Light-directed 5′→3′ synthesis of complex oligonucleotide microarrays

Light-directed synthesis of high-density microarrays is currently performed in the 3′→5′ direction due to constraints in existing synthesis chemistry. This results in the probes being unavailable for many common types of enzymatic modification. Arrays that are synthesized in the 5′→3′ direction could be utilized to perform parallel genotyping and resequencing directly on the array surface, dramatically increasing the throughput and reducing the cost relative to existing techniques. In this report we demonstrate the use of photoprotected phosphoramidite monomers for light-directed array synthesis in the 5′→3′ direction, using maskless array synthesis technology. These arrays have a dynamic range of >2.5 orders of magnitude, sensitivity below 1 pM and a coefficient of variance of <10% across the array surface. Arrays containing >150 000 probe sequences were hybridized to labeled mouse cRNA producing highly concordant data (average R² = 0.998). We have also shown that the 3′ ends of array probes are available for sequence-specific primer extension and ligation reactions.     

Aminonucleosides and their derivatives. IVI Synthesis of the 3′-amino-3′-deoxynucleoside 5′-phosphates

A new procedure has been developed for the synthesis of 3′-amino-3′-deoxyribonucleosides of adenine, cytosine and uracil by condensing the trimethylsilylated bases with peracylated 3-azido-3-deoxyribose derivative. The azido group could subsequently be reduced to amino. The 5′-phosphates of these nucleosides have been prepared and the analogues have been tested for their ability to stimulate the ribosome-catalyzed reaction of 3′(2′)-O-(N-formylmethionyl)adenosine 5′-phosphate with phenylalanyl-tRNA.     

Transformation of 2,2′-Bimorphine to the Novel Compounds 10-α-S-Monohydroxy-2,2′-Bimorphine and 10,10′-α,α′-S,S′-Dihydroxy-2,2′-Bimorphine by Cylindrocarpon didymum

Whole-cell suspensions of Cylindrocarpon didymum were observed to transform 2,2′-bimorphine to the compounds 10-α-S-monohydroxy-2,2′-bimorphine and 10,10′-α,α′-S,S′-dihydroxy-2,2′-bimorphine. Mass spectrometry and ¹H nuclear magnetic resonance spectroscopy confirmed the identities of these new morphine alkaloids.     

Identification and characterization of a short 2′–3′ bond-forming ribozyme

A large number of natural and artificial ribozymes have been isolated since the demonstration of the catalytic potential of RNA, with the majority of these catalyzing phosphate hydrolysis or transesterification reactions. Here, we describe and characterize an extremely short ribozyme that catalyzes the positionally specific transesterification that produces a 2′–3′ phosphodiester bond between itself and a branch substrate provided in trans, cleaving itself internally in the process. Although this ribozyme was originally derived from constructs based on snRNAs, its minimal catalytic motif contains essentially no snRNA sequence and the reaction it catalyzes is not directly related to either step of pre-mRNA splicing. Our data have implications for the intrinsic reactivity of the large amount of RNA sequence space known to be transcribed in nature and for the validity and utility of the use of protein-free systems to study pre-mRNA splicing.     

The Antibacterial Properties of 4, 8, 4′, 8′-Tetramethoxy (1,1′-biphenanthrene) -2,7,2′,7′-Tetrol from Fibrous Roots of Bletilla striata

Biphenanthrene compound, 4, 8, 4′, 8′-tetramethoxy (1, 1′-biphenanthrene)—2, 7, 2′, 7′-tetrol (LF05), recently isolated from fibrous roots of Bletilla striata, exhibits antibacterial activity against several Gram-positive bacteria. In this study, we investigated the antibacterial properties, potential mode of action and cytotoxicity. Minimum inhibitory concentrations (MICs) tests showed LF05 was active against all tested Gram-positive strains, including methicillin-resistant Staphylococcus aureus (MRSA) and staphylococcal clinical isolates. Minimum bactericidal concentration (MBC) tests demonstrated LF05 was bactericidal against S. aureus ATCC 29213 and Bacillus subtilis 168 whereas bacteriostatic against S. aureus ATCC 43300, WX 0002, and other strains of S. aureus. Time-kill assays further confirmed these observations. The flow cytometric assay indicated that LF05 damaged the cell membrane of S. aureus ATCC 29213 and B. subtilis 168. Consistent with this finding, 4 × MIC of LF05 caused release of ATP in B. subtilis 168 within 10 min. Checkerboard test demonstrated LF05 exhibited additive effect when combined with vancomycin, erythromycin and berberine. The addition of rat plasma or bovine serum albumin to bacterial cultures caused significantly loss in antibacterial activity of LF05. Interestingly, LF05 was highly toxic to several tumor cells. Results of these studies indicate that LF05 is bactericidal against some Gram-positive bacteria and acts as a membrane structure disruptor. The application of biphenanthrene in the treatment of S. aureus infection, especially local infection, deserves further study.     

