Mutant profiles of selectable genetic elements.

A method is presented that allows simultaneous analysis of the effects of all possible point mutations within a specific mutation window of at least 50 base pairs on a DNA fragment that codes for a selectable function. It relies on the detection of mismatched base pairs with hydroxylamine and osmium tetroxide. A mutant plasmid library of randomly distributed point mutations within the lacZ' gene of Escherichia coli was selected for functional alpha-complementation by growth on lactose. The DNA fragments of the selected and unselected library were each heat denatured and again renatured, thereby generating a randomly distributed set of all possible mismatches within the mutagenesis window. Cytidine-containing mismatches were then detected with hydroxylamine, and thymidine-containing mismatches were detected with osmium tetroxide. When this procedure was performed for both DNA strands, all mismatches could be detected. A comparison of the results of the unselected and selected library leads to an estimation of the effects of each detectable mutation on alpha-complementation in vivo. This method, called "mutant profiling," should be applicable to all selectable genetic elements.

The influence of an alternate template conformation on elongating phage T7 RNA polymerase.

We investigated the effect of left-handed Z-DNA on transcription by bacteriophage T7 RNA polymerase in vitro and, surprisingly, found that the enzyme can efficiently utilize a template containing a stretch of left-handed DNA close to the promoter. Analysis of transcription products revealed that only a small fraction of elongating polymerases abort transcription either at the promoter proximal or at the distal B-to-Z junction and, even less frequently, within the stretch of left-handed DNA. Our results indicate that, unlike E. coli RNA polymerase, T7 RNA polymerase can utilize a template with a CG stretch in an alternate conformation. In contrast, polymerases are completely blocked at the promoter proximal junction by a monoclonal antibody directed against Z-DNA. This blockage remains stable over a remarkable time, even when negative supercoiling is released by linearization of the template. Together with our recent finding of transcription-induced formation of Z-DNA (3), our data provide an example for a possible auto-regulatory mechanism that employs a change in DNA conformation.

Field inversion gel electrophoresis with different pulse time ramps.

The influence of different pulse time ramps on the separation of yeast chromosomes with field inversion gel electrophoresis (FIGE) was investigated by the means of two dimensional gel electrophoresis. The problem of band inversion, which makes it difficult to distinguish DNA molecules of different size, has been solved by using double randomized pulse times. A major disadvantage of the field inversion technique is thereby overcome, making this system comparable to other pulsed field techniques.

A systematic study of field inversion gel electrophoresis.

The mobilities of oligomers of phage lambda DNA and of yeast chromosomes in agarose gels during field inversion gel electrophoresis (FIGE) were measured at different pulse times and electric fields. Also the ratios between forward and backward pulse times and/or field gradients were varied. The problem of 'band inversion' during FIGE, leading to an ambiguity in the mobility of large DNA fragments, was solved by using two dimensional gel electrophoresis with different parameters in the first and second dimension. The results are compared with those obtained with other pulsed electrophoresis systems and with a theoretical model.

DNA sequencing with direct blotting electrophoresis and colorimetric detection.

We describe optimized procedures for colorimetrically-detected DNA sequencing with direct blotting electrophoresis. One-step protocols for Sequenase and Klenow enzyme are given. The clapping technique has been adapted to allow convenient casting of very thin gels with an optimal lower gel (transfer) surface. This gives very sharp band patterns, enabling more than 350 bases from a single loading to be read with confidence. The crucial points for direct blotting electrophoresis are discussed. Background problems resulting from unspecific binding of streptavidin to the nylon membranes have been eliminated by the use of high concentrations of SDS in the incubation buffer; and using a single large glass tube for all incubation and washing steps is a very convenient and effective development protocol. Automation of the colorimetric development process is described.

Hysteretic behaviour of a Z-DNA-antibody complex.

