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Permeabilization of Beauveria bassiana blastospores for in situ enzymatic assays

     Department of Applied Microbiology and Food Science, College of Agriculture, University of Saskatchewan, Saskatoon, Saskatchewan, Canada S7N 5A8

	ABSTRACT

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Vigorous agitation of an aqueous suspension of blastospores (BS) of Beauveria bassiana mixed with nine volumes of a 1:4 (v/v) mixture of toluene:ethanol (95%) for 10 min permits blastospore permeabilization. Agitation results in greater membrane permeabilization than heating blastospores in the presence of toluene:ethanol or the detergents Triton X-100, sodium dodecyl sulfate, hexadecyltrimethylammonium bromide and Brij-35. The ß-galactosidase activity in permeabilized blastospores was determined with these methods. The effectiveness of permeabilization in detecting enzyme activity was assessed by comparison to whole BS lysates prepared by mechanical disruption and pressurized disruption of BS biomass. The toluene:ethanol method was applied to study the incorporation of ³H-thymidine triphosphate into blastospore DNA. Whole BS permeabilization allows the examination of enzyme activity and DNA synthesis at a cellular level in this important mycoinsecticide.

Key words: Beauveria bassiana, ß-galactosidase, deoxythymidine triphosphate, in situ, permeabilization

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Beauveria bassiana is an entomopathogen with the potential to have a wider market niche in the area of biological pest control (Feng et al 1994). Although the use of B. bassiana has increased in accordance with increased understanding of the biochemistry and molecular biology of entomopathogenicity, additional tools are needed to study B. bassiana in the laboratory and in the field (Hegedus and Khachatourians 1995, Khachatourians 1996, Khachatourians et al 2002). A challenge in the study of B. bassiana physiology has been rapid assay methods to investigate enzyme kinetics and function.

Whole-cell permeabilization has been applied in filamentous fungi and yeast for enzyme assay methods. Interest in whole-cell permeabilization was the result of an inability to extrapolate in vitro results to the enzyme concentrations found in vivo (Serrano et al 1973). Methods vary, based on the microorganisms and compatibility of the permeabilization system. Whole-cell permeabilization for in situ assays has been achieved with organic solvents such as chloroform, ether or a solution of toluene:ethanol (Felix 1982, Oertel and Goulian 1979, Serrano et al 1973, Quigley et al 1987, Basabe et al 1979, Maggesse et al 1982, Sorol et al 2001). Treatments, such as heating the cells in detergent solutions of 1–2% (v/v), also have been reported (Felix 1982). However, the use of alcohols, antibiotics, detergents, ethylenediaminetetraacetic acid (EDTA) and methods such as osmotic shock and freezing and thawing also have enabled in situ assays in yeast and filamentous fungi (Quigley et al 1987, Liu et al 1999, Kippert 1995, Seip and Di Cosimo 1992, Laouar et al 1992, Miozzari et al 1978, Förster et al 1998, Shailasree et al 1999, esták and Farka 2001, Oliveira et al 1981, Wellman and Pendayala 1979, Felix 1982). The comparability of permeabilization methods based on different cell treatments becomes difficult because each method produces cells with different permeability alterations, each of which has applicability under different conditions.

Permeabilization of fungi first was developed in yeast using a solvent-based method. A solution of toluene:ethanol was used as the permeabilizing agent for enzymatic studies in Saccharomyces cerevisiae and Candida albicans to determine cellular levels of nitrate reductase, glucose-6-phosphatase, alkaline/acid phosphatase, ß-galactosidase, hexokinase and pyruvate kinase (Serrano et al 1973, Choudary and Rao 1976, Ram et al 1983, Choudary 1984). Using solvent and detergent-based methods, several investigators have permeabilized S. cerevisiae cells to be used as industrial biocatalysts in either immobilized or suspension modes (Seip and Di Cosimo 1992, Shailasree et al 1999, Liu et al 1999). Similarly, toluene:ethanol permeabilization has been advantageous for in situ enzyme assays of filamentous fungi (Basabe et al 1979, Maggesse et al 1982, Quigley et al 1987, Sorol et al 2001).

