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PubMed1995

Substrate specificities of poly(hydroxyalkanoate)-degrading bacteria and active site studies on the extracellular poly(3-hydroxyoctanoic acid) depolymerase of Pseudomonas fluorescens GK13.

The isolation of poly(3-hydroxyoctanoic acid)- and poly(6-hydroxyhexanoic acid)-degrading bacteria yielded 28 strains with abilities to degrade various polymers. The most versatile strains hydrolyzed five different polyesters comprising short chain length and medium chain length poly(hydroxyalkanoates). The new isolates together with previously isolated poly(hydroxyalkanoate)-degrading bacteria were classified into 11 groups with respect to their polymer-degrading specificities. All PHA depolymerases studied so far have been characterized by the lipase consensus sequence Gly-X-Ser-X-Gly in their amino acid sequence, which is a known sequence for serine hydrolases. When we replaced the central residue, Ser-172, in the corresponding sequence Gly-Ile-Ser-Ser-Gly of the extracellular poly(3-hydroxyoctanoic acid) depolymerase of Pseudomonas fluorescens GK13, with alanine the enzyme lost its activity completely. This result of the mutational experiment indicates that the poly(3-hydroxyoctanoic acid) depolymerase belongs to the family of serine hydrolases.

Amino Acid Sequence↗
PubMed1987

Relationship of the terminal sequences to the length of poly-N-acetyllactosamine chains in asparagine-linked oligosaccharides from the mouse lymphoma cell line BW5147. Immobilized tomato lectin interacts with high affinity with glycopeptides containing long poly-N-acetyllactosamine chains.

To investigate the factors regulating the biosynthesis of poly-N-acetyllactosamine chains containing the repeating disaccharide [3Gal beta 1,4GlcNAc beta 1] in animal cell glycoproteins, we have examined the structures and terminal sequences of these chains in the complex-type asparagine-linked oligosaccharides from the mouse lymphoma cell line BW5147. Cells were grown in medium containing [6-3H]galactose, and radiolabeled glycopeptides were prepared and fractionated by serial lectin affinity chromatography. The glycopeptides containing the poly-N-acetyllactosamine chains in these cells were complex-type tri- and tetraantennary asparagine-linked oligosaccharides. The poly-N-acetyllactosamine chains in these glycopeptides had four different terminal sequences with the structures: I, Gal beta 1,4GlcNAc beta 1,3Gal-R; II, Gal alpha 1,3Gal beta 1,4GlcNac beta 1,3Gal-R; III, Sia alpha 2,3Gal beta 1,4GlcNAc beta 1,3Gal-R; and IV, Sia alpha 2,6Gal beta 1,4GlcNAc beta 1,3Gal-R. We have found that immobilized tomato lectin interacts with high affinity with glycopeptides containing three or more linear units of the repeating disaccharide [3Gal beta 1,4GlcNAc beta 1] and thereby allows for a separation of glycopeptides on the basis of the length of the chain. A high percentage of the long poly-N-acetyllactosamine chains bound by immobilized tomato lectin were not sialylated and contained the simple terminal sequence of Structure I. In addition, a high percentage of the sialic acid residues that were present in the long chains were linked alpha 2,3 to penultimate galactose residues (Structure III). In contrast, a high percentage of the shorter poly-N-acetyllactosamine chains not bound by the immobilized lectin were sialylated, and most of the sialic acid residues in these chains were linked alpha 2,6 to galactose (Structure IV). These results indicate that there is a relationship in these cells between poly-N-acetyllactosamine chain length and the degree and type of sialylation of these chains.

Animals↗
PubMed1993

4-Hydroxyaminoquinoline 1-oxide can convert the Z-form of poly(dG-m5dC).poly(dG-m5dC) back to B-form-DNA.

Reactions between the chemical carcinogen 4-hydroxyaminoquinoline 1-oxide (4HAQO) and poly(dG-dC).poly(dG-dC), poly(dG-m5dC).poly(dG-m5dC) and poly(dG-br5dC).poly(dG-br5dC) were studied under B-form and Z-form conformation conditions. 4HAQO bound covalently to both B-form and Z-form-DNA. The extent of the B-form to Z-form-DNA transition was diminished by 4HAQO, as inferred by spectroscopic methods. Moreover, conversion of the Z-form-DNA back to a right handed DNA close to the B-form was observed with this carcinogen.

