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1.
RNA ligase has been extensively purified by a new procedure in high yield from T4-infected Escherichia coli. The enzyme consists of a single polypeptide chain of molecular weight 47,000. It catalyzes the formation of a phosphodiester bond between a 5′-PO4-terminated oligonucleotide and a 3′-OH terminated oligonucleotide. The purified enzyme catalyzes both the intramolecular formation of single-stranded circles with longer oligonucleotides of the type pAp(Ap)nA?OH, where n is about 15 or greater and the intermolecular joining of pAp(Ap)3AOH (where the 5′-PO4-terminated oligonucleotide is short enough to prevent apposition of its 3′ and 5′ ends) to UpUpUOH when high concentrations of the 3′-OH-terminated acceptor oligonucleotide are present. Preparations of RNA ligase at all stages of purification show an unusual dependence of specific activity of the enzyme on the concentration of enzyme present in the assay. However, when care is taken to determine meaningful specific activities at each step, the ligase is found to be very stable during chromatography on various ion-exchange columns and may be purified by conventional techniques.  相似文献   

2.
Double digestion of oligonucleotides obtained from ribonuclease T1 or pancreatic ribonuclease A fingerprints results in the following series of products: (Ap)0-nCp, (Ap)0-nUp, and (Ap)0-nGp. A new micromethod is described for the rapid analysis of these digests. The procedure consists of two-dimensional chromatography on a small PEI thin-layer plate. In the first dimension, the oligonucleotides are separated independent of their Ap content into three groups, which represent the Cp-, Gp-, and Up- 3′-terminal oligonucleotide series, respectively. In the second dimension, the products are fractionated according to their chain length. This method, which allows direct identification of even the longer Ap tracts in a double digest of an oligonucleotide or an RNA chain, is very rapid and highly sensitive and can be applied to the simultaneous analysis of a large number of samples in a single run. The procedure has also been adapted to the analysis of pancreatic ribonuclease A digests of small RNA fragments.  相似文献   

3.
A series of peptidyl-tRNA analogs with varying peptide chain length, BrAc(Gly) nPhe-tRNAphe, n = 0 to 16 has been prepared. When bound to Escherichia coli 70 S ribosomes these all react covalently with certain ribosomal proteins. The overwhelming majority of the reaction is with 50 S ribosomal proteins L2, L16, L24, L26–L27 and L32–L33. The extent of reaction with each protein is a function of peptide chain length, making it possible to estimate the relative proximity of these proteins to the 3′-terminus of tRNA bound in the ribosomal P site. This fact, coupled with the findings of others about the length dependence of the binding and peptide donor activity of peptidyl-tRNAs suggests that there is actually a binding site for the growing peptide chain. If this is true, the results presented here permit the ordering of the proteins in this site: L2 is closest to the 3′-end of tRNA followed by L26–L27, L32–L33 and last L24. Evidence is also given that the direction of the growing peptide chain must point away from the A site.  相似文献   

4.
Vicia faba root ribonucleases are bound to Cibacron blue F3GA. A Blue dextran-Sepharose column was used to purify RNase A3, the more abundant enzyme from V. faba root. Using dinucleoside monophosphate as substrates, it appears that this enzyme behaves as a cyclizing phosphotransferase. With high enzyme/substrate ratios on prolonged digestion a partial release of a nucleoside 3′ phosphate occurs. The specificity is relatively high since only the purine-purine phosphodiester linkages out of 16 types of possible links are easily cleaved. When a pyrimidine is involved in the phosphodiester bond, a much slower rate of attack (Py in 5′) or no attack (Py in 3′) was detected.  相似文献   

5.
A rapid method for hemoglobin chain recombination which gives a homogeneous product was developed. The method utilizes a small carboxymethylcellulose column as a medium for chain recombination and concentration of the hemoglobin. Equimolar amounts of p-hydroxymercuribenzoate derivatives of α- and β-chains were mixed with 300× molar excess of β-mercaptoethanol over the p-hydroxy mercuribenzoate groups. After 10 min of incubation in an ice bath, the mixture was adjusted to pH 5.85, and was loaded on a carboxymethylcellulose column. The column was washed with 10 mm phosphate buffer-1 mm Na2EDTA-47 mm β-mercaptoethanol, pH 5.85 and then with 10 mm phosphate buffer, pH 5.85. The hemoglobin was eluted from the column by use of 15 mm K2HPO4. The hemoglobin was homogeneous on polyacrylamide gel electrophoresis and had a visible spectrum, electrophoretic mobility, and number of -SH groups comparable to those shown by control hemoglobin.  相似文献   

