Control of Isoleucine, Valine, and Leucine Biosynthesis VI. Effect of 5′,5′,5′-Trifluoroleucine on Repression in Salmonella typhimurium

The leucine analogue 5',5',5',-trifluoroleucine (fluoroleucine) replaced leucine for repression of the isoleucine-valine biosynthetic enzymes in Salmonella typhimurium. In contrast, the analogue had no effect on derepression of the leucine biosynthetic enzymes in leucine auxotrophs grown on limiting amounts of leucine. The effect of fluoroleucine on repression appeared to be specific for leucine since derepression of the isoleucine-valine enzymes due to an isoleucine or valine limitation was not affected by the analogue. The prevention of derepression by fluoroleucine was probably due to repression and not to the formation of false proteins, since the analogue had no effect on the derepression of a number of enzymes unrelated to the isoleucine-valine pathway. Fluoroleucine was able to attach to leucine transfer ribonucleic acid (tRNA) as evidenced by the ability of the analogue to protect about 70% of leucine tRNA from oxidation by periodate. We propose that the differential effects of fluoroleucine on repression are due to differences in the ability of the analogue to bind to the various species of leucine tRNA.     

Hepatic microsomal enzyme induction by 2, 2', 3, 3', 4, 4'- and 2, 2', 3', 4, 4', 5-hexachlorobiphenyl

In an attempt to resolve conflicting reports in the literature, the effects of 2, 2', 3, 3', 4, 4'-hexa- and 2, 2', 3', 4, 4', 5-hexachlorobiphenyl on the hepatic microsomal drug-metabolizing enzymes were evaluated in the immature male rat. By comparison with the effects of the classical enzyme inducers, phenobarbitone (PB) and 3-methylcholanthrene (MC), 2, 2', 3, 3', 4, 4'-hexachlorobiphenyl, irrespective of its synthetic route, exhibited PB- and MC-type characteristics; the most prominent feature of the latter being a seven-fold increase in 4-chlorobiphenyl hydroxylase activity. 2, 2', 3', 4, 4', 5-Hexachlorobiphenyl also resembled a mixed (PB + MC)-type inducer although its MC-type characteristics were more pronounced than those of 2, 2', 3, 3', 4, 4'-hexachlorobiphenyl.     

Synthesis and properties of 2'-O,4'-C-methyleneoxymethylene bridged nucleic acid

A novel bridged nucleic acid (BNA) analogue, 2'-O,4'-C-methyleneoxymethylene bridged nucleic acid (2',4'-BNA(COC)), was synthesized and incorporated into oligonucleotides. The 2',4'-BNA(COC) modified oligonucleotides showed high binding affinity with an RNA complement and significant enzymatic stability against snake venom phosphodiesterase.     

Synthesis and properties of oligonucleotides containing 5-formyl-2′-deoxycytidine: in vitro DNA polymerase reactions on DNA templates containing 5-formyl-2′-deoxycytidine

Oligodeoxynucleotides (ODNs) containing 5-formyl-2′-deoxycytidine (fC) were synthesized by the phosphoramidite method and subsequent oxidation with sodium periodate. The stabilities of duplexes containing A, G, C or T opposite fC were studied by thermal denaturation. It was found that fC:A, fC:C or fC:T base pairs significantly reduce the thermal stabilities of duplexes. Next, single nucleotide insertion reactions were performed using ODNs containing fC as templates and the Klenow fragment of Escherichia coli DNA polymerase I. It was found that: (i) insertion of dGMP opposite fC appears to be less efficient relative to insertion opposite 5-methyl-2′-deoxycytidine (mC); (ii) dAMP is misincorporated more frequently opposite fC than mC, although the frequency of misincorporation seems to be dependent on the sequence; (iii) TMP is misincorporated more frequently opposite fC than mC. These results suggest that fC may induce the transition mutation C·G→T·A and the transversion mutation C·G→A·T during DNA synthesis.     

Metabolism and biological potency of 5,6-monoepoxy-β-carotene and 5,6:5′,6′-diepoxy-β-carotene

1. 5,6-Monoepoxy-beta-carotene and 5,6:5',6'-diepoxy-beta-carotene were partially converted into the furanoid forms during passage through the rat stomach. 2. The monoepoxide was converted into vitamin A in the small intestine and showed a biological potency 21% of that of beta-carotene. Neither beta-carotene nor 5,6-monoepoxyvitamin A was formed. 3. Intraperitoneal administration of the monoepoxide led to the accumulation of the unchanged compound in the liver and other tissues. 4. The diepoxide gave no beta-carotene or vitamin A or 5,6-monoepoxyvitamin A when given orally and showed no biological potency. 5. The significance of these results with special reference to the mechanism of formation of vitamin A from beta-carotene is discussed.     