The amount of complex observed between Z-DNA in the supercoiled DNA from plasmid pFP332 (with d(C-G)16 cloned into pUC8) with the radiolabelled monoclonal antibody Z-D11 (with very high affinity for left-handed Z-DNA) depends on the temporal order of addition of the ligands. If the antibody is bound first a 20-30-fold higher cloroquine concentration is necessary to dissociate half of the complex compared to the case where the suprahelical density is changed first and the complex formed afterwards. This hysteretic behavior is observed for weeks and presents a kind of molecular memory system, which is based on the topological and conformational properties of DNA and the high-affinity protein binding to Z-DNA.

Searching for potential Z-DNA in genomic Escherichia coli DNA.

The Clarke-Carbon library with Escherichia coli DNA cloned into plasmid ColE1 was partially screened for Z-DNA with the monoclonal antibody Z-D11 using the retardation of the covalently closed circular DNA-protein complex by nitrocellulose filters. About 85% of the plasmids tested at "natural" supercoil density bound to the filter. Together with binding studies of the iodinated antibody, one Z-DNA segment per about 18,000 base-pairs of E. coli DNA is observed. One clone containing the region around the lactose operon, pLC20-30, was studied in detail. Subcloning a partial Sau3A digest and selection with antibodies gave three different Z-forming sites. They were mapped to within about +/- 20 base-pairs by preparing unidirectional deletion clones, selection of protein binding plasmids on nitrocellulose filters and subsequent sizing on agarose gels. The size of the Z-DNA-forming segments was estimated from two-dimensional gels of topoisomer mixtures. Together with results from sequencing of the plasmid DNA using exonuclease III to create single-stranded templates, stretches of alternating purine-pyrimidine tracts of 12 to 15 base-pairs were found to be responsible for Z-DNA formation. One of the sites was found in the middle of the lacZ gene, where it might be an obstacle for RNA polymerase. The methods used here should also be helpful for studying other DNA-protein sites, especially if they exist only in supercoiled DNA.

Dynamics of the B-to-Z transition in supercoiled DNA.

The sequence (dC-dG)16, inserted into the polylinker of plasmid pUC8, adopts a left-handed Z-DNA conformation at "natural" supercoil density. The radioactively labeled monoclonal antibody Z-D11, which has a very high affinity for this DNA conformation, provides a convenient sensitive tool to measure selectively the amount of Z-DNA. Chloroquine reversibly changes the supercoil density of plasmid DNA and thereby the equilibrium between right- and left-handed double-helical DNA. The time-dependent formation or disappearance of Z-DNA was measured by using the antibody either as a fast indicator of Z-DNA or as an additional effector of the B-to-Z equilibrium. In the middle of the transition, a relaxation time of about 1 hr is observed in 0.1 M NaCl at 22 degrees C. The kinetic data are compatible with an all-or-none transition between the two conformations. The overall rate constant for Z-DNA formation, kBZ, decreases with the square of the chloroquine concentration, while the reverse one, kZB, increases with about the fourth power.

DNA sequencing with direct blotting electrophoresis.

A method for transferring the DNA molecules of sequencing reaction mixtures onto an immobilizing matrix during electrophoresis has been developed. A blotting membrane moves with constant speed across the end of a very short, denaturing gel and collects the molecules according to size. A constant distance between bands for molecules differing in length by one nucleotide is obtained over a large range (approximately 600 nucleotides with a 5% gel), simplifying the determination of DNA sequences considerably. Reliable sequences of 500 nucleotides can be read and sequence features up to greater than 1000 nucleotides are revealed in a single experiment. The sequencing of a potential Z-DNA-forming fragment from Escherichia coli DNA is given as an example and possible further developments are discussed.

Isolation of Z-DNA-containing plasmids.

A purified, monoclonal antibody, specific for the left-handed Z-form of poly(dG-dC), was coupled covalently to Sephacryl S-1000 beads. Such an antibody column provides a convenient method to isolate and purify those plasmid DNAs that contain Z-DNA from a large excess of other DNAs, RNA, etc. From a library of Escherichia coli DNA, cloned into the vector plasmid pUC-8, several recombinant plasmids were isolated, which bind to this antibody. Thus, E. coli contains sequences, which in "natural" negatively supercoiled DNA, adopt a left-handed Z-DNA-like conformation.

