Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.1.3.1 (alkaline phosphatase)
47,916 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

RNA ligase in eukaryotic mammalian cells was studied by using mouse brain and liver cell extracts as enzyme sources and Oligo A as substrates. RNA ligase activity was determined by measuring the formation of alkaline phosphate-resistant product from 5'-32P-terminated Oligoribonucleotides. Under appropriate conditions, the activity of this enzyme in brain and liver cells may vary between 16-49 mU/ml. The joining way between donor and acceptor is 5'-P----3'-OH. Further studies were carried out by using synthetic UpCpU and 32pNp as substrates and crude enzyme preparations from extracts of cell nuclei of brain and liver as enzyme sources. RNA ligase activity was examined by homochromatography and autoradiography. A clear joining product was demonstrated and then isolated from the reaction mixture by DEAE-Sephadex A25 column chromatography. The eluted fractions were identified by DEAE-cellulose thin layer chromatography. The joining product was hydrolyzed either with KOH or with alkaline phosphatase, the autoradiographic spot of the product disappeared. In this case the joining way between donor and acceptor is 3'-P----5'-OH instead of 5'P----3'-OH. All this indicated that in extracts of mouse brain and liver cells most probably exists some other kind of RNA ligase, which differs from the T4 RNA ligase in the joining way.
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PMID:Studies on RNA ligase activity in the brain and liver cells of mouse. 654 94

The 5'-terminal sequence of hen ovalbumin mRNA was investigated using a novel labeling method. Ovalbumin mRNA was purified by hybridization to complementary DNA coupled to cellulose. The mRNA thus purified was shown to be 97.9% pure by hybridization with plasmid DNA containing sequences to the messengers coding for conalbumin and ovomucoid, the next two most abundant messengers of oviduct. After digestion with RNase T1 and alkaline phosphatase, 5'-terminal capped oligonucleotides were selected by binding to anti-m7G-Sepharose. These were then labeled using RNA ligase and [5'-32P]pCp, separated by two-dimensional gel electrophoresis, and sequenced by partial digestion with base-specific ribonucleases. A nested set of three capped oligonucleotides was identified. Their structures and relative abundances were m7GpppAUACAG, 3% m7GpppACAUACAG, 61+; and m7GpppGUACAUACAG, 36%.
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PMID:Heterogeneity of the 5' terminus of hen ovalbumin messenger ribonucleic acid. 678 28

2'(3')-O-DL-Alanyl (Ip)5I was synthesized by a new method. An alanine ortho ester of inosine 5'-phosphate was added to (Ip)4I using the ATP-independent reaction of T4 RNA ligase, and the product was converted smoothly to the desired ester. The enzymic reverse transfer reaction was conveniently suppressed by the dephosphorylation of the adenosine 5'-phosphate coproduct, catalyzed in situ by alkaline phosphatase.
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PMID:Synthesis of 2'(3')-O-DL-alanyl hexainosinic acid using T4 RNA ligase: suppression of the enzymic reverse transfer reaction by alkaline phosphatase. 685 53

