Nucleotide sequence analysis of the gene specifying the bifunctional 6''-aminoglycoside acetyltransferase 2"-aminoglycoside phosphotransferase enzyme in Streptococcus faecalis and identification and cloning of gene regions specifying the two activities.

The gene specifying the bifunctional 6'-aminoglycoside acetyltransferase [AAC(6')] 2"-aminoglycoside phosphotransferase [APH(2")] enzyme from the Streptococcus faecalis plasmid pIP800 was cloned in Escherichia coli. A single protein with an apparent molecular weight of 56,000 was specified by this cloned determinant as detected in minicell experiments. Nucleotide sequence analysis revealed the presence of an open reading frame capable of specifying a protein of 479 amino acids and with a molecular weight of 56,850. The deduced amino acid sequence of the bifunctional AAC(6')-APH(2") gene product possessed two regions of homology with other sequenced resistance proteins. The N-terminal region contained a sequence that was homologous to the chloramphenicol acetyltransferase of Bacillus pumilus, and the C-terminal region contained a sequence homologous to the aminoglycoside phosphotransferase of Streptomyces fradiae. Subcloning experiments were performed with the AAC(6')-APH(2") resistance determinant, and it was possible to obtain gene segments independently specifying the acetyltransferase and phosphotransferase activities. These data suggest that the gene specifying the AAC(6')-APH(2") resistance enzyme arose as a result of a gene fusion.     

5'-modified agonist and antagonist (2'-5')(A)n analogues: synthesis and biological activity

Two 5'-modified (2'-5')(A)4 oligomers with an increased resistance to phosphatase degradation were synthesized and evaluated for their ability to develop an antiviral response when introduced into intact cells by microinjection or by chemical conjugation to poly(L-lysine). The enzymatic synthesis of 5'-gamma-phosphorothioate and beta,gamma-difluoromethylene (2'-5')(A)4 from adenosine 5'-O-(3-thiotriphosphate) and adenosine beta,gamma-difluoromethylenetriphosphate by (2'-5')-oligoadenylate synthetase is described. The isolation and characterization of these (2'-5')(A)4 analogues were achieved by high-performance liquid chromatography. The structures of 5'-modified tetramers were corroborated by enzyme digestion. These two 5'-modified tetramers compete as efficiently as natural (2'-5')(A)4 for the binding of a radiolabeled (2'-5')(A)4 probe to ribonuclease (RNase) L. Nevertheless, at the opposite to 5'-gamma-phosphorothioate (2'-5')(A)4, beta,gamma-difluoromethylene (2'-5')(A)4 failed to induce an antiviral response after microinjection in HeLa cells. In addition, it behaves as an antagonist of RNase L as demonstrated by its ability to inhibit the antiviral properties of 5'-gamma-phosphorothioate (2'-5')(A)4 when both are microinjected in HeLa cells. The increased metabolic stability of 5'-gamma-phosphorothioate (2'-5')(A)4 as compared to that of (2'-5')(A)4 was first demonstrated in cell-free extracts and then confirmed in intact cells after introduction in the form of a conjugate to poly(L-lysine). Indeed, 5'-gamma-phosphorothioate (2'-5')(A)4-poly(L-lysine) conjugate induces protein synthesis inhibition and characteristic ribosomal RNA cleavages for longer times than unmodified (2'-5')(A)4-poly(L-lysine) in the same cell system.(ABSTRACT TRUNCATED AT 250 WORDS)     

The chromosomal 3',5"-aminoglycoside phosphotransferase in Streptococcus pneumoniae is closely related to its plasmid-coded homologs in Streptococcus faecalis and Staphylococcus aureus.

The apparently chromosomally encoded 3',5"-aminoglycoside phosphotransferase (type III), from the high-level aminoglycoside-resistant Streptococcus pneumoniae BM4200, was compared with homologous enzymes coded for by the plasmids pJH1 and pSH2, originally isolated from Streptococcus faecalis and Staphylococcus aureus, respectively, and also found in a wild strain of S. aureus, BM4600. The enzymes appeared to be indistinguishable, and we conclude that the gene encoding 3',5"-aminoglycoside phosphotransferase (type III) can cross generic barriers within gram-positive cocci.     

