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I'm currently learning about using PCR techniques to make fluorescently labelled DNA probes, and the textbook mentions "conjugated deoxyribonucleotides"
Can someone explain what these are? Nothing too in-depth and detailed. Just a decent description/explanation so I can get somewhat of an understanding.
To "conjugate" means to join or couple something together. Deoxyribonucleotides are the "letters" of DNA - A, T, C, and G. In biology/biochemistry, when you conjugate something you chemically modify it by adding something to it. Given the context of your question, the conjugated deoxyribonucleotides are fluorescently-labeled - a fluorescent dye has been covalently attached to it.
Nucleic Acid Bases in Anionic 2′-Deoxyribonucleotides: A DFT/B3LYP Study of Structures, Relative Stability, and Proton Affinities
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In just over two decades since the discovery of the first microRNA (miRNA), the field of miRNA biology has expanded considerably. Insights into the roles of miRNAs in development and disease, particularly in cancer, have made miRNAs attractive tools and targets for novel therapeutic approaches. Functional studies have confirmed that miRNA dysregulation is causal in many cases of cancer, with miRNAs acting as tumour suppressors or oncogenes (oncomiRs), and miRNA mimics and molecules targeted at miRNAs (antimiRs) have shown promise in preclinical development. Several miRNA-targeted therapeutics have reached clinical development, including a mimic of the tumour suppressor miRNA miR-34, which reached phase I clinical trials for treating cancer, and antimiRs targeted at miR-122, which reached phase II trials for treating hepatitis. In this article, we describe recent advances in our understanding of miRNAs in cancer and in other diseases and provide an overview of current miRNA therapeutics in the clinic. We also discuss the challenge of identifying the most efficacious therapeutic candidates and provide a perspective on achieving safe and targeted delivery of miRNA therapeutics.
The aim of the present study was to test the antigenicity of α-deoxyribonucleotides in order to develop a new tool for the detection of nucleic acid sequences for use in diagnostic applications. We describe four monoclonal antibodies (Mabs) which recognize α-deoxyribonucleotides. Two were raised against a poly(α-dT) sequence and specifically recognized the α-dT nucleotide. Two were raised against a sequence containing all four common nucleotides as α-nucleotides and, surprisingly, only recognized the α-dG nucleotide. For all four Mabs, no cross reactivity was observed with /3-oligonucleotides. These Mabs were reactive with aoligonucleotide sequences whether these sequences were single-stranded or hybridized to DNA or RNA. The four Mabs were tested in a sandwich hybridization assay that consisted of an α-oligonucleotide (for target sequence recognition), one of the four Mabs (for recognition of the hybridized a-oligonucleotide), and goat anti-mouse antibody conjugated to horse radish peroxydase (HRP) (for detection). One of the monoclonal antibodies, Mab 2E11D7, was directly conjugated to HRP and used in sandwich hybridization to detect PCR fragments of HPV 18 DNA. The sensitivity of this reaction was 1 pg of plasmid DNA containing the HPV 18 fragment. The specificity of the detection was demonstrated using HPV 6/11 and 16 DNA sequences.
Conjugated linoleic acid is a fatty acid found in nature, and does not have any known negative side effects. CLA was known from 1930 but it was isolated in 1987 as an antimutagenic agent by Dr Pariza and opened door for new research in the arena of fatty acids. Conjugated linoleic acid (CLA, 18:2) is a mixture of positional and geometric isomers of linoleic acid with two conjugated double bonds at various carbon positions in the fatty acid (FA) chain (figure 1) such as (7,9), (8,10) (9,11]), (10,12), and (11,13). Each double bond can be either cis or trans, but those with one trans double bond are the bioactive isomers (1). There are 28 different isomers with slightly different from each other with their chemical bonds but cis-9, trans-11 and trans-10, cis-12 is the most studied isomers (2). The double bonds positioned between the 9th and 11th carbon atoms as cis 9, trans-11 is referred rumenic acid on the basis of its ruminant origin (2).
The most abundant source of natural CLA is the meat and dairy products of grass-fed ruminants such as cows, sheep and goats. The average CLA content in meat products of ruminant and non-ruminant is reported as 0.46% and 0.16% of fat by Dhiman et al (3) whereas Shantha et al,, Mir et al (4-5) reported a higher range of CLA (1.7 to 8.5 mg CLA/g lipid) in meat products of ruminants. The CLA content in meat depends on variable factors such as species, breed, feeding regimes (5-6) (and management strategies used to raise cattle (3, 7). Breeds of cattle that deposit high amounts of fat in muscle will evidently provide a higher amount of CLA in their products (7). The total CLA content of specific foods may vary widely (3) and it is relatively easy to raise the concentration of cis-9, trans-11 CLA in ruminant lipids through manipulation of animal diet (8). Grass-fed animals are rich in CLA in comparison to stall fed animals. Substantially higher concentration of CLA was found in spring and summer season (when cows were pastured) than in fall and winter (when cows were stall-fed) (9). Further research has shown that the cows that graze at relatively higher altitudes may produce the healthiest milk of all compared with lowland grazers milk from high altitude grazers (3700-6200 ft) has even more omega-3 fatty acids and CLA and significantly less saturated fat. Because Plants growing in higher altitudes have more omega-3 fatty acids which solidify at lower temperatures than other fats and therefore acts as a form of anti-freeze. The cows graze this enriched pasture and pass the nutrients on to their milk (10).
Grazing beef steers on pasture or increasing the amount of forage (grass or legume hay) in the diet has been shown to increase the CLA content in the fat of cattle. Supplementation of high-grain diets of beef cattle with oils (e.g., soybean oil, linseed oil, and sunflower oil) may also increase the CLA content of beef, high fat diet such as soybean significantly increased CLA in cow milk (11). Beef fat from steers fed control diet or the same diet supplemented with 2% or 4% soybean oil, respectively, contained 0.1, 1.2, or 1%, of trans-10, cis-12 CLA, respectively (11). CLA in meat is stable under normal cooking and storage condition (3). Different species of animal have different amount of CLA such as horses have the lowest CLA content and sheep the highest. Human milk is in the middle and the CLA contain in milk is as mare’s milk < sow’s milk < human milk < goat’s milk < cow’s milk < ewe’s milk (12).
