Showing papers on "Lysis published in 2000"

Antimicrobial mechanism of action of surfactant lipid preparations in enteric Gram-negative bacilli.

Abstract: Two surfactant lipid preparations (SLPs) were investigated to determine their mechanism of antimicrobial action. 8N8, a water-in-oil emulsion, and W60C, a liposome, both have bactericidal activity against Gram-positive bacteria and non-enteric Gram-negative bacteria. Additionally, W60C is bactericidal for enteric Gram-negative bacilli when suspended in deionized water. Zeta potential measurements suggested that the resistance of Gram-negative bacilli to 8N8 might be caused by ionic repulsion. Addition of 50 micromol 1(-1) ethylene diamine tetra acetic acid in 100 mmol 1(-1) Tris buffer to either SLPs yielded efficient bactericidal activity against Gram-negative bacilli. This appeared to be due to disruption of the outer membrane and the chelation of divalent cations, as the addition of excess calcium inhibited the antimicrobial effect. Electron microscopy studies documented that 8N8 disrupts the bacterial cell wall, lysing the bacteria, while W60C fuses and internalizes within the cell, causing damage without immediate cell lysis. Understanding the mechanisms of action of these biocidal formulations will help to produce improved formulations with broader spectra of activity.

Differential damage in bacterial cells by microwave radiation on the basis of cell wall structure.

Abstract: Microwave radiation in Escherichia coli and Bacillus subtilis cell suspensions resulted in a dramatic reduction of the viable counts as well as increases in the amounts of DNA and protein released from the cells according to the increase of the final temperature of the cell suspensions. However, no significant reduction of cell density was observed in either cell suspension. It is believed that this is due to the fact that most of the bacterial cells inactivated by microwave radiation remained unlysed. Scanning electron microscopy of the microwave-heated cells revealed severe damage on the surface of most E. coli cells, yet there was no significant change observed in the B. subtilis cells. Microwave-injured E. coli cells were easily lysed in the presence of sodium dodecyl sulfate (SDS), yet B. subtilis cells were resistant to SDS.

Granulysin, a T Cell Product, Kills Bacteria by Altering Membrane Permeability

Abstract: Granulysin, a protein located in the acidic granules of human NK cells and cytotoxic T cells, has antimicrobial activity against a broad spectrum of microbial pathogens. A predicted model generated from the nuclear magnetic resonance structure of a related protein, NK lysin, suggested that granulysin contains a four α helical bundle motif, with the α helices enriched for positively charged amino acids, including arginine and lysine residues. Denaturation of the polypeptide reduced the α helical content from 49 to 18% resulted in complete inhibition of antimicrobial activity. Chemical modification of the arginine, but not the lysine, residues also blocked the antimicrobial activity and interfered with the ability of granulysin to adhere to Escherichia coli and Mycobacterium tuberculosis . Granulysin increased the permeability of bacterial membranes, as judged by its ability to allow access of cytosolic β-galactosidase to its impermeant substrate. By electron microscopy, granulysin triggered fluid accumulation in the periplasm of M. tuberculosis , consistent with osmotic perturbation. These data suggest that the ability of granulysin to kill microbial pathogens is dependent on direct interaction with the microbial cell wall and/or membrane, leading to increased permeability and lysis.

Genetic evidence that the bacteriophage φX174 lysis protein inhibits cell wall synthesis

Abstract: Protein E, a 91-residue membrane protein of fX174, causes lysis of the host in a growth-dependent manner reminiscent of cell wall antibiotics, suggesting E acts by inhibiting peptidoglycan synthesis. In a search for the cellular target of E, we previously have isolated recessive mutations in the host gene slyD (sensitivity to lysis) that block the lytic effects of E. The role of slyD, which encodes a FK506 binding protein-type peptidyl-prolyl cis-trans isomerase, is not fully understood. However, E mutants referred to as Epos (plates on slyD) lack a slyD requirement, indicating that slyD is not crucial for lysis. To identify the gene encoding the cellular target, we selected for survivors of Epos. In this study, we describe the isolation of dominant mutations in the essential host gene mraY that result in a general lysis-defective phenotype. mraY encodes translocase I, which catalyzes the formation of the first lipid-linked intermediate in cell wall biosynthesis. The isolation of these lysis-defective mutants supports a model in which translocase I is the cellular target of E and that inhibition of cell wall synthesis is the mechanism of lysis.