Nucleotide sequence of the Streptococcus faecalis plasmid gene encoding the 3'5"-aminoglycoside phosphotransferase type III

We have cloned in Escherichia coli and sequenced a 1489-bp DNA fragment conferring resistance to kanamycin and originating from the streptococcal plasmid pJH1. The resistance gene was located by analysis of the initiation and termination codons in an open reading frame (ORF) of 792 bp. The deduced gene product, a 3'5'-aminoglycoside phosphotransferase of type III, has an Mr of 29,200. Comparison of its amino acid sequence with those of type I (Oka et al., 1981) and type II (Beck et al., 1982) 3' phosphotransferase, from transposable elements Tn903 and Tn5, respectively, indicated a statistically significant structural relationship between these enzymes from phylogenetically remote bacterial genera. The degree of homology observed indicate that phosphotransferase type III and type I genes have diverged from a common ancestor and that the phosphotransferase type II gene has emerged more recently from the type I evolutionary pathway.     

An adenosine 3':5'-monophosphate-dependent protein kinase from the human erythrocyte membrane. Purification and characterization.

An adenosine 3':5'-monophosphate-dependent protein kinase (ATP:protein phosphotransferase, EC 2.7.1.37) has been isolated from the human erythrocyte memebrane and the phosphotransferase activity exhibited by this enzyme has been purified 800-fold. In concentrated solutions, the membrane-derived protein kinase undergoes aggregation with a concomitant loss in observed phosphotransferase activity. This loss of activity can be restored by means of inducing deaggregation. The phosphotransferase activity of the protein kinase is virtually obliterated in the presence of high (300 mM) concentrations of sodium chloride. This effect is also reversible. The pH optimum for the phosphotransferase reaction that is catalyzed by the membrane-derived protein kinase is approximately 8. Micromolar concentrations of cAMP are optimal with respect to promoting the phosphotransferase reaction. Initial velocity and product inhibition studies were conducted on the cAMP-independent protein kinase derived from the cAMP-dependent enzyme. These studies indicate that the phosphotransferase reaction proceeds by a sequential kinetic mechanism.     

Comparative study of apramycin-resistant microorganisms isolated from man and animals

Apramycin-modifying strains isolated from pigs with coli bacteriosis, from humans and hospital environment were studied comparatively. Production of enzymes modifying the aminoglycoside was estimated with the radioactive cofactor procedure. E. coli isolates from the animals were phenotypically resistant to apramycin and a number of other aminoglycosides. They produced acetyltransferase AAC(3)IV, phosphotransferase APH(3')(5"), APH(3") and other enzymes. Resistance of the strains to gentamicin was also conditioned by AAC(3)IV since these strains did not produce AAD(2") and AAC(6'). In the resistant strains of E. coli and their transconjugates there were detected plasmids with a relative molecular weight of 60-80 MD. Some of the belonged to the compatibility group I1, the others belonged to the compatibility group H1. Strains of S. marcescens, K. pneumoniae. K. oxytoca and S. aureus isolated from humans and hospital environment were sensitive to apramycin. Only isolates of P. aeruginosa were resistant to this antibiotic. However, all the isolates produced AAC(3)IV. Some of them additionally produced AAC(6'), an enzyme modifying amikacin, kanamycin and other antibiotics and not acetylating apramycin. Almost all the strains produced kanamycin- and streptomycin phosphotransferases. Possible coselection of strains resistant to apramycin and gentamicin using one of these aminoglycosides is discussed.     