Cyclic Adenosine 3′,5′-Monophosphate in Escherichia coli

The concentration of cyclic adenosine 3',5'-monophosphate (c-AMP) in Escherichia coli growing on different sources of carbon was studied. Cultures utilizing a source of carbon that supported growth relatively poorly had consistently higher concentrations of c-AMP than did cultures utilizing sugars that supported rapid growth. This relationship was also observed in strains defective in c-AMP phosphodiesterase and simultaneously resistant to catabolite repression; in such strains the c-AMP concentration was slightly higher for several sources of carbon tested. Cultures continued to synthesize c-AMP and secreted it into the medium, under conditions that brought about an inhibition of the intracellular accumulation of the cyclic nucleotide. Transient repression of the synthesis of beta-galactosidase was not associated with an abrupt decrease in the cellular concentration of c-AMP.     

Effects of Adenosine 3′,5′-Monophosphate and Adenosine 5′-Monophosphate on Glycogen Degradation and Synthesis in Dictyostelium discoideum

Data are presented demonstrating that the presence in vivo of adenosine 3',5'-monophosphate (3',5'-AMP) causes a rapid depletion of glycogen storage material in the cellular slime mold. The effect of adenosine 5'-monophosphate (5'-AMP) is twofold, stimulating both glycogen degradation and synthesis. In pseudoplasmodia, cell-free extracts appear to contain at least two species of glycogen phosphorylase, one of which is severely inhibited by glucose-1-phosphate and another which is only partially inhibited by this hexose-phosphate. In some cases, 5'-AMP partially overcomes the inhibition by glucose-1-phosphate. Data presented here also indicate the existence of two forms of glycogen synthetase, the total activity of which does not change during 10 hr of differentiation from aggregation to culmination. During this period there is a quantitative conversion of glucose-6-phosphate-independent enzyme activity to glucose-6-phosphate-dependent activity. It is suggested that one effect of 3',5'-AMP is closely related to enzymatic processes involved in the rapid conversion of glycogen to cell wall material and other end products accumulating during sorocarp construction.     

Role of PCNA-dependent stimulation of 3′-phosphodiesterase and 3′–5′ exonuclease activities of human Ape2 in repair of oxidative DNA damage

Human Ape2 protein has 3′ phosphodiesterase activity for processing 3′-damaged DNA termini, 3′–5′ exonuclease activity that supports removal of mismatched nucleotides from the 3′-end of DNA, and a somewhat weak AP-endonuclease activity. However, very little is known about the role of Ape2 in DNA repair processes. Here, we examine the effect of interaction of Ape2 with proliferating cell nuclear antigen (PCNA) on its enzymatic activities and on targeting Ape2 to oxidative DNA lesions. We show that PCNA strongly stimulates the 3′–5′ exonuclease and 3′ phosphodiesterase activities of Ape2, but has no effect on its AP-endonuclease activity. Moreover, we find that upon hydrogen-peroxide treatment Ape2 redistributes to nuclear foci where it colocalizes with PCNA. In concert with these results, we provide biochemical evidence that Ape2 can reduce the mutagenic consequences of attack by reactive oxygen species not only by repairing 3′-damaged termini but also by removing 3′-end adenine opposite from 8-oxoG. Based on these findings we suggest the involvement of Ape2 in repair of oxidative DNA damage and PCNA-dependent repair synthesis.     

Adenosine 3′,5′-Cyclic Monophosphate-Deficient Mutants of Vibrio cholerae

Two adenosine 3',5'-cyclic monophosphate (AMP)-deficient mutants of Vibrio cholerae (biotype El Tor) were successfully isolated by nitrosoguanidine treatment followed by pencillin screening for pleiotropic sugar-negative clones. Exogenous cyclic AMP is required for the fermentation of sucrose, trehalose, fructose, maltose, and mannose but not of glucose, as well as for the formation of normal flagella and specific somatic antigens. A striking characteristic of the mutants is their growth behavior at higher temperatures. They cannot grow on TCBS selective plates at 37 C or higher unless they are provided with a supply of exogenous cyclic AMP, although they are capable of producing colonies on the same medium, even without cyclic AMP, at temperatures lower than 30 C. Since the mutants are converted to spheroplasts, spindle forms, and spiral filaments in cyclic AMP-free media at 37 C, and this phenomenon is stopped by the addition of cyclic AMP or a combination of 20% sucrose and 0.2% magnesium chloride, it is assumed that cyclic AMP is essential for the synthesis of the cell wall of V. cholerae at higher temperatures.     