Bactericidal and tumoricidal activities of synthetic peptides derived from granulysin

Abstract: Granulysin, a 9-kDa protein localized to human CTL and NK cell granules, is cytolytic against tumor cells and microbes. Molecular modeling predicts that granulysin is composed of five alpha-helices separated by short loop regions. In this report, synthetic peptides corresponding to the linear granulysin sequence were characterized for lytic activity. Peptides corresponding to the central region of granulysin lyse bacteria, human cells, and synthetic liposomes, while peptides corresponding to the amino or carboxyl regions are not lytic. Peptides corresponding to either helix 2 or helix 3 lyse bacteria, while lysis of human cells and liposomes is dependent on the helix 3 sequence. Peptides in which positively charged arginine residues are substituted with neutral glutamine exhibit reduced lysis of all three targets. While reduction of recombinant 9-kDa granulysin increases lysis of Jurkat cells, reduction of cysteine-containing granulysin peptides decreases lysis of Jurkat cells. In contrast, lysis of bacteria by recombinant granulysin or by cysteine-containing granulysin peptides is unaffected by reducing conditions. Jurkat cells transfected with either CrmA or Bcl-2 are protected from lysis by recombinant granulysin or the peptides. Differential activity of granulysin peptides against tumor cells and bacteria may be exploited to develop specific antibiotics without toxicity for mammalian cells.

Radiation-Induced Heat-Labile Sites that Convert into DNA Double-Strand Breaks

Abstract: The yield of DNA double-strand breaks (DSBs) in SV40 DNA irradiated in aqueous solution was found to increase by more than a factor of two as a result of postirradiation incubation of the DNA at 50 degrees C and pH 8.0 for 24 h. This is in agreement with data from studies performed at 37 degrees C that were published previously. Importantly, similar results were also obtained from irradiation of mammalian DNA in agarose plugs. These results suggest that heat-labile sites within locally multiply damaged sites are produced by radiation and are subsequently transformed into DSBs. Since incubation at 50 degrees C is typically employed for lysis of cells in commonly used pulsed-field gel assays for detection of DSBs in mammalian cells, the possibility that heat-labile sites are present in irradiated cells was also studied. An increase in the apparent number of DSBs as a function of lysis time at 50 degrees C was found with kinetics that was similar to that for irradiated DNA, although the magnitude of the increase was smaller. This suggests that heat-labile sites are also formed in the cell. If this is the case, a proportion of DSBs measured by the pulsed-field gel assays may occur during the lysis step and may not be present in the cell as breaks but as heat-labile sites. It is suggested that such sites consist mainly of heat-labile sugar lesions within locally multiply damaged sites. Comparing rejoining of DSBs measured with short and long lysis procedure indicates that the heat-labile sites are repaired with fast kinetics in comparison with repair of the bulk of DSBs.

Dna isolation method

Abstract: Disclosed is a method and apparatus for the isolation of DNA or cell nuclei or a mixture thereof from cell samples in a CD device. The method includes treating a suspension of whole cells with a lysis reagent so as to lyse the cytoplasmic membrane and at least some of the nuclear membranes, and introducing the lysate into micro-channels of a microfabricated apparatus in which each of the micro-channels is provided with a barrier disposed in the channel to impede the passage or flow of DNA and cell nuclei while allowing the passage of liquid through the micro-channel so that a mesh comprising DNA is formed in the channel.

Influence of scale-up on the quality of recombinant human growth hormone.

Abstract: The aerobic fed-batch production of recombinant human growth hormone (rhGH) by Escherichia coli was studied. The goal was to determine the production and protein degradation pattern of this product during fed-batch cultivation and to what extent scale differences depend on the presence of a fed-batch glucose feed zone. Results of laboratory bench-scale, scale-down (SDR), and industrial pilot-scale (3-m(3)) reactor production were compared. In addition to the parameters of product yield and quality, also cell yield, respiration, overflow, mixed acid fermentation, glucose concentration, and cell lysis were studied and compared. The results show that oxygen limitation following glucose overflow was the critical parameter and not the glucose overflow itself. This was verified by the pattern of byproduct formation where formate was the dominating factor and not acetic acid. A correlation between the accumulation of formate, the degree of heterogeneity, and cell lysis was also visualized when recombinant protein was expressed. The production pattern could be mimicked in the SDR reactor for all parameters, except for product quantity and quality, where 30% fewer rhGH-degraded forms were present and where about 80% higher total yield was achieved, resulting in 10% greater accumulation of properly formed rhGH monomer.