Activation of ribonuclease L by (2'-5')(A)4-poly(L-lysine) conjugates in intact cells

Molecular hybrids were synthesized by coupling (2'-5')(A)n oligoadenylates or 2-5A, an intracellular mediator involved in antiviral activity of interferons (IFNs), with poly(L-lysine) used as a membrane carrier. (2'-5')(A)n in its free form was not taken up by cells, probably because of its ionic character. Conjugation with the polypeptide carrier overcame this problem and enabled its pharmacological properties to be developed. The alpha-glycol group of individual (2'-5')(A)n oligomers was oxidized by periodate oxidation and conjugated by an amino reductive reaction to poly(L-lysine), Mr 14 000, in a molar ratio of 5:1. These hybrid molecules left the biologically active 5' end moiety of the (2'-5')(A)n molecule unchanged, and in particular its triphosphate group, and stabilized the molecule by increasing its resistance to phosphodiesterase hydrolysis. A dose-dependent inhibition of virus growth was observed on concomitant incubation of (2'-5')(A)n-poly(L-lysine) conjugates with vesicular stomatitis virus infected L1210 cell cultures. This was a result of the activation of the (2'-5')(A)n-dependent endoribonuclease (RNase L) by intracellularly delivered (2'-5')(A)n as in some IFN-treated virus-infected cells. Indeed, (2'-5')(A)n-poly(L-lysine) conjugates bind RNase L effectively as can be seen from their ability to compete with authentic (2'-5')(A)n in a cell-free radiobinding assay. Moreover, (2'-5')(A)n-poly(L-lysine) conjugates promote transient inhibition of protein synthesis and a characteristic cleavage pattern of ribosomal RNAs in intact cells.(ABSTRACT TRUNCATED AT 250 WORDS)     

Elongation of palindromic repetitive DNA by DNA polymerase from hyperthermophilic archaea: a mechanism of DNA elongation and diversification

DNA polymerase from hyperthermophilic bacteria can elongate tandem repetitive oligoDNA with a complete or incomplete palindromic sequence under isothermal conditions by "hairpin elongation". However, the product of the reaction has not yet been sufficiently characterized. Here, I demonstrate that when palindromic repetitive oligoDNA, e.g., (5'AGATATCT3')(6), was added as a "seed" to the DNA synthesis reaction catalyzed by DNA polymerase from the archaea Thermococcus litoralis (Vent polymerase) at 74 degrees C, the product was (5'AGATATCT3')(n). The product itself was palindromic and repetitive, and its motif (unit) sequence was exactly the same as that of the seed oligoDNA. On the other hand, when a pseudopalindrome, which contains a palindrome-breaking nucleotide (underlined), was present in seed oligoDNA, e.g., (5'GATTC3')(6), the product was (5'GATATC3')(n), which had a different motif sequence from that of the seed oligoDNA. When a pseudopalindrome (5'AGATATCA3')(6) was added to the reaction, the products were 5'TATCA . (AGATATCA)(3) . AGATATCT . (TGATATCT)(5) . TGATA3', etc. When 5'AGATATCA . (AGATATCT3')(5) was added, products were 5'TATCT . (AGATATCT)(2).TGATATCT . AGATATCT . AGATATCA . AGATATCT . AGA3', etc., demonstrating the generation of many "mutations" in the product DNA. I conclude that a tandem repetitive sequence is faithfully elongated (amplified) by hyperthermophilic DNA polymerase if it is completely palindromic, but is elongated with many errors if it is incompletely palindromic (pseudopalindromic) or mixed with a pseudopalindrome. The results suggest a protein-catalyzed elongation/diversification mechanism of short repetitive DNAs on the early earth.     

The effect of base sequence on the stability of RNA and DNA single base bulges

Forty-eight RNA duplexes were constructed that contained all common single base bulges at six different locations. The stabilities of the RNAs were determined by temperature gradient gel electrophoresis (TGGE). The relative stability of a single base bulge was dependent on both base identity and the nearest neighbor context. The single base bulges were placed into two categories. A bulged base with no identical neighboring base was defined as a Group I base bulge. Group II-bulged bases had at least one neighboring base identical to it. Group II bulges were generally more stable than Group I bulges in the same nearest neighbor environments. This indicates that position degeneracy of an unpaired base enhances stability. Differences in the mobility transition temperatures between the RNA fragments with bulges and the completely base-paired reference RNAs were related to free energy differences. Simple models for estimating the free energy contribution of single base bulges were evaluated from the free energy difference data. The contribution of a Group I bulge 5'-(XNZ)-3'.5'-(Z'-X')-3' where N is the unpaired base and X.X' and Z.Z' the neighboring base pairs, could be well-represented (+/-0.34 kcal/mol) by the equation, DeltaG((X)(N)()(Z))(.)((Z)(')(-)(X)(')()) = 3.11 + 0. 40DeltaG(s)()((XZ))(.)((Z)(')(X)(')()). DeltaG(s)()((XZ))(. )((Z)(')(X)(')()) is the stacking energy of the closing base pair doublet. By adding a constant term, delta = -0.3 kcal/mol, to the right side of the above equation, free energies of Group II bulges could also be predicted with the same accuracy. The term delta represents the stabilizing effect due to position degeneracy. A similar equation/model was applied to previous data from 32 DNA fragments with single base bulges. It predicted the free energy differences with a similar standard deviation.     

