Showing papers on "Lysis published in 1983"

Induction of degranulation and lysis of haemocytes in the freshwater crayfish, Astacus astacus by components of the prophenoloxidase activating system in vitro.

Abstract: To study the role of the prophenoloxidase activating system, an enzyme cascade located in the haemocytes of crustaceans, in the cellular defences of the freshwater crayfish, Astacus astacus in vitro, monolayer cultures of mixed or separated haemocyte populations, isolated by density gradient centrifugation, were challenged with the bacterium, Moraxella sp. pre-coated with phenoloxidase and the other attaching proteins in crayfish haemocyte lysate (HLS), or in the case of controls, with saline or Moraxella sp. pre-incubated in saline alone. Examination of the coverslips 1 h after inoculation revealed that, in the mixed haemocyte cultures, most of the cells had undergone profound degranulation and lysis following exposure to the HLS-coated bacteria. Cell lysis was also evident in the experimental semigranular cell monolayers, but not in the controls, although in those controls treated with the saline-incubated bacteria, the semigranular haemocytes had undergone degranulation without lysis. In contrast, the granular cells appeared to be unaffected by the saline-incubated Moraxella sp., and with the HLS-coated bacteria displayed only marked degranulation. Greater numbers of bacteria were always associated with the cells or cell remnants in the experimental cultures compared to the controls. We suggest that the attaching proteins of the prophenoloxidase cascade are strong nonself signals for the haemocytes, causing them to degranulate and release previously cell-bound recognition factors into the haemolymph, where they are free to trigger activation of adjacent haemocytes.

Cytolysis of nucleated cells by complement: cell death displays multi-hit characteristics

Abstract: Lysis of nucleated cells by complement was studied to determine whether the lytic process by C5b-9 conforms to a one-hit mechanism as in the case of erythrocytes. Two nucleated cell lines, Molt 4 and U937, derived from human T lymphocytes and histiocytes, respectively, were employed as targets. The antibody-sensitized cells were used to develop the titration curves, measuring cell death as a function of limiting quantities of human C6 or C5,6 complex in the presence of an excess of other complement components. The cytolysis curves generated in both experiments were sigmoidal, in sharp contrast to the monotonic curves observed in lysis of erythrocytes treated similarly. The sigmoidal curves of cytolysis indicate a cooperative action of several molecules of C6 or acid-activated C5,6 complex, C(56)a. In contrast to the multi-hit characteristics of cytolysis, dose-response measurements of the release of 86Rb indicated that only one effective molecule of C6 per cell is required for assembly of a 86Rb-releasing channel. This divergence indicates that lysis requires formation of several channels or, alternatively, assembly of large channels that are formed by several molecules of C6. Because prior studies with erythrocyte ghosts have shown that only a single effective molecule of C6 is required for assembly of a transmembrane channel, regardless of size, we prefer to interpret the multi-hit characteristics of nucleated cell lysis as an indication of a multi-channel requirement, rather than channel enlargement.

A study of the antibacterial activity of some polyhexamethylene biguanides towards Escherichia coli ATCC 8739.

Abstract: The antibacterial action of some polyhexamethylene biguanides upon Escherichia coli has been investigated. An amine ended dimer (AED) (n= 2), a polydisperse mixture sold by I.C.I. Limited as the active ingredient of Vantocil IB (PHMB, n= 5.5) and a high molecular weight (HMW) fraction (n≤ 10) of PHMB were used. PHMB and the high molecular weight fraction totally inhibited growth and motility of Esch. coli in liquid culture, whilst amine ended dimer never totally inhibited either function, irrespective of concentration. Growth inhibition and bacteriocidal activity increased with increasing levels of biocide-polymerization. The three compounds, whilst being active over different concentration ranges, possessed similar concentration exponents and temperature coefficients. Cytoplasmic membrane damage and disruption of the cell envelope, indicated by loss from the cells of 260 nm absorbing materials, inorganic phosphate, and changes in permeability towards the dye 2-p-toluidinylnaphthylene-6-sulphonate, was observed at concentrations which were markedly bactericidal whilst bacteristatic concentrations caused only loss of potassium. Loss of cytoplasmic materials from treated cells in all instances followed first-order kinetics. This indicated that irreversible damage was initiated and completed within a short time of contact between cells and biocide. Alteration of cytoplasmic membrane permeability towards various cations was assessed by measuring rates of lysis of sphaeroplasts in isotonic solutions of Na, Li and Cs acetate. For a given biocide concentration, the rates of lysis were inversely proportional to the hydrated ionic radii of the cation. This indicated that the damage to the cytoplasmic membrane was non-specific and proportional to biocide concentration. Temperature coefficients (Q10) of approximately 2.0 for the loss of 260 nm absorbing material, inorganic phosphate and potassium from treated cell-suspensions and bacteriocidal activity suggested that death of cells and cytoplasmic membrane damage are directly associated and are a direct result of biocide action, rather than mediated through the induction of autolytic enzymes.