CLA occurs in eggs and fat of fish and sea animals as well (Table 1). Besides animal products white button Mushrooms (13) and pomegranate seed oil (14) is also reported as natural food sources of CLA. The low concentrations of CLA have also been reported in vegetable oils (Table 1) and infant formulas (15-16)
CLA is synthesized endogenously by two pathways. In the first pathways the biohydrogenation of ingested forage-derived dietary unsaturated fatty acids such as oleic acid and lenoleic acid (LA) convert to Stearic acid (17) by the rumen bacteria (18). The intermediates produced during biohydrogenation are cis -9, trans 11 (C18:2) (the main CLA isomer in milk) and trans 11 (C18:1) trans vaccenic acid. These intermediates are accumulated and absorbed in the intestine and incorporated to various tissues. The second pathway occurs in adipose tissue and in mammary gland of lactating cows by D9-desaturase (19). The trans-Δ11-vaccenic acid (t-VA), produced as a rumen biohydrogenation intermediate from both linoleic acid and α-linolenic acid and other PUFAs (20) provides a major alternate route for CLA biosynthesis in mammalian cells, including humans, viaΔ9 desaturation by stearoyl-CoA desaturase (SCD). The mammary gland and adipose tissue of ruminants have substantial D9-desaturase activity to perform the whole process (21-24). In ruminants Δ9-desaturase activity is high in adipose tissue of growing animals, mammary tissue and adipose tissue of lactating animals mRNA and protein for this enzyme are negligible in liver. The second most prevalent CLA isomer in milk fat is trans-7, cis-9 and it originates almost exclusively from endogenous synthesis involving Δ9-desaturase and trans-7 (C18:1) produced in the rumen.
Though some bacterial species in the large intestine of simple stomach animals can synthesize CLA (25) but the CLA formed in the large intestine does not be subsequently absorbed. After formation in the rumen cis-9, trans-11 CLA may be directly absorbed and further metabolized (biohydrogenated) by rumen microorganisms to trans-11-octadecenoic acid. Trans-11-octadecenoic acid may then be converted back to cis-9, trans-11 CLA within mammalian cells (26-27) by stearoyl–CoA desaturase (SCD). This is the major pathway in the formation of cis-9, trans-11 CLA in cow’s milk (20, 28). Cow’s milk also contains trans-10, cis-12 CLA, and trans-10-octadecenoic acid as well (20).
Dietary supplementation of CLA has been effective in reducing the percentage of body fat and increasing the percentage of body protein (29). Several in-vivo studies with rodents, pigs and cattle have shown that total body fat was reduced when animals were fed on a mixture of the two CLA isomers from synthetic sources (5, 30-33), may be due to inhibition of proliferation of adipocytes by the bio-formed CLA. CLA also helps in lessening leptin a hormone associated with weight gain and fat storage (34). CLA inhibits cancer by blocking the growth and metastatic spread of tumors. CLA inhibit both malignant and benign tumors almost immediately (35). The 10-CLA isomer seems to work preferentially through modulation of apoptosis and cell cycle control, while 9-CLA isomer affects arachidonic acid metabolism (36). CLA isomers have also been shown to have variable effects on bone formation (osteosynthesis) and resorption in animals. Dietary CLA inhibits eddosteal bone resorption, increases endocortical bone formation, and modulates the action and expression of COX enzymes, thereby decreasing prostaglandin-dependent bone resorption (37-38).
Since CLA can affect inflammatory cytokines, it is hypothesized that CLA may be a good tool for prevention or reduction of rheumatoid arthritis symptoms in humans (39). CLA is a potent anti-atherogenic dietary fatty acid in animal models of atherosclerosis by activating PPARs (40-41). CLA is very helpful in diabetic management and glycemic control, especially type II diabetes. Many studies strongly suggest that the 10-CLA isomer may be the bioactive isomer of CLA to influence the body weight changes observed in subjects with type II diabetes, a mixture of CLA (9- and 10-CLAs) rather than single could be more beneficial for the management of insulin resistance (42). However human studies have not shown this effect.
CLAs can alter the growth of neoplastic cells by influencing cell replication, interfering with components of cell cycle, or increasing cell death by promoting necrosis or/and apoptosis. Necrosis generally result from insult or toxicity reaction and triggers inflammation, whereas apoptosis is a distinct energy requiring process of programmed cell death, characterized by DNA fragmentation, chromosome condensation, nuclear fragmentation, formation of apoptotic bodies, and inversion of phosphatidylserine in the plasma membrane. CLA can reduce cell proliferation by blocking DNA synthesis (43) and cell cycle proteins (31, 44) that regulate this process and may support elevated apoptosis primarily by suppressing the expression of antiapoptotic bcl-2 gene (45). With different cell lines, CLA was able to increase the IL-2 and IFN-γ via modulation of protein kinase activity and production of oxidant species, which significantly inhibited proliferation (46). Thus, CLA has potential health benefits and can be used as prevention and treatment of many pathogenic conditions, we here will emphasize only on immunomodulatory effect of CLA.
The immune system plays a crucial role for good health and wellbeing. It protects the body from potentially harmful substances by recognizing and responding to antigens. Antigens can be both living and non-living. Living antigens are molecules (usually proteins) on the surface of cells, viruses, fungi, or bacteria. Nonliving substances are toxins, chemicals, drugs, and foreign particles (such as a splinter). The immune system recognizes and destroys substances that contain these antigens. The body cell proteins also act as an antigen and called HLA antigen. The antigens HLAs are found in large amounts on the surface of white blood cells. They help the immune system to recognize the difference between body tissue and foreign substances. There are two types of closely connected immunities present in human defense system, the innate or nonspecific and the adaptive immunity. Innate, or nonspecific, immunity is the defense system with which a person is born. It protects against all antigens through the barriers that keep harmful materials from entering in to the body. These barriers form the first line of defense in the immune response (47-48). It includes cough reflex, enzymes in tears and skin oils, mucus, which traps bacteria and small particles, skin, stomach acid and of broad pattern recognition that leads to phagocytosis and extracellular killing of invading agents. The cells of the innate immune response such as monocytes or macrophages and natural killer cells secrete cytokines (TNF-_, IL-1, and IL-6), prostaglandins, and leukotrienes early during the nonspecific immune response and are associated with inflammatory reaction. Prostaglandins have local effects and mainly perform in the tissues where they are synthesized.
Another kind of immunity is acquired or adaptive immunity develops with exposure to various antigens. Body’s immune system builds a defense that is specific to that antigen. The adaptive immune system consists of B and T lymphocytes, which elicit their effect or functions in an antigen-specific fashion. Development of effector cells is driven by the action of T helper cells, which can be divided on the basis of the cytokines they produce into Th1 (interferon-_ (IFN-), interleukin 2 (IL- 2)] and Th2 (IL-4, IL-5, IL-10) cells. After antigenic stimulation, a subset of lymphocytes become memory cells, which can elicit faster and more potent immune responses at a subsequent exposure with the same antigen (47). Lymphocytes and natural killer cells are derived from undifferentiated, self-renewing hemopoietic stem cells through highly regulated differentiation and maturation processes. These processes are mediated by micro-environmental factors, including cell-to-cell interactions, cytokines, and growth factors (48-49).