Simplified Protocol for Pulsed-Field Gel Electrophoresis Analysis of Streptococcus pneumoniae

Abstract: A variety of pulsed-field gel electrophoresis (PFGE) protocols for the molecular subtyping of Streptococcus pneumoniae have been reported; most are time-consuming and complex. We sought to modify reference PFGE protocols to reduce the time required while creating high-quality gels. Only protocol modifications that resulted in high-quality banding patterns were considered. The following protocol components were modified. Lysis enzymes (lysozyme, mutanolysin, and RNase A) were deleted in a stepwise fashion, and then the lysis buffer was deleted. Lysis and digestion were accomplished in a single step with EDTA and N-lauroyl sarcosine (ES; pH 8.5 to 9.3) incubation at 50°C in the absence of proteinase K. All enzymes except the restriction enzyme were omitted. A minimum incubation time of 6 h was required to achieve high-quality gels. All of the reactions were performed within 9 h, and the total protocol time from lysis to gel completion was reduced from 3 days to only 36 h. Combining lysis and digestion into a single step resulted in a substantial reduction in the time required to perform PFGE for S. pneumoniae. The ES solution may have caused cell lysis by activating N-acetylmuramyl-l-alanine amidase, the pneumococcal autolysin.

Development of a new lysis solution for releasing genomic DNA from bacterial cells for DNA amplification by polymerase chain reaction.

Abstract: A new lysis solution designated TZ, consisting of 2.0% Triton X-100 plus 2.5 mg sodium azide/ml in 0.1 M Tris-HCl buffer at pH 8.0, yielded higher levels of genomic DNA from Escherichia coli O157:H7 cells compared with a number of other commonly used cell lysis methods. Ethidium bromide stained DNA bands resulting from PCR amplification of target DNA from 100 CFU of E. coli O157:H7 were readily detected following electrophoresis of agarose gels. In contrast, conventional cell lysis methods failed to detect target DNA from 100 CFU after PCR amplification. The new solution was highly effective for lysing cell suspensions of Salmonella enteritidis, Pseudomonas putida, Lysteria monocytogenes and Psychrobacter immobilis.

Lactococcin 972, a bacteriocin that inhibits septum formation in lactococci.

Abstract: Addition of lactococcin 972 to exponentially growing sensitive cultures of Lactococcus lactis resulted in cell elongation and widening. Thin sections revealed that septum invagination was blocked. Cell growth progressed until most cells showed equatorial constriction and even initial deposition of material at the septum ring, although cell division did not proceed any further. The increase in the incorporation of labelled precursors into the cell wall shifted from an exponential to a linear mode in treated cultures, subsequently being arrested. Gross degeneration of the cells was observed prior to cell death, followed by slow lysis of the culture. In contrast, stationary-phase cultures remained unaffected.

Saccharomicins, Novel Heptadecaglycoside Antibiotics Produced by Saccharothrix espanaensis: Antibacterial and Mechanistic Activities

Abstract: Saccharomicins A and B, two new heptadecaglycoside antibiotics, were isolated from the fermentation broth of the rare actinomycete Saccharothrix espanaensis. They represent a novel class of bactericidal antibiotics that are active both in vitro and in vivo against bacteria and yeast (MICs: Staphylococcus aureus, 128; and yeast, >128 microg/ml), including multiply resistant strains. Saccharomicins protected mice from lethal challenges by staphylococci (subcutaneous 50% effective dose range of 0.06 to 2.6 mg/kg of body weight, depending on the S. aureus strain). The 50% lethal dose by the subcutaneous route was 16 mg/kg. Mechanistic studies with Escherichia coli imp and Bacillus subtilis suggested complete, nonspecific inhibition of DNA, RNA, and protein biosynthesis within 10 min of drug treatment. Microscopic examination of drug-treated cells also suggested cell lysis. These data are consistent with a strong membrane-disruptive activity. The antibacterial activities of the saccharomicins against gram-positive bacteria were unaffected by the presence of Ca(2+) or Mg(2+), but activity against gram-negative bacteria was substantially reduced.