T4 RNA ligase joins a 3'-hydroxyl-terminated acceptor oligoribonucleotide to a 5'-phosphate-terminated donor oligoribonucleotide. An analogous reaction with single-strand DNA oligonucleotides would be useful for the synthesis of defined sequences of DNA because it would eliminate the need to synthesize complementary sequences to form the duplex substrates required by DNA ligase. We have studied the model reaction dA(pdA)5 + [5'-32P] (pdT)4pdCp leads to dA(pdA)5 [3' leads to 5'-32P]pdT(pdT)3pdCp and have obtained 40-60% yields at equimolar concentrations (100 microM to 1 mM) of the two substrates. Higher yields have been obtained when acceptor concentrations in excess of those of the donor are used. The use of a 5'-hydroxyl, 3'-hydroxyl terminated acceptor and a 5'-phosphate, 3'-phosphate terminated donor limits the reaction to a unique product. The 3'-phosphate-terminated donor was prepared by using RNA ligase to add a single deoxyribonucleoside 3',5'-bisphosphate donor to an oligo(deoxyribonucleotide) acceptor [Hinton, D.M., Baez, J.A., & Gumport, R.I. (1978) Biochemistry 17, 5091]. The DNA oligomer joining reaction requires low concentrations of ATP and an ATP regenerating system, Mn2+, high levels of nuclease-free RNA ligase (30 microM), and incubation for several days at 17 degrees C. The product of the reaction was characterized by its resistance to alkaline phosphatase, degradation by micrococcal nuclease to the expected product [3'-32P]dAMP, and mobility during high-pressure liquid chromatography on RPC-5. The joining of several other deoxyoligomers was also demonstrated. We anticipate that this reaction of RNA ligase will contribute to its usefulness as a reagent for the synthesis of DNA of defined sequence.
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PMID:T4 ribonucleic acid ligase joins single-strand oligo(deoxyribonucleotides). 698 3

The deletion of nucleoside 37 from yeast tRNAPhe was accomplished in a three-step procedure that involved (i) specific depurination of this purine under acidic conditions and removal of the carbohydrate moiety with aniline-2-aminopyridine, (ii) removal of phosphate monoesters from the 5'-half-molecule with alkaline phosphatase, and (iii) resealing of the anticodon loop with T4 RNA ligase. The course of the resealing reaction was monitored by polyacrylamide gel electrophoresis and found to be complete within a few hours when incubated at 37 degrees C in the presence of 215 units/ml of RNA ligase. Although the half-molecules used to prepare the modified tRNA had substantial phenylalanine acceptance when assayed at low ionic strength, the modified species itself was essentially devoid of phenylalanine acceptance. We conclude that the anticodon loop of tRNAPhe is involved in the activation of this tRNA and that both the presence of specific nucleotides in this loop and their ability to assume an appropriate spatial or conformational arrangement may be important for enzyme recognition.
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PMID:A structurally modified yeast tRNAPhe with six nucleotides in the anticodon loop lacks significant phenylalanine acceptance. 705 Jan 15

We have devised a method to replace the cap structure of a mRNA with an oligoribonucleotide (r-oligo) to label the 5' end of eukaryotic mRNAs. The method consists of removing the cap with tobacco acid pyrophosphatase (TAP) and ligating r-oligos to decapped mRNAs with T4 RNA ligase. This reaction was made cap-specific by removing 5'-phosphates of non-capped RNAs with alkaline phosphatase prior to TAP treatment. Unlike the conventional methods that label the 5' end of cDNAs, this method specifically labels the capped end of the mRNAs with a synthetic r-oligo prior to first-strand cDNA synthesis. The 5' end of the mRNA was identified quite simply by reverse transcription-polymerase chain reaction (RT-PCR).
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PMID:Oligo-capping: a simple method to replace the cap structure of eukaryotic mRNAs with oligoribonucleotides. 812 98