Vectorial synthesis of a polysaccharide by isolated plasma membranes.

Abstract: To ascertain the directionality of chitin synthesis by yeast plasma membranes, the external surface of Saccharomyces cerevisiae protoplasts was labeled with ferritin--concanavalin A. After protoplast lysis, plasma membranes were isolated and treated with trypsin to activate chitin synthase (UDP-2-acetamido-2-deoxy-D-glucose:chitin 4-beta-acetamidodeoxy-D-glucosyl-transferase, EC 2.4.1.16). The membranes were then enrobed in agar and allowed to synthesize chitin from UDP-N-acetylglucosamine. After fixation and embedding in Epon, thin sections were stained for chitin with wheat germ agglutinin--colloidal gold complexes. The chitin marker was found near the ferritin-labeled external face of the membrane--i.e., the polysaccharide was located on the outside of the membrane, as it is in the intact cell. Chitin synthase activity was not detected in intact protoplasts before or after treatment with trypsin. The enzyme became available to trypsin activation after lysis of the protoplasts. Together with similar, previously reported experiments on the inactivation of chitin synthase by glutaraldehyde, these results indicate that the enzyme faces the interior of the cell. We conclude that, both in vivo and in vitro, the synthase receives N-acetylglucosamine residues from UDP-N-acetylglucosamine at the cytoplasmic face of the membrane and transfers them vectorially to a growing chain of chitin that is concomitantly extruded to the outside.

Inhibition of the Lytic Action of Cell-bound Terminal Complement Components by Human High Density Lipoproteins and Apoproteins

Abstract: Human serum lipoproteins are known to participate in or modify several immunologically relevant responses, including the inhibition of target cell lysis initiated by fluid-phase C5b-7 (reactive lysis). We now report that human high density lipoproteins (HDL) can inhibit the complement (C) lytic mechanism after C5b-7, C5b-8, and even C5b-9 have been bound to the target membrane. This inhibitory activity of serum or plasma copurifies in hydrophobic chromatography with antigenically detected apolipoprotein A-I (apoA-I), the major HDL apoprotein, and with HDL in CsCl density gradient ultracentrifugation. Although HDL is more active than its apoproteins in fluid-phase inhibition of C5b-7-initiated reactive lysis, the HDL apoproteins are more effective after C5b-7, C5b-8, or C5b-9 have become bound to human or sheep erythrocytes (E). Highly purified HDL apoproteins, apoA-I and apoA-II, both have greater inhibitory activity than whole HDL on a protein weight basis, and some evidence has been obtained that apoA-I dissociating spontaneously from HDL may be the principal inhibitory moiety in physiological situations. HDL lipids themselves are inactive. The HDL-related inhibitors are ineffective when incubated with EC5b-7 and removed before C8 and C9 are added, and only minimally effective on cell-bound C5b-8 sites before C9 is added. They exert their most prominent inhibitory activity after C9 has been bound to EC5b-8 at low temperature, but before the final temperature-dependent, Zn(++)-inhibitable membrane damage steps have occurred. Therefore, HDL or its apoproteins do not act to repair already established transmembrane channels, but might interfere either with insertion of C9 into the lipid bilayer or with polymerization of C9 at C5b-8 sites. This heat-stable inhibitory activity can be demonstrated to modify lysis of erythrocytes in whole serum, i.e., it does not depend upon artificial interruption of the complement membrane attack sequence at any of the above-mentioned stages. Contributions of the target membrane itself to the mechanism of inhibition are suggested by the observations that, in contrast to sheep or normal human E, lysis of guinea pig E or human E from patients with paroxysmal nocturnal hemoglobinuria is inhibited poorly. This is the first description of a naturally occurring plasma inhibitor acting on the terminal, membrane-associated events in complement lysis. Although further study is required to assess the physiologic or immunopathologic significance of this new function of HDL, the HDL apoproteins or their relevant fragments should be useful experimentally as molecular probes of the lytic mechanism.