CLA Influence on Thymocyte Differentiation
CLA can influence critical pathways of thymocyte differentiation. Thermocytes are hematopoetic progenitor cells present in thymus. With regard to the effects of CLA on immune cell development, phenotypic analysis of T cell subsets in the thymus and peripheral blood revealed that CLA acted first on immature thymocytes (ie, day 35 of dietary supplementation) and later modulated mature peripheral blood T cells (i.e. days 49 to 72 of dietary supplementation) in a pig model (50) .
Innate Response of CLA
CLA in the diet of animals affect different immune-related mechanisms involved in both allergic reactions and infections (51). O’Shea et al (51) further reported that the decrease in leukotriene and prostaglandin production in organs from CLA-fed animals. Leukotrienes mediate many of the inflammatory phenomena that are characteristic of immediate hypersensitivity reactions such as direct effects on smooth muscle cells and blood vessels contributing to the recruitment of inflammatory cells. The effects of CLA on aspects of the innate response to viral infection such as increased organ weight at the sight of infection because of increased production and recruitment of inflammatory cells coupled with the reduction in viral particles in CLA-fed animals indicate a reduction in the adverse effects resulting from influenza infection.
CLA Effect on Adaptive Immunity
CLA can modulate both innate and adaptive immunity (52). It is well documented that CLA enhanced certain immune responses while reducing the adverse effects of immune-mediated catabolism (53-54). Dietary CLA has been shown to increase immunoglobulin production in rat spleen lymphocytes (55) and to reduce antigen-induced histamine and PGE2 release from sensitized guinea pig tracheae (55). The precise roles of the individual CLA isomers in these effects are not yet known, but it has been reported that trans-10, cis-12 CLA increases lymphocyte proliferation in vitro (56).
CLA enhances immune function in-vitro and in-vivo conditions. O’Shea, et al (57) investigated the potential of CLA to modulate the humoral and cell mediated immune responses in human immune system using the two main isomers in different ratios (50:50 and 80:20 of cis-9, trans-11 and trans-10, cis-12 CLA, respectively). Hepatitis B (Hbs) vaccination was used as an infection model to investigate the humoral and cell mediated immune response. Hepatitis B antibody titres were evaluated for each subject on day 0 and 2 weeks post initial vaccination and final booster. Mean serum Hbs antibody concentration at day 85 was twice as high for subjects consuming CLA 50:50 compared with the control or the 80:20 groups. The seroprotection rate (SPR, i.e. the number of subjects with anti-Hbs concentrations >10 IU/L compared to the number of subjects with titers <10 IU/L) was significantly higher (P>0.05) for the 50:50 group compared with the control or the 80:20 group.
The cell mediated immune response was measured using the CMI multi test for Delayed-Type Hypersensitivity (DTH). Evaluation of the DTH responses on 7 recall antigens, at different time points showed no statistically significant differences in all groups. Sugano et al (58) observed increased concentration of IgA, IgG, and IgM and a drop in IgE concentration in the serum of 7-week-old rats fed with 1% CLA (50:50 isomers) mix, may be due to a shift towards Th-1 cytokine profile in. A similar effect in humans after supplementation with a 50:50 CLA isomer mixture for 12 weeks was observed by Song et al (59). CLA supplementation also decreased the levels of the proinflammatory cytokines, TNF-alpha and IL-1beta (P < 0.05), but increased the levels of the anti-inflammatory cytokine, IL-10 (P < 0.05). Another aspect of immune function, delayed type hypersensitivity (DTH) response, was decreased during and after CLA supplementation (P < 0.05). However, plasma glucose, lipids, lymphocyte phenotypic results were not affected significantly by CLA (59). Hayek et al (60) observed that dietary CLA enhances in- vitro T cell function but has no effect on in-vivo T cell-mediated function as measured by the DTH skin reaction or B cell and NK activity. The immunostimulatory effect was more pronounced in young than in old mice and was not mediated through a change in PGE2 or IL-1 production.
However, Yamasaki et al (55) found no significant effects on serum IgA, IgG, or IgM concentration after feeding 5-wk-old rats a 50:50 CLA isomer mixture for 3 weeks at doses ranging from 0.05% to 0.5%. A study carried out in another species during gestation and lactation has reported an increment concentration of serum IgG (61), a fact that supports humoral enhancement effects of CLA in early age. The specific mechanism by which CLA enhances IgA levels at mucosal sites remains unknown. But since CLA has been shown to suppress IL-4 production in-vitro (61), attenuate Th2 responses in challenged animals and regulate the number and effectors functions of several lymphocytes (50), CLA is modulating the effects of TGFb on IgA production, it is probably due to posttranscriptional and/or translational regulation, which are important in this cytokine, because it has been suggested that TGFb mRNA levels do not completely correlate with the quantity of protein produced (62). On the other hand, the increase of IgA as result of CLA supplementation might be independent of the isotype switching mechanism produced by TGFb, which has been described, but is not completely defined (63) .
Immune Mechanism of CLA
The immunological effects of CLA are not well known. Several studies have suggested that it may be due to the antioxidant properties of CLA, which were demonstrated in cell free systems (64), liver microsomes (65) and in mammary glands (66). It was suggested that CLA may be a more potent antioxidant than α-tocopherol and almost as effective as butylated hydroxytoluene (BHT) (64, 67).
Two main hypotheses to explain the immuno-enhancing effects of dietary CLA have been proposed. The first one is that CLA interact with peroxisome proliferators activated receptors (PARS). PPARs (α, β, γ and δ) are nuclear receptors that translate nutritional and /or pharmacological stimuli into changes in gene expression (68) and it has a pivotal role in the stimulation of IgA responses at mucosal sites (69).The specific mechanism by which CLA enhances IgA levels at mucosal sites is unknown, but since CLA has been shown suppress IL-4 production in-vitro (70), attenuate Th2 responses in challenged animals (71) and regulates the number and effectors functions of several lymphocyts (50). PPARs bind to PPAR receptors and suppress or induce the transcription of target genes. The change in gene expression shows effect in myriad of cellular metabolic pathways such as lipid, carbohydrate and energy metabolism in immune and non-immune cells (72). The CLA isomers are active modulators of PPARs (73-74). These receptors enhance immune response and regulate gene expression. The synthetic PPAR-γ agonist inhibits the proinflammatory cytokines affecting the differentiation of monocytes and macrophages (75).