Comparison between properties of 2'-O,4'-C-ethylene-bridged nucleic acid (ENA) phosphorothioate oligonucleotides and N3'-P5' thiophosphoramidate oligonucleotides

Synthesis and properties of an oligonucleotide uniformly modified with 2'-O,4-C-ethylene-bridged nucleic acid (ENA) units were compared with those of GRN163, which is modified with N3'-P5' thiophosphoramidates, with the sequence targeting human telomerase RNA subunit. Although an ENA phosphorothioate oligonucleotide, ENA-13, could be synthesized using ENA phosphoramidites on a 100-mg scale, synthesis of GRN163 was very hard even on a 1-micomol scale. In view of both stability of the duplex formation with complementary RNA and the efficiency of cellular uptake by endocytosis, ENA-13 was superior to GRN163. These findings suggest that ENA-13 has useful properties for antisense therapeutic application.     

Release of the β-Galactosidase-Synthesizing System from Ultraviolet Catabolite Repression by Cyclic 3′,5′-Adenosine Monophosphate, Dark Repair, Photoreactivation, and Cold Treatment

Recovery from the inhibitory effect of ultraviolet irradiation on the induced synthesis of beta-galactosidase was studied in Escherichia coli B/r. When irradiated cells (520 ergs/mm(2) at 254 nm) were induced and incubated in minimal medium supplemented with Casamino Acids (conditions of catabolite repression), the ability to form enzyme was greatly reduced for about 100 min and then recovery began. The inhibition observed immediately after ultraviolet irradiation was partially reversed by cyclic 3',5'-adenosine monophosphate (cyclic AMP) or by photoreactivation treatment. Inhibition was reduced if the cells were given cold treatment (5 C) before or during irradiation; the kinetics of induced enzyme formation in each case were similar to those of irradiated cells receiving cyclic AMP. These kinetics suggest that the cold treatments, like cyclic AMP, cause the release of the beta-galactosidase-synthesizing system from catabolite repression. When irradiated cells were incubated for various times before cyclic AMP or photoreactivation treatment, some reversal of the inhibition of induced enzyme formation was obtained, but by 100 min the treatments were ineffective. Because 100 min was also the time at which dark recovery of enzyme formation began, the recovery process was interpreted to be the result of completion of DNA repair, which, in turn, released the beta-galactosidase-synthesizing system from catabolite repression.     

Synthesis and properties of 3'-amino-2',4'-BNA, a bridged nucleic acid with a N3'-->P5' phosphoramidate linkage

The synthesis and properties of a bridged nucleic acid analogue containing a N3'-->P5' phosphoramidate linkage, 3'-amino-2',4'-BNA, is described. A heterodimer containing a 3'-amino-2',4'-BNA thymine monomer, and thymine and methylcytosine monomers of 3'-amino-2',4'-BNA and their 5'-phosphoramidites, were synthesized efficiently. The dimer and monomers were incorporated into oligonucleotides by conventional 3'-->5' assembly, and 5'-->3' reverse assembly phosphoramidite protocols, respectively. Compared to a natural DNA oligonucleotide, modified oligonucleotides containing the 3'-amino-2',4'-BNA residue formed highly stable duplexes and triplexes with single-stranded DNA (ssDNA), single-stranded RNA (ssRNA), and double-stranded DNA (dsDNA) targets, with the average increase in melting temperature (T(m)) against ssDNA, ssRNA and dsDNA being +2.7 to +4.0 degrees C, +5.0 to +7.0 degrees C, and +5.0 to +11.0 degrees C, respectively. These increases are comparable to those observed for 2',4'-BNA-modified oligonucleotides. In addition, an oligonucleotide modified with a single 3'-amino-2',4'-BNA thymine residue showed extraordinarily high resistance to nuclease degradation, much higher than that of 2',4'-BNA and substantially higher even than that of 3'-amino-DNA and phosphorothioate oligonucleotides. The above properties indicate that 3'-amino-2',4'-BNA has significant potential for antisense and antigene applications.     

