Specificity of DNA base release by bleomycin.

DNA was treated with bleomycin in the presence of Fe2+ and 2-mercaptoethanol under conditions where only a few percent of the bases were released. Release of all four bases was a linear function of bleomycin concentration, but the amount of thymine released was twice that of cytosine, 7 times that of adenine, and twelve times that of guanine. Unidentified minor products of thymine, of cytosine and of a purine were also released. Bromouracil did not sensitize DNA to bleomycin-induced breakage, and was released at the same rate as thymine.

The characterisation of liposomes with covalently attached proteins.

The problem of characterising liposomes with covalently attached proteins has been analysed theoretically in terms of a normal weight distribution of liposome diameters. The polydispersity of protein conjugation is considered in terms of the width (standard deviation) of the liposome size distribution. It is shown that the weight-average number of proteins per liposome is a convenient parameter to use to define the protein content of proteoliposomes. Two types of proteoliposome have been prepared (small unilamellar vesicles and reverse phase evaporation vesicles) in which wheat germ agglutinin is covalently coupled to the liposomal surface. The liposomes cover a range of weight average diameter from 65 to 240 nm and of polydispersity (weight to number average diameter (dw/dn) from 2.6 to 11.4. The liposomes have been characterised by chemical analysis and photon correlation spectroscopy and the results are discussed in terms of the theoretical consequences of an equivalent normal weight distribution of diameters.

Targeting and delivery of bactericide to adsorbed oral bacteria by use of proteoliposomes.

Proteoliposomes having surface-bound succinylated concanavalin A (s-conA) have been prepared from a range of phospholipid mixtures by sonication (SUV) and reverse phase evaporation (REV) covering a range of size (weight-average diameter (dw)) from approx. 35 to 310 nm and weight-average number of protein molecules per liposomes (Pw) from approx. 50 to 3000. The targeting of the proteoliposomes to adsorbed biofilms of the bacteria Streptococcus sanguis and Streptococcus mutans has been assessed from the extent of inhibition of an enzyme-linked immunosorbent assay (ELISA) for bacterial cell surface antigens. The surface-bound lectin enhances targeting relative to 'naked' liposomes of comparable concentration by factors of 2-50 depending on the liposomal lipid composition and Pw. The effect of the bactericide Triclosan on the thermal properties and permeability characteristics of liposomes has been studied. At and above a molar ratio of Triclosan to lipid of 0.6, Triclosan eliminates the gel to liquid-crystalline phase transition in dipalmitoylphosphatidylcholine (DPPC) containing liposomes and increases the bilayer permeability of both liposomes and proteoliposomes to D-glucose. The proteoliposomes have been used to deliver Triclosan to S. sanguis biofilms and the inhibition of growth of the bacteria after treatment with liposomally delivered Triclosan has been determined using a microtitre plate re-growth assay and compared with growth inhibition by 'free' Triclosan. It is shown that for short exposure times (1 to 2 min) proteoliposomally delivered Triclosan is a more effective growth inhibitor than free Triclosan. The results are discussed in terms of the targeting, retention and subsequent release of Triclosan into the bacterial biofilms.

Analysis of deletions induced in the genome of mammalian cells by ionizing radiation.

A theory is presented for the distribution in size of deletions induced by ionizing radiation, based on three assumptions: (1) deletions that are observed delete part or all of a gene to make a mutation, but not adjacent DNA sequences essential for survival of the mutant; (2) deletions are distributed at random along the DNA; (3) the probability of formation is proportional to the rate at which the two endpoints, which must meet to form the deletion, collide with each other. Experimental data for radiation-induced deletions in human and hamster hprt genes are in good agreement with calculations that assume the inducing lesion does not break the intracellular chromatin fiber; calculations assuming the inducing lesion is a break are not a good fit to the data. The low frequency of deletions observed in the hamster aprt gene is shown to be a consequence of the small gene size and the presence of a nearby essential DNA sequence, ensuring that most deletions affecting the gene also delete the essential sequence and are thus not observed.

Deletions induced by gamma rays in the genome of Escherichia coli.