2',3'-seco pyrimidine nucleosides and nucleotides, including structural analogues of 3':5'-cyclic CMP and UMP, and their behaviour in several enzyme systems

Cyclization of 2',3'-seco-5'- CMP and UMP with dicyclohexylcarbodiimide leads to 2',3'-seco-3':5'- cCMP and cUMP, formal structural analogues of 3':5'- cCMP and cUMP. POCl3 phosphorylation of 2',3'-secocytidine gave the same product in 50% yield, plus three additional seco nucleotides, one of which was independently obtained by enzymatic phosphorylation with the wheat shoot phosphotransferase system. The behaviour of these nucleotides has been examined in several enzyme systems. In particular, the seco 3':5'- cyclic phosphates are resistant to beef heart cyclic nucleotide phosphodiesterase, but are slowly hydrolyzed to the monophosphates by higher plant cyclic nucleotide phosphodiesterase.     

Kinetic scheme for intermolecular RNA cleavage by a ribozyme derived from hepatitis delta virus RNA

A minimal kinetic mechanism for a trans-acting ribozyme derived from the HDV antigenomic RNA self-cleaving element was established from steady-state, pre-steady-state, single-turnover, and binding kinetics. Rate constants for individual steps, including substrate binding and dissociation, cleavage, and product release and binding, were measured at 37 degrees C at pH 8.0 in 10 mM Mg(2+) using oligonucleotides as either substrates, noncleavable analogues or 3' product mimics. A substrate containing a normal 3',5'-linkage was cleaved with a first-order rate constant (k(2)) of 0.91 min(-)(1). The association rate constant for the substrate to the ribozyme (2.1 x 10(7) M(-)(1) min(-)(1)) was at the lower range of the expected value for RNA duplex formation, and the substrate dissociated with a rate constant (1.4 min(-)(1)) slightly faster than that for cleavage. Thus the binary complex was not at equilibrium with free enzyme and substrate prior to the cleavage step. Following cleavage, product release was kinetically ordered in that the 5' product was released rapidly (>12 min(-)(1)) relative to the 3' product (6.0 x 10(-)(3) min(-)(1)). Rapid 5' product release and lack of a demonstrable binding site for the 5' product could contribute to the difficulty in establishing the ribozyme-catalyzed reverse reaction (ligation). Slow release of the 3' product was consistent with the extremely low turnover under steady-state conditions as 3' product dissociation was rate-limiting. The equilibrium dissociation constant for the substrate was 24-fold higher than that of the 3' cleavage product. A substrate with a 2',5'-linkage at the cleavage site was cleaved with a rate constant (k(2)) of 1.1 x 10(-)(2) min(-)(1). Thus, whereas cleavage of a 3',5'-linkage followed a Briggs-Haldane mechanism, 2', 5' cleavage followed a Michaelis-Menten mechanism.     

cDNA genes formed after infection with retroviral vector particles lack the hallmarks of natural processed pseudogenes.