Direct adenylation from 5′-OH-terminated oligonucleotides by a fusion enzyme containing Pfu RNA ligase and T4 polynucleotide kinase

5′-Adenylated oligonucleotides (AppOligos) are widely used for single-stranded DNA/RNA ligation in next-generation sequencing (NGS) applications such as microRNA (miRNA) profiling. The ligation between an AppOligo adapter and target molecules (such as miRNA) no longer requires ATP, thereby minimizing potential self-ligations and simplifying library preparation procedures. AppOligos can be produced by chemical synthesis or enzymatic modification. However, adenylation via chemical synthesis is inefficient and expensive, while enzymatic modification requires pre-phosphorylated substrate and additional purification. Here we cloned and characterized the Pfu RNA ligase encoded by the PF0353 gene in the hyperthermophilic archaea Pyrococcus furiosus. We further engineered fusion enzymes containing both Pfu RNA ligase and T4 polynucleotide kinase. One fusion enzyme, 8H-AP, was thermostable and can directly catalyze 5′-OH-terminated DNA substrates to adenylated products. The newly discovered Pfu RNA ligase and the engineered fusion enzyme may be useful tools for applications using AppOligos.     

Circular dichroism of nucleic acid monomers. I. Calculated adenosine and 2′-deoxyadenosine CD spectra

A combination of the DeVoe and Kirkwood polarizability concepts is developed to calculate CD spectra of nucleic acid monomers. The method is perfectly general and applies to any system where the constituents have absorption properties which are widely separated in terms of frequency. The theory is applied to calculate the CD spectra of adenosine and 2′-deoxyadenosine conformers. Bond polarizabilities are evaluated for the ribosyl moiety of adenosine, as a function of glycosidic rotational angles and polarizability anisotropies. It is found that a wide range of C-C and C-O bond polarizabilities give similar CD results. Isotropic atom polarizabilities are also evaluated. It is found that the CD results using these polarizabilities do not differ significantly from those obtained with bond polarizabilities. The CD spectra of adenosine and 2′-deoxyadenosine are calculated for three x-ray diffraction determined geometries: A-form RNA, B-form DNA, and C-form DNA. The results indicate that the monomer CD spectra are strongly dependent on the precise geometry and appear to be of importance in understanding the spectra of oligomers and polymers. The deoxyadenosine conformers are found to have calculated CD spectra which are less intense than those of the ribosyl conformers. These results indicate that the measured differences between the CD magnitudes of ribo- and deoxyriboadenosine are due to the presence or absence of the 2′-hydroxyl. Weighted averaged adenosine CD spectra are calculated with the aid of probability distributions from conformational energy calculations. The results suggest a new method for obtaining empirical monomer parameters for use in optical calculations. The calculations in this paper indicate for the first time that DeVoe theory, in combination with the Kirkwood theory, provides a useful method for the calculation of the CD spectra of nonpolymeric molecules.     

Triplex formation involving 2'-O,4'-C-methylene bridged nucleic acid (2',4'-BNA) with 2-pyridone base analogue: efficient and selective recognition of C:G interruption

For the effective recognition of C:G interruption in homopurine-homopyrimidine duplex DNA, we examined triplex-forming ability and sequence-selectivity of a triplex-forming oligonucleotide (TFO) involving of 2'-O,4'-C-methylene bridged nucleic acid with 2-pyridone base analogue. We found that the modified TFO formed stable triplex with high binding affinity and sequence-selectivity.     

Significance of Enhanced Morphological Detection of Cryptosporidium sp. Oocysts in Water Concentrates Determined by Using 4′,6′-Diamidino-2-Phenylindole and Immunofluorescence Microscopy

Of 2,361 water concentrates analyzed for the presence of Cryptosporidium spp. oocysts between January 1992 and May 1998, 269 (11.4%) were positive, of which 235 (87.4%) were raw and 34 were final water concentrates. Of 740 oocysts enumerated in positive samples, 656 oocysts (88.7%) were detected in raw and 84 oocysts (11.3%) were detected in final water concentrates by using a commercially available fluorescein isothiocyanate-labeled anti-Cryptosporidium sp. monoclonal antibody and the nuclear fluorogen 4′,6′-diamidino-2-phenylindole (DAPI). Of raw water positive samples, 66.8% had oocysts that contained nuclei, while 58.8% of final water samples had oocysts that contained nuclei. The most frequently identified oocysts had either no DAPI-positive nuclei and no internal morphology according to Nomarski differential interference-contrast microscopy (DIC) or four DAPI-positive nuclei together with internal contents according to DIC (39.5 and 32.8% of raw and 42.9 and 30.9% of final water positives, respectively). By use of the presence of DAPI-stained nuclei to support oocyst identification based upon oocyst wall fluorescence, 56.5% of oocysts were identified when at least one nucleus was present, while increasing the number of nuclei necessary for identification to four reduced the percentage identifiable to 32.8% in raw water concentrates. In final water concentrates, 51% of oocysts were identified using oocyst wall fluorescence and the presence of at least one nucleus, while increasing the number of nuclei necessary for identification to four reduced the percentage identifiable to 30.9%. By consolidating our identification criteria from the presence of at least one nucleus to the presence of four nuclei, we excluded approximately 20% of oocysts in either water type. Approximately 40% of oocysts detected in these United Kingdom samples were empty and could not be detected by alternative methods, including the PCR and fluorescence in situ hybridization.     

