Chiral electrostatics breaks the mirror symmetry of DNA supercoiling.

DNA supercoiling plays a fundamental role in regulating cellular activity and in the packaging of genetic material. In this communication, we analyse the effect of attractive chiral forces on the conformation of a closed circular DNA molecule, arising due to the helical patterns of charges on the DNA. We propose a model for closed loop DNA which uses the results of the recent theory of electrostatic interactions of a braid of two free-ended DNA molecules. Our model reproduces the known features of DNA supercoiling in an environment of low ionic strength. In high salt conditions, and in the presence of counterions that have high affinity to the DNA grooves, helix-specific forces significantly affect the conformation of the molecule by favouring a state characterized by a central left-handed braided section where there is close contact between distant portions of the loop. In such an environment we predict a previously unexplored possibility that nicked or topologically relaxed DNA molecules adopt a writhed state. This prediction suggests an alternative explanation for experiments in which it was assumed that the most stable topoisomer is always an open circle. Our results also give the first plausible explanation for the occurrence of tightly interwound molecules observed in cryo-electron microscopy and atomic force microscopy in a high ionic strength environment. We suggest several new experiments to test the predictions of this theory.

Complete sequence analyses show that two defective interfering influenza viral RNAs contain a single internal deletion of a polymerase gene.

Defective interfering (DI) influenza viral RNAs arise by internal deletion of progenitor RNAs. By using recombinant DNA cloning and DNA sequence analysis techniques, we have deduced the complete sequence of two such RNAs (L2b and L3), both arising from the same polymerase (P1) gene of WSN influenza virus. We have also partially determined the sequence of the P1 polymerase gene, including the sequence at the point of deletion and the flanking regions. Our sequence study shows the following. (i) Both L2b and L3 arise by a simple deletion in the P1 gene. (ii) L2b and L3 are 683 and 441 nucleotides long, respectively. (iii) The first 413 and 244 nucleotides of the 5' ends of L2b and L3, respectively, are identical to those of the 5' end of the P1 gene. (iv) The last 270 nucleotides of L2b and 197 nucleotides of L3 are the same as those of the 3' end of the P1 gene. (v) The entire sequence of L3 is present in the sequence of L2b. (vi) Both the 5' and the 3' termini, including the transcription stop and poly(A) addition signals of the progenitor P1 gene, are present in both L2b and L3. (vii) The sequences at the deletion point and the flanking region of the P1 gene do not resemble the consensus splicing sequence of spliced mRNA suggesting that a replicational event rather than splicing is involved in the formation of influenza defective interfering RNAs.

Physical maps for HSV type 2 DNA with five restriction endonucleases.

The ordering of restriction endonuclease fragments of HSV-2 DNA for physical maps has been studied using molecular hybridization techniques and the cleavage of isolated restriction endonuclease fragments with further restriction endonucleases. Physical maps for the fragments produced by EcoRI, Hind III, Bgl II, Xba and Hpa I have been constructed. The mol. wt. of the various regions which constitute HSV-2 genome are very similar to the corresponding mol. wt. in the HSV-1 genome.

Physical mapping of herpes simplex virus-coded functions and polypeptides by marker rescue and analysis of HSV-1/HSV-2 intertypic recombinants.

A number of temperature-sensitive (ts) mutants and one pyrimidine deoxyribonucleoside kinase-deficient mutant of herpes simplex virus (HSV), have been located on the physical map of the genome by means of marker rescue experiments and by the analysis of the crossover points in intertypic recombinants between HSV types 1 and 2. The physical map is compared to the genetic map and certain anomalies identified. Analysis of infected-cell polypeptides specified by intertypic recombinants has allowed tentative map co-ordinates to be assigned to the structural genes (or genes which cause post-translational modification) for many of the polypeptides. Immediate-early, phosphorylated, glycosylated and structural as well as non-structural polypeptides have been analysed in this way and it can be concluded that there is no restriction of any of these groups of polypeptides to either the long or the short regions of the genome. One of the recombinants, 2853, is at least partially "frozen" in one orientation of the long region. This orientation is also the one which exhibits a minimum number of crossovers in three other recombinants.

Sequence arrangement in herpes simplex virus type 1 DNA: identification of terminal fragments in restriction endonuclease digests and evidence for inversions in redundant and unique sequences.

It has been proposed by Sheldrick and Berthelot (1974) that the terminal sequences of herpes simplex virus type 1 (HSV-1) DNA are repeated in an internal inverted form and that the inverted redundant sequences delimit and separate two unique sequences, S and L. In this study the sequence arrangement in HSV-1 DNA has been investigated with restriction endonuclease cleavage, end-labeling studies, and molecular hybridization experiments. The terminal fragments in digests with restriction endonucleases Hind III, Hpa-1, EcoRI and Bum were identified and shown to be consistent with the Sheldrick and Berthelot model. Inverted fragments which contain unique sequences as well as redundant sequences, and which the model predicts, were identified by DNA-DNA hybridization studies. Further cleavage of Bum fragments with Hpa-1 also revealed inversions of the terminal sequences that contained unique sequences. The results obtained showed that the unique sequences S and L are relatively inverted in different DNA molecules in the population, resulting in the presence of four related genomes with rearranged sequences in apparently equal amounts. The redundant sequences bounding S do not share complete sequence homology with those bounding L, but hybridization studies are presented which show that the terminal 0.3% of the genome is repeated in every redundant sequence.

Analysis of herpesvirus DNA substructure by means of restriction endonucleases.

The mol. wt. and molar ratios of the Hind III and Hpa I fragments of HSV-1 DNA and the Eco RI fragments of HSV-2 DNA have been determined. Results obtained suggest that DNA isolated from both HSV-1 and HSV-2 consists of molecules with four different sequence arrangements which are present in similar amounts. Our explanation of the cleavage patterns of these four genome arrangements with the different restriction enzymes is presented. Some of the possible implications of these four genome arrangements for genetic recombination are discussed.

Control of gene expression in bacteriophage lambda: suppression of N mutants by mutations of the antirepressor.

The lysogenization and induction properties of phages lambdasusN7CI857Ai7 and lambdasusN53cro27 are described. Both phages, at 32 degrees kill little, but show only a moderate frequency of lysogenization whether an amber suppressor is present or absent in the host bacterium. In the latter case, lysogens for lambdasusN7CI857Ai7 or lambdasusN53CI857cro27 can exist in two different regulatory states, here called P r- and Pr+. The Pr+ phase is characterized by phage release and cell death at 40 degrees; conversely, cells in the Pr- phase are similarly killed but release no or very little phage. Pr- is the phase usually obtained at lysogenization. Each phase may be transmitted at 32 degrees for an unlimited number of generations, however, shifts to the opposite phase take place from time to time with a low probability. Two previously described antirepressor defective mutants. Ai7 and cro27, were found to suppress specifically the growth defect caused by an amber mutation in gene N. This suppression is observed in non-suppressing hosts at 40 or 42 degrees. Apparent revertants of N- mutants were shown to be often (80%) caused by a second mutation, in the Ai gene (also called tof, cro and fed). All the revertants so far examined appeared to be recessive. Lambda phages bearing a double amber mutation in gene N did not acquire full N independence by the acquisiton of an Ai mutation; this could be achieved, however, in the presence of a CII mutation. The above findings are discussed in terms of a direct interaction between the N, Ai and CII products.