PPAR-γ comprises two isoforms, PPAR-γ 1 and PPAR- γ 2. Both are expressed in adipocytes, but PPAR- γ 1 is expressed in T and B cells, monocytes, dendritic cells, and epithelial cells (76). There are several possible options through which CLA might act. Ponferrada et al (77) reported that PPAR-γ agonists can revert stress-induced decrease of IgA production in the colon mucosa, even beyond the IgA controlled basal concentration. Infect PPAR- γ acts through modulation of transcriptional factors such as NF-kB, AP1, and STAT1 (78), which are involved in B-cell regulatory processes.A close links between intestinal-microbial interactions and regulation of PPAR-γ expression by epithelial cells of colon tissue (79). CLA may be modulating the entry of luminal antigen, the capacity for direct antigenic presentation, or even the transmission of antigen to dendritic cells from the intestinal mucosa. These hypotheses are supported by the fact that dendritic cell immunogenicity is regulated by PPAR-γ CLA dietary supplementation with CLA increases the intestinal immune defenses of Wistar rats during the first stages of life (suckling and extended to early infancy) (61). CLA-dependent enhancement of humoral mucosal immune response was demonstrated by the striking increase of intestinal IgA expression in 28-day-old rats fed CLA for 4 weeks during early life. The effects of CLA are more pronounced the earlier and more long-lasting CLA dietary supplementation.
Secondly CLA may modify mediators of immunity such as eicosanoids, prostaglandins, cytokines, and immunoglobulins (51, 80-81). CLA may act in part by competing with linoleic acid in the biosynthesis of arachidonic acid (82). Cook et al (52) showed that feeding chicks a diet containing 0.5% CLA significantly reduced the level of arachidonic acid. Belury and Kempa-Steczko (83) also demonstrated that feeding rats 0.5, 1.0 and 1.5% CLA resulted in decreased arachidonic acid levels Arachidonic acid is the precursor for PGE2 thus, increased CLA intake may decrease PGE2 production and suppressive effect on IL-2 production and T cell proliferation (68, 84). In- vitro and in- vivo studies in various animal models demonstrate that CLA influences cytokine and prostaglandin production, which could influence the inflammatory response (80).
In experimental animals, CLA has been shown to modulate the immune system and prevent immune-induced wasting (51) as well as cancer-induced wasting (85). Some studies in humans suggest a potentially beneficial effect of CLA on immune function (81). A randomized controlled trial in 28 healthy adults showed that 3g/day of CLA (mixed isomer) fed for 12 weeks, improved cell mediated immunity which allows the body to resist viruses, bacteria, fungi, and tumors (59). Improvements in other markers of immune function with CLA supplementation indicated that CLA was capable of reducing inflammatory responses as well as allergic reactions (59). A primary mechanism for immune-modulation is the multiple antioxidant capability which can reduce the deleterious effects of reactive oxygen species and free radicals, leads to premature death of immune cells (86).CLA can enhance intracellular enzyme concentration and consequently to enhance immune system.
CLA also has the ability to increase total GSH (GSH+GSSG) amount and gamma-glutamylcysteine ligase (gammaGCL) protein expression and is associated with the inhibition of typical pathological signs in mice. Glutathione (GSH) is often referred to as the body’s master antioxidant, synthesized endogenously all throughout the body and can be found in virtually every cell of the human body. Glutathione is also important in detoxification of electrophilic xenobiotics, modulation of redox regulated signal transduction, storage and transport of cysteine, regulation of cell proliferation, synthesis of deoxyribonucleotide synthesis, regulation of immune responses, and regulation of leukotriene and prostaglandin metabolism (87). Bergamo, et al (88) studied the effect of CLA on antioxidant status of pregnant mice. Significantly higher total GSH and Trolox equivalent antioxidant capacity (TEAC) levels were measured in serum of CLA-treated dams (and their pups), as compared with controls. GSH homeostasis plays a key role in the treatment of diseases in which cytokines are major participants in their pathophysiology (89).
MATERIALS AND METHODS
Deoxyuridine triphosphates labelled with various Cy3 and Cy5 fluorophore analogues
The chemical structures and optical properties of eight deoxyuridine triphosphates labelled with analogues of Cy3 and Cy5 fluorophores are summarized in Table Table1. 1 . The methods used in the syntheses were partially described previously (17). Detailed synthetic procedures and spectroscopic characteristics, including data on the intermediates, are provided in the Supplementary Data (Part A: Reaction Schemes of Modified Nucleoside Triphosphates and Part B: Procedures and Spectroscopic Data). Amersham Cy3-dUTP and Amersham Cy5-dUTP were purchased from GE Healthcare (Little Chalfont, UK).
Oligodeoxyribonucleotides, PCR, primer extension and extra nucleotide addition to the 3′ ends of DNA fragments
The sequences of oligonucleotides listed in Supplementary Table S1 were selected to reduce the formation of hairpin structures. Possible hairpin structures were calculated using the DI-nucleic acid hybridization and melting prediction web server (http://unafold.rna.albany.edu). Detailed synthetic and analytical procedures are provided in the Supplementary data (Part C: Oligodeoxyribonucleotides, Supplementary Tables S1 and 2).
Detailed descriptions of PCRs, primer extension reactions and adding extra nucleotides to the 3′ ends of DNA fragments are in the Supplementary Data (Part D: PCR, primer extension and extra nucleotide addition to the 3′ ends of DNA fragments).
Electrophoresis and gel image acquisition
Before being loaded on denaturing 20% polyacrylamide gels for electrophoresis, the samples were desalted and purified from an excess of dNTPs and fluorescently labelled dUTPs on a Sephadex G25 Superfine gel filtration column (Pharmacia, Sweden volume 1 ml, length 50 mm). The column was washed with Milli-Q water using a peristaltic pump at a speed of 1 mm 3 per second. Sample elution was monitored with a Pharmacia Dual Path Monitor UV2 double-beam sensor (Pharmacia, Sweden). The eluted samples were reprecipitated in 10 volumes of 2% LiClO4 in acetone, diluted in 5 μl of 7 М urea and then loaded into gel wells.
Electrophoresis was carried out in denaturing 20% polyacrylamide gels (19:1 (w/w) acrylamide/bis-acrylamide, 7 M urea 600 V thermostabilized 15 × 15 cm glass sandwich with 1-mm gel thickness TBE buffer (89 mM Tris-borate and 2 mM ethylenediaminetetraacetic acid, pH 8.3)).
After electrophoresis, gel images were obtained using a research fluorescence Gel Imager with an image field of 15 × 15 cm equipped with an RTE/CCD-1536-K/1 CCD camera (Roper Scientific, Sarasota, FL, USA) and a mercury lamp. Fluorescence was recorded in the range of the Cy3 dye using a 535DF35 and 580DF27 filter pair, in the range of the Cy5 dye using a 630DF30 and 690DF40 filter pair, and in the range of the fluorescein isothiocyanate (FITC) dye using a 470DF40 and 535DF35 filter pair. In the broad spectral range of Cy3 and Cy5 dyes, fluorescence was recorded using a 535DF35 and 690DF40 filter pair. Additionally, the visualization of oligonucleotide bands in polyacrylamide gels was performed using an aluminium sheet coated with TLC Silica gel 60 F254 (Merck KGaA, Darmstadt, Germany) as a gel support, a TCP-20 LC transilluminator with an excitation wavelength of 254 nm (Vilber Lourmat, Marne-la-Vallພ, France) as a UV source and a 470DF40 filter for observation. All filters were purchased from Omega Optical, Brattleboro, VT, USA. The microscope was equipped with a computer running ImaGel Research software (18,19).