An Escherichia coli lysogen was constructed with a lambda phage bearing a lacZ gene surrounded by about 100 x 10(3) base-pairs of dispensable DNA. The lacZ mutants induced by gamma rays in this lysogen were more than 10% large deletions, ranging in size from 0.6 x 10(-3) to 70 x 10(3) base-pairs. These deletions were centered, not on lacZ, but on a ColE1 origin of DNA replication located 1.2 x 10(3) bases downstream from lacZ. This suggested that this origin of replication was involved in the process by which the deletions were formed. In agreement with this hypothesis, a lysogen of the same phage without the ColE1 origin showed a very much lower percentage of radiation-induced deletions, as did a second lysogen of a lambda phage without any known plasmid origin of replication. Indirect evidence is presented for radiation-induced deletions centered on the lambda origin of DNA replication in a lysogen. It is suggested that high percentages of large deletions may occur among radiation-induced mutations in mammalian cells because deletions centered on some of the thousands of origins of replication in these genomes do not kill the cells.

Ultraviolet light-induced mutagenesis in the Escherichia coli chromosome. Sequences of mutants in the cI gene of a lambda lysogen.

DNA sequences were determined for 56 mutations induced by ultraviolet light in the lambda cI gene of an Escherichia coli uvr+ lysogen, which should reflect those occurring in the E. coli chromosome. The spectrum of mutagenesis was similar to that found in the cI gene of irradiated phase assayed in uvr- host cells, except that the fraction of transversions is about 35% in prophage and about 15% in phage. The cause of this difference is not known. Of 17 frameshifts in phage and prophage, six have an accompanying base substitution. These double mutational events are consistent with a model in which a photoproduct in a template can cause a DNA polymerase to insert a wrong base and destabilize the next few bases added, thus leading to a frameshift by a slippage mechanism.

Non-targeted mutagenesis of unirradiated lambda phage in Escherichia coli host cells irradiated with ultraviolet light.

Non-targeted mutagenesis of lambda phage by ultraviolet light is the increase over background mutagenesis when non-irradiated phage are grown in irradiated Escherichia coli host cells. Such mutagenesis is caused by different processes from targeted mutagenesis, in which mutations in irradiated phage are correlated with photoproducts in the phage DNA. Non-irradiated phage grown in heavily irradiated uvr+ host cells showed non-targeted mutations, which were 3/4 frameshifts, whereas targeted mutations were 2/3 transitions. For non-targeted mutagenesis in heavily irradiated host cells, there were one to two mutant phage per mutant burst. From this and the pathways of lambda DNA synthesis, it can be argued that non-targeted mutagenesis involves a loss of fidelity in semiconservative DNA replication. A series of experiments with various mutant host cells showed a major pathway of non-targeted mutagenesis by ultraviolet light, which acts in addition to "SOS induction" (where cleavage of the LexA repressor by RecA protease leads to din gene induction): (1) the induction of mutants has the same dependence on irradiation for wild-type and for umuC host cells; (2) a strain in which the SOS pathway is constitutively induced requires irradiation to the same level as wild-type cells in order to fully activate non-targeted mutagenesis; (3) non-targeted mutagenesis occurs to some extent in irradiated recA recB cells. In cells with very low levels of PolI, the induction of non-targeted mutagenesis by ultraviolet light is enhanced. We propose that the major pathway for non-targeted mutagenesis in irradiated host cells involves binding of the enzyme DNA polymerase I to damaged genomic DNA, and that the low polymerase activity leads to frameshift mutations during semiconservative DNA replication. The data suggest that this process will play a much smaller role in ultraviolet mutagenesis of the bacterial genome than it does in the mutagenesis of lambda phage.

Changes in DNA base sequence induced by targeted mutagenesis of lambda phage by ultraviolet light.

In targeted mutagenesis of lambda phage by ultraviolet light, the mutations are caused by radiation-induced lesions in the phage DNA. Of 62 mutations in the lambda cI gene that were sequenced, 41 (63%) of the targeted mutations were transitions, with similar numbers of C X G to T X A and T X A to C X G base changes. The remaining 21 mutations were about equally divided among eight transversions, seven frameshifts (5 additions and 2 deletions), and six double events with either two nearby base changes or a base change and a nearby frameshift. Of the 62 mutations, 60 could be associated with -Pyr-Pyr- sequences in the DNA, sites of likely photoproducts. For more information on this point, lambda phage were irradiated with 313 nm light in the presence of acetophenone, for which the major photoproduct is reported to be the thymine-thymine cyclobutyl dimer, with no measurable Pyr(6-4)Pyo photoproducts. Of 22 mutations sequenced, 19 were transversions and only one was a transition, permitting the conclusion that thymine-thymine cyclobutyl dimers are not the primary cause of ultraviolet light-induced transitions. A consideration of all the data strongly suggests that Pyr(6-4)Pyo photoproducts are mutagenic lesions.