Evidence for an active role of donor cells in natural transformation of Pseudomonas stutzeri.

Abstract: The transfer of chromosomal genes in a cell mat of Pseudomonas stutzeri was ca. 10(3) times more efficient per microgram of DNA if DNA was added as a constituent of intact donor cells rather than as a solution. Such intact cell-mediated transfer appears to depend on cell contact. It is independent of the presence of plasmids in donor strains and is DNase I sensitive, thus fitting the usual definition of transformation. It is bidirectional: cells of either strain in a transformation mixture served as the donor and recipients. The donor function in cell contact transformation was inhibited by nalidixic acid but was unaffected by rifampin and streptomycin at growth-inhibiting concentrations. Concentrations of nalidixic acid sufficient to inhibit donor function completely had no effect on the ability of nalidixic acid-resistant recipients to take up DNA from solution. These experiments suggest that certain cells donate DNA to others in the cell mat: they argue against the hypothesis that the function of donor cells is merely cell lysis.

Detection and partial characterization of antibacterial factor(s) in alveolar lining material of rats.

Abstract: Intracellular killing of Staphylococcus aureus by alveolar macrophages is known to be enhanced by exposure to alveolar lining material. Because this material may have a role in pulmonary host defenses, we have studied its effect on pneumococci and other nonstaphylococcal organisms. Alveolar lining material from rats caused rapid killing and lysis of pneumococci. The antipneumococcal activity was localized to the surfactant-containing fraction of the fluid and was not affected by trypsin. Phospholipid extracts of the surfactant fraction or purified lamellar bodies killed pneumococci. Lysis of pneumococci by the surfactant fraction appeared to be mediated by a detergent-like activation of pneumococcal autolysin, in that bacteriolysis was prevented by substitution of ethanolamine for choline in pneumococcal cell walls, and a pneumococcal transformant that lacked autolysin was not lysed. The surfactant fraction readily killed pneumococci containing ethanolamine or the autolysin-defective transformant, and studies with tritiated methyl-D-glucose loading and release showed that killing was associated with increased bacterial cell membrane permeability. Bactericidal activity (without lysis) was observed with several nonpneumococcal gram-positive bacteria, including Streptococcus viridans, unspeciated respiratory streptococci, Streptococcus pyogenes, Streptococcus bovis, and Bacillus species. Purified diacylphospholipids had no antibacterial activity, however, a lysophospholipid, palmitoyl lysophosphatidylcholine, had many properties resembling the surfactant-containing fraction of lavage, including autolysin-mediated pneumococcal lysis, altered cell membrane permeability, and antibacterial activity against several gram-positive bacteria.

The mechanism of synergistic complement‐mediated lysis of rat red cells by monoclonal IgG antibodies

Abstract: The mechanism of synergistic complement-mediated lysis of rat red cells was investigated using rat monoclonal antibodies against class I RT1Aa antigens. The increased lytic activity when using two antibodies simultaneously is due to the increase in the number of activated C1 molecules on the cell surface and this results from (a) an increase in the number of binding sites for C1q, (b) an increase in the functional affinity constant for C1q binding and (c) an increase in the rate of activation of C1. Complete lysis of red cells was only achieved if one member of the synergistic pair was of the gamma 2b isotype, and this isotype was the only one to which binding of 125I-labeled C1q could be detected. A partial synergistic effect was seen using an F(ab')2 fragment of antibody. Increased uptake and activation of C1 probably results both from the presence of two antibodies attached to each antigen molecule and from the formation of antigen-antibody catenars.