Quantitative analysis of electrophoretic bands containing PCR-amplified full-length DNA fragments
The relative quantities of PCR-amplified full-length DNA fragments containing dUMPs labelled with Cy5 dye analogues were considered proportional to the relative fluorescence intensities of the Cy3-labelled primers. These quantities were calculated separately for each of lanes 3𠄸 in Figure Figure1A (Results 1A (Results and Discussion section). For this purpose, the summed Cy3 fluorescence intensities of the upper bands in each of lanes 3𠄸 in Figure Figure1A 1A (relative to the level of the doublet band in lane 2) that arose due to the incorporation of dUMPs labelled with the rather heavy Cy5 dye analogues were quantified using virtual rectangular frames that surrounded the upper bands. The fluorescence intensities of all the pixels surrounded by the frame were summed to obtain the fluorescence intensity within the frame. The fluorescence intensities of the blank gel regions within the same frames were then subtracted from the obtained values. The obtained quantities were normalized to the fluorescence intensity of the full-length DNA in lane 2 in Figure Figure1A, 1A , which contained only natural nucleotides.
Effect of the charge of dUTP-conjugated Cy3 and Cy5 analogues on PCR performed by Taq polymerase using a 68-nt M0 template that contained varying numbers of consecutive adenine nucleotides: a single A, an AA doublet and an AAA triplet (Supplementary Table S1). (A and B) Electrophoretic separation of PCR products obtained in the presence of dNTPs and 5% fluorescently labelled dUTPs. (Table (Table1, 1 , Supplementary Schemes S1𠄳 and Table S2). The experiment shown in A and B was performed two times. Lane 1: P1-(Cy3a−) and P2-(Cy3a−) primers lane 2: PCR products prepared using natural nucleoside triphosphates and P1-(Cy3a−) and P2-(Cy3a−) primers lanes 3𠄸: PCR products obtained after the addition of 5% dU(Cy5a+)TP, dU(Cy5a1±)TP, dU(Cy5a2±)TP, dU(Cy5a3±)TP, dU(Cy5a−)TP or Amersham Cy5-dUTP, respectively. Lane 9: PCR products prepared using natural nucleoside triphosphates and P1-(Cy5a−) and P2-(Cy5a−) primers lanes 10: PCR products obtained after the addition of 5% dU(Cy3a+)TP, dU(Cy3a1±)TP, dU(Cy3a−)TP or Amersham Cy3-dUTP, respectively. Lane 14: P1-(Cy5a−) and P2-(Cy5a−) primers. (C) Histogram of the relative quantities (grey columns) of PCR-amplified full-length DNA fragments containing fluorescently labelled dUMPs and normalized fluorescence signals characterizing the brightness of these incorporated dUMPs (see also Supplementary Table S3). The brightness of incorporated dUMPs in the Cy3 and Cy5 ranges are shown in blue and orange, respectively. The values averaged over the two experiments are shown. The bars indicate the absolute deviations.
The same quantitative analysis was performed for lanes 9 in the Cy5 fluorescence range (Figure (Figure1B) 1B ) to separately estimate the relative quantities of synthesized full-length DNA fragments containing dUMPs labelled with Cy3 dye analogues. The corresponding values characterizing the efficiency of the enzymatic incorporation of dUMPs labelled with Cy3 or Cy5 dye analogues are shown in Supplementary Table S3 and are also plotted in the histogram in Figure Figure1C 1C (grey columns).
Additionally, the normalized fluorescence intensities characterizing the brightness of incorporated dUMPs labelled with Cy3 and Cy5 dye analogues were calculated in the Cy3 (lanes 10, Figure Figure1A) 1A ) and Cy5 (lanes 3𠄸, Figure Figure1B) 1B ) fluorescence ranges, respectively. For this purpose, the Cy3 fluorescence intensities of the bands containing full-length DNA products in each of lanes 10 (Figure (Figure1A) 1A ) were summarized using the virtual rectangular frames surrounding the bands (the fluorescence intensities of the blank gel regions within the same frames were subtracted from the obtained values). In a similar manner, the summed Cy5 fluorescence intensities of the bands containing full-length DNA products in each of lanes 3𠄸 (Figure (Figure1B) 1B ) were calculated. The obtained two sets of fluorescence intensities were normalized to their maximum values (separately for the Cy3 and Cy5 fluorescence ranges) and plotted in the histogram shown in Figure Figure1C 1C (blue and orange columns, respectively).
Quantitative analysis of electrophoretic bands containing full-length DNA fragments obtained during primer extension
To estimate the relative quantities of the full-length products of primer extensions in Figures Figures2 2 and 3 and Supplementary Figure S1, we calculated the fluorescence intensity of each analysed band in the fluorescence range of the labelled primer using a virtual rectangular frame surrounding the band (Results and Discussion section). Fluorescence intensities of all the pixels surrounded by the frame were summed to obtain the fluorescence intensity within the frame. The fluorescence intensity of a blank gel region within the same frame was then subtracted from the obtained value. The obtained fluorescence signals of the analysed bands within each gel image were normalized to the fluorescence intensity of the band containing the full-length natural product of primer extension. Within each gel image in Supplementary Figure S14, the results were normalized to the full-length natural product of primer extension obtained in the presence of dATP, dGTP and dCTP.
Incorporation of deoxyuridines labelled with Cy3 fluorophore analogues carrying positive, neutral or negative charges by Taq polymerase in a P1-(Cy5a−) primer extension reaction using the M1 template. (A) Primer extension reaction scheme. (B and C) Electrophoretic separation of primer extension products obtained in the presence of dTTP, dU(Cy3a+)TP, dU(Cy3a1±)TP and dU(Cy3a−)TP. The experiment shown in B and C was performed two times. Lane 1: P1-(Cy5a−) primer lane 2: reaction products prepared using natural nucleoside triphosphates. Lanes 3𠄶, 7 and 11: reaction products prepared using dATP, dCTP, dGTP, increasing amounts of modified dUTPs (10, 50, 90 and 100%) and the corresponding decreasing amounts of dTTP (90, 50, 10 and 0%). Lanes 3𠄶: dU(Cy3a+)TP, lanes 7: dU(Cy3a1±)TP and lanes 11: dU(Cy3a−)TP.