DNA base sequence changes induced by ultraviolet light mutagenesis of a gene on a chromosome in Chinese hamster ovary cells.

The DNA base sequence changes induced by mutagenesis with ultraviolet light have been determined in a gene on a chromosome of cultured Chinese hamster ovary (CHO) cells. The gene was the Escherichia coli gpt gene, of which a single copy was stably incorporated and expressed in the CHO cell genome. The cells were irradiated with ultraviolet light and gpt- colonies were selected by resistance to 6-thioguanine. The gpt gene was amplified from chromosomal DNA by use of the polymerase chain reaction (PCR), and the amplified DNA sequenced directly by the dideoxy method. Of the 58 sequenced mutants of independent origin 53 were base change mutations. Forty-one base substitutions were single base changes, ten had two adjacent (or tandem) base changes, and one had two base changes separated by a single base-pair. Only one mutant had a multiple base change mutation with two or more well separated base changes. In contrast much higher levels of such mutations were reported in ultraviolet mutagenesis of genes on a shuttle vector in primate cells. Two deletions of a single base-pair were observed and three deletions ranging from 6 to 37 base-pairs. The mutation spectrum in the gpt gene had similarities to the ultraviolet mutation spectra for several genes in prokaryotes, which suggests similarities in mutational mechanisms in prokaryotes and eukaryotes.

Effect of photoreactivation on mutagenesis of lambda phage by ultraviolet light.

There is disagreement in the literature as to whether the major mutagenic photoproduct induced in DNA by ultraviolet light is the cyclobutane dipyrimidine dimer, the most common product, or the [6-4] photoproduct, the next most frequent. In the experiments reported here, cyclobutane dimers were removed from irradiated lambda phage DNA by enzymatic photoreactivation, a process thought to affect no other photoproduct. Photoreactivation of lambda phage in host cells and of lambda DNA in solution reduced clear plaque mutants per plaque-forming unit by two-thirds, in host cells with a constant and near-maximal expression of the SOS functions required for mutagenesis. This result is interpreted to mean that removal of cyclobutane dimers in or near the mutated gene reduces mutation induced by ultraviolet light by two-thirds; therefore, cyclobutane dimers in the phage DNA are responsible for most observed mutations. DNA sequences of mutations in photoreactivated phage showed a smaller fraction of G.C to A.T transitions and a larger fraction of A.T to G.C transitions, compared to phage that were not photoreactivated. This suggests that cyclobutane dimers at TC and CC sites are particularly mutagenic.

Changes in DNA base sequence induced by gamma-ray mutagenesis of lambda phage and prophage.

Mutations in the cI (repressor) gene were induced by gamma-ray irradiation of lambda phage and of prophage, and 121 mutations were sequenced. Two-thirds of the mutations in irradiated phage assayed in recA host cells (no induction of the SOS response) were G:C to A:T transitions; it is hypothesized that these may arise during DNA replication from adenine mispairing with a cytosine product deaminated by irradiation. For irradiated phage assayed in host cells in which the SOS response had been induced, 85% of the mutations were base substitutions, and in 40 of the 41 base changes, a preexisting base pair had been replaced by an A:T pair; these might come from damaged bases acting as AP (apurinic or apyrimidinic) sites. The remaining mutations were 1 and 2 base deletions. In irradiated prophage, base change mutations involved the substitution of both A:T and of G:C pairs for the preexisting pairs; the substitution of G:C pairs shows that some base substitution mechanism acts on the cell genome but not on the phage. In the irradiated prophage, frameshifts and a significant number of gross rearrangements were also found.

Lectin-mediated targeting of liposomes to a model surface. An ELISA method.

Wheat germ agglutinin has been conjugated to the surfaces of sonicated phospholipid liposomes by reacting the protein derivatised with N-succinimidyl-S-acetylthioacetate (SATA) with the m-maleimidobenzoyl-N-hydroxysuccinimide (MBS) derivative of dipalmitoylphosphatidylethanolamine (DPPE) incorporated into the liposomal bilayers. The liposomes as characterised by photon correlation spectroscopy had a weight-average radius of 44 +/- 10 nm and the number of WGA molecules per liposome was in the range up to approx. 120. An ELISA method has been developed to assess the efficiency of targeting the conjugated liposomes to the antigenic determinants on a surface coated with glycophorin A (blood group B). For liposomes in which the degree of conjugation was controlled by varying the mol% DPPE-MBS from 3 to 27% targeting efficiency as assessed from the extent of inhibition of the ELISA increased by a factor of 10.