4-Hydroxyaminoquinoline-1-oxide↗
PubMed1993

Reovirus protein lambda 3 is a poly(C)-dependent poly(G) polymerase.

Reovirus protein lambda 3 has been isolated from cells infected with two recombinant vaccinia viruses into the TK gene of which the reovirus serotype3 strain Dearing L1 genome segment under the control of the bacteriophage T7 RNA polymerase promoter, or the T7 polymerase gene itself, had been cloned. Highly purified protein lambda 3 does not transcribe double-stranded reovirus RNA into single-stranded RNA, or plus-stranded reovirus RNA into minus-stranded RNA, but it does transcribe poly(C) into poly(G). It prefers Mn2+ to Mg2+. A polymer consisting of poly(C) linked linearly to poly(U) provided template activity only for its poly(C) moiety. Protein lambda 3 forms complexes with protein lambda 1, as well as with protein lambda 2, and with both lambda 1 and lambda 2, which are sufficiently stable to be precipitated by monospecific antisera. None of these complexes are capable of transcribing either ds- or ssRNA.

Base Sequence↗
PubMed1996

Ribosomal association of poly(A)-binding protein in poly(A)-deficient Saccharomyces cerevisiae.

Poly(A)-binding protein, the most abundant eukaryotic mRNP protein, is known primarily for its association with polyadenylate tails of mRNA. In the yeast, Saccharomyces cerevisiae, this protein (Pabp) was found to be essential for viability and has been implicated in models featuring roles in mRNA stability and as an enhancer of translation initiation. Although the mechanism of action is unknown, it is thought to require an activity to bind poly(A) tails and an additional capacity for an interaction with 60 S ribosomal subunits, perhaps via ribosomal protein L46 (Rpl46). We have found that a significant amount of Pabp in wild-type cells is not associated with polyribosome complexes. The remaining majority, which is found in these complexes, maintains its association even in yeast cells deficient in polyadenylated mRNA and/or Rpl46. These observations suggest that Pabp may not require interaction with poly(A) tails during translation. Further treatment of polyribosome lysates with agents known to differentially disrupt components of polyribosomes indicated that Pabp may require contact with some RNA component of the polyribosome, which could be either non-poly(A)-rich sequences of the translated mRNA or possibly a component of the ribosome. These findings suggest that Pabp may possess the ability to bind to ribosomes independently of its interaction with poly(A). We discuss these conclusions with respect to current models suggesting a multifunctional binding capacity of Pabp.

Poly A↗
PubMed1978

Interaction of tobacco mosaic virus protein with synthetic polynucleotides containing a fluorescent label: optical properties of poly(A,epsilonA) and poly(C,epsilonC) copolymers and energy migration from the tryptophan to 1,N6-ethenoadenine or 3,N4-ethenocytosine residues in RNP.

A spectrophotometric method for determination of the modification degrees and molar extinction coefficients for poly(A, epsilonA) and poly(C, epsilonC) copolymers has been developed. Dependence of some absorption and fluorescence parameters of the copolymers on the modification degree has been studied. Distribution of modified residues in copolymers differs from random and depends on modification conditions. Interaction between the TMV protein and copolymers has been investigated. The protein interacts with poly(A, epsilonA) of low or medium modification degree and displays no activity with respect to poly(epsilonA). On the contrary, introduction of epsilonC to the polynucleotide promotes complex formation between poly(C, epsilonC) and TMV protein. Analysis of the fluorescence emission and excitation spectra has revealed energy transfer from tryptophan to epsilonA or epsilon C in the RNP to occur and permits one to estimate the average distance between Trp (presumably Trp 52)o and the RNA base binding region in the virus to be 17 to 20 A.

Adenine↗
PubMed1981

DNase A, a poly(dA) and poly(dT)-specific deoxyribonuclease from Achatina fulica. Purification and characterization.

