A two-state conformational equilibrium for alternating (A-T)n sequences in negatively supercoiled DNA.

We have made a study of the pattern of osmium tetroxide modification in supercoiled plasmids containing alternating (A-T)n tracts. Two distinct alternative patterns may be obtained, depending upon conditions. At moderate salt concentrations, or at low temperature, only thymine bases close to the centres of the tracts were modified, consistent with the presence of a cruciform structure. At higher temperatures in the absence of cations, uniform modification throughout the tracts was observed. The cationic concentration required to stabilize cruciform structure depends markedly on its charge, and a number of transition metal ions were totally ineffective. The results are interpreted in terms of a two-state equilibrium between the cruciform and a perturbed helical structure, the position of which is temperature- and salt-dependent. For longer (A-T)n tracts, a third pattern of osmium tetroxide modification is found at intermediate salt concentrations, consistent with a cruciform having an extensively disrupted four-way junction.

Structural alteration in alternating adenine-thymine sequences in positively supercoiled DNA.

An alternating adenine-thymine tract in a relaxed closed circular plasmid was found to become strongly reactive to osmium tetroxide in the presence of actinomycin D. We suggest that this is due to a local overwinding of the alternating tract as a result of positive supercoiling induced by intercalation of the antibiotic at GpC sequences elsewhere in the DNA. We have previously shown that (A.T)n sequences undergo a local underwinding in response to negative supercoiling, and it appears that such sequences are especially torsionally deformable in both directions.

Identification and developmental expression of a 5'-3' exoribonuclease from Drosophila melanogaster.

In multicellular organisms, very little is known about the role of mRNA stability in development, and few proteins involved in degradation pathways have been characterized. We have identified the Drosophila homologue of XRN1, which is the major cytoplasmic 5'-3' exoribonuclease in Saccharomyces cerevisiae. The protein sequence of this homologue (pacman) has 59% identity to S. cerevisiae XRN1 and 67% identity to the mouse homologue (mXRN1p) in certain regions. Sequencing of this cDNA revealed that it includes a trinucleotide repeat (CAG)9 which encodes polyglutamine. By directly measuring pacman exoribonuclease activity in yeast, we demonstrate that pacman can complement the yeast XRN1 mutation. Northern blots show a single transcript of approximately 5.2 kb which is abundant only in 0-8-h embryos and in adult males and females. In situ hybridization analysis revealed that the pcm transcripts are maternally derived, and are expressed at high levels in nurse cells. During early embryonic syncytial nuclear divisions, pcm transcripts are homogenously distributed. pcm mRNA is expressed abundantly and ubiquitously throughout the embryo during gastrulation, with high levels in the germ band and head structures. After germ band retraction, pcm transcripts are present at much lower levels, in agreement with the Northern results. Our experiments provide the first example of an exoribonuclease which is differentially expressed throughout development.

Compact structures of d(CNG)n oligonucleotides in solution and their possible relevance to fragile X and related human genetic diseases.

We show that oligonucleotides of CNG tracts readily adopt compact DNA structures that move unusually fast on gels. Base composition does not explain this, and non-CNG triplets (including GNC) do not form such structures. Chemical probing and melting experiments suggest that the structures probably are not hairpins. Although both long and short tracts can adopt compact structures, the structure formed by longer tracts is more compact than that formed by shorter ones. We note the possibility that such structures may form in vivo, and be instrumental in normal and/or abnormal function of human genes.

(A-T)n tracts embedded in random sequence DNA--formation of a structure which is chemically reactive and torsionally deformable.

Alternating d(A-T)n sequences which are contiguous with DNA of effectively random sequence have an abnormal conformation in linear DNA molecules. These regions are strongly reactive towards chemical modification by osmium tetroxide, and are preferentially cleaved by micrococcal nuclease. Both the chemical modification and the enzymic cutting occur uniformly through the alternating tract, and there is no evidence for enzyme or chemical sensitivity in the interfaces between the tract and DNA of normal conformation. These reactivities have a requirement for an alternating sequence. In addition to chemical reactivity, alternating (A-T)n sequences exhibit anomalously small twist changes on cruciform formation, suggesting that the pre-extruded DNA is underwound. We propose that the alternating sequences adopt an altered conformation which is subject to easy torsional deformation.

Superhelical torsion in cellular DNA responds directly to environmental and genetic factors.

Superhelical tension of DNA in living bacteria is believed to be partially constrained by interaction with proteins. Yet DNA topology is a significant factor in a number of genetic functions and is apparently affected by both genetic and environmental influences. We have employed a technique that allows us to estimate the level of unconstrained superhelical tension inside the cell. We study the formation of cruciform structures by alternating adenine-thymine sequences in plasmid DNA by in situ chemical probing. This structural transition is driven by superhelical torsion in the DNA and thus reports directly on the level of such tension in the cellular DNA. We observe that the effect of osmotic shock is an elevation of superhelical tension; quantitative comparison with changes in plasmid linking number indicates that the alteration in DNA topology is all unconstrained. We also show that the synthesis of defective topoisomerase leads to increased superhelical tension in plasmid DNA. These experiments demonstrate that the effect of environmental and genetic influences is felt directly at the level of torsional stress in the cellular DNA.

A highly conserved plasmid from the extreme thermophile Thermotoga maritima MC24 is a member of a family of plasmids distributed worldwide.

We have screened Thermotoga strains, isolated from hydrothermal vents near the Kuril Islands, for the presence of plasmid DNA. The miniplasmid pMC24 was isolated from the extreme thermophilic eubacteria Thermotoga maritima and sequenced, showing it to be a plasmid of 846 bp. It was found, from a search of the databases, to be closely related to the previously described Thermotoga miniplasmid pRQ7, isolated from a strain found on the Azore Islands, and was distinguished by only two point mutations. These changes resulted in two consecutive frameshifts altering a region encoding 9 amino acids in the Rep-coding region. We have also shown that pMC24, as with pRQ7, is negatively supercoiled. It seems that negatively supercoiled miniplasmids related to pRQ7 are spread worldwide and strongly maintained among Thermotoga strains.