Incorporation of deoxyuridines labelled with Cy5 fluorophore analogues carrying positive, neutral or negative charges by Taq polymerase in a P1-(Cy3a−) primer extension reaction using the M1 template. (A) Primer extension reaction scheme. (B and C) Electrophoretic separation of primer extension products obtained in the presence of dTTP, dU(Cy5a+)MP, dU(Cy5a1±)MP and dU(Cy5a−)MP. The experiment shown in B and C was performed two times. Lane 1: P1-(Cy3a−) primer lane 2: reaction products prepared using natural nucleoside triphosphates. Lanes 3𠄶, 7 and 11: reaction products prepared using dATP, dCTP, dGTP, increasing amounts of modified dUTPs (10, 50, 90 and 100%) and the corresponding decreasing amounts of dTTP (90, 50, 10 and 0%). Lanes 3𠄶: dU(Cy5a+)TP, lanes 7: dU(Cy5a1±)TP and lanes 11: dU(Cy5a−)TP. (D and E) Electrophoretic separation of primer extension products obtained in the presence of dTTP, dU(Cy5a1±)TP and Amersham Cy5-dUMP. The experiment shown in D and E was performed two times. Lane 1: P1-(Cy3a−) primer lane 2: reaction products prepared using natural nucleoside triphosphates. Lanes 3𠄶 and 7: reaction products prepared using dATP, dCTP, dGTP, increasing amounts of modified dUTPs (10, 50, 90 and 100%) and the corresponding decreasing amounts of dTTP (90, 50, 10 and 0%). Lanes 3𠄶: dU(Cy5a1±)TP and lanes 7: Amersham Cy5-dUTP.
Within each gel image in each of the remaining quantitatively analysed figures (Figure (Figure6 6 and Supplementary Figures S2, 6, 8, 9 and 11), the fluorescence signals were obtained in the same way and normalized to the maximum value.
Determining the Michaelis–Menten constants (Km) and the maximum substrate incorporation rates (Vmax) for dTMP and dU(Cy5a1±)MP incorporation by Taq polymerase. (A–C) Primer extension reaction schemes. (D–I) Electrophoretic separation of the reaction products. Lane 1: P1-(Cy3a−) primer. Lane 2: products of a 40-min reaction in the presence of dATP, dCTP and dGTP (5 × 10 𢄤 M each) 5 × 10 𢄦 M M2 (D and G), M11 (E and H) or M12 (F and I) template 5 × 10 𢄦 M P1-(Cy3a−) primer and 1.5 units of Taq polymerase. D and G: lanes 3, 5, 7, 9, 11, 13 and 15—the same mixture as for lane 2 with an increasing concentration of dTTP: 5 × 10 𢄨 , 5 × 10 𢄧 , 10 𢄦 , 5 × 10 𢄦 , 2.5 × 10 𢄥 , 5 × 10 𢄥 and 5 × 10 𢄤 M, respectively. E, F, H and I: lanes 3, 5, 7, 9, 11 and 13—the same mixture as for lane 2 with an increasing concentration of dTTP: 5 × 10 𢄨 , 5 × 10 𢄧 , 10 𢄦 , 5 × 10 𢄦 , 5 × 10 𢄥 and 5 × 10 𢄤 M, respectively. D and G: lanes 4, 6, 8, 10, 12, 14 and 16—the same mixture as for lane 2 with an increasing concentration of dU(Cy5a1±)TP: 5 × 10 𢄨 , 5 × 10 𢄧 , 10 𢄦 , 5 × 10 𢄦 , 2.5 × 10 𢄥 , 5 × 10 𢄥 and 5 × 10 𢄤 M, respectively. E, F, H and I: lanes 4, 6, 8, 10, 12 and 14—the same mixture as for lane 2 with an increasing concentration of dU(Cy5a1±)TP: 5 × 10 𢄨 , 5 × 10 𢄧 , 10 𢄦 , 5 × 10 𢄦 , 5 × 10 𢄥 and 5 × 10 𢄤 M, respectively. Lane 17 in D and lane 15 in E and F contained the reaction products obtained in 2-h primer extension reactions in the presence of dATP, dCTP, dGTP and dTTP (5 × 10 𢄤 M each) 5 × 10 𢄦 M M2 (D), M11 (E) or M12 (F) template 5 × 10 𢄦 M P1-(Cy3a−) primer and 15 units of Taq polymerase. The experiments shown in the figure were performed two times. A quantitative analysis of the gel images shown in the figure is presented in Figure Figure7 7 .
The fluorescence intensities characterizing the brightness of incorporated dUMPs labelled with Cy3 and Cy5 dye analogues were also similarly calculated in the Cy3 and Cy5 fluorescence ranges, respectively. Values were normalized to the maximum value.
The details of mass spectroscopic analysis are provided in the Supplementary data (Part E: Mass spectrometry).
Michaelis–Menten kinetics measurements
When measuring the kinetic parameters, including the Michaelis–Menten constant (Km), maximum substrate incorporation rate (Vmax), and substrate incorporation rate at [S] = 5 × 10 𢄤 M (V([S] = 5 × 10 𢄤 M)), 1.5 units of Taq polymerase were added to the primer extension reaction mixtures. The dTTP, dU(Cy5a1±)TP, dU(Cy5a2±)TP, dU(Cy5a3±)TP, dU(Cy5a+)TP and dU(Cy5a−)TP concentrations were varied from 5 × 10 𢄨 M to 5 × 10 𢄤 M, and the reactions proceeded for 40 min (Figures (Figures6 6 and 7 Supplementary Figures S9). The other conditions were the same as described in the Primer extension and extra nucleotide addition to the 3′ ends of DNA fragments subsection of the Supplementary data (Part D: PCR, primer extension and extra nucleotide addition to the 3′ ends of DNA fragments).
Determination of Km and Vmax values characterizing the incorporation of dTMP and dU(Cy5a1±)MP by Taq polymerase. (A–C) P1-(Cy3a−) primer extension reaction schemes. (D–F) Plots of the incorporation rates, d[P]/dt, for one (D), two (E) and three (F) consecutively incorporated dTMP(s) (black circles and curves) as a function of dTTP concentration. (G–I) Plots of d[P]/dt for one (G), two (H) and three (I) consecutively incorporated dU(Cy5a1±)MP(s) (orange circles and curves) as a function of dU(Cy5a1±)TP concentration. The values averaged over the two experiments are shown. The bars indicate absolute deviations.
Each electrophoretic gel included a single lane containing the product of a control reaction. The control reaction mixture contained 15 units of Taq polymerase and 5 × 10 𢄤 M dTTP. In this case, the primer extension reaction proceeded for 2 h, until the entire primer was extended. The fluorescence signals from other full-length reaction products on the gel image were normalized to the fluorescence signal from abovementioned control reaction product.