DNases A1 and A2 have been purified to homogeneity from the hepatopancreas of Achatina fulica by a series of steps: acetate buffer extraction, ammonium sulfate precipitation and column chromatography on hydroxylapatite, phosphocellulose, Blue-Sepharose, and poly(A)-Sepharose. The purified enzymes are free of acidic phosphomonoesterase, phosphodiesterase, and RNase activities. They are slightly acidic glycoproteins with identical isoelectric point (6.90). On 0.1% SDS gel electrophoresis, DNase A2 had a molecular weight of 30,000 when dissolved in 1% SDS, but it had molecular weights of 17,500, 8,000, and 4,800 when dissolved in 1% SDS and 1% 2-mercaptoethanol. This was evidence that the enzyme consists of three different subunits joined by interchain disulfide bonds. DNases A1 and A2 are endonucleases working at acidic pH (3.5--6.0) and do not require divalent cations for their activities. The enzymes degrade poly(dA) 5 times faster and poly(dT) 3 times faster than heat-denatured DNA under optimal conditions but do not appreciably digest poly(dG) and poly(dC). We developed an analytical procedure for oligodeoxynucleotides by high-performance liquid chromatography. The phosphomonoester end group and the mode of degradation were examined by the method. The termini produced by the enzymes have 3'-phosphoryl and 5'-hydroxy end groups. The products of exhaustive hydrolysis contain di-, tri-, tetra-, and pentanucleotides and mononucleotide was barely detected. The hydrolyzing activities of DNases A1 and A2 are stimulated by polyamines such as spermine, spermidine, and putrescine, but are inhibited by synthetic polynucleotides and various drugs. Adenosine deaminase highly active on oligoadenylic acids was found in a crude DNase A fraction. The enzyme preparation has higher activity on 3'-adenylic acid than on 5'-adenylic acid. The first adenosine residue of oligoadenylic acids was deaminated considerably more rapidly than the second or succeeding ones.

Animals↗
PubMed1998

mRNA stabilization by poly(A) binding protein is independent of poly(A) and requires translation.

Translation and mRNA stability are enhanced by the presence of a poly(A) tail. In vivo, the tail interacts with a conserved polypeptide, poly(A) binding protein (Pab1p). To examine Pab1p function in vivo, we have tethered Pab1p to the 3' UTR of reporter mRNAs by fusing it to MS2 coat protein and placing MS2 binding sites in the 3' UTR of the reporter. This strategy allows us to uncouple Pab1p function from its RNA binding activity. We show that mRNAs that lack a poly(A) tail in vivo are stabilized by Pab1p, and that the portions of Pab1p required for stabilization are genetically distinct from those required for poly(A) binding. In addition, stabilization by Pab1p requires ongoing translation of the mRNA. We conclude that the primary, or sole, function of poly(A) with respect to mRNA stability is simply to bring Pab1p to the mRNA, and that mRNA stabilization is an intrinsic property of Pab1p. The approach we describe may be useful in identifying and assaying 3' UTR regulatory proteins, as it uncouples analysis of function from RNA binding.

3' Untranslated Regions↗
PubMed1986

Reconstitution and poly(ADP-ribosyl)ation of proteolytically fragmented poly(ADP-ribose) synthetase.

Calf thymus poly(ADP-ribose) synthetase (Mr = 120,000) is cleaved with papain into two fragments of M(r) = 74,000 and 46,000 and also split with chymotrypsin into two fragments of M(r) = 66,000 and 54,000. Each fragment purified to homogeneity is enzymatically inactive, but combined incubation of the 74,000 and 46,000 fragments in the presence of DNA restored 20% of the enzyme activity. In contrast, combined incubation of the 66,000 and 54,000 fragments does not restore any enzyme activity. In the former incubation, autopoly(ADP-ribosyl)ation reaction occurs exclusively on the 74,000 fragment. When each fragment is incubated with [adenine-U-14C]NAD in the presence of DNA and a catalytic amount of the native enzyme, poly(ADP-ribosyl)action occurs in the overlapped portion (22,000) of the 66,000 fragment and the 74,000 fragment. Nevertheless, the purified 22,000 fragment is a poor acceptor for poly(ADP-ribosyl)ation. The degree of poly(ADP-ribosyl)ation of the proteolytic fragments is significantly reduced by increasing NaCl concentration, probably due to the lack of the interaction between the enzyme fragments and DNA. These results, taken together, indicate that DNA is indispensable for the reconstitution of the catalytic activity as well as the poly(ADP-ribosyl)ation of the fragmented enzyme.