Requirement of Autolytic Activity for Bacteriocin-Induced Lysis

Abstract: The bacteriocin produced by Lactococcus lactis IFPL105 is bactericidal against several Lactococcus and Lactobacillus strains. Addition of the bacteriocin to exponential-growth-phase cells resulted in all cases in bacteriolysis. The bacteriolytic response of the strains was not related to differences in sensitivity to the bacteriocin and was strongly reduced in the presence of autolysin inhibitors (Co 21 and sodium dodecyl sulfate). When L. lactis MG1363 and its derivative deficient in the production of the major autolysin AcmA (MG1363acmAD1) were incubated with the bacteriocin, the latter did not lyse and no intracellular proteins were released into the medium. Incubation of cell wall fragments of L. lactis MG1363, or of L. lactis MG1363acmAD1 to which extracellular AcmA was added, in the presence or absence of the bacteriocin had no effect on the speed of cell wall degradation. This result indicates that the bacteriocin does not degrade cell walls, nor does it directly activate the autolysin AcmA. The autolysin was also responsible for the observed lysis of L. lactis MG1363 cells during incubation with nisin or the mixture of lactococcins A, B, and M. The results presented here show that lysis of L. lactis after addition of the bacteriocins is caused by the resulting cell damage, which promotes uncontrolled degradation of the cell walls by AcmA. Bacteriocins are antimicrobial polypeptides synthesized ribosomally by bacteria (34). Most bacteriocins from lactic acid bacteria exert their antibacterial effect by permeabilizing the target cell membrane, whereby the cells lose their viability (1, 5, 29). Apart from damaging cell membranes, some bacteriocins have also been reported to cause bacteriolysis. Bierbaum and Sahl (4) were among the first to show the involvement of autolysins in the bacteriolytic effect of a bacteriocin. Autolysins are peptidoglycan hydrolases that are capable of causing bacterial autolysis (39). The authors showed that the bacteriocins Pep5, produced by Staphylococcus epidermidis, and nisin, produced by Lactococcus lactis, activate an N-acetylmuramoyl-Lalanine amidase and an b-N-acetylglucosaminidase of S. simulans (4). Plantaricin C has been shown to be bacteriolytic for Lactobacillus fermentum LM 13554 and L. delbrueckii subsp. bulgaricus LMG 13551, while no reduction of the optical densities (ODs) of mid-exponential-phase cultures of L. sake CECT 906, L. helveticus LMG13555, or Leuconostoc mesenteroides was observed (14, 15). Microscopic analysis of L. fermentum cells treated with plantaricin C showed that changes had taken place in the cell wall. The authors suggested that cell lysis could be a secondary effect of the bacteriocin caused by a deregulation of the autolytic system of the sensitive cells resulting in destruction of the peptidoglycan layer. While no lysis of Lc. mesenteroides cells treated with plantaricin C was seen, a clear reduction of the OD was observed when these cells were incubated with pediocin AcH (3). This effect was not observed with Lactobacillus plantarum, although intracellular components were released. Transmission electron micrographic analysis of cells of both bacterial species revealed the presence of lysed ghost cells upon treatment with pediocin AcH (18). The action of nisin against Listeria monocytogenes

In-situ sampling and separation of RNA from individual mammalian cells.

Abstract: In this investigation RNA was directly sampled and separated at the single-cell level (without extraction) by capillary electrophoresis (CE). Laser-induced fluorescence (LIF) was employed to detect ethidium bromide-labeled RNA molecules under native conditions. Hydroxypropylmethylcellulose was used as a matrix for molecular sieving. Additives to the polymer solution included poly(vinylpyrrolidone) to eliminate the electroosmotic flow and mannitol to enhance the separation. Peak identities were confirmed as RNA by enzymatic treatment with RNase I. The individual Chinese Hamster Ovary (CHO-K1) cells were injected into a capillary and the cells were lysed on-line with sodium dodecyl sulfate (SDS) solutions before running electrophoresis. Low molecular mass (LMM) RNAs as well as larger fragments (tentatively identified as 18S and 28S ribosomal RNA by comparison with the literature) were detected with this system, which corresponds to a detected amount of ≈10−20 pg of RNA/cell. A Proteinase K study showed that...

Heterotrimeric G-proteins of a filamentous fungus regulate cell wall composition and susceptibility to a plant PR-5 protein.