Plots of the product synthesis rate, d[P]/dt, against substrate concentration, [S], were approximated by curves according to the Michaelis–Menten equation (20):
where Vmax is the maximum substrate incorporation rate and Km is the Michaelis–Menten constant. The approximation was performed using OriginPro 8.6 software (OriginLab Corp., Northampton, MA, USA), which provided estimations of the parameters Vmax and Km and the coefficient of determination, R 2 .
For dU(Cy5a1±)TP, dU(Cy5a2±)TP, dU(Cy5a3±)TP, dU(Cy5a+)TP and dU(Cy5a−)TP, the d[P]/dt values at a substrate concentration of 5 × 10 𢄤 M were five to ten times smaller than those obtained at substrate concentrations of 5 × 10 𢄦 − 5 × 10 𢄥 M. Therefore, 5 × 10 𢄤 M fluorescently labelled dUTPs greatly inhibited the reactions and the corresponding points were not considered in the approximations.
UTP also has the role of a source of energy or an activator of substrates in metabolic reactions, like that of ATP, but more specific. When UTP activates a substrate (like Glucose-1-phosphate), UDP-glucose is formed and inorganic phosphate is released.  UDP-glucose enters the synthesis of glycogen. UTP is used in the metabolism of galactose, where the activated form UDP-galactose is converted to UDP-glucose. UDP-glucuronate is used to conjugate bilirubin to a more water-soluble bilirubin diglucuronide. UTP is also used to activate amino sugars like Glucosamine-1-phosphate to UDP-glucosamine, and N-acetyl-glucosamine-1-phosphate to UDP-N-acetylglucosamine. 
UTP also has roles in mediating responses by extracellular binding to the P2Y receptors of cells. UTP and its derivatives are still being investigated for their applications in human medicine.
- ^ Meisenberg, Gerhard Meisenberg (2017). Principles of Medical Biochemistry. Philadelphia, PA, USA: Elsevier. p. 505. ISBN978-0-323-29616-8 .
- Victor, Rodwell (2015). Haper's illustrated Biochemistry. USA: McGraw-Hill. p. 118. ISBN978-0-07-182537-5 .
- Meisenberg, Gerhard Meisenberg (2017). Principles of MEDICAL BIOCHEMISTRY, 4th edition. Philadelphia, PA, USA: Elsevier. p. 59. ISBN978-0-323-29616-8 .
- ^ ab
- Voet, Donald (2011). Biochemistry, 4th edition. USA: JOHN WILEY & SONS, INC. p. 645. ISBN978-0470-57095-1 .
- Rodwell, Victor (2015). Harper's illustrated Biochemistry. USA: McGraw-Hill. p. 176. ISBN978-0-07-182537-5 .
- Rodwell, Victor (2015). Harper's illustrated biochemistry, 13th edition. USA: McGraw-Hill. p. 204. ISBN978-0-07-182537-5 .
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Monday, 19 April 2021
STPM Biology Biological Molecules Part 20 Osmotic, Turgor, Wall Pressure and Water Potential
- When a solution is separated from pure water by semi-permeable membrane , there will be net water moving across into the solution.
- The minimum pressure that has to be exerted by the solution to prevent water from moving in is called the osmotic pressure of the solution.
|Osmotic pressure examples|
- Turgor pressure = the pressure of cytoplasm exerted against the walls of a turgid cell.
- This pressure is counteracted by the wall pressure.
- Wall pressure = the pressure of the cell wall exerted against the cytoplasm of the plant cell.
- The wall pressure is also known as pressure potential (Ψ p) for plant cells.
- Pressure potential usually has a positive value.
|The relationship between turgor pressure and wall pressure|
- Water potential = the potential of water to move out of a solution by osmosis.
- The water potential of a cell is the potential of water to move out of a cell through osmosis.
- Symbol = (Ψ) Unit = kPa (kiloPascal 1kPa = 1000Pa) or MPa (MegaPascal), 1MPa = 100,000Pa)
- Pure water has the highest water potential . The water potential of pure water is 0 kPa at atmosphere pressure (101325 kPa).
- The water potential of a plant cell (Ψ) = solute potential (Ψ s) + pressure potential (Ψ p)
|The water movement from a dilute to a concentrated solution|
- Solute potential = the potential of a solution to take in water by osmosis due to the presence of solute materials.
- Solute potential is also known as osmotic potential .
Water Potential for Solution (Ψ sol)
- (Ψ sol) = the potential of water to move out of a solution by osmosis .
- The water potential of a solution is negative in value.
- This is because water potential for pure water is 0 kPa and pure water has the highest water potential.
- Solution with larger negative water potential value have low water potential.
- For example, cell A with water potential of -0.5 kPa has higher water potential than cell B with water potential value of -0.9 kPa. Thus, water will flow from A to B.
|The movement of water through different types of solution|
STPM Biology Biological Molecules Part 19 Mineral Ions and Vitamins
- Mineral ions and vitamins are generally needed in minute amounts.
- Lack of them in diet can lead to a variety of disorders.
- The importance of mineral ions and vitamins are shown in the tables below.
SPM Biology 4 Chemical Composition of the Cell Part 4 Organic Compounds in the Cell - Lipids
1. Contain carbon, hydrogen, oxygen .
2. Proportion of oxygen is lower than in carbohydrates. For example: stearic acid C18H36O2 .
3. Insoluble in water (non-polar molecule), but dissolve in other lipids and non-polar solvents (ether, ethanol, etc.).
4. Four main types of lipids:
- Store large amount of energy
- Sources of energy
- A major part of the structure of cell membranes
Fats and Oils (triglycerides)
1. Fats are solid at room temperature (20ºC).
3. Triglyceride is formed from a condensation reaction between 1 molecule of glycerol and 3 molecules of fatty acids. The bonds formed are called ester bonds .
|Formation of triglyceride|
4. Fats often contain only saturated fatty acid (single bond).
5. Oils usually contain unsaturated fatty acid (double bond).
6. Importance of fats and oil:
- Function as energy reserve & storage materials. They provide 38kJ per gram, while carbohydrates provide only 17kJ per gram.
- Fats act as an insulator against the loss of heat.
|Similarities and differences of saturated fat and unsaturated fat|
1. Similar to triglycerides.
2. Produced by both plants & animals.
3. Usually hard solids at room temperature.
- Used to waterproof the external surfaces of plants & animals. E.g: cuticle of leaf, protective covering on an insect’s body.
- Also a constituent of the honeycomb of bees.