Torsional stress induces left-handed helical stretches in DNA of natural base sequence: circular dichroism and antibody binding.

Above a threshold of torsional stress, the c.d. spectrum of covalently closed circular DNA of natural base sequence acquires a Z-like contribution and antibodies raised against Z-DNA are bound. Mapping of the antibody binding sites by electron microscopy reveals sites which correlate with stretches enriched in alternating purine-pyrimidine sequences and GC base pairs.

Salt-induced transition between two double-helical forms of oligo (dC-dG).

The change between a right-handed and a left-handed double-helical structure is certainly an extreme example of the conformational variability of DNA. Studying the energetics and dynamics with defined oligomers provides a convenient way to obtain a quantitative understanding of such processes and can serve as a model for possible regulatory mechanisms in biological systems. Monoclonal antibodies with different specificities to Z-DNA offer a promising technique to search for such structures in more-complicated systems and represent a model system for the quantitative investigation of the interaction of proteins with particular conformations of DNA.

Temperature dependence of the activity of DNA-modifying enzymes: endonucleases and DNA ligase.

The activities of 17 endonucleases: the restriction endonucleases AvaI, Bam HI, EcoRI, HindIII, PstI and SalI, which cleave pBR322 DNA once: AluI, AvaII, CfoI, HaeIII, HhaI, HinfI, HpaII and TaqI, which cut pBR322 DNA several times, and three 'unspecific' nucleases (S1 nuclease, staphylococcal nuclease and DNase I from bovine pancreas) were determined between 0 degrees and 65 degrees C. The reaction was followed by the disappearance of covalently closed circular pBR322 DNA, using the alkaline ethidium fluorescence assay of Morgan et al. [Nucleic Acids Res. (1979) 7, 547-594]; the activity of T4 DNA ligase was similarly measured by the conversion of nicked circular DNA to closed circular DNA. For each enzyme, small aliquots of the same solution were incubated at different temperatures simultaneously in a temperature gradient device, resulting in a high relative precision. The experimental results are summarized by the simplest possible theoretical description, using linear or exponential kinetics and apparent activation energies Ea for the enzymatic reaction, Ei for the enzyme inactivation and Ti for the inactivation temperature. To a good approximation these three parameters suffice for describing the temperature dependence of the activity of most of the enzymes.

"Alternating B-DNA" conformation for the oligo(dG-dC) duplex in high-salt solution.

The high resolution 1H and 31P NMR spectra of the (dG-dC)8 duplex have been recorded in low- and high-salt solutions in order to evaluate the structural aspects of the salt-induced transition of oligo(dG-dC) in solution [Pohl, F. M. & Jovin, T. M. (1972) J. Mol. Biol. 67, 375-396]. The NMR data require that the (dG-dC)8 duplex in 4 M NaCl adopt an "alternating B-DNA" conformation for which the symmetry unit repeats every two base pairs. By contrast, the oligomer duplex in low-salt solution is of the regular B-DNA type in solution. The chemical shift parameters for oligo(dG-dC) in high-salt solution demonstrate that every other glycosidic torsion angle and phosphodiester linkage adopts a different conformation from that observed in regular B-DNA. We demonstrate further that the generation of the "alternating B-DNA" structure is facilitated by introduction of halogen atoms at the 5 position of pyrimidine and that this probably reflects the greater overlap of this position with adjacent base pairs in high salt solution. An "alternating B-DNA" model has recently been proposed for alternating deoxy purine-deoxy pyrimidine polynucleotides based on the x-ray structure of pdA-dT-dA-dT [Klug, A., Jack, A., Viswamitra, M.A., Kennard, O., Shakked, Z. & Steitz, T.A. (1979) J. Mol. Biol., in press].