Long-distance RNA-RNA interactions and conserved sequence elements affect potato virus X plus-strand RNA accumulation.

Conserved octanucleotide sequences located upstream of two major potato virus X (PVX) subgenomic RNAs (sgRNAs), as well as elements in the 5' end of the genome, affect accumulation of sgRNA. To determine if complementarity between these sequences is important for PVX RNA accumulation, we analyzed the effects of mutations within these elements and compensatory mutations in a tobacco protoplast system and in plants. Mutations in the 5' nontranslated region (NTR mutants) that reduced complementarity resulted in lower genomic RNA (gRNA) and sgRNA levels, whereas mutations to the octanucleotide elements affected only the corresponding sgRNA levels. However, for both the NTR and octanucleotide mutants, the extent of reductions in RNA levels did not directly correlate with the degree of complementarity, suggesting that the sequences of these elements are also important. Mutants containing changes in the NTR and compensatory changes in one of the octanucleotide elements restored levels of gRNA and the other sgRNA species with an unaltered octanucleotide element to those of wild-type. Although compensatory changes significantly increased levels of the sgRNA species with the modified octanucleotide element, levels were not restored to those of wild-type. Our data indicate that long distance RNA-RNA interactions and the sequences of the interacting elements are required for PVX plus-strand RNA accumulation.     

Probing intercalation and conformational effects of the anticancer drug 2-methyl-9-hydroxyellipticinium acetate in DNA fragments with circular dichroism.

Circular dichroism was applied to the analysis of drug-DNA associations. With the octanucleotide d(TGACGTCA) (octanucleotide I), which is the cAMP-responsive element (CRE) in gene promoters and its reverse d(ACTGCAGT) (octanucleotide II), it was demonstrated that the anticancer polyaromatic agent celiptium intercalates into DNA base pairs with its long direction perpendicular to both the DNA-helix axis and the base-pair long axis and induces larger conformational changes in the CpG-containing octanucleotide I CRE than in its reverse-sequence octanucleotide II. It was concluded that CD is a powerful and sensitive technique to discriminate between drug-binding modes of DNA, to define the geometry of the chromophore inserted into base pairs and, finally, to measure sequence-dependent conformational changes induced by intercalation in DNA. We anticipate that these studies will contribute to a better understanding of the molecular bases that underlie the mechanism of action of those cytotoxic drugs which interfere with the DNA-nuclear-protein recognition.     

Helix-coil transition of the self-complementary dG-dG-dA-dA-dT-dT-dC-dC duplex.

The helix-coil transition of the octanucleotide self-complementary duplex dG-dG-dA-dA-dT-dT-dC-dC has been monitored at the Watson-Crick protons, the base and sugar nonexchangeable protons and the backbone phosphates by high-resolution nuclear magnetic resonance (NMR) spectroscopy. The melting transition of the octanucleotide monitored by ultraviolet absorbance spectroscopy is characterized by the thermodynamic parameters delta H degree = -216.7 kJ/mol and delta S degree (25 degrees C) = -0.632 KJ mol-1 K-1 in 0.1 M NaCl, 10 mM phosphate solution. Correlation of the transition midpoint values monitored by the ultraviolet absorbance studies at strand concentrations below 0.2 mM and by NMR studies at 5.3 mM suggest that both methods are monitoring the octanucleotide duplex-to-strand transition. The NMR spectra of the Watson-Crick ring NH protons of the octanucleotide duplex have been followed as a function of temperature. The resonance from the terminal dG.dC base pairs broadens out at room temperature while the resonances from the other base pairs broaden simultaneously with the onset of the melting transition. The nonexchangeable base and sugar H-1' protons are resolved in the duplex and strand states and shift as average peaks through the melting transition. The experimental shifts on duplex formation have been compared with calculated values based on ring-current and atomic diamagnetic anisotropy contributions for a B-DNA base-pair-overlap geometry in solution. Several nonexchangeable proton resonances broaden in the fast-exchange region during the duplex-to-strand transition and the excess widths yield a duplex dissociation rate constant for the octanucleotide of 1.9 x 10(3) s-1 at 32 degrees C (fraction of duplex = 0.86) in 0.1 M NaCl, 10 mM phosphate buffer. The 31P resonances of the seven internucleotide phosphates are distributed over 0.6 ppm in the duplex state, shift downfield during the duplex-to-strand transition and undergo additional downfield shifts during the stacked-to-unstacked strand transition with increasing temperature.     

