Selection and characterization of pre-mRNA splicing enhancers: identification of novel SR protein-specific enhancer sequences.
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Splicing enhancers are RNA sequences required for accurate splice site recognition and the control of alternative splicing. In this study, we used an in vitro selection procedure to identify and characterize novel RNA sequences capable of functioning as pre-mRNA splicing enhancers. Randomized 18-nucleotide RNA sequences were inserted downstream from a Drosophila doublesex pre-mRNA enhancer-dependent splicing substrate. Functional splicing enhancers were then selected by multiple rounds of in vitro splicing in nuclear extracts, reverse transcription, and selective PCR amplification of the spliced products. Characterization of the selected splicing enhancers revealed a highly heterogeneous population of sequences, but we identified six classes of recurring degenerate sequence motifs five to seven nucleotides in length including novel splicing enhancer sequence motifs. Analysis of selected splicing enhancer elements and other enhancers in S100 complementation assays led to the identification of individual enhancers capable of being activated by specific serine/arginine (SR)-rich splicing factors (SC35, 9G8, and SF2/ASF). In addition, a potent splicing enhancer sequence isolated in the selection specifically binds a 20-kDa SR protein. This enhancer sequence has a high level of sequence homology with a recently identified RNA-protein adduct that can be immunoprecipitated with an SRp20-specific antibody. We conclude that distinct classes of selected enhancers are activated by specific SR proteins, but there is considerable sequence degeneracy within each class. The results presented here, in conjunction with previous studies, reveal a remarkably broad spectrum of RNA sequences capable of binding specific SR proteins and/or functioning as SR-specific splicing enhancers. (+info)
Regulation of de novo purine biosynthesis in human lymphoblasts. Coordinate control of proximal (rate-determining) steps and the inosinic acid branch point.
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Purine nucleotide synthesis de novo has been studied in a permanent tissue culture line of human splenic lymphoblasts with particular attention to coordination of control of the proximal (rate-determining) steps with the distal branch point of the pathway. An assay was used which permits simultaneous determination of the overall rate of labeling of all intracellular purines with sodium [14C]formate, as well as the distribution of isotope into all intracellular guanine- and adenine-containing compounds. The guanine to adenine labeling ratio was used as an index of IMP branch point regulation. It was found that exogenous adenine and guanine produce feedback-controlling effects not only on the first step in the de novo pathway, but also on the IMP branch point. Concentrations of adenine which produce less than 40% inhibition of the overall rate of de novo purine synthesis do so by selectively inhibiting adenine nucleotide synthesis de novo by 50 to 70% while stimulating guanine nucleotide synthesis de novo by up to 20%. A reciprocal effect is seen with exogenous guanine. The adenosine analog 6-methylmercaptopurine ribonucleoside selectivity inhibits adenine nucleotide synthesis via the de novo pathway but not from exogenous hypoxanthine. Thus, the reactions of purine nucleotide interconversion, in particular adenylosuccinate synthetase, may be regulated differently in cells deriving their purine nucleotides solely from de novo synthesis than when deriving them via "salvage" of preformed hypoxanthine. (+info)
Utilization of exogenous purine compounds in Bacillus cereus. Translocation of the ribose moiety of inosine.
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Intact cells of Bacillus cereus catalyze the breakdown of exogenous AMP to hypoxanthine and ribose 1-phosphate through the successive action of 5'-nucleotidase, adenosine deaminase, and inosine phosphorylase. Inosine hydrolase was not detectable, even in crude extracts. Inosine phosphorylase causes a "translocation" of the ribose moiety (as ribose 1-phosphate) inside the cell, while hypoxanthine remains external. Even though the equilibrium of the phosphorolytic reaction favors nucleoside synthesis, exogenous inosine (as well as adenosine and AMP) is almost quantitatively transformed into external hypoxanthine, since ribose 1-phosphate is readily metabolized inside the cell. Most likely, the translocated ribose 1-phosphate enters the sugar phosphate shunt, via its prior conversion into ribose 5-phosphate, thus supplying the energy required for the subsequent uptake of hypoxanthine in B. cereus. (+info)
In vitro reactions of butadiene monoxide with single- and double-stranded DNA: characterization and quantitation of several purine and pyrimidine adducts.
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We have previously shown that butadiene monoxide (BM), the primary metabolite of 1,3-butadiene, reacted with nucleosides to form alkylation products that exhibited different rates of formation and different stabilities under in vitro physiological conditions. In the present study, BM was reacted with single-stranded (ss) and double-stranded (ds) calf thymus DNA and the alkylation products were characterized after enzymatic hydrolysis of the DNA. The primary products were regioisomeric N-7-guanine adducts. N-3-(2-hydroxy-3-buten-1-yl)adenine and N-3-(1-hydroxy-3-buten-2-yl)adenine, which were depurinated from the DNA more rapidly than the N-7-guanine adducts, were also formed. In addition, N6-(2-hydroxy-3-buten-1-yl)deoxyadenosine and N6-(1-hydroxy-3-buten-2-yl)deoxyadenosine were detected and evidence was obtained that these adducts were formed by Dimroth rearrangement of the corresponding N-1-deoxyadenosine adducts, not while in the DNA, but following the release of the N-1-alkylated nucleosides by enzymatic hydrolysis. N-3-(2-hydroxy-3-buten-1-yl)deoxyuridine adducts, which were apparently formed subsequent to deamination reactions of the corresponding deoxycytidine adducts, were also detected and were stable in the DNA. Adduct formation was linearly dependent upon BM concentration (10-1000 mM), with adduct ratios being similar at the various BM concentrations. At a high BM concentration (750 mM), the adducts were formed in a linear fashion for up to 8 h in both ssDNA and dsDNA. However, the rates of formation of the N-3-deoxyuridine and N6-deoxyadenosine adducts increased 10- to 20-fold in ssDNA versus dsDNA, whereas the N-7-guanine adducts increased only slightly, presumably due to differences in hydrogen bonding in ssDNA versus dsDNA. These results may contribute to a better understanding of the molecular mechanisms of mutagenesis and carcinogenesis of both BM and its parent compound, 1,3-butadiene. (+info)
Hypotension induced by exercise is associated with enhanced release of adenyl purines from aged rat artery.
