Preparation of highly labeled (32P)nucleic acids from yeasts; isolation of "denaturable" leucine acceptor transfer RNA.

Culture conditions were developed that permit efficient incorporation of [(32)P]phosphate into nucleic acids of baker's yeast. RNA sufficiently labeled for sequence determination was obtained in this way. Pure "denaturable" leucine acceptor transfer RNA (tRNA(Leu)(3)) was isolated for this purpose.

Laboratory culturing of a thermophilic alga at high temperature.

Conditions for laboratory growth of the thermophilic alga Cyanidium caldarium at elevated temperatures have been developed. Growth characteristics of the organisms are described.

Nucleotide sequence of the "denaturable" leucine transfer RNA from yeast.

The nucleotide sequence of " denaturable"leucine acceptor transfer RNA (tRNA(Leu)(3)) from baker's yeast was determined on (32)P-labeled material. The molecule is 85 nucleotides long and can be folded into the "cloverleaf" model for secondary structure. The basis on which the sequence was deduced from the products of complete enzymatic digestion, prior to its unambiguous determination, is presented.

Functional pleiotropy of an intramolecular triplex-forming fragment from the 3'-UTR of the rat Pigr gene.

A microsatellite-containing 359-bp restriction fragment, isolated from the rat Pigr gene (murine polymeric immunoglobulin receptor gene) 3'-untranslated region (3'-UTR) and inserted into 3'-UTR or 3' flanking positions in transcription units of supercoiled plasmids, attenuates luciferase reporter gene expression in orientation- and position-dependent ways following transient transfection of human 293 cells. The same fragment stimulates orientation-dependent gene expression in a 5' flanking position. Plasmid linearization abrogates both orientation- and position-dependent responses. Cell-free translation reveals that 5' and 3' flanking expression responses are proportional to increased and decreased luciferase mRNA levels, whereas 3'-UTR expression is associated with control mRNA levels. Hypersensitivity to nucleases S1 and P1, gel mobility differences between supercoiled plasmids carrying opposing microsatellite orientations, and anomalous melting profiles of this fragment are also observed. These results suggest that functional pleiotropy of this fragment depends on the DNA context of its purine-rich microsatellite strand and on DNA supercoiling. Intramolecular triplexes stabilized by supercoiling and secondary structures of purine repeat-rich mRNAs may also confer regulatory properties to similar genomic elements.

Third strand-mediated psoralen-induced correction of the sickle cell mutation on a plasmid transfected into COS-7 cells.

A significant level of correction of the mutation responsible for sickle cell anemia has been achieved in monkey COS-7 cells on a plasmid containing a beta-globin gene fragment. The plasmid was treated in vitro with a nucleic acid 'third strand' bearing a terminal photoreactive psoralen moiety that binds immediately adjacent to the mutant base pair. Following covalent attachment of the psoralen by monoadduct or diadduct formation to the mutant T-residue on the coding strand, the treated plasmid was transfected into the cells, which were then incubated for 48 h to allow the cellular DNA repair mechanisms to remove the photoadducts. Upon re-isolation and amplification of the transfected plasmid, sickle cell mutation correction, as determined by sequence analysis of both complementary strands, was established in a full 1%. This result encourages extension of the approach to correct the mutation directly on the chromosome.

Fluorescence of terbium ion-nucleic acid complexes: a sensitive specific probe for unpaired residues in nucleic acids.

The interaction of the lanthanide cation Tb3+ with the phosphate moieties of non-hydrogen-bonded residues of nucleic acids has been shown to result in substantial enhancement of the fluorescence of this cation. The excitation spectrum for this fluorescence is characteristic of the base moiety of the residue to which the Tb3+ is bound, while the emission spectrum is characteristic of the cation itself. The intensity of the fluorescence enhancement, however, is dependent upon the base of the ligand moiety, with G inducing the strongest enhancement, C and T rather less, and A very little. Base-paired residues of nucleic acids induce no such fluorescence enhancement, even though the cation is more tightly bound to double helical regions than to residues in single strands. The enhancement of Tb3+ fluorescence upon binding to non-hydrogen-bonded residues therefore provides a highly specific conformational probe for such residues. This probe has been exploited successfully for the purpose of analyzing the kinetics of reassociation of DNAs (C0t analysis) and as a specific stain for single-strand DNA bands on polyacrylamide gels.

UV spectroscopic identification and thermodynamic analysis of protonated third strand deoxycytidine residues at neutrality in the triplex d(C(+)-T)6:[d(A-G)6.d(C-T)6]; evidence for a proton switch.

Near-UV difference spectral analysis of the triplex formed from d(C-T)6 and d(A-G)6.d(C-T)6 in neutral and acidic solution shows that the third strand dC residues are protonated at pH 7.0, far above their intrinsic pKa. Additional support for ion-dipole interactions between the third strand dC residues and the G.C target base pairs comes from reduced positive dependence of triplet stability on ionic strength below 0.9 M Na+, inverse dependence above 0.9 M Na+ and strong positive dependence on hydrogen ion concentration. Molecular modeling (AMBER) of C:G.C and C+:G.C base triplets with the third strand base bound in the Hoogsteen geometry shows that only the C+:G.C triplet is energetically feasible. van't Hoff analysis of the melting of the triplex and target duplex shows that between pH 5.0 and 8.5 in 0.15 M NaCl/0.005 M MgCl2 the enthalpy of melting (delta H degree obs) varies from 5.7 to 6.6 kcal.mol-1 for the duplex in a duplex mixture and from 7.3 to 9.7 kcal.mol-1 for third strand dissociation in the triplex mixture. We have extended the condensation-screening theory of Manning to pH-dependent third strand binding. In this development we explicitly include the H+ contribution to the electrostatic free energy and obtain [formula: see text]. The number of protons released in the dissociation of the third strand from the target duplex at pH 7.0, delta n2, is thereby calculated to be 5.5, in good agreement with approximately six third strand dc residues per mole of triplex. This work shows that when third strand binding requires protonated residues that would otherwise be neutral, triplex formation and dissociation are mediated by proton uptake and release, i.e., a proton switch. As a by-product of this study, we have found that at low pH the Watson-Crick duplex d(A-G)6.d(C-T)6 undergoes a transition to a parallel Hoogsteen duplex d(A-G)6.d(C(+)-T)6.

Ultraviolet-induced 8,8-adenine dehydrodimers in oligo- and polynucleotides.

Characteristic fluorescence excitation and emission is induced by either acetone-sensitized 313 nm irradiation of mixtures of 8-bromoadenosine and adenosine or 254 nm irradiation of oligo- and polynucleotides containing adenine neighbors. The acetone-sensitized reaction involves cleavage of bromine from 8-bromoadenosine with activation of C-8, leading to formation of an 8,8-adenosine dehydrodimer. Comparable fluorescence properties arise in the unsensitized photoreaction of dApdA, pdApdA, ApA, poly(dA), poly(A), poly(dA.dT), and poly(dA.U). The previously unidentified adenine ultraviolet photoproduct described by Porschke has been isolated as several variants from solutions of pdApdA and poly(dA) irradiated at 254 nm. Based upon fluorescence spectra and mass spectra, these variants are shown to contain the 8,8-adenine dehydrodimer moiety.