- Major component of plasma membranes
- Made up of 1 glycerol, 2 fatty acid and 1 phosphate
1. Complex ring structure . Do not contain fatty acids .
2. Occur in plants and animals.
MATERIALS AND METHODS
Polθ 3′ terminal extension activity
An amount of 200 nM Polθ (or other concentrations as indicated) was incubated with 50 nM of the indicated 5′ 32 P-labeled RNA or ssDNA at 37° (or other temperatures as noted) in the presence of 20 mM Tris–HCl pH 8.2, 0.01% NP-40, 0.1 mg/ml bovine serum albumin (BSA) and 10% glycerol. Figure Figure1A 1A and B contained either 2 mM MnCl2 or 2 mM MgCl2 as indicated. All other reactions contained 2 mM MnCl2. Twenty units of Ambion ™ RNase Inhibitor (Thermo-Scientific) was added to reactions containing RNA. An amount of 500 μM of either ribonucleoside triphosphates (NTPs) or deoxyribonucleoside triphosphates (dNTPs) (or individual dNTP or NTPs as noted) was present in reactions as indicated. Nucleotide analogs were added at 100 µM. Reactions were terminated after 60 min (unless otherwise noted) by the addition of 25 mM ethylenediaminetetraacetic acid (EDTA) and 45% formamide and were resolved by electrophoresis in 15% urea polyacrylamide gels, then visualized by phosphorimager or autoradiography. Percentage extension was determined by dividing the sum of the intensities of the extended product bands by the sum of the intensities of the extended and unextended bands. ImageJ was used to determine the intensities of individual bands.
Comparison of Polθ and TdT RNA 3′ terminal extension activities. (A and B) Denaturing gels showing Polθ extension activity on DNA (A) and RNA (B) in the presence of the indicated dNTPs, NTPs and divalent cations. (C) Denaturing gels showing Polθ RNA 3′ terminal extension activity on the indicated substrates in the presence of Mn 2+ and dNTPs or NTPs. (D) Denaturing gel showing lack of TdT RNA terminal nucleotidyl transferase activity in the presence of Co 2+ and the indicated substrates (left). Denaturing gel showing TdT DNA terminal nucleotidyl transferase activity in the presence of Co 2+ and the indicated substrate (right). (E) Denaturing gels showing a time course of Polθ extension activity on the indicated DNA substrate in the presence of dNTPs (lanes 2𠄵) and NTPs (lanes 6𠄹). (F) Denaturing gels showing a time course of Polθ extension activity on the indicated DNA and RNA substrates in the presence of dNTPs. (G and H) Denaturing gels showing a time course of Polθ extension activity on the indicated RNA substrate in the presence of dNTPs with and without excess unlabeled RNA trap added 30 s after reactions were initiated.
Polθ 3′ terminal extension processivity assays
Figure Figure1G: 1G : Reactions were performed with 200 nM Polθ at 37° in the presence of 50 nM of the indicated 5′ 32 P-labeled RNA, 2 mM MnCl2, 500 µM dNTPs, 20 mM Tris–HCl pH 8.2, 0.01% NP-40, 0.1 mg/ml BSA, 10% glycerol and 20 units of AmbionTM RNase Inhibitor (Thermo-Scientific). Cold trap (7.5 µM 454R) was added 30 s later. Reactions were terminated at the indicated time points by the addition of 25 mM EDTA and 45% formamide and were resolved by electrophoresis in 15% urea polyacrylamide gels, then visualized by phosphorimager or autoradiography.
Figure Figure1H: 1H : An amount of 2 nM Polθ was incubated with 100 nM of the indicated 5′ 32 P-labeled RNA at 37° in the presence of 2 mM MnCl2, 20 mM Tris-HCl, pH 8.2, 0.01% NP-40, 0.1 mg/ml BSA, 10% glycerol and 20 units of AmbionTM RNase Inhibitor (Thermo-Scientific). Reactions were initiated with 500 µM dNTPs. Thirty seconds later, 10 µM cold trap (454R) was added to the reaction. Reactions were terminated at the indicated time points by the addition of 25 mM EDTA and 45% formamide and were resolved by electrophoresis in 15% urea polyacrylamide gels, then visualized by phosphorimager or autoradiography.
TdT terminal transferase activity
An amount of 200 nM of TdT was incubated with 50 nM of the indicated 5′ 32 P-labeled RNA or ssDNA in conditions recommended by New England Biolabs (50 mM potassium acetate, 20 mM Tris acetate, 10 mM magnesium acetate, pH 7.9, with 0.25 mM cobalt) at 37ଌ. Twenty units of Ambion™ RNase Inhibitor (Thermo-Scientific) was added to reactions containing RNA. NTPs or dNTPs were present at 500 μM and reactions were incubated for 1 h. Reactions were terminated and processed as above.
Polθ was purified as described using a newly generated SUMOstar (Life Sensors) expression vector and SUMOstar protease I (Life Sensors) (9). TdT was obtained from New England Biolabs (NEB).
Nucleic acids (5′-3′) (Integrated DNA Technologies)
RNA and DNA were 32 P 5′-labeled using 32 P-γ-adenosine triphosphate (ATP) (Perkin Elmer) and bacteriophage T4 polynucleotide kinase (New England Biolabs).
Caspase-dependent Proteolysis of Human Ribonucleotide Reductase Small Subunits R2 and p53R2 during Apoptosis
Ribonucleotide reductase (RnR) is a key enzyme synthesizing deoxyribonucleotides for DNA replication and repair. In mammals, the R1 catalytic subunit forms an active complex with either one of the two small subunits R2 and p53R2. Expression of R2 is S phase-specific and required for DNA replication. The p53R2 protein is expressed throughout the cell cycle and in quiescent cells where it provides dNTPs for mitochondrial DNA synthesis. Participation of R2 and p53R2 in DNA repair has also been suggested. In this study, we investigated the fate of the RnR subunits during apoptosis. The p53R2 protein was cleaved in a caspase-dependent manner in K-562 cells treated with inhibitors of the Bcr-Abl oncogenic kinase and in HeLa 229 cells incubated with TNF-α and cycloheximide. The cleavage site was mapped between Asp(342) and Asn(343). Caspase attack released a C-terminal p53R2 peptide of nine residues containing the conserved heptapeptide essential for R1 binding. As a consequence, the cleaved p53R2 protein was inactive. In vitro, purified caspase-3 and -8 could release the C-terminal tail of p53R2. Knocking down these caspases, but not caspase-2, -7, and -10, also inhibited p53R2 cleavage in cells committed to die via the extrinsic death receptor pathway. The R2 subunit was subjected to caspase- and proteasome-dependent proteolysis, which was prevented by siRNA targeting caspase-8. Knocking down caspase-3 was ineffective. Protein R1 was not subjected to degradation. Adding deoxyribonucleosides to restore dNTP pools transiently protected cells from apoptosis. These data identify RnR activity as a prosurvival function inactivated by proteolysis during apoptosis.
Keywords: apoptosis caspase nucleoside/nucleotide biosynthesis protein degradation ribonucleotide reductase.
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