Amino Acids↗
PubMed1982

Polynucleotide helix geometry and stability. Spectroscopic, antigenic and interferon-inducing properties of deoxyribose-, ribose-, or 2'-deoxy-2'-fluororibose-containing duplexes of poly(inosinic acid) . poly(cytidylic acid).

Circular dichroism, absorbance temperature profiles, antigenic properties, and interferon-inducing capacity of the nine double-stranded complexes between poly(inosinic acid) and poly(cytidylic acid) [(rI)n . (rC)n] and their deoxy- and 2'-deoxy-2'-fluoro-analogues were studied. The complexes containing only ribose or 2'-fluororibose chains showed similar CD spectra and thermal stabilities. Anti-(rI)n . (rC)n antibodies were well recognized by the duplexes containing 2'-fluororibose in either one strand. These two duplexes were also efficient interferon inducers. Presence of fluororibose in both strands decreased the affinity of anti-(rI)n . (rC)n antibodies slightly and abolished interferon-inducing activity. All polydeoxyriboside-containing complexes showed CD spectra significantly different from the previous group; they showed also 40- to 100-times lower affinity for the anti-(rI)n . (rC)n antibodies, and did not induce interferon. It is concluded that the structure of (rI)n . (rC)n, an A-type helix, is little perturbed by substitution of one or both strands by 2'-fluororibose. Substitution by deoxyribose in either strand considerably changes the structure of the helices. The hybrids containing poly(deoxycytidylic acid) retain at least some of the structural features of the B-type helix poly(deoxyinosinic acid) . poly(deoxycytidylic acid).

Animals↗
PubMed1980

Initiation of poly(ADP-ribosyl) histone synthesis by poly(ADP-ribose) synthetase.

Initiation of poly(ADP-ribosyl) histone synthesis was achieved in vitro using an apparently homogeneous preparation of poly(ADP-ribose) synthetase. When poly(ADP-ribose) was synthesized in the presence of DNA and increase amounts of histone H1, increasing portions (up to about 55%) of the product were found associated with the histone, judging from solubility in 5% HClO4 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Most of the polymers were directly attached to the histone protein and not produced by elongation from pre-existing ADP-ribose; the cohesive end of poly(ADP-ribose), isolated as ribose 5-phosphate with snake venom phosphodiesterase digestion, was labeled almost quantitatively with [ribose (NMN)-14C]NAD. The poly(ADP-ribose) . histone linkage was labile in mild alkali and neutral NH2OH, suggesting that the same bond, probably ester, was formed in this system as in crude chromatin or isolated nuclei. Elongation of a histone-bound monomer into a polymer by this enzyme was previously demonstrated (Ueda, K., Kawaichi, M., Okayama, H., and Hayaishi, O. (1979) J. Biol. Chem. 254, 679-687), but initiation of ADP-ribose chains on histone has never been shown with a purified enzyme. This appeared to be due to the low concentrations of histone so far used. These findings indicated that a single enzyme catalyzes two different types of reaction, i.e. an attachment of ADP-ribose to histone and its elongation into a polymer.

Animals↗
PubMed2006

Inhibition of telomerase activity by reduction of poly(ADP-ribosyl)ation of TERT and TEP1/TP1 expression in HeLa cells with knocked down poly(ADP-ribose) polymerase-1 (PARP-1) gene.

Inhibitors of poly(ADP-ribose) polymerase (PARP) have been shown to reduce the telomerase activity in cultured cells. To find out the specific member of the PARP family, which participates in the regulation of telomerase activity, in the present investigation, we knocked down the PARP-1 gene by siRNA in HeLa cells and studied the telomerase activity. Reduction of expression of PARP-1 by siRNA increased cellular NAD(+) level and decreased general poly(ADP-ribosyl)ation of proteins. Telomerase activity decreased in cells with knocked down PARP-1 gene. Besides, we observed that telomerase reverse transcriptase (hTERT) was poly(ADP-ribosyl)ated in HeLa cells and such modification was decreased in cells with reduced PARP-1 expression. In addition, the expression of telomerase-associated protein 1 (TEP1/TP1) subunit of human telomerase holoenzyme reduced significantly in PARP-1 knock down HeLa cells. Thus, PARP-1 modulates the telomerase activity by altering poly(ADP-ribosyl)ation of TERT and/or the expression of TEP1/TP1.