The lysis function of RNA bacteriophage Qbeta is mediated by the maturation (A2) protein.

Abstract: Complete or partial cDNA sequences of the RNA bacteriophage Qbeta were cloned in plasmids under the control of the lambdaP(L) promoter to allow regulated expression in Escherichia coli harbouring the gene for the temperature-sensitive lambdaCI857 repressor. Induction of the complete Qbeta sequence leads to a 100-fold increase in phage production, accompanied by cell lysis. Induction of the 5'-terminal sequence containing the intact maturation protein (A2) cistron also causes cell lysis. Alterations of the A2 cistron, leading to proteins either devoid of approximately 20% of the C-terminal region or of six internal amino acids, abolish the lysis function. Expression of other cistrons in addition to the A2 cistron does not enhance host lysis. Thus, in Qbeta, the A2 protein, in addition to its functions as maturation protein, appears to trigger cell lysis. This contrasts with the situation in the distantly related group I RNA phages such as f2 and MS2 where a small lysis polypeptide is coded for by a region overlapping the end of the coat gene and the beginning of the replicase gene.

S gene product: identification and membrane localization of a lysis control protein.

Abstract: The product of the bacteriophage S gene has been previously shown to be required for an essential step in triggering host cell lysis. By using two different protein labeling systems, maxicells and UV-irradiated infected cells, we identified the S gene product as an 8,500-molecular-weight polypeptide associated with the cell envelope. The apparent molecular weight is significantly less than the 11,500 predicted from the S gene sequence. We were unable to confirm two previous identifications of S gene products, an acidic 15,000-molecular-weight polypeptide found by two-dimensional gel electrophoresis of infected cells and a 5,500-molecular-weight polypeptide in purified phage particles.

Expression of a gene in a 400-base-pair fragment of colicin plasmid ColE2-P9 is sufficient to cause host cell lysis.

Abstract: The colicin E2 immunity (ceiB) and lysis (celB) genes of colicin plasmid ColE2-P9 were cloned as a 900-base-pair insert under the control of the lac promoter in high-copy-number plasmid pUR222. Hosts carrying this plasmid were immune to colicin E2, produced increased amounts of immunity protein (molecular weight, 9,000) and two smaller proteins (molecular weights, 5,000 and 3,000), and lysed when incubated in medium containing isopropyl-beta-D-thiogalactopyranoside (IPTG). A 400-base-pair lacp-distal fragment derived from the insert in this plasmid was recloned in the same orientation into pUR222. Although hosts carrying this plasmid also lysed when grown in the presence of IPTG, they were sensitive to colicin E2 and produced increased amounts of the 5,000- and 3,000-molecular-weight proteins (but not the full-length immunity protein) when treated with IPTG. The results were consistent with the idea that expression of celB (production of the 5,000- and 3,000-molecular-weight proteins) is sufficient to cause host cell lysis in the absence of colicin production and derepression of the host cell SOS system. Images

Arylsulfatase in natural killer cells: its possible role in cytotoxicity

Abstract: Ultrastructural cytochemistry of natural killer cells enriched by Percoll gradient centrifugation showed them to possess arylsulfatase (aryl-sulfate sulfohydrolase, EC 3.1.6.1). The enzyme was located in vesicles, granules, and the parallel tubular arrays, organelles characteristic for cytotoxic lymphocytes. Biochemically, peak enzyme activity correlated with the Percoll fractions containing cells with cytotoxicity for melanoma target cells. Treatment of natural killer cells with Na2SO4, a competitive inhibitor of arylsulfatase, suppressed cytotoxicity by almost 50%. Electron microscopy of effector-target cell conjugates, which had been permitted to incubate for only 30 min, disclosed numerous arylsulfatase-positive sites at the points of contact between the effector/target cell membranes. Thus, the enzyme was translocated to the surface before lysis of the target cell was morphologically evident. It is postulated that the parallel tubular arrays play a role in this translocation and that arylsulfatase may function in the degradation of cerebroside sulfate ester components of the target cell membrane to initiate the lytic event.