Effect of nucleoside 5′-di- and 5′-tri-phosphates on pancreatic ribonuclease activity

1. ADP, ATP and GDP inhibited the phosphotransferase activity, the release of cyclic nucleotides from RNA, of ribonuclease. No significant inhibition was elicited by pyrimidine 5'-nucleoside diphosphates, CDP and UDP. 2. Inhibition by ADP, AMP, adenosine, adenine, NAD and NADP was insignificant at the concentrations tested. Small inhibition was observed with high concentrations of AMP and only when soluble RNA was the substrate. 3. Inhibition by ADP was found to be ;uncompetitive'. 4. Results seem to indicate that at least for optimum inhibition the polyphosphate of the purine nucleoside is essential. They further suggest that the inhibitor acts by combining with the enzyme only when the enzyme is bound to the substrate.     

3′,5′-Cyclic Adenosine Monophosphate-Requiring Mutants of Escherichia coli

Mutants that require exogenous 3',5'-cyclic adenosine monophosphate (cAMP) for exponential growth were isolated from strains deficient in adenyl cyclase. Studies of one strain showed that cAMP is not incorporated into macromolecules; instead, it seems to have a regulatory function, i.e., in media lacking cAMP, cells form ribonucleic acid (RNA) and protein at linear rather than exponential rates. The exact lesion is not known; ribosomes, messenger RNA, and the beta and beta' subunits of RNA polymerase continue to be made in absence of added cAMP.     

Cyclic 3′,5′-Adenosine Monophosphate Phosphodiesterase of Escherichia coli

The cyclic 3',5'-adenosine monophosphate (c-AMP) phosphodiesterase from Escherichia coli has been partially purified. The enzyme has an apparent molecular weight of 30,000, a Michaelis constant of 0.5 mM c-AMP, and a pH optimum of 7. The partially purified enzyme requires for activity the presence of a reducing compound and of either iron or a protein which seemingly acts as iron carrier.     

Uncoupling of Protein and Ribonucleic Acid Synthesis by 5′,5′,5′-Trifluoroleucine in Salmonella typhimurium

The addition of 5',5',5'-trifluoroleucine (fluoroleucine) to leucine auxotrophs of Salmonella typhimurium permitted protein but not ribonucleic acid (RNA) synthesis to continue after leucine depletion. The uncoupling of the formation of these macromolecules by fluoroleucine was apparent if RNA and protein synthesis was measured either by the uptake of radioactive precursors or by direct chemical determinations. The analogue did not appear to be an inhibitor of RNA formation, since it was as effective as leucine in permitting RNA synthesis in a leucine auxotroph upon the addition of small amounts of chloramphenicol. In contrast to these data, fluoroleucine allowed continued protein and RNA formation in a leucine auxotroph of Escherichia coli strain W. In addition, contrary to the results obtained with S. typhimurium, the analogue replaced leucine for repression of the leucine bio-synthetic enzymes as well as the isoleucine-valine enzymes. We propose that these ambivalent effects of fluoroleucine on repression and RNA and protein synthesis in the two strains are due to differences in the ability of the analogue to attach to the various species of leucine transfer RNA.     

Cyclic Adenosine 3′,5′-Monophosphate and Morphogenesis in Mucor racemosus

Yeastlike cells of Mucor racemosus grown under 100% CO(2) underwent morphogenesis to hyphae after exposure to air. The addition of dibutyryl cyclic adenosine monophosphate (dbcAMP) to yeastlike cultures inhibited this morphogenesis in media containing 2% glucose. The maintenance of uniformly spherical, budding cells required 1 mM dbcAMP in a defined medium containing Casamino Acids, and 3 mM dbcAMP in a medium containing yeast extract and peptone. At these concentrations, dbcAMP also induced yeastlike development in young aerobic hyphae grown in media containing 2% glucose. Removal of dbcAMP resulted in hyphal development. The endogenous cyclic AMP (cAMP) content of yeastlike cultures was measured after a shift from CO(2) to air. A fourfold decrease in intracellular cAMP preceded the appearance of hyphal germ tubes. These results indicate that cAMP plays a role in the control of morphogenesis in Mucor racemosus.     