6.
In this study, Rv2613c, a protein that is encoded by the open reading frame Rv2613c in Mycobacterium tuberculosis H37Rv, was expressed, purified, and characterized for the first time. The amino acid sequence of Rv2613c contained a histidine triad (HIT) motif consisting of H-phi-H-phi-H-phi-phi, where phi is a hydrophobic amino acid. This motif has been reported to be the characteristic feature of several diadenosine 5′,5′′′-P1,P4-tetraphosphate (Ap4A) hydrolases that catalyze Ap4A to adenosine 5′-triphosphate (ATP) and adenosine monophosphate (AMP) or 2 adenosine 5′-diphosphate (ADP). However, enzymatic activity analyses for Rv2613c revealed that Ap4A was converted to ATP and ADP, but not AMP, indicating that Rv2613c has Ap4A phosphorylase activity rather than Ap4A hydrolase activity. The Ap4A phosphorylase activity has been reported for proteins containing a characteristic H-X-H-X-Q-phi-phi motif. However, no such motif was found in Rv2613c. In addition, the amino acid sequence of Rv2613c was significantly shorter compared to other proteins with Ap4A phosphorylase activity, indicating that the primary structure of Rv2613c differs from those of previously reported Ap4A phosphorylases. Kinetic analysis revealed that the Km values for Ap4A and phosphate were 0.10 and 0.94 mM, respectively. Some enzymatic properties of Rv2613c, such as optimum pH and temperature, and bivalent metal ion requirement, were similar to those of previously reported yeast Ap4A phosphorylases. Unlike yeast Ap4A phosphorylases, Rv2613c did not catalyze the reverse phosphorolysis reaction. Taken together, it is suggested that Rv2613c is a unique protein, which has Ap4A phosphorylase activity with an HIT motif.  相似文献   

7.
8.
Spr1479 from Streptococcus pneumoniae R6 is a 33-kDa hypothetical protein of unknown function. Here, we determined the crystal structures of its apo-form at 1.90 Å and complex forms with inorganic phosphate and AMP at 2.30 and 2.20 Å, respectively. The core structure of Spr1479 adopts a four-layer αββα-sandwich fold, with Fe3+ and Mn2+ coordinated at the binuclear center of the active site (similar to metallophosphoesterases). Enzymatic assays showed that, in addition to phosphodiesterase activity for bis(p-nitrophenyl) phosphate, Spr1479 has hydrolase activity for diadenosine polyphosphate (ApnA) and ATP. Residues that coordinate with the two metals are indispensable for both activities. By contrast, the streptococcus-specific residue Trp-67, which binds to phosphate in the two complex structures, is indispensable for the ATP/ApnA hydrolase activity only. Moreover, the AMP-binding pocket is conserved exclusively in all streptococci. Therefore, we named the protein SapH for streptococcal ATP/ApnA and phosphodiester hydrolase.  相似文献   

9.
《Life sciences》1989,45(24):iii-x
(2′–5′)An-dependent RNase functions as a translational regulatory protein which mediates interferon action. Levels of this enzyme are decreased in barrier-reared Balb/c (+/+), Balb/c (+/nu), and Balb/c (nu/nu) mice when compared to conventionally reared Balb/c (+/+) mice. This suggests that high levels of (2′–5′)An-dependent RNase in conventionally reared mice are maintained by continuous exposure to microbial flora which may induce interferons. Interferon treatment of barrier-reared mice does not, however, result in an increase in (2′–5′)An-dependent RNase levels. This suggests that responsiveness to interferons is decreased in barrier-reared mice. The high levels of (2′–5′)An-dependent RNase which are maintained in normal mice under physiological conditions may be important for rapid and effective defense against viral pathogens.  相似文献   