Photosensitized lysis of liposomes by hematoporphyrin derivative

Abstract: The spectral properties and efficiency for photosensitizing the lysis of phosphatidylcholine liposomes have been measured for the components of hematoporphyrin derivative (Hpd) after alkaline hydrolysis and fractionation by polyacrylamidc gel chromatography. Two major and two minor Hpd fractions have been identified whose spectral properties correlate with the anoxic sensitizing efficiency and the oxygen enhancement ratio (OER). The fastest moving fraction, which is the putative biologically active component, comprised one-third of the starting material and had OER = 2.7. Liposome lysis by this fraction was inhibited in the presence of human serum albumin at concentration ratios comparable to those employed for photoradiation therapy. The present results show that Hpd can act as an oxic and anoxic photosensitizer of a model biomembrane and suggest that separation from serum proteins is required for in vivo photosensitization.

Ca2+-sensitive isolation of a cortical actin matrix fromDictyostelium amoebae

Abstract: A cortical actin matrix has been isolated from amoebae ofDictyostelium discoideum grown in liquid culture. The existence of this actin matrix in whole cells is indicated in electron micrographs as an area free of cytoplasmic organelles. The actin beneath the membrane is more clearly visible in sections of cells that are lysed gently with 0.5% Triton X-100 and fixed with 1% glutaraldehyde. Such Triton-lysed cells have fragments of plasma membrane associated with the cortical actin matrix. Isolation of the actin matrix, which sediments at 400g, is inhibited by Ca2+. As much as 50% of the actin of the cell and about 12% of the total protein is found in the matrix isolated in lysis buffer containing no added Ca2+ and 2.5mm EGTA, whereas less than 15% of the actin of the cell is recovered in a 400g pellet when cells are lysed in buffer containing 2.5mm Ca2+ and 2.5mm EGTA. A 40 000 molecular weight protein that fragments F-actin in a Ca2+-dependent manner is not found in the isolated cortical actin matrix.

Recognition and lysis of altered-self cells by macrophages. I. Modification of target cells by 2,4,6-trinitrobenzene sulphonic acid.

Abstract: Peritoneal exudate macrophages from normal, untreated or thioglycollate-elicited mice, lysed syngeneic fibroblasts and lymphoblasts modified by 2,4,6-trinitrobenzene sulphonic acid (TNBS) in vitro. Optimal lysis of the hapten-modified cells by elicited macrophages was usually seen after 18 hr of co-cultivation at E:T ratios of 10:1-30:1. Cytotoxicity was expressed by macrophages depleted of T cells, and was not potentiated by LPS. Allogeneic TNBS-modified cells were lysed by non-immune, non-activated macrophages to the same extent as syngeneic modified targets, indicating that genetic restriction does not appear to play a role in macrophage-mediated cytolysis of TNBS-modified cells.

Does hydroxyurea inhibit DNA replication in mouse cells by more than one mechanism

Abstract: Cell-free DNA synthesis was performed in a lysed cell system from mouse cell cultures. The in vitro reaction was totally inhibited by N-ethylmaleimide but unaffected by hydroxyurea or fluorodeoxyuridine when these compounds were added to the incubation mixture. However, in a preparation obtained from cells which had been blocked by hydroxyurea before lysis, the rate of DNA synthesis was markedly reduced. This effect could not have been caused by the depletion of the precursor pools as all necessary triphosphates were added to the in vitro incubation mixture. Analysis by alkaline density gradients showed that the ligation of primary synthesis products is retarded in hydroxyurea-pretreated lysed cells and that small fragments accumulate. These results suggest that hydroxyurea interferes with the processing of early replication products, preventing the formation of longer intermediates. Its mechanism is either independent from the well-known inhibition of ribonucleoside diphosphate reductase or it may be the result of an as-yet-unknown function of this enzyme in a later step of replication. This observation could help to explain why cells appear to be blocked by hydroxyurea in the early part of the S phase (rather than at the G1/S border proper) and also why DNA repair synthesis is relatively insensitive to the drug.

Effect of growth conditions on production of rhamnose-containing cell wall and capsular polysaccharides by strains of Lactobacillus casei subsp. rhamnosus.

