotrophy. The use of these cells in hybridoma derivation adds practical utility to a detailed appreciation of cholesterol metabolism in these cultures.     

Towards automation: Radiata pine shoot hedges in vitro

A novel system for in vitro shoot production has been developed whereby shoot hedges are maintained in one vessel. Monthly crops of shoots are produced for rooting. Radiata pine shoot hedges were maintained on Lepoivre (LP) nutrient agar medium for 18 months using a weekly liquid-nutrient replenishment system. In a separate experiment liquid-LP-nutrient replenishment of shoots twice weekly without transfers (D) resulted in better shoot growth and health than monthly transfers to fresh agar medium (B), monthly transfers to fresh agar medium plus aeration twice weekly (C), or no transfers and no liquid nutrient addition (A). Liquid nutrient replenishment twice weekly was better than 2 weekly or 4 weekly replenishment. The percentage of normal waxy (abundant tubular epicuticular wax) shoots harvested monthly increased significantly over the culture period from 41% at the first harvest to 93% at the eight harvest, and remained high at 97% from the ninth to twelfth harvest. The percentage of wet (no tubular epicuticular wax, small amounts of globular epicuticular wax) shoots harvested showed a corresponding decline—from 59%, to 7% at the eighth harvest. Shoots were harvested at a rate of 672/h (1.19 cents/shoot at a labour cost of NZ$8.00/h) and approximately 1100 shoots were produced per square metre of agar surface per month. Initial problems of contamination and crowding were overcome. These results will greatly facilitate progress towards automation of shoot production and reduction of costs of micropropagated trees. An automated system used in combination with other cost-saving techniques or robotics could potentially result in a substantial reduction in costs. This is the first report of a method of culturing shoots as hedges for a period of up to 18 months without manual subculturing.     

Effects of temperature,darkness and gelling agent on long-term storage of in vitro shoot cultures of Australian woody plant species

Storage of in vitro shoot cultures under reduced temperatures was investigated for eight Australian native woody plant species. Survival of multiple-shoot cultures for up to 12 months was best at 10°C in the light. Darkness increased the extent of vitrification and lower temperatures reduced survival. Acclimation at an intermediate temperature following storage at 10°C was sometimes beneficial. Survival was greater on Gelrite-based medium compared with agar, particularly at 22°C.     

Zonal immobilization of proteins

A gel matrix that can be used in sequence to separate proteins and then immobilize them was obtained by incorporating into agarose an aldehydic ligand with readily controllable reactivity. The gel was prepared by etherifying agarose with glycidol and subsequently oxidizing with periodate. It provided an inert matrix equivalent to ordinary agarose for separating proteins at neutral or acidic pH, but rapidly absorbed them through formation of stable alkyl amine linkages on exposure to either alkaline or concentrated NaCNBH₃. Thus, the protein could be fixed without use of denaturants. The ability to array proteins electrophoretically on an immobilizing substrate opens new possibilities for analysis of complex mixtures by providing means for carrying out affinity binding assays in relation to physical properties of the protein, and for performing multiple tests of composition without loss or spread.