The Emergence of Alternative 3′ and 5′ Splice Site Exons from Constitutive Exons

Alternative 3′ and 5′ splice site (ss) events constitute a significant part of all alternative splicing events. These events were also found to be related to several aberrant splicing diseases. However, only few of the characteristics that distinguish these events from alternative cassette exons are known currently. In this study, we compared the characteristics of constitutive exons, alternative cassette exons, and alternative 3′ss and 5′ss exons. The results revealed that alternative 3′ss and 5′ss exons are an intermediate state between constitutive and alternative cassette exons, where the constitutive side resembles constitutive exons, and the alternative side resembles alternative cassette exons. The results also show that alternative 3′ss and 5′ss exons exhibit low levels of symmetry (frame-preserving), similar to constitutive exons, whereas the sequence between the two alternative splice sites shows high symmetry levels, similar to alternative cassette exons. In addition, flanking intronic conservation analysis revealed that exons whose alternative splice sites are at least nine nucleotides apart show a high conservation level, indicating intronic participation in the regulation of their splicing, whereas exons whose alternative splice sites are fewer than nine nucleotides apart show a low conservation level. Further examination of these exons, spanning seven vertebrate species, suggests an evolutionary model in which the alternative state is a derivative of an ancestral constitutive exon, where a mutation inside the exon or along the flanking intron resulted in the creation of a new splice site that competes with the original one, leading to alternative splice site selection. This model was validated experimentally on four exons, showing that they indeed originated from constitutive exons that acquired a new competing splice site during evolution.     

Termination of Deoxyribonucleic Acid in Escherichia coli by 2′,3′-Dideoxyadenosine

2′,3′-Dideoxyadenosine was previously shown to be lethal to Escherichia coli and to inhibit deoxyribonucleic acid (DNA) synthesis irreversibly in this organism. It was also shown that triphosphate of this analogue terminates DNA chains in an in vitro system. Data presented here show that the nucleoside is relatively insensitive to E. coli adenosine deaminase and is converted intracellularly into the dideoxynucleotide, including the triphosphate. Thymine nucleotide pools were not reduced in inhibited bacteria, nor did preformed DNA break down. Some adenine was liberated from the dideoxyadenosine on incubation, and the latter was incorporated into ribonucleic acid. Nevertheless, about 4,000 molecules of the dideoxynucleoside were incorporated into DNA per cell. The dideoxynucleotide occurred in DNA chains in a terminal position, liberated selectively by venom phosphodiesterase. The possible nature of the lethal event is discussed.     

Cyclic Guanosine 3′,5′-Monophosphate in the Dimorphic Fungus Mucor racemosus

The dimorphic fungus Mucor racemosus was found to contain the cyclic nucleotide guanosine 3′,5′-monophosphate (cGMP). Approximately equivalent amounts of the compound were found in ungerminated spores, yeastlike cells, and mycelia. Germinating spores contained severalfold higher amounts of cGMP than the other cell forms. cGMP levels did not change significantly during the morphogenetic conversion of yeast to mycelia. Added exogenous cGMP or the dibutyryl derivative did not influence cell morphology in any way and did not alter the effect that cyclic adenosine 3′,5′-monophosphate has upon cell morphology.     

Requirement of Cyclic Adenosine 3′,5′-Monophosphate for the Thermosensitive Effects of Rts1 in a Cyclic Adenosine 3′,5′-Monophosphate-Less Mutant of Escherichia coli

Previous publications showed that a covalently closed circular (CCC) Rts1 plasmid deoxyribonucleic acid (DNA) that confers kanamycin resistance upon the host bacteria inhibits host growth at 42 degrees C but not at 32 degrees C. At 42 degrees C, the CCC Rts1 DNA is not formed, and cells without plasmids emerge. To investigate the possible role of cyclic adenosine 3',5'-monophosphate (cAMP) in the action of Rts1 on host bacteria, Rts1 was placed in an Escherichia coli mutant (CA7902) that lacks adenylate cyclase or in E. coli PP47 (a mutant lacking cAMP receptor protein). Rts1 did not exert the thermosensitive effect on these cells, and CCC Rts1 DNA was formed even at 42 degrees C. Upon addition of cAMP to E. coli CA7902(Rts1), cell growth and formation of CCC Rts1 DNA were inhibited at 42 degrees C. The addition of cAMP to E. coli PP47(Rts1) did not cause inhibitory effects on either cell growth or CCC Rts1 DNA formation at 42 degrees C. The inhibitory effect of cAMP on E. coli CA7902(Rts1) is specific to this cyclic nucleotide, and other cyclic nucleotides such as cyclic guanosine 3',5'-monophosphate did not have the effect. For this inhibitory effect, cells have to be preincubated with cAMP; the presence of cAMP at the time of CCC Rts1 DNA formation is not enough for the inhibitory effect. If the cells are preincubated with cAMP, one can remove cAMP during the [(3)H]thymidine pulse and still observe its inhibitory effect on the formation of CCC Rts1 DNA. The presence of chloramphenicol during this preincubation period abolished the inhibitory effect of cAMP. These observations suggest that cAMP is necessary to induce synthesis of a protein that inhibits CCC Rts1 DNA formation and cell growth at 42 degrees C.