Abstract: Strains of Lactobacillus casei subsp. rhamnosus possessing two cell wall polysaccharides, a hexosamine-containing H-polysaccharide and a rhamnose-containing R-polysaccharide, were examined for the effect of growth conditions on the production of these two components. In strain NCTC 6375, R- and H-polysaccharides accounted for an estimated 44 and 20%, respectively, of the cell wall for organisms grown in batch culture with glucose as the carbohydrate source. Growth on fructose-containing media reduced the amount of R-polysaccharide by approximately 50% without affecting the amount of H-polysaccharide. Subculture of fructose-grown organisms in glucose restored the original proportions of the two polysaccharides. Galactose- and sucrose-grown cells behaved similarly to glucose-grown cells with respect to polysaccharide production, whereas growth in rhamnose or ribose showed values close to those for fructose-grown cells. Continuous culture of strain NCTC 6375 for more than 100 generations showed a gradual and irreversible reduction of the R-polysaccharide to less than 5% of the cell wall and an increase of the H-polysaccharide to 40% of the cell wall. Other type culture strains of L. casei subsp. rhamnosus, NCIB 7473 and ATCC 7469, behaved similarly in batch and continuous culture. In contrast, strains of L. casei subsp. rhamnosus isolated at the Institute of Dental Research showed phenotypic stability with respect to the relative proportions of R- and H-polysaccharides in both batch and continuous culture. Changes in polysaccharide composition of type culture strains were also mirrored in changes in the immunogenicity of the two components and resistance to the rate of enzymic lysis of whole organisms. For L. casei subsp. rhamnosus strain NCTC 10302 the R-polysaccharide is present entirely as capsular material. The amount of R-polysaccharide produced was also markedly dependent on the carbohydrate component of the medium in batch culture and both dilution rate and nature of the limiting carbohydrate in continuous culture, varying over a 10-fold range, whereas the cell wall H-polysaccharide remained constant.

A method for the purification of E. coli plasmid DNA by homogeneous lysis and polyethylene glycol precipitation.

Abstract: A procedure is described for the isolation and purification of E coli plasmid DNA by polyethylene glycol precipitation The method is rapid, simple, inexpensive and amenable to both small and large scale manipulation This procedure involves lysis of bacterial cells by treatment with pronase in sodium dodecyl sulfate, removal of chromosomal DNA by centrifugation, precipitation of residual nucleic acids with polyethylene glycol and removal of RNA by precipitation with LiCl Plasmid DNA purified as described is pure enough for restriction endonuclease analysis, for use as a vector for the cloning of cDNA or synthetic DNA, or for use as a template in an E coli transcription-translation cell-free system

The kinetics and mechanics of ultrasonically-induced cell lysis produced by non-trapped bubbles in a rotating culture tube

Abstract: The kinetics of ultrasonically-induced cell lysis are examined in terms of classical radiation biology target theory. A theoretical expression relating the concentration of intact cells remaining after a given period of sonication in a rotating culture tube to the number of non-trapped bubbles, l , which a cell must encounter in order to be lysed is obtained. The expression is compared to experimental results in order to determine the actual value of l . It is found that l equals one (1). The concentration of non-trapped bubbles which is responsible for the observed cell lysis is calculated to be 250–500 cm −3 . Finally, it is proposed that non-trapped bubbles tunnel into cells while undergoing stable cavitation and that cell lysis is produced by one or more transient events inside the cell.

Protein synthesis by intact Coxiella burnetii cells.

Abstract: Coxiella burnetii was isolated from persistently infected fibroblast host cells by a rapid mechanical lysis technique. Macromolecular synthesis was initiated in these otherwise dormant cells by incubation at pH 4.5. The synthesis of protein proceeded for as long as 24 h. Initiation of protein synthesis in C. burnetii was dependent upon RNA synthesis. Approximately 24 species of polypeptides were synthesized, and some of these appeared to be major synthetic products. Increases in protein biomass of 15 to 30% were calculated to occur during incubation. Inhibition of DNA synthesis affected protein synthesis after 12 h of incubation. The results suggest that although these parasitic bacteria did not grow in the axenic media devised, significant biosynthetic processes occurred.