Several dinucleoside polyphosphates accept cytidine-3', 5'-bisphosphate from the adenylylated donor 5'-adenylylated cytidine 5',3'-bisphosphate in the T4 RNA ligase catalyzed reaction. The 5'-adenylylated cytidine 5',3'-bisphosphate synthesized in a first step, from ATP and cytidine-3',5'-bisphosphate, is used as a substrate to transfer the cytidine-3',5'-bisphosphate residue to the 3'-OH group(s) of diguanosine tetraphosphate (Gp4G) giving rise to Gp4GpCp and pCpGp4GpCp in a ratio of approximately 10 : 1, respectively. The synthesized Gp4GpCp was characterized by treatment with snake venom phosphodiesterase and alkaline phosphatase and analysis (chromatographic position and UV spectra) of the reaction products by HPLC. The apparent Km values measured for Gp4G and 5'-adenylylated cytidine 5',3'-bisphosphate in this reaction were approximately 4 mM and 0.4 mM, respectively. In the presence of 0.5 mM ATP and 0.5 mM cytidine-3',5'-bisphosphate, the relative efficiencies of the following nucleoside(5')oligophospho(5')nucleosides as acceptors of cytidine-3',5'-bisphosphate from 5'-adenylylated cytidine 5', 3'-bisphosphate are indicated in parentheses: Gp4G (100); Gp5G (101); Ap4G (47); Ap4A (39). Gp2G, Gp3G and Xp4X were not substrates of the reaction. Dinucleotides containing two guanines and at least four inner phosphates were the preferred acceptors of cytidine-3', 5'-bisphosphate at their 3'-OH group(s).
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PMID:Several dinucleoside polyphosphates are acceptor substrates in the T4 RNA ligase catalyzed reaction. 1071 2

The genome of Japanese iris necrotic ring virus (JINRV) consists of a positive-sense ssRNA of 4014 nucleotides with six major open reading frames (ORFs). A 5'-non-coding region of 31 nucleotides precedes the first initiation codon. Like Carnation mottle virus (CarMV), the 5'-proximal three ORFs encode a 26 kDa protein (p26) and two readthrough proteins, i.e. an 85 kDa putative RNA replicase (p85) and a 99 kDa protein (p99). The central ORF encodes a small 8 kDa protein (p8). The 3'-proximal ORF encodes a 38 kDa capsid protein (p38). Another ORF encoding a 12 kDa protein (p12) overlaps the p99 ORF.JINRV RNA treated with bacterial alkaline phosphatase and tobacco acid pyrophosphatase could not be ligated to an oligoribonucleotide using T4 RNA ligase, indicating that the 5' end of the viral RNA is uncapped. The 3' end is not polyadenylated. Comparison of the genomic organization and the predicted amino acid sequences with those of other viruses confirmed that JINRV should be classified as a member of the genus Carmovirus, family Tombusviridae.
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PMID:The nucleotide sequence and genome organization of Japanese iris necrotic ring virus, a new species in the genus Carmovirus. 1079 30

ras GTPase activating protein (rasGAP) is highly conserved among mammalian species and is required for normal cardiovascular system development. Expression of this protein exhibits both quantitative and qualitative variability among tissues. Using a combination of DNA sequencing and database analyses, we have determined that the human rasGAP gene spans 122 kb and is composed of 25 exons; the size of each intron and the intron/exon junctions also have been elucidated. With one exception, all intron/exon boundaries conform to the GT/AG rule; the splice donor site of intron 3 is GC/AG. Results of RNA ligase mediated rapid amplification of cDNA ends followed by sequence determination indicate that the transcription start point (TSP) is approximately 588 bp upstream from the translational start site and is uninterrupted by introns; this extremely long 5' untranslated region is continuous with the first coding exon. Analysis of 1 kb of sequence upstream of the TSP did not identify any of the typical promoter elements (TATA or CAAT boxes). Sequential deletions of this 1 kb region followed by secreted alkaline phosphatase reporter gene analysis revealed that transcription is supported by this region of the rasGAP gene. Because the highest efficiency is demonstrated by a 213 bp sequence just upstream from the TSP (-786 to -584), this region is identified as containing the rasGAP minimal promoter. Sequence analysis of this 213 bp sequence shows few candidate sites for transcription factor binding. A 406 bp fragment surrounding the TSP exhibits characteristics of a CpG island (68% C+G; observed/expected ratio of CpG=0.95). RapidScan analysis revealed that high levels of rasGAP transcript are present in placenta and testis, but transcript is not detectable in kidney and intestinal tract. These data suggest that rasGAP transcription is regulated by an atypical mechanism capable of producing quantitative variability among tissue types.
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PMID:Organization and regulation of the human rasGAP gene. 1203 41


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