Melting Temperatures Of Duplexes Research Articles

Improved free-energy parameters for predictions of RNA duplex stability.

Thermodynamic parameters for prediction of RNA duplex stability are reported. One parameter for duplex initiation and 10 parameters for helix propagation are derived from enthalpy and free-energy changes for helix formation by 45 RNA oligonucleotide duplexes. The oligomer sequences were chosen to maximize reliability of secondary structure predictions. Each of the 10 nearest-neighbor sequences is well-represented among the 45 oligonucleotides, and the sequences were chosen to minimize experimental errors in delta GO at 37 degrees C. These parameters predict melting temperatures of most oligonucleotide duplexes within 5 degrees C. This is about as good as can be expected from the nearest-neighbor model. Free-energy changes for helix propagation at dangling ends, terminal mismatches, and internal G X U mismatches, and free-energy changes for helix initiation at hairpin loops, internal loops, or internal bulges are also tabulated.

Deoxyadenosine-deoxycytidine pairing in the d(C-G-C-G-A-A-T-T-C-A-C-G) duplex: conformation and dynamics at and adjacent to the dA X dC mismatch site.

Deoxyadenosine-deoxycytidine pairing at symmetrically related dA X dC mismatch sites in the d(C1-G2-C3-G4-A5-A6-T6-T5-C4-A3-C2-G1) self-complementary duplex (henceforth called 12-mer AC) has been investigated by proton and phosphorus NMR studies in aqueous solution. We demonstrate that base pairing is maintained on either side of the mismatch site in the 12-mer AC duplex at low temperature. The proton chemical shifts and NOE measurements rule out models in which the H-2 proton of adenosine at the mismatch site is stacked over adjacent dG X dC base pairs. A comparison of the hydrogen-exchange kinetics in the d(C-G-C-G-A-A-T-T-C-G-C-G) duplex (henceforth called 12-mer) with standard dG X dC base pairs at position 3 from either end with the 12-mer AC duplex, which contains dA X dC mismatches at these positions, demonstrates kinetic destabilization at dG X dC base pair 4 adjacent to the mismatch site and at dA X dT base pairs 5 and 6 far from this site. This contrasts with previous hydrogen-exchange studies on the 12-mer GT and 12-mer GA duplexes where the kinetic destabilization was localized to base pair 4, which is adjacent to the mispairing site. The melting temperature of the 12-mer AC duplex in 0.1 M phosphate is approximately 30 degrees C lower than the corresponding value for the 12-mer duplex.(ABSTRACT TRUNCATED AT 250 WORDS)

Prokaryote invertible DNA systems are highly conserved

The expression of virulence-associated genes in Bordetella pertussis can be lost in three ways: phase variation, antigenic modulation, or serotype conversion. The mechanism(s) of these alterations in gene expression is unclear. B. pertussis chromosomal DNA was probed with cloned pin genes from Escherichia coli and cloned hin genes from Salmonella typhimurium. DNA duplex melting temperature experiments indicated significant homology between B. Pertussis chromosamal DNA and both DNA inversion genes. Southern blots using the hin gene probe showed homology with a 15 kb EcoRI fragment of B. pertussis chromosomal DNA. We postulate here that B. pertussis contains a DNA inversion system which may be responsible for serotype conversion or virulence phase change in this organism.

Hemoglobin switching in sheep and goats. Preparation and characterization of complementary DNAs specific for the alpha-, beta-, and gamma-globin messenger RNAs of sheep.

Specific complementary DNAs (cDNAs) for the messenger RNAs coding for sheep alpha-, betaA-, betaB-, betaC-, and gamma-globins were prepared by thermal denaturation of heterologous hybrids (e.g. alphabetaB-cDNA-alphagamma-mRNA) followed by hydroxylapatite chromatography. Each cDNA represented a nearly full-length copy of its globin mRNA complement as determined by electrophoretic analysis in polyacrylamide gels containing 98% formamide. The purity of each cDNA fraction was estimated by hybridization analysis and thermal denaturation. The beta- and gamma-cDNAs contained 5 to 20% contaminating alpha-cDNA while the alpha-cDNA was 25 to 30% contaminated with non-alpha-cDNA. The melting temperatures (Tm) of homologous duplexes between each non-alpha chain cDNA and its mRNA complement ranged from 69.5-71.5 degrees in 50% formamide while alpha-alpha duplexes melted with a Tm of 75-76 degrees. The Tm values of heterologous duplexes formed between each non-alpha-cDNA and the various globin mRNAs (e.g. betaB-cDNA-Hb C mRNA) ranged between 64.5 degrees and 68 degrees and thus were only 1.5-5.0degrees below that of homologous duplexes. These results suggest that the nucleotide sequence divergence among the various non-alpha-mRNAs (or cDNAs) is not greatly different from the minimum predicted from the amino acid sequence differences of the corresponding globins. When annealing reactions were performed above the Tm of the heterologous hybrids (68 degrees), each non-alpha-cDNA hybridized only to its own complementary mRNA. Thus the purified cDNAs provide molecular probes for the quantitation of alpha-, beta-, and gamma-globin-specific nucleotide sequences.

Stepwise biosynthesis in vitro of globin genes from globin mRNA by DNA polymerase of avian myeloblastosis virus.

Two approaches have been explored for the synthesis of double-stranded DNA from single-stranded DNA template complementary to rabbit 9S globin mRNA (cDNA). (i) cDNA was elongated with dCMP or dTMP homopolymeric tracts using terminal deoxynucleotidyltransferase (EC 2.7.7.31; nucleosidetriphosphate:DNA deoxynucleotidylexotransferase). cDNA-dC, in the presence of an oligo(dG)10 primer, was an efficient template with either DNA polymerase of Escherichia coli (EC 2.7.7.7; deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase) or RNA-directed DNA polymerase of avian myeloblastosis virus. cDNA-dT [ with an oligo(dA)10 primer] functioned as template only with E. coli polymerase. (ii) cDNA, without homopolymeric tails, was also efficiently copied in the absence of oligonucleotide primer, by DNA polymerase of avian myeloblastosis virus or of E. coli. The product of the reaction consisted of long hairpin molecules which could be converted into DNA duplex (melting temperature, 93 degrees) by digestion with single-strand nuclease S1. The data indicate that a loop structure on the 3' end of cDNA allowed DNA synthesis to take place by a "self-priming" mechanism. Some of the double-stranded DNA synthesized corresponded to the entire sequence of the 9S mRNA template. The synthesis of full-length double-stranded DNA from mouse globin mRNA and immunoglobulin light chain mRNA is also discussed.