Triplex formation involving 2'-O,4'-C-methylene bridged nucleic acid (2',4'-BNA) with 1-isoquinolone base analogue: efficient and selective recognition of C:G interruption

For the effective recognition of C x G interruption in homopurine-homopyrimidine duplex DNA, we examined triplex-forming ability and sequence-selectivity of a triplex-forming oligonucleotide (TFO) involving of 2'-O, 4'-C-methylene bridged nucleic acid with 1-isoquinolone base analogue. We found that the modified TFO formed stable triplex with high binding affinity and sequence-selectivity.     

Stereoselective synthesis of fully protected (2S,4S,6S)‐2‐amino‐6‐hydroxy‐4‐methyl‐8‐oxodecanoic acid (AHMOD)

The stereocontrolled synthesis of fully protected (2S,4S,6S)‐2‐amino‐6‐hydroxy‐4‐methyl‐8‐oxodecanoic acid was accomplished using a glutamate derivative as starting material. The key steps of this stereochemical synthetic pathway involved an Evans asymmetric alkylation, a Sharpless asymmetric epoxidation, and a Grignard reaction. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

RNA 5′-Triphosphatase, Nucleoside Triphosphatase, and Guanylyltransferase Activities of Baculovirus LEF-4 Protein

Autographa californica nuclear polyhedrosis virus late and very late mRNAs are transcribed by an RNA polymerase consisting of four virus-encoded polypeptides: LEF-8, LEF-9, LEF-4, and p47. The 464-amino-acid LEF-4 subunit contains the signature motifs of GTP:RNA guanylyltransferases (capping enzymes). Here, we show that the purified recombinant LEF-4 protein catalyzes two reactions involved in RNA cap formation. LEF-4 is an RNA 5′-triphosphatase that hydrolyzes the γ phosphate of triphosphate-terminated RNA and a guanylyltransferase that reacts with GTP to form a covalent protein-guanylate adduct. The RNA triphosphatase activity depends absolutely on a divalent cation; the cofactor requirement is satisfied by either magnesium or manganese. LEF-4 also hydrolyzes ATP to ADP and P_i (K_m = 43 μM ATP; V_max = 30 s⁻¹) and GTP to GDP and P_i. The LEF-4 nucleoside triphosphatase (NTPase) is activated by manganese or cobalt but not by magnesium. The RNA triphosphatase and NTPase activities of baculovirus LEF-4 resemble those of the vaccinia virus and Saccharomyces cerevisiae mRNA capping enzymes. We suggest that these proteins comprise a novel family of metal-dependent triphosphatases.     

Promotion of triplex formation by 2'-O,4'-C-methylene bridged nucleic acid (2',4'-BNA) modification: thermodynamic and kinetic studies

We analyzed the effect of 2'-O,4'-C-methylene bridged nucleic acid (2',4'-BNA) modification of triplex-forming oligonucleotide (TFO) on pyrimidine motif triplex formation at neutral pH, a condition where pyrimidine motif triplexes are unstable. The binding constant of the pyrimidine motif triplex formation at pH 6.8 with 2',4'-BNA modified TFO was about 20 times larger than that observed with unmodified TFO. The observed increase in the binding constant at neutral pH by the 2',4'-BNA modification resulted from the considerable decrease in the dissociation rate constant.     

AarI, a restriction endonuclease from Arthrobacter aurescens SS2-322, which recognizes the novel non-palindromic sequence 5′-CACCTGC(N)4/8-3′

A new type II restriction endonuclease AarI has been isolated from Arthrobacter aurescens SS2-322. AarI recognizes the non-palindromic heptanucleotide sequence 5′-CACCTGC(N)_4/8-3′ and makes a staggered cut at the fourth and eighth bases downstream of the target duplex producing a four base 5′-protruding end. AarI activity is stimulated by oligodeoxyribonucleotide duplexes containing an enzyme-specific recognition sequence.