10.
The cytomolecular discrimination of the Am- and A-genome chromosomes facilitates the selection of wheat-Triticum monococcum introgression lines. Fluorescence in situ hybridisation (FISH) with the commonly used DNA probes Afa family, 18S rDNA and pSc119.2 showed that the more complex hybridisation pattern obtained in T. monococcum relative to bread wheat made it possible to differentiate the Am and A chromosomes within homoeologous groups 1, 4 and 5. In order to provide additional chromosomal landmarks to discriminate the Am and A chromosomes, the microsatellite repeats (GAA)n, (CAG)n, (CAC)n, (AAC)n, (AGG)n and (ACT)n were tested as FISH probes. These showed that T. monococcum chromosomes have fewer, generally weaker, simple sequence repeat (SSR) signals than the A-genome chromosomes of hexaploid wheat. A differential hybridisation pattern was observed on 6Am and 6A chromosomes with all the SSR probes tested except for the (ACT)n probe. The 2Am and 2A chromosomes were differentiated by the signals given by the (GAA)n, (CAG)n and (AAC)n repeats, while only (GAA)n discriminated the chromosomes 3Am and 3A. Chromosomes 7Am and 7A could be differentiated by the lack of (GAA)n and (AGG)n signals on 7A. As potential landmarks for identifying the Am chromosomes, SSR repeats will facilitate the introgression of T. monococcum chromatin into wheat.  相似文献   

11.
3-O-Immobilized and 6-immobilized pyridoxal 5′-phosphate analogs of Sepharose were bound to the allosteric site of nucleoside diphosphatase with very high affinity. Active immobilized nucleoside diphosphatase was prepared by reduction of the Schiff base linkage between the enzyme and pyridoxal 5′-phosphate bound to Sepharose with NaBH4. 3-O-Immobilized pyridoxal 5′-phosphate analog gave more active immobilized enzyme than the 6-analog; the immobilized enzyme on the 3-O-immobilized pyridoxal 5′-phosphate analog showed about 90% of activity of free enzyme. The immobilized enzyme thus prepared was less sensitive to ATP, an allosteric effector, and showed a higher heat stability than the free enzyme. When an assay mixture containing inosine diphosphate and MgCl2 was passed through a column of the immobilized enzyme at 37 °C, inosine diphosphate liberated inorganic phosphate almost quantitatively. Properties of the immobilized enzyme on the pyridoxal 5′-phosphate analog were compared with those of the immobilized enzyme on CNBr-activated Sepharose.  相似文献   

12.
Synthetic and natural allotetraploid Brassica napus (2n?=?38, AACC) have been widely used as a model to study the genetic changes associated with allopolyploidization; however, there has been little research on the homoeolog expression patterns and the roles of cis and trans regulation. Herein, homoeolog expression patterns were assessed by using RNA-seq for two interspecific hybrids (AnCo with the extracted A subgenome from natural B. napus, and ArCo with the A subgenome from extant B. rapa), synthetic and natural allopolyploids (CoCoArAr and AnAnCnCn), and the diploid parents. The ranges of homoeolog expression bias decreased after hybridization, whereas the extents of homoeolog expression bias and non-conserved expression, especially transgressive expression, increased over evolutionary time. Despite sharing the same C subgenome parent, these two hybrids showed different homolog expression patterns in many respects. In AnCo, the trans-regulatory factors from Co subgenome tended to cause downregulation of An subgenome homoeologs, but trans-regulatory factors from the An subgenome acted as both activators and repressors, and such asymmetric effects of trans-regulatory factors might explain why the homoeolog expression was biased toward the C subgenome after genome merger. No significant asymmetric effects of trans-regulatory factors were found in ArCo, which was consistent with the overall balanced expression of homoeologs. These results suggested that A subgenomes with different regulatory systems might act differently in modulating homoeolog expression after merger with the C subgenome, resulting in either balanced or unbalanced homoeolog expression biases.  相似文献   