Abstract: Membrane permeabilizing plant defensive proteins first encounter the fungal cell wall that can harbor specific components that facilitate or prevent access to the plasma membrane. However, signal transduction pathways controlling cell wall composition in filamentous fungi are largely unknown. We report here that the deposition of cell wall constituents that block the action of osmotin (PR-5), an antifungal plant defense protein, against Aspergillus nidulans requires the activity of a heterotrimeric G-protein mediated signaling pathway. The guanidine nucleotide GDPbetaS, that locks G-proteins in a GDP-bound inactive form, inhibits osmotin-induced conidial lysis. A dominant interfering mutation in FadA, the alpha-subunit of a heterotrimeric G-protein, confers resistance to osmotin. A deletion mutation in SfaD, the beta-subunit of a heterotrimeric G-protein also increases osmotin resistance. Aspergillus nidulans strains bearing these mutations also have increased tolerance to SDS, reduced cell wall porosity and increased chitin content in the cell wall.

Application of polymerase chain reaction-denaturing gradient gel electrophoresis for comparison of direct and indirect extraction methods of soil DNA used for microbial community fingerprinting.

Abstract: We used polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE) to compare bacterial community patterns obtained with target DNA extracted from a soil by direct and indirect methods For this purpose, two direct extraction methods, ie cell lysis by bead beating and cell disruption by grinding in liquid N, and two indirect methods, ie cell extraction followed by DNA extraction, and combined RNA/DNA extraction from the bacterial cell fraction, were performed Crude extracts were purified and amplified using universal bacterial primers PCR products were then analysed by DGGE, and similarity between the profiles obtained was determined by unweighted pair group with mathematical averages clustering The results showed clear profiles that presumably represented the dominant bacterial fractions in the samples The profiles generated by all four methods were similar, indicating that the methods were of approximately equal efficiency in the extraction of target DNA representative of the soil bacterial community However, the patterns of clustering also indicated that different populations of bacteria could be detected in the same soil using different soil DNA extraction methods The application of two dilution levels of DNA in PCR-DGGE showed that the most stable profile of the soil bacterial community could be generated by the direct methods The indirect methods gave clustered profiles at both dilution levels It is likely that these methods extracted DNA from a major, easily desorbed, bacterial fraction, consisting of low-density populations PCR-DGGE was found to be a suitable technique with which to assess differences in methods for DNA extraction from soil, which can be further used for the determination of microbial community diversity at the molecular level

The cytolytic effect of a glycoconjugate extracted from corms of saffron plant (Crocus sativus) on human cell lines in culture.

Abstract: Corms of Crocus sativus L. (Iridaceae) contain a glycoconjugate that shows cytotoxic activity on tumoral cells in culture. Studies of intracellular calcium fluctuations, and release of lactate dehydrogenase in human cervical epitheloid carcinoma cells, showed that this compound caused plasma membrane damage, allowing movements of both calcium and macromolecules, and leading to cell lysis. Analysis of DNA fragmentation showed that cell death was not mediated by apoptosis. This molecule is active against human tumoral cells derived from fibrosarcoma, cervical epithelioid carcinoma and breast carcinoma, with IC50 values of 7, 9 and 22 micrograms/ml, respectively. The proteoglycan is about 8 times more cytotoxic for malignant cells than for their normal counterparts. In addition, 100 micrograms/ml of proteoglycan produced 50% in vitro lysis of normal human erythrocytes, whereas 320 micrograms/ml induced about 60% cell death on cultured human hair follicles. Altogether, these results suggests a distinctive cytotoxic activity of this molecule on different human cell types.

Use of sonication for measuring acid phosphatase activity in soil

Abstract: Extracellular enzymes in soil often occur in immobilised forms, a state that may alter their interactions with substrates in comparison with enzymes in the solution phase. Sonication was evaluated for its usefulness in studying immobilised acid phosphatase by dispersing soil aggregates. Factors affecting soil dispersion during ultrasound application were soil extraction ratio, total applied energy and power output ml−1 of sonicated soil slurry. For the clay loam soil used, optimal values for these variables were, respectively, 1:6 (w/v) and, at least, 1800 J ml−1 and 15 W ml−1. At the optimal sonication conditions for soil dispersion a substantial increase in phosphatase activity (up to 156% greater than the non-sonicated control) was induced by sonication. This increase in activity with sonication, which coincided with a release of soil chromophores, might be related to the exposure or release from aggregates of the extracellular enzyme fraction immobilised on humic colloids. Analysis of multiple regression between the phosphatase activity (dependent variable) and chromophore solubilisation and ATP release (independent variables) suggested the increased activity was from complexed enzymes that were released and not due to cell lysis. Soil treatment with sonication appeared to have liberated a large dormant portion of acid phosphatase activity. Coefficients of variation of the activity decreased greatly (from 20% in control soil to 4% as an average after sonication).