Base Sequence↗
PubMed1994

DNA binding of a spermine derivative: spectroscopic study of anthracene-9-carbonyl-N1-spermine with poly[d(G-C).(d(G-C)] and poly[d(A-T).d(A-T)].

The binding of polyamines, including spermidine (1) and spermine (2), to poly[d(G-C).d(G-C)] was probed using spectroscopic studies of anthracene-9-carbonyl-N1-spermine (3); data from normal absorption, linear dichroism (LD), and circular dichroism (CD) are reported. Ligand LD and CD for transitions located in the DNA region of the spectrum were used. The data show that 3 binds to DNA in a manner characteristic of both its amine and polycyclic aromatic parts. With poly[(dG-dC).(dG-dC)], binding modes are occupied sequentially and different modes correspond to different structural perturbations of the DNA. The most stable binding mode for 3 with poly[d(G-C).d(G-C)] has a site size of 6 +/- 1 bases, and an equilibrium binding constant of (2.2 +/- 1.1) x 10(7) M-1 with the anthracene moiety intercalated. It dominates the spectra from mixing ratios of approximately 133:1 until 6:1 DNA phosphate: 3 is reached. The analogous data for poly[d(A-T).d(A-T)] between mixing ratios 36:1 and 7:1 indicates a site size of 8.3 +/- 1.1 bases and an equilibrium binding constant of (6.6 +/- 3.3) x 10(5) M-1. Thus, 3 binds preferentially to poly[d(G-C).d(G-C)] at these concentrations.

Circular Dichroism↗
PubMed1979

Observations on the kinetics of action of polyspermine-ribonuclease on poly(A).poly(U).

Starting from the observation that double-stranded ribonucleic acids are hydrolyzed more rapidly by bovine pancreatic ribonuclease that has been cross-linked to polyspermine, we have made an initial examination of the kinetics of the process. The addition of eight residues of the polyamine serves to strengthen the binding to poly(A).poly(U) 100-fold (Km changes from 2.7 . 10(-4) to 2.7 . 10(-6) M in total U) and to increase V for hydrolysis of the susceptible poly(U) strand from 2.5 to 16.2 delta A250 . min-1 per mg enzyme. There is evidence for inhibition by the RNAase-resistant poly(A) tracts in the substrate; free poly(A) shows a Ki of about 8 . 10(-6) M in total A.

Animals↗
PubMed1988

The preparation of poly (dT)-5'-transferrin conjugates and hybridisation studies with poly (dA)-tailed linearised pBR322 plasmid DNA.

The formation of transferrin-DNA complexes intended for ligand-directed transfection studies has been achieved through a hybridisation technique involving complementary homodeoxypolynucleotide chains attached to the participating protein and DNA species. Oligothymidylate residues (pT)n obtained by dicyclohexylcarbodiimide (CDI) polymerisation of thymidine-5'-monophosphate (5'-TMP) were activated to the 5'-imidazolides which on incubation with transferrin yielded the 5'linked phosphoramidates (pT)n-5'-transferrin. Homopolymeric chain extension of (pT)5-5'-transferrin by terminal transferase and dTTP at 30 degrees for 30 min yielded (pT) 300-5'-transferrin. Cleavage of the phosphoramide link in the polymer modified transferrin at 37 degrees was pronounced after 30 min although at 25 degrees hydrolysis was less than 5% after 4 hr. Poly(dT)-5'-transferrin readily hybridised with [3H]poly(dA)-tailed Pst 1 linearised pBR322 DNA. Resultant complexes were demonstrated by nitrocellulose filter binding and immunoprecipitation with anti-transferrin antibody. In contrast with poly(dT)-5'-transferrin, poly(dT)-5'-transferrin-poly(dA)-tailed pBR322 DNA complexes were stable at 37 degrees suggesting that annealing is followed by further stabilising interactions between the DNA and protein components.