Development of a colorimetric test system for detection of point mutations via ligation of a tandem of short oligonucleotides on methacrylate beads

A new approach for detection of point mutations has been developed. The nonradioactive test system proposed is based on enzymatic ligation of a tandem of three short oligonucleotides B∼pN₈+pN₄+pN′₈ Bio in the presence of a complementary DNA template. The 5′-terminal octanucleotide B∼pN₈ is immobilized on polymer methacrylate beads (B) and the 3′-terminal octanucleotide pN′₈ Bio contains a biotin residue at the 3′-phosphate. Ligation of the tandem produces a 20-mer biotinylated oligonucleotide on a polymer bead, which is then visualized via subsequent treatments with streptavidin-alkaline phosphatase conjugate and chromogenic substrates. Intense staining of the polymer beads is observed when the amount of DNA template (20-mer oligonucleotide) is as low as ∼10⁻¹⁴ mol. It is shown that practically no polymer staining is observed when the complex formed by the tandem and the 20-mer DNA template contains a mismatch either in the tetranucleotide duplex or in the duplex of octanucleotide immobilized on the beads. This suggests a possibility of using the presented approach in test systems for detection of point mutations in PCR-amplified DNA fragments.     

Basepairing potential of the 3' terminus of 16S RNA: dependence on the functional state of the 30S subunit and the presence of protein S21.

The deoxyoctanucleotide 5'd (AAGGAGGT) which is complementary to the 3' terminus of 16S RNA has been used as a probe to measure the potential of this rRNA region to engage in intermolecular basepairing. The site specific binding of the octanucleotide is shown by labeling 16S RNA in situ at its 3' end with [32P]pCp and T4 RNA ligase (EC 6.5.1.3.). The label can be released as pA[32P]pCp by the simultaneous action of RNAse H (EC 3.1.4.34) and 5'd(AAGGAGGT). WE show that (1) 30S subunits prepared according to standard procedures, bind less than one copy of 5'd(AAGGAGGT); (2) isolated 16S RNA and 30S subunits inactivated by transcient exposure to 0.5 mM Mg2+ do not bind the octanucleotide; (3) binding to inactive subunits can be restored by a brief heat treatment; (4) 30S subunits lacking protein S21 do not bind 5'd(AAGGAGGT) even when submitted to heat treatment; (5) addition of protein S21 to subunits lacking S21 restores octamer binding; (6) the apparent exposure of the 16S RNA 3' terminus brought about by protein S21 is accompanied by the potential of the subunits to accept MS2 RNA as messenger; (7) the presence or absence of S1 on 30S subunits has no effect on their octanucleotide binding property.     

Sequence-dependent conformation of DNA duplexes. The AATT segment of the d(G-G-A-A-T-T-C-C) duplex in aqueous solution

The nonexchangeable base and sugar protons of the octanucleotide d(G-G-A-A-T-T-C-C) have been assigned by two-dimensional correlated (COSY) and nuclear Overhauser effect (NOESY) methods in aqueous solution. The assignments are based on distance connectivities of less than 4.5 A established from NOE effects between base and sugar protons on the same strand and occasionally between strands, as well as, coupling connectivities within the protons on each sugar ring. We observe the NOEs to exhibit directionality and are consistent with the d(G-G-A-A-T-T-C-C) duplex adopting a right-handed helix in solution. The relative magnitude of the NOEs between base and sugar H2' protons of the same and 5'-adjacent sugars characterizes the AATT segment to the B-helix type in solution.     

Determination of a nucleotide sequence in bacteriophage f1 DNA by primed synthesis with DNA polymerase

A method is described for the determination of nucleotide sequences in DNA by using specific oligonucleotides as primers for copying specific regions by DNA polymerase. The method was applied to bacteriophage f1 DNA using the synthetic octanucleotide A-C-C-A-T-C-C-A as primer and a sequence (sequence A) of 81 nueleotides was determined. Synthesis was carried out in the presence of manganese and with one of the deoxyribotriphosphates (dCTP or dGTP) replaced by the corresponding ribotriphosphate so that mixed oligonucleotides were found which could be specifically split at the ribonucleotide residues by the appropriate ribonuclease or by alkali. The relative order of the digestion products was determined by fractionating the undigested oligonucleotides according to size on a two-dimensional system and digesting the isolated products. In the presence of rGTP the octanucleotide appeared to prime at a second site giving rise to a second sequence (B) besides sequence A. The complementary sequence to sequence A, which corresponds to the plus strand of f1 DNA and to the messenger RNA, contains five nonsense codons, four of which are in the same phase, and two possible initiation codons. It also contains a repetitive sequence which suggests its evolutionary origin by duplication.     