Failure to detect extranuclear DNA in Trichomonas vaginalis and Tritrichomonas foetus.

Abstract: The biological affinities of hydrogenosomes, characteristic organelles of trichomonad flagellates, remain obscure (Miiller, 1980, Symp. Soc. Gen. Microbiol. 30: 127-142). Based on metabolic similarities they have been compared with anaerobic bacteria lacking cytochromes (Cerkasovovfi et al., 1980. In Industrial and clinical enzymology, L. Vitale and V. Simeon (eds.), pp. 257-275; Muller, 1980, loc. cit.) and it was suggested that they might be the anaerobic equivalents of mitochondria and might have a similar relationship to anaerobic bacteria as do mitochondria to aerobic bacteria and chloroplasts to cyanobacteria. This suggestion led to a search for DNA in these organelles. Cerkasovovfa et al. (1976, Folia Parasitol. Praha 23: 33-37) published electron microscopic observations on circular DNA from Tt. foetus hydrogenosomes isolated by density gradient centrifugation and subsequently treated with DNAse (Nass, 1969, J. Mol. Biol. 42: 521-528). To test this problem by an independent approach, we applied to trichomonads methods used to detect and study mitochondrial DNA (mtDNA) in various eukaryotic cells, including eukaryotic microorganisms (Williamson and Fennel, 1975. In Methods in Cell physiology, D. Prescott (ed.), 12: 235-351). Trichomonas vaginalis ATCC 30001 strain and Tt. foetus KVi strain were grown in TYM medium supplemented with 10% horse serum. Following the methods described by Williamson and Fennel (1975, loc. cit.) unfixed cells and cells fixed in formaldehyde were exposed to 4',6-diamidine2-phenylindole (DAPI), a fluorochrome strongly interacting with DNA, especially with A+T rich DNA. Under the fluorescence microscope such cells showed a clear fluorescence of the nucleus only, but none in the cytoplasm nor in any cytoplasmic organelle. Prior to extraction of DNA, cells were washed twice by gentle centrifugation with 250 mM sucrose solution containing 200 ,iM aurintricarboxylic acid (ATA). DNA was extracted either from whole, washed cells or from a large-particle fraction enriched fivefold in hydrogenosomes by differential centrifugation (Lindmark and Miiller, 1974, J. Biol. Chem. 248: 7724-7728). ATA was present in all solutions used in the fractionation procedure. Two methods were used for preparation of DNA. The first method was based on that of Cummings et al. (1979, Molec. Gen. Genet. 171: 229-238). To a pellet of whole cells or large particles of T. vaginalis resuspended in minimum volume of sucrose about 10 volumes of lysis buffer was added (250 mM Tris HC1, pH 8.0, 100 mM EDTA, 200 ,tM ATA, and 1% SDS) at 65 C, and the lysate was maintained at this temperature for about 15 min. The cells or subcellular organelles lysed within a few seconds and the suspension became viscous. The lysate was then centrifuged at 20,000 g for 15 min at 0 C and the supernatant solution retained. After addition of CsCl, the solution was recentrifuged at 20,000 g for 15 min to remove most of the remaining SDS and protein. When this method was applied to Tt. foetus, the DNA was rapidly degraded after heating to 65 C, so lysis was carried out at 0 C and within 30 sec the lysate was extracted with an equal volume of buffer-saturated phenol, followed by chloroform extraction and ethanol precipitation of the nucleic acids. The precipitate was dissolved in 250 mM Tris HC1, 100 mM EDTA (pH 8.0), and CsCl added. The second method is similar to that used previously by Mandel and Honigberg (1964, J. Protozool. 11: 114-116) to obtain high MW DNA from Trichomonas spp. To 4 ml of solution prepared by either method, 4.2 g CsCl and 500 jtg DAPI were added, and the samples were centrifuged for 24 hr at 42,000 rev min-l in the angle head of an MSE Superspeed 50 ultracentrifuge. Only one fluorescent band was observed in gradients containing DNA isolated from whole cells or hydrogenosome enriched fractions of either trichomonad species, and it probably represents nuclear DNA (Fig. 1A). A DNA species significantly lighter or