Nucleic Acid Hybridization↗
PubMedDate not supplied

The formation of acetylaminofluorene adducts in poly(dC-dG) and poly(dA-dT) on reaction with N-acetoxy-2-acetylaminofluorene and the effect of such modification upon the polymers as templates for DNA polymerases.

N-Acetoxy-2-acetylaminofluorene (AcO-AAF) reacts with the alternating DNA-like polynucleotides poly(dC-dG) and poly(dA-dT) in vitro to give adducts of the guanine and adenine bases similar to those reported to be formed in DNA. A previously unobserved guanine adduct was detected in the poly(dC-dG). Using a double-labelled [U-14C-dG, 8-3H-G]-poly(dC-dG) we show that this adduct does not involve the 7- or 8-positions of the guanine. Similarly a thymine adduct of unknown structure was observed in poly(dA-dT). Modification of the polymers with AcO-AAF inhibits their capacity to act as templates for Escherichia coli DNA polymerase I and mammalian DNA polymerase alpha although the binding of the polymerases to the polynucleotides is unaffected. Such modification also leads to an increase in the levels of non-complementary nucleotides incorporated into newly synthesised DNA.

2-Acetylaminofluorene↗
PubMed1990

Multiplication of beet necrotic yellow vein virus RNA 3 lacking a 3' poly(A) tail is accompanied by reappearance of the poly(A) tail and a novel short U-rich tract preceding it.

Beet necrotic yellow vein virus RNAs 1 and 2 but not RNAs 3 and 4 are required for viral multiplication in Chenopodium quinoa leaves. Elimination of the 3' poly(A) tail from RNA 3 transcripts markedly attenuated their ability to be amplified when co-inoculated with RNAs 1 and 2 to this host. Successful multiplication of the tailless RNA 3 was accompanied by the reappearance of new 3' poly(A) tails on the progeny. The evidence suggests that the newly acquired poly(A) sequence results from the action of a poly(A) polymerase rather than recombination with the homologous 3' terminal domains of RNAs 1 or 2. An unexpected feature of these progeny RNA 3 molecules was the presence of a novel short heterogenous U-rich tract separating the poly(A) tail from the 3' end of the heteropolymeric RNA 3 sequence proper.

Base Composition↗
PubMed1985

Relationship between large granular lymphocytes and NK-1.2+ cells from normal and poly(inosinic:cytidylic acid) (poly(I:C]-treated mice.

The present work analyzes the relationship between large granular lymphocytes (LGL), NK-1.2+ cells, and natural killer (NK) activity of C3H/HeN mice. Different hematic cell fractions were obtained according to their nylon-wool adherence and density on Percoll gradients. NK-1.2+ cells (8% of nucleated cells) were more numerous than LGL (3% of nucleated cells) in the input blood population. Eighty-five percent of LGL were recovered from the sorted NK-1.2+ cell fraction. After incubation on nylon-wool column, 63% of LGL and 36% of NK-1.2+ were eluted in the nonadherent fraction. Eighteen percent of NK-1.2+ cells were recovered from the most adherent elutable cell fraction. After the discontinuous Percoll gradient most LGL were present in the low-density fractions while 20% of NK-1.2+ cells were recovered from the highest-density fraction. NK activity was significant both in the nylon-wool-nonadherent and -adherent fractions. After the Percoll gradient most NK activity was present in the low-density fractions. In the present experimental conditions treatment poly(inosinic:cytidylic acid) (poly(I:C] did not increase the numbers of LGL and NK-1.2+ cells either in the blood or in the spleen. However it increased significantly the NK activity of the input cell populations and of the nonadherent and low-density fractions. Similarly, exposure of specific pathogen-free (SPF) mice to non-SPF conditions stimulated NK cytotoxicity but did not alter the percentage of LGL in the blood or in the spleen. Poly(I:C) treatment induced a shift of LGL and NK-1.2+ cells toward the low-density fractions. In poly(I:C)-treated mice images of granule secretion from LGL were detected. Taken together, the present results indicate that LGL and NK-1.2+ cell populations do not totally overlap. Moreover subpopulations of LGL and NK-1.2+ cells can differ in NK activity, morphology, density, adherence to nylon wool, and response to poly(I:C).

Animals↗