Retroviral proteins can encapsidate RNAs without retroviral cis-acting sequences. Such RNAs are reverse transcribed and inserted into the genomes of infected target cells to form cDNA genes. Previous investigations by Southern blot analysis of such cDNA genes suggested that they were truncated at the 3' and the 5' ends (R. Dornburg and H. M. Temin, Mol. Cell. Biol. 8:2328-2334, 1988). To analyze such cDNA genes further, we cloned three cDNA genes (derived from a hygromycin B phosphotransferase gene) in lambda vectors and analyzed them by DNA sequencing. We found that they did not correspond to the full-length mRNA: they were truncated at both the 3' and the 5' ends, did not contain a poly(A) tract, and were not flanked by direct repeats. The 3'-end junctions to chromosomal DNA of five more cDNA genes were amplified by polymerase chain reaction, cloned in pUC vectors, and sequenced. All of these cDNA genes had 3'-end truncations, and no poly(A) tracts were found. Further polymerase chain reaction experiments were performed to detect hygromycin B phosphotransferase cDNA genes with a poly(A) tract in DNA extracted from a pool of about 500 colonies of cells containing cDNA genes. No hygromycin B phosphotransferase cDNA gene with a poly(A) tract was found. Investigation of two preintegration sites by Southern analysis revealed that deletions were present in chromosomal DNA at the site of the integration of the cDNA genes. Naturally occurring processed pseudogenes correspond to the full-length mRNA, contain a poly(A) sequence, and are flanked by direct repeats. Our data indicate that cDNA genes formed by infection with retrovirus particles lack the hallmarks or natural processed pseudogenes. Thus, it appears that natural processed pseudogenes were not generated by retrovirus proteins.     

Thymidine phosphotransferase and nucleotide phosphohydrolase of the fern Asplenium nidus. General properties and inhibition by adenosine 3'':5''-cyclic monophosphate.

1. Extracts of several plant species contained nucleoside-AMP phosphotransferase activity. The ratio of activity with thymidine to that with uridine as nucleoside substrate was essentially constant, both between species and throughout plant development. Evidence is presented that the total thymidine-AMP phosphotransferase activity of the leaves of Asplenium nidus (bird's-nest fern) and of Helianthus tuberosus (Jerusalem artichoke) increases during maturation. 2. Thymidine-AMP phosphotransferase was purified 22-fold from a very rich source of this activity, extracts of A. nidus. 3. A broad specificity towards both nucleoside and nucleoside 5'-monophosphate substrates is displayed by this preparation, and the evidence suggests that all could be due to a single enzyme. 4. Nucleosides that act as substrates will also inhibit phosphotransfer to other nucleosides, with Ki values close to the corresponding Km values found when utilized as substrates. 5. Ca2+-activated ATP phosphohydrolase was separated from the phosphotransferase by differential complexing to Blue Dextran in the presence of urea, whereas an AMP phosphohydrolase activity was closely associated with thymidine-AMP phosphotransferase through all separation techniques used. 6. Metal ions did not activate either of the latter two activities, and 1,10-phenanthroline was found to inhibit the phosphotransferase. 7. Km values for AMP for the respective activities were 0.11 mM (thymidine phosphotransferase) and 0.20 mM (AMP phosphohydrolase) and for thymidine (phosphotransferase only) 0.88 mM. 8. 3':5'-Cyclic AMP was found to inhibit both phosphotransferase and AMP phosphohydrolase activities, with Ki values of 0.056 mM and 0.15 mM respectively. It is suggested that this inhibitor would be of value in revealing the existence of thymidine kinase in plant extracts with high thymidine phosphotransferase activity.     

Phosphorothioate analogues of (2'-5')(A)4: agonist and antagonist activities in intact cells

Metabolically stable phosphorothioate tetramer analogues of (2'-5')(A)n with Rp and/or Sp chirality in the 2'-5'-phosphodiester linkages constitute a new class of antiviral agents since they mimic the effects of interferons. Three of the diastereomeric 5'-monophosphates (i.e., pRpRpRp, pSpRpRp, and pRpSpSp) bind to and activate RNase L from extracts of HeLa cells. However, the pSpSpSp (2'-5')-(A)4-phosphorothioate is unique in that it binds to, but cannot activate, RNase L to cleave rRNA. When microinjected into the cytoplasm of HeLa cells followed by virus infection, the pRpRpRp, pSpRpRp, and pRpSpSp (2'-5')(A)4-phosphorothioates demonstrate antiviral activity, as does (2'-5')(A)4ox-red, an active (2'-5')(A)n analogue. When microinjected simultaneously with (2'-5')(A)nox-red, an active the pSpSpSp (2'-5')(A)4-phosphorothioate inhibits activation of RNase L in HeLa cells, thereby blocking direct protection of vesicular stomatitis virus. The agonist and antagonist properties of pRpRpRp and pSpSpSp, respectively, are transient probably as a consequence of the hydrolysis of the 5'-monophosphate and formation of the less active (2'-5')(A)4-phosphorothioate cores. The possible use of these (2'-5')(A)4-phosphorothioates as tools for dissecting the biological significance of the (2'-5')(A)n system or in antiviral chemotherapy is discussed.     