In studies of molecular biology of DNA synthesis, in fungi, whole-cell permeabilization is an important tool. In terms of precursors of DNA replication, thymine or deoxythymidine are not incorporated into DNA because they are not transported well into the cytoplasm and fungi do not possess thymidine kinase (Kornberg and Baker 1992). This prevents thymidine labeling of DNA in fungi unless a recombinant thymidine kinase is expressed (Sachs et al 1977, Dien and Srienc 1991). Permeabilization enables uptake of deoxythymidine triphosphate (dTTP), which can be incorporated directly into DNA and removes the need for thymidine kinase for specific labeling of DNA. Chromosomal and mitochondrial DNA synthesis in S. cerevisiae was investigated using ether permeabilized cells and osmotically shocked spheroplasts (Oertel and Goulian 1979, 1977).

In this paper we describe a toluene- and ethanol-based permeabilization method to assay ß-galactosidase and study DNA synthesis using blastospores (BS) of the entomopathogenic filamentous fungus, Beauveria bassiana.

	MATERIALS AND METHODS

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Strain and growth condition – Beauveria bassiana, strain GK2016 (BioInsecticide Research Laboratory, Department of Applied Microbiology and Food Science, College of Agriculture, University of Saskatchewan, Canada) was grown in minimal medium (9 mM NH₄Cl, 12.5 mM NH₄NO₃, 14 mM Na₂SO₄, 22 mM KH₂PO₄, 52 mM K₂HPO₄, 0.4 mM MgSO₄·7 H₂O) broth with 0.1% (w/v) gelatin and 0.1% (w/v) sucrose. A 1 x 10⁸/mL conidiospore suspension was used to inoculate the broth at 1% (v/v). Growth was permitted for 192 h in a water bath shaker at 27 C and 180 rpm. Blastospores were harvested by filtration through glass wool, as described in Hegedus et al (1990).

Cell permeabilization – Filtered BS were centrifuged at 3000 x g for 15 min and resuspended in permeabilization buffer (75 mM imidazole, 0.1 M KCl, 10 mM MgCl₂, 0.02% (v/v) Tween 80) modified from Serrano et al (1973). Blastospore concentration was 10⁹/mL, as determined by a hemocytometer count. The BS suspension was aliquoted into 1.5 mL microfuge tubes in the volume ranges 200 µL, 500 µL or 1000 µL, depending on experimental conditions. Permeabilization also was carried out in microtiter plates, in which 100 µL or 200 µL of a BS suspension was aliquoted into the microtiter plate wells. Permeabilization required the addition of a solution of toluene (T): 95% (v/v) ethanol (E) at a ratio of 1:4 (T:E) at a concentration of 10% (v/v). BS were agitated at 1400 rpm in an Eppendorf Thermomixer 5436 set at 27 C for 5, 10 or 15 min, depending on experimental conditions. Microtiter plates were agitated in a temperature-controlled Labnet Vortemp 56EVC at 1200 rpm and 27 C.

Alternate permeabilization methods tested involved heating the blastospore suspension for 30 min in the presence of the selected permeabilization reagent. The reagents tested at 0.2% (w/v) were Triton X-100, sodium dodecyl sulfate (SDS), hexadecyltrimethylammonium bromide (CTAB), and Brij-35. A combination of Triton X-100 and CTAB each at 0.1% (w/v) also was used. The effect of heating the BS in the presence of 10% (v/v) T:E was assessed.

The extent of BS permeabilization was qualitatively determined by adding 0.4% (w/v) trypan blue to an aliquot of the BS suspension at a ratio of 1:5. A wet mount was viewed at 1000x under oil immersion with untreated BS representing no permeabilization.

B-galactosidase assay – The assay was preformed by using the chromogenic substrate method of Mac Pherson and Khachatourians (1991), modifying BS permeabilization to the method outlined above. Reaction tubes and microtiter plates were mixed at 600 rpm and 300 rpm, respectively, at 27 C during the assay. The reaction was stopped by addition of Na₂CO₃ to a final concentration of 0.2 M, and BS were pelleted by centrifugation at 3000x g for 15 min. ß-galactosidase activity was determined by reading the absorbance of the supernatant with a Spectronic 20 spectrophotometer at 420 nm. To determine whether BS permeabilization caused leakage of BS components ß-galactosidase activity was assayed in two fractions. The first fraction determined the activity within the BS (pellet) from the absorbance of the supernatant obtained from centrifugation immediately after the reaction was stopped. The second fraction determined the ß-galactosidase activity remaining within the cells able to leak out into the surrounding buffer by resuspending pelleted BS, without further addition of substrate, in buffer and assaying a second time. The BS then were repelleted by centrifugation, as described above, and the absorbance of the resulting supernatant fraction read at 420 nm.