13.
Large oligonucleotide fragments from tRNA were separated on PEI-cellulose tle using stepwise gradients of increased concentrations of LiCl (containing 0.3 m Tris-HCl and 7.5 m urea at pH 7.9) or Li-formate (containing 7.5 m urea at pH 3.5). These large oligonucleotides, obtained by cleavage of tRNA with nuclease S1, aniline-NaOH, or partial ribonuclease T1 digestion and separated on PEI-cellulose, were analyzed by three different methods. The first method entailed elution and total base analysis by the tritium-postlabeling technique; the second involved complete ribonuclease T1 digestion in situ, contact transfer to another PEI-cellulose tle plate, and two-dimensional tle fingerprinting; the third employed complete digestion in situ with ribonuclease T1 and bacterial alkaline phosphatase, followed by the elution, periodate oxidation, introduction of a tritium into 3′-terminus, and subsequent two-dimensional PEI-cellulose fingerprinting. These techniques can aid in the determination of the complete nucleotide sequence of tRNA when only small quantities of pure tRNAs (less than 10 A260 units) are available or when the tRNAs are not amenable to in vivo radioactive labeling.  相似文献   

14.
15.
Precursors of 5 S ribosomal RNA in Bacillus subtilis   总被引:8,自引:0,他引:8  
Bacillus subtilis 168 accumulates subnormal quantities of mature 5 S ribo-somal RNA in the presence of inhibitors of protein synthesis, such as chloramphenicol, or during pulse-labeling experiments. However, two RNA species, evidently precursors of m5 rRNA and therefore designated as p5A and p5B, do accumulate under these conditions. These RNA species are substantially longer than B. subtilis m5 rRNA: p5A is about 179 nucleotides in length and p5B is composed of approximately 152 nucleotides. The sum of p5A, p5B and m5 rRNA accumulating in the absence of protein synthesis, less excess chain length associated with p5A and p5B, equals the expected quantities of m5 rRNA in growing cells. p5A and p5PB both contain all t1 RNase-generated oligonucleotides characteristic of m5 rRNA plus additional sequences. At least the 5′ termini of p5A and p5B differ from that of m5. If chloramphenicol is removed from a culture in which p5A and p5B have accumulated and further RNA synthesis is inhibited, then a quantitative reciprocal loss of p5A and p5B occurs as m5 rRNA accumulates. No evidence suggests any p5A to p5B transition under these conditions.  相似文献   

16.
A simple and effective method of the methylation on the 2′-O position of adenosine is described. Adenosine is treated with CH3I in an anhydrous alkaline medium at 0°C for 4 h. The major products of this reaction are monomethylated adenosine at either the 2′-O or 3′-O position (total of 64%) and the side products are dimethylated adenosine (2′,3′-O-dimethyladenosi, 21%, and N6-2′-O-dimethyladenosine, 11%). The ratio of 2′-O- and 3′-O-methyladenosine has been found to be 8 to 1. Therefore, this reaction preferentially favors the synthesis of 2′-O-methyladenosine. The monomethylated adenosine is isolated from reaction mixture by a silica gel column chromatography. Then the pure 2′-O-methyladenosine can be separated by crystallization in ethanol from the mixture of 2′-O and 3′-O-methylated isomers. The overall yield of 2′-O-methyladenosine is 42%.  相似文献   

17.
Molecular-mechanics calculations have been carried out on the base-paired hexanucleoside pentaphosphates d(TATATA)2, d(ATATAT)2, d(A6)·d(T6), d(CGCGCG)2, d(GCGCGC)2, and d(C6)·d(G6) in both A- and B-DNA geometries. The calculated relative energies of these polymers are consistent with the relative stabilities of the polymers found experimentally. In particular, the results of our calculations support the observation that the homopolymer d(A)n·d(T)n is more stable in a B-DNA conformation, while the homopolymer d(G)n·d(C)n is more stable in an A-DNA conformation. The molecular interactions responsible for these differential stabilities include both inter- and intrastrand base stacking, as well as base–phosphate interactions. While definitive experiments on the heteropolymer stabilities have not yet been carried out, the results of our calculations also suggest a greater stability of the purine-3′,5′-pyrimidine sequence over the pyrimidine-3′,5′-purine sequence in both the A- and B-conformations. The reason for this greater stability lies in the importance of the inherent directionality (5′ → 3′ vs 3′ → 5′) of phosphate–base and base–base interactions. The largest conformation change observed on energy refinement is sugar repuckering, which occurs mainly on pyrimidine-attched sugars and only in the B-DNA geometry. We suggest a molecular mechanism, specifically, differential base–sugar steric interactions involving neighboring sugars, to explain why this repuckering occurs more with d(A6)·d(T6) than with other isomers.  相似文献   