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To determine whether the antihypertensive effects of exercise are associated with release of ATP and its metabolites from arteries, we assayed blood pressure and the release of adenine nucleotides and nucleosides from the caudal arteries of exercised and sedentary aged hypercholesterolemic rats. Exercise on a treadmill for 12 wk significantly decreased the rise in systolic and diastolic blood pressure by 7.5 and 15.9%, respectively, with advanced age. The concentrations of oleic, linoleic, and linolenic acids in the caudal artery decreased significantly with exercise, demonstrating an association between exercise and the unsaturation index of caudal arterial fatty acids. The amounts of total adenyl purines released by the arterial segments from exercised rats, both spontaneously and in response to norepinephrine, were significantly greater by 80.0 and 60.7%, respectively, than those released by tissues from sedentary rats. These results suggest that exercise alters the membrane fatty acid composition in aged rats as well as the release of ATP from vascular endothelial cells and that these factors are associated with the regression of the rise in blood pressure normally observed with advanced age. (+info)
Pokeweed antiviral protein cleaves double-stranded supercoiled DNA using the same active site required to depurinate rRNA.
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Ribosome-inactivating proteins (RIPs) are N-glycosylases that remove a specific adenine from the sarcin/ricin loop of the large rRNA in a manner analogous to N-glycosylases that are involved in DNA repair. Some RIPs have been reported to remove adenines from single-stranded DNA and cleave double-stranded supercoiled DNA. The molecular basis for the activity of RIPs on double-stranded DNA is not known. Pokeweed antiviral protein (PAP), a single-chain RIP from Phytolacca americana, cleaves supercoiled DNA into relaxed and linear forms. Double-stranded DNA treated with PAP contains apurinic/apyrimidinic (AP) sites due to the removal of adenine. Using an active-site mutant of PAP (PAPx) which does not depurinate rRNA, we present evidence that double-stranded DNA treated with PAPx does not contain AP sites and is not cleaved. These results demonstrate for the first time that PAP cleaves supercoiled double-stranded DNA using the same active site that is required for depurination of rRNA. (+info)
1H-NMR spectroscopy of body fluids: inborn errors of purine and pyrimidine metabolism.
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BACKGROUND: The diagnosis of inborn errors of purine and pyrimidine metabolism is often difficult. We examined the potential of 1H-NMR as a tool in evaluation of patients with these disorders. METHODS: We performed 1H-NMR spectroscopy on 500 and 600 MHz instruments with a standardized sample volume of 500 microL. We studied body fluids from 25 patients with nine inborn errors of purine and pyrimidine metabolism. RESULTS: Characteristic abnormalities could be demonstrated in the 1H-NMR spectra of urine samples of all patients with diseases in the pyrimidine metabolism. In most urine samples from patients with defects in the purine metabolism, the 1H-NMR spectrum pointed to the specific diagnosis in a straightforward manner. The only exception was a urine from a case of adenine phosphoribosyl transferase deficiency in which the accumulating metabolite, 2,8-dihydroxyadenine, was not seen under the operating conditions used. Similarly, uric acid was not measured. We provide the 1H-NMR spectral characteristics of many intermediates in purine and pyrimidine metabolism that may be relevant for future studies in this field. CONCLUSION: The overview of metabolism that is provided by 1H-NMR spectroscopy makes the technique a valuable screening tool in the detection of inborn errors of purine and pyrimidine metabolism. (+info)
Specific contacts between residues in the DNA-binding domain of the TyrR protein and bases in the operator of the tyrP gene of Escherichia coli.
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In the presence of tyrosine, the TyrR protein of Escherichia coli represses the expression of the tyrP gene by binding to the double TyrR boxes which overlap the promoter. Previously, we have carried out methylation, uracil, and ethylation interference experiments and have identified both guanine and thymine bases and phosphates within the TyrR box sequences that are contacted by the TyrR protein (J. S. Hwang, J. Yang, and A. J. Pittard, J. Bacteriol. 179:1051-1058, 1997). In this study, we have used missing contact probing to test the involvement of all of the bases within the tyrP operator in the binding of TyrR. Our results indicate that nearly all the bases within the palindromic arms of the strong and weak boxes are important for the binding of the TyrR protein. Two alanine-substituted mutant TyrR proteins, HA494 and TA495, were purified, and their binding affinities for the tyrP operator were measured by a gel shift assay. HA494 was shown to be completely defective in binding to the tyrP operator in vitro, while, in comparison with wild-Type TyrR, TA495 had only a small reduction in DNA binding. Missing contact probing was performed by using the purified TA495 protein, and the results suggest that T495 makes specific contacts with adenine and thymine bases at the +/-5 positions in the TyrR boxes. (+info)