Subcellular distribution of 2'',5''-oligoadenylate synthetase in differentiating Friend leukemia cells

The occurrence of distinct (2'-5')(A)n-synthetase activities has recently been documented in cytoplasmic and nuclear extracts of several interferon (IFN)-treated cell lines. Since a role has been proposed for (2'-5')(A)n synthetase in the control of cell growth and differentiation, we examined the subcellular distribution of (2'-5')(A)n-synthetase activity both in IFN-treated undifferentiated Friend leukemia cells (FLCs) and during dimethyl-sulfoxide (DMSO)-induced erythroid differentiation of FLCs. Both the nuclear and cytoplasmic (2'-5')(A)n activities were modulated to the same extent by IFNs and DMSO. No evidence for a causal relationship between enzyme activation and FLC differentiation was found.     

Membrane-bound nucleotidase of Bacillus cereus.

A membrane-bound nucleotidase of Bacillus cereus T was solubilized by digestion with trypsin and subsequently purified more than 300-fold. The purified nucleotidase was most active on ribonucleoside 5'-monophosphates and was slightly less active (40 to 60%) on deoxyribonucleoside 5'-monophosphates and ribonucleoside 3'-monophosphates. In addition to hydrolytic activity, the nucleotidase preparation possessed phosphotransferase activity by which phosphate is transferred from a phosphate donor to the 5' position of nucleosides.     

Detection of (2'-5')oligoadenylate binding proteins by nondenaturing polyacrylamide gel electrophoresis and affinity blotting onto nitrocellulose

A latent endoribonuclease, RNase L, binds to and is activated by (2'-5')oligoadenylates ((2'-5')(A)n, n = 2-15). Binding to a labeled derivative of (2'-5')(A)n, [32P](2'-5')(A)3pCp, is detected as a protein-ligand complex observed following nondenaturing polyacrylamide gel electrophoresis. One major binding complex and two minor binding complexes are readily seen in cytoplasmic extracts from Ehrlich ascites tumor cells, murine tissue extracts and rabbit liver tissue extracts. At least one of the more rapidly migrating complexes appears to be a proteolytic degradation product of the larger [32P](2'-5')(A)3pCp binding protein. Cell and tissue extracts containing [32P](2'-5')(A)3pCp binding activity can be immobilized onto nitrocellulose filters and [32P](2'-5')(A)3pCp binding activity detected using a simple, rapid, economical affinity blot assay. Detection of [32P](2'-5')(A)3pCp binding proteins following electrophoresis on nondenaturing polyacrylamide gels and the affinity blot assay significantly improve and simplify the analysis of (2'-5')(A)n binding proteins.     

Flavonoids from seeds of Camellia semiserrata Chi. and their estrogenic activity

Two new flavonol glycosides and three known flavonoids were isolated from seeds of Camellia semiserrata Chi. The structures of these new flavonol glycosides were established as kaempferol 3-O-[(2',3',4'-triacetyl)-alpha-L-rhamnopyranosyl(1-->3)(2',4'-diacetyl)-alpha-L-rhamnopyranosyl (1-->6)-beta-D-glucopyranoside] and kaempferol 3-O-[(3',4'-diacetyl)-alpha-L-rhamnopyranosyl(1-->3)(2',4'-diacetyl)-alpha-L-rhamnopyranosyl(1-->6)-beta-D-glucopyranoside] by spectroscopic methods. The estrogenic activity of these compounds was investigated by a recombinant yeast screening assay.