Sequence specificity of mutagenesis in the cI gene of bacteriophage lambda.

Studies of DNA base sequence alterations have shown that for every agent the mutagenic process is specific with respect to the types of base changes induced and the location of the changes in the DNA. Analysis of the types of mutations produced by mutagenic agents can provide insight into the mechanism of mutation and can suggest which DNA lesions may be involved in the actual mutagenic event. We have developed a system for the analysis of chemically induced base sequence alterations in the cI repressor gene of bacteriophage lambda using DNA sequencing techniques. To illustrate the utility of this type of analysis, we present the results obtained with ultraviolet light (UV). Irradiation of target DNA with UV alone, or UV followed by photoreactivating light (which removes dimers), produces mostly transitions at pyrimidine-pyrimidine sites. Conversely, irradiation with 313 nm light plus acetophenone (which produces only thymine dimers) produces mostly transversions at low efficiency. This and other evidence suggests that the actual premutagenic UV lesion in E. coli may not be pyrimidine-pyrimidine dimers, but rather pyr(6-4)pyo photoproducts.

A new extra long acting depot preparation of the LHRH analogue Zoladex. First endocrinological and pharmacokinetic data in patients with advanced prostate cancer.

A new depot formulation of the LHRH analogue Zoladex (goserelin acetate) has been developed which releases the drug over a period of at least 3 months as judged by measurement of drug content in depots at intervals after insertion in male rats and by the suppression of oestrogen secretion and oestrus in female rats. This formulation is based on the lactide/glycolide polymer system used for the standard 1-month Zoladex depot, but the dose has been increased to 10.8 mg and the characteristics have been modified to enable a longer release of drug to be achieved. Thirty-eight patients with histologically proven, locally advanced (stage T3 or T4) and/or metastatic prostate cancer were treated with this new longer acting LHRH analogue depot formulation containing 10.8 mg Zoladex. After initial increase of serum testosterone in the first week of therapy, castration levels were reached in all patients after 4 weeks and this was maintained for more than 14 weeks. At the time of depot exhaustion, when escape from castration levels of androgen occurred, all patients received a single injection of a standard 1-month depot containing 3.6 mg Zoladex which restored castration levels of androgen thus showing that the pituitary gland was again suppressed. The tolerance and acceptability of the longer-acting depot is high and comparable to the 1-month depot. Taking into account social and psychological factors, patients with advanced prostate carcinoma will soon be able to be treated with a longer acting LHRH depot formulation every 3 months an alternative of the 1-month depot now widely used clinically.

Comparison of the action of nonoxynol-9 and chlorhexidine on sperm.

The effect on sperm motility of nonoxynol-9 chlorhexidine diacetate was compared in semen and cervical mucus. Both compounds had similar spermicidal potency in semen, abolishing sperm motility within 3 minutes at 0.5 mg/ml. When these compounds were allowed to diffuse into mucus, the subsequent survival of sperm in the mucus was different. Restricted penetration and loss of motility occurred rapidly after treatment with 0.1 mg/ml chlorhexidine, whereas sperm survived normally in mucus after prolonged contact with 200 mg/ml chlorhexidine. When the compounds were mixed directly with the mucus before sperm penetration was attempted, chlorhexidine still immobilized sperm, but concentrations of nonoxynol-9 that would be spermicidal in semen had no effect in mucus.

PIP: This study compared the effect of nonoxynol-9 and chlorhexidine in sperm motility in both semen and cervical mucus. Both compounds had similar spermicidal potency in semen, abolishing sperm motility within 3 minutes at 0.5 mg/ml, although the reduction of the proportion of motile sperm at lower concentrations was more marked for chlorhexidine. When these compounds were diffused into mucus, sperm survival showed different patterns. Restricted penetration and loss of motility occurred rapidly after treatment with 0.1 mg/ml of chlorhexidine, whereas sperm survived normally in mucus after prolonged contact with 200 mg/ml of nonoxynol-9. When the compounds were mixed directly with mucus before sperm penetration, chlorhexidine still immobilized sperm, but concentrations of nonoxynol-9 that would be spermicidal in semen had no effect. These findings suggest that nonoxynol-9 does not have access to the domains of the cervical mucus occupied by the swimming sperm; moreover, there is no evidence that this compound is able to diffuse into mucus. The nonoxynol-9 type of spermicide is probably active only in the vagina and sperm that enter the cervical mucus before being immobilized by the spermicide are protected from further action. In contrast, chlorhexidine may structurally alter the cervical mucus, preventing the entry of sperm, or may actively accumulate in mucus to spermicidal levels. The effectiveness of spermicidal vaginal contraception could be improved by extending the area of the female genital tract available to the contraceptive agent. Chlorhexidine provides such an extension.