18.
Oligo(2'-5')adenylate synthetase in human lymphoblastoid cells   总被引:1,自引:0,他引:1  
The enzyme oligo(2′–5′)adenylate synthetase, when activated by double-stranded RNA, polymerizes ATP into the novel oligonucleotide (2′–5′)ppp(Ap)nA. We describe conditions for assay of this enzyme in crude extracts of a human lymphoblastoid cell line, Namalwa. The production of (2′–5′)ppp(Ap)nA by Namalwa extracts was 3–5 times greater than the production by extracts of interferon pretreated mouse L cells, and 700 fold higher than the production by extracts of untreated mouse L cells. The relatively high level of oligo(2′–5′)adenylate synthetase in Namalwa cells was not attributable solely to their constitutive secretion of low levels of interferon. Analysis of the size distribution of the oligomers formed at different times suggested that the enzyme can add ATP to a free pppApA. Infection by Newcastle disease virus or treatment with interferon raised the apparent synthetase levels only marginally. Experiments that employed antibody to interferon suggested that the interferon must be externalized from the NDV-infected cell to induce maximal synthetase levels.  相似文献   

19.
20.
Acridine orange and proflavine form complexes with the dinucleoside monophosphate, 5-iodocytidylyl(3′–5′)guanosine. The acridine orange-iodoCpG2 crystals are monoclinic, space group P21, with unit cell dimensions a = 14.36 A?, b = 19.64 A?, c = 20.67 A?, β = 102.5 °. The proflavine-iodoCpG crystals are monoclinic, space group C2, with unit cell dimensions a = 32.14 A?, b = 22.23 A?, c = 18.42 A?, β = 123.3 °. Both structures have been solved to atomic resolution by Patterson and Fourier methods, and refined by full matrix least-squares.Acridine orange forms an intercalative structure with iodoCpG in much the same manner as ethidium, ellipticine and 3,5,6,8-tetramethyl-N-methyl phenanthrolinium (Jain et al., 1977, Jain et al., 1979), except that the acridine nucleus lies asymmetrically in the intercalation site. This asymmetric intercalation is accompanied by a sliding of base-pairs upon the acridine nucleus and is similar to that observed with the 9-aminoacridine-iodoCpG asymmetric intercalative binding mode described in the previous papers (Sakore et al., 1977, Sakore et al., 1979). Basepairs above and below the drug are separated by about 6.8 Å and are twisted about 10 °; this reflects the mixed sugar puckering pattern observed in the sugar-phospate chains: C3′ endo (3′–5′) C2′ endo (i.e. each cytidine residue has a C3′ endo sugar comformation, while each guanosine residue has a C2′ endo sugar conformation), alterations in glycosidic torsional angles and other small but significant conformational changes in the sugar-phosphate backbone.Proflavine, on the other hand, demonstrates symmetric intercalation with iodoCpG. Hydrogen bonds connect amino groups on proflavine with phosphate oxygen atoms on the dinucleotide. In contrast to the acridine orange structure, base-pairs above and below the intercalative proflavine molecule are twisted about 36 °. The altered magnitude of this angular twist reflects the sugar puckering pattern that is observed: C3′ endo (3′–5′) C3′ endo. Since proflavine is known to unwind DNA in much the same manner as ethidium and acridine orange (Waring, 1970), one cannot use the information from this model system to understand how proflavine binds to DNA (it is possible, for example, that hydrogen bonding observed between proflavine and iodoCpG alters the intercalative geometry in this model system).Instead, we propose a model for proflavine-DNA binding in which proflavine lies asymmetrically in the intercalation site (characterized by the C3′ endo (3′–5′) C2′ endo mixed sugar puckering pattern) and forms only one hydrogen bond to a neighboring phosphate oxygen atom. Our model for proflavine-DNA binding, therefore, is very similar to our acridine orange-DNA binding model. We will describe these models in detail in this paper.  相似文献   

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