Ubiquitination of mRNA cycling sequence binding protein from Leishmania donovani (LdCSBP) modulates the RNA endonuclease activity of its Smr domain

In trypanosomatid parasites, an octanucleotide sequence (C/A)AUAGAA(G/A) in the UTRs primarily determines the stability of S-phase specific mRNAs. A multi-domain protein LdCSBP from Leishmania donovani interacts with the UTR of an S-phase RNA containing the octanucleotide sequence through its unique CCCH-type Zn-finger motifs. Interestingly, the RNA binding protein contains a previously characterized DNA endonuclease domain - Smr. It has been demonstrated here that the LdCSBP Smr domain independently possesses both DNA and RNA endonuclease activities, but the full-length LdCSBP exhibits only riboendonuclease activity. Moreover, LdCSBP protein has been shown to be ubiquitinated, resulting in the down-regulation of its riboendonuclease activity. In conclusion, the results described here suggest a novel regulatory mechanism of mRNA degradation through ubiquitination in eukaryotes.     

Sse8387I, a new type-II restriction endonuclease that recognizes the octanucleotide sequence 5''-CCTGCAGG-3''.

A type II restriction endonuclease designated Sse8387I was partially purified from Streptomyces sp. 8387. This enzyme cleaved adenovirus 2 DNA at three sites, lambda phage DNA at five sites, and pUC18 and M13mp18 RF DNA at one site each, but did not cleave the DNAs from pBR322, SV40, or phi X174. Sse8387I recognized the octanucleotide sequence 5'-CCTGCA decreases GG-3', cleaving where shown by the arrow. Sse8387I is the first restriction endonuclease to be reported that recognizes an octanucleotide sequence consisting of all four nucleotides, G, A, T, and C. The frequency of occurrence of Sse8387I sites within sequenced regions of primate genomes was 2.4 times that of NotI sites.     

Chemical synthesis of a self-complementary octanucleotide, dG-G-T-T-A-A-C-C by a modified triester method.

The synthesis of a self-complementary octanucleotide, d(G-G-T-T-A-A-C-C-), using a modified triester approach is described. The protected dinucleotides, d(Me2O)TribG(C1C6H4) ibG, d(Me2O)TrT(ClC6H4)T, d(Me2O)TrbzA(ClC6H4)bzA, and d(Me2O)TranC(ClC6H4)anC were synthesized by a one step triester procedure. After removal of the trityl group, the dinucleotides, dT(ClC6H4)T and danC (ClC6H4)anC were coupled to d(Me2O)TribG(ClC6H4)ibG and d(Me2O)TrbzA(ClC6H4)bzA, respectively to yield the respective tetranucleotides. The tetranucleotide, d(Me2O)TrbzA(ClC6H4)bzA(ClC6H4) and (ClC6H4)anC was detritylated and then coupled with d(Me2O)TribG(ClC6H4)ibG(ClC6H4)T(Cl6H4)T to give octanucleotide. The fully protected octanucleotide was deblocked by treatment with aqueous NH3 followed by acid and was characterized by nucleotide sequence analysis.     

rRNA topography in ribosome. IV. The accessibility of the 5'-end region of 16S rRNA

The accessibility of the 5'-end region of 16S rRNA (A₈GAGUUUG₁₅) inEscherichia coli ribosomes for complementary binding with the synthetic octanucleotide d(CAAACTCT) has been studied. Nonequilibrium gel-filtration was used to evaluate parameters of the binding of this oligonucleotide with free 16S rRNA, ribosomal subunits, and 70S ribosomes. A simple approach is presented to calculate the apparent association constants and the number of binding sites based upon the data obtained under those conditions. Free 16S rRNA, 30S subunits, and 70S ribosomes were found to form rather stable complexes with the octanucleotide, the association constants being similar in all three cases. These data strongly suggest the surface location of the 16S rRNA 5'-end inE. coli ribosomes.