Dependence of the sedimentation of high molecular weight DNA on centrifuge speed.

A theory by Zimm [B.H.Zimm, Biophys. Chem. 1-(1974)279] predicts that for a given centrifuge speed, there is a broad maximum in a plot of the sedimentation coefficient of DNA against molecular wight. Experimental measurements of these maxima for various centrifuge speeds were made for double-helical DNA in neutral sucrose gradients and singlestrand DNA in alkaline gradients. The measurements are in quantitative agreement with the theory, providing good evidence for its validity. The existence of the maximum shows that there is a limit to the sedimentation rate under specified conditions for KNA in the linear form. By implication, DNA observed to sediment faster than this limit is not in the linear form to which most sedimentation theory is applicable.

Formation of two double-strand breaks in the same DNA molecule by a single high-energy photon or ionizing particle.

I calculate the probability that a single high-energy ionizing particle or photon makes two widely spaced double-strand breaks in the same DNA molecule. Deletions (or inversions) between two breaks formed by the same incident particle are linear in radiation dose and occur even at extremely low dose-rates; deletions between breaks induced by separate particles are quadratic in dose and are much fewer at very low dose-rate. The calculations show that for a few grays of sparsely ionizing radiations such as fast electrons, X-rays of gamma-rays, the formation of two double-strand breaks in a DNA molecule 1 megabase in size should be nearly entirely quadratic in dose. For heavily ionizing particles such as alpha particles from radon products, the linear and quadratic terms are comparable in size. These conclusions are robust and insensitive to details of the calculations. The results are essentially the same for DNA in a random coil configuration and for DNA uniformly and randomly distributed within a sphere.

Calculation by microdosimetric methods of the formation by a single high-energy photon or electron of two lesions in the same DNA molecule.

The formation by a single high-energy photon or electron of two lesions (such as double-strand breaks) in the same DNA molecule is calculated by microdosimetric methods. The result is similar to a previous calculation using a target theory approach. Thus, it is reasonably certain that, for > or = 1 Gy of sparsely ionizing radiation acting on a DNA molecule of > or = 10(6) base pairs, < or = 3% of two-hit events such as deletions are from a single radiation event.

The effect of bleomycin on DNA in Escherichia coli K12 cells.

In Escherichia coli cells treated to reduce colony-forming ability to about 10%, bleomycin causes fewer than six randomly located DNA single-strand breaks or three double-strand breaks per genome. This is many fewer than produced by strand-breaking agents such as ionizing radiations in cells with similar loss of colony-forming ability. Bleomycin treatment to this level of colony-forming ability does affect the intracellular DNA, as shown by a change in the sedimentation rate of the chromosomal structure found in lysates made with sodium dodecyl sulfate. Bleomycin may act on only a limited part of the chromosome of such cells, perhaps the part associated with the outer cell membrane, or it may make strand breaks that are less repairable than those formed by ionizing radiations. Extensive DNA degradation in heavily treated cells (colony-forming ability 1% or less) could be from the action on DNA of bleomycin entering freely through membranes which are no longer intact, or from enzymatic degradation in heavily damaged cells.

Published data on mutagenesis by ionizing radiation of plasmids in solution probably reflect in part the specificity of adventitious transition metal ions complexed to the DNA.

A number of recent papers show that single base changes induced by mutagenesis with ionizing radiation of genes on plasmids in solution, followed by transfection into mammalian or bacterial cells for assay, are mostly at G:C base pairs, with mutagenic hot spots. Genes irradiated in mammalian or bacterial cells, on the other hand, have comparable numbers of base changes at all sites, with no evidence for hot spots. The differences are ascribed to induction of many base change mutations in vitro by reactions catalyzed by adventitious transition metal ions complexed to the DNA. Reasons are given why this process should play a much smaller role in vivo.