One step sample preparation and detection of nucleic acids in complex biological samples

Abstract: A method for simultaneous release and detection of nucleic acids from complex biological samples is described. The invention relates to the combined use of lysis buffers containing strong chaotropic agents such as guanidine thiocyanate to facilitate cell lysis and release of cellular nucleic acids and to the use of a novel type of bicyclic nucleotide analogues, locked nucleic acid (LNA) to detect specific nucleic acids released during lysis by nucleic acid hybridisation. In particular methods are described for the covalent attachment of the catching LNA-oligo. Novel methods for sample preparation of e.g. polyadenylated mRNA species are also presented. The invention further addresses reagents for performing the methods as well as reagents and applications of the method.

The Streptococcus thermophilus autolytic phenotype results from a leaky prophage.

Abstract: Streptococcus thermophilus autolytic strains are characterized by a typical bell-shaped growth curve when grown under appropriate conditions. The cellular mechanisms involved in the triggering of lysis and the bacteriolytic activities of these strains were investigated in this study. Lactose depletion and organic solvents (ethanol, methanol, and chloroform) were shown to trigger a premature and immediate lysis of M17 exponentially growing cells. These factors and compounds are suspected to act by altering the cell envelope properties, causing either the permeabilization (organic solvents) or the depolarization (lactose depletion) of the cytoplasmic membrane. The autolytic character was shown to be associated with lysogeny. Phage particles, most of which were defective, were observed in the culture supernatants after both mitomycin C-induced and spontaneous lysis. By renaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a bacteriolytic activity was detected at 31 kDa exclusively in the autolytic strains. This enzyme was detected during both growth and spontaneous lysis with the same intensity. We have shown that it was prophage encoded and homologous to the endolysin Lyt51 of the streptococcal temperate bacteriophage φ01205 (M. Sheehan, E. Stanley, G. F. Fitzgerald, and D. van Sinderen, Appl. Environ. Microbiol. 65:569–577, 1999). It appears from our results that the autolytic properties are conferred to the S. thermophilus strains by a leaky prophage but do not result from massive prophage induction. More specifically, we propose that phagic genes are constitutively expressed in almost all the cells at a low and nonlethal level and that lysis is controlled and achieved by the prophage-encoded lysis proteins.

Extraction and Purification of Plasmid DNA

Abstract: Plasmids may be isolated by a variety of methods many of which rely on the differential denaturation and reannealing of plasmid DNA compared to chromosomal DNA. Many of these are rapid, small-scale "minipreps" that may be used effectively for plasmid analysis and further manipulation. The methods differ primarily in the means by which cells are lysed. One commonly used technique developed by Birnboim and Doly involves alkaline lysis (1). Although many slight modifications of the original protocol exist, it essentially relies on bacterial lysis by sodium hydroxide and sodium dodecyl sulfate (SDS), followed by neutralization with a high concentration of low-pH potassium acetate. This gives selective precipitation of the bacterial chromosomal DNA and other highmolecular-weight cellular components. The plasmid DNA remains in suspension and is precipitated with isopropanol. An alternative to alkaline lysis is the rapid boiling method developed by Holmes and Quigley (2). Here, the cells are lysed partially allowing plasmids to escape, whereas the bacterial chromosomal DNA remains trapped in the cell debris. High temperature is then used to denature the chromosomal DNA, after which reannealing allows the plasmids to reassociate. Centrifugation removes the chromosomal DNA along with the cell debris, leaving the plasmid in suspension, from where it is recovered by isopropanol precipitation. After the initial characterization, it is possible to purify further some or all of the plasmid DNAs by RNase digestion and extraction with organic solvents. This further purified DNA is suitable for techniques such as DNA sequencing, subcloning or the production of gene probes (3).