Whole BS lysates – ß-galactosidase activity of whole BS lysates of B. bassiana was determined by two methods. The BS biomass obtained from glass wool filtration subsequently was collected by vacuum on a double layer of Whatman No.1 filter paper. A French press method described by Moussou et al (2000) was used with the following changes. A 500 mL broth culture of B. bassiana was prepared as described above and resuspended in 60 mL Z-buffer (Sambrook et al 1989) to 10⁹ fungal propagules/mL. The phenylmethyl sulfonyl fluoride (PMSF) stock solution used was prepared as described in Sambrook et al (1989). In addition, a small-scale whole BS lysate preparation was used that mechanically disrupted the BS. The BS biomass of a 50 mL culture, prepared as previously described, was resuspended in a 15 mL polystyrene tube to 10⁹ fungal propagules/mL with 1 mL Z-buffer and 10 µL 100 mM PMSF to which 0.30 g glass beads (diameter = 425–600 µm, acid washed) were added. Blastospore breakage was achieved by vortexing at maximum speed for 5 min constantly or a total of 5 min in 30 s bursts with 30 s on ice in between. The lysates were centrifuged for 5 min at 10 000 x g to pellet BS debris. A 1-mL aliquot of each supernatant was removed and the absorbance read at 420 nm. The ability of each method to rupture BS was assessed by viewing a wet mount of the whole BS lysates under 1000x oil immersion.

Radioisotope uptake assay – Blastospores were permeabilized, as described, with 5% (w/v) glucose added to the permeabilization buffer, then ³H-deoxythymidine triphosphate ammonium salt (³H-dTTP) was added to a final concentration of 30 nM (30 Ci/mmol, Amersham Biosciences). Reaction tubes were incubated 2.5–120 min at 27 C while being mixed at 600 rpm in an Eppendorf Thermomixer 5436. Linearity of uptake was determined by dilution of 0.33 nM ³H-dTTP with unlabeled PCR Grade dTTP (100 mM, GibcoBRL Life Technologies) using concentrations ranging from 10 nM to 1000 nM. Samples were incubated 15 min. Reactions were stopped by addition of two volumes of ice-cold 5% (w/v) trichloroacetic acid (TCA) in relation to sample volume and held on ice 30–60 min. The contents of the reaction then were filtered through a Millipore HA filter, pore size 0.45 µm (13 mm diameter) to capture and retain BS. The filters were washed twice with 3 mL of 5% (w/v) ice-cold TCA and twice with 3 mL of 40 C distilled water. Total counts per minute (CPM) in reaction tubes were determined by applying 5 µL of ³H-dTTP (0.15 µCi/mM) to Whatman No. 1 filter paper. Millipore and Whatman filters were dried in scintillation vials at 45 C. Beckman Ready-Solv scintillation liquid was added to the vials that were counted using a Beckman LS1801 scintillation counter.

DNA extraction – The BS were mixed in a 1.5 mL microfuge tube for 5 min by a vortex using 0.30 g glass beads (diameter = 425–600 µm, acid washed) in 200 µL of extraction buffer (2% (v/v) Triton X-100, 1% (w/v) SDS, 100 mM NaCl, 10 mM Tris HCl, 1 mM EDTA, pH 8), 200 µL phenol and 200 µL chloroform. This treatment caused BS breakage. The supernatant was recovered after centrifugation at 10 000x g for 5 min, and DNA was extracted by a subsequent phenol:chloroform extraction (Sambrook et al 1989). The DNA was treated with ribonuclease before 95% (v/v) ethanol precipitation (Sambrook et al 1989). The radioactivity of DNA samples was determined by applying samples to Whatman Reeves Angel glass fiber filter, Grade 934AH.

Statistical methods – Throughout this study, triplicate samples were employed and, for each experiment, three or more independent repeat trails were conducted. Statistical reliability of the data was measured by the calculation of the mean for the replicates to obtain standard deviations. The values for standard deviations were under 10%. Statistical significance was determined by T test, and values were consistently more than 2 between the treatments. The SigmaPlot software (SPSS Inc., Chicago, Illinois) was used.

	RESULTS

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We tested various permeabilizing agents, as well as agitation and heating, individually or in combination, to determine the best method of permeabilizing B. bassiana BS. Permeabilized BS were shown to be nonviable but retained ß-galactosidase activity (Fig. 1) to enable in situ determination of activity levels. The extent of permeabilization concurrently was assessed by microscopy with the vital staining by trypan blue (results not shown). The stain permeation of treated BS was significantly higher than no treatment and showed concordance with permeabilization for BS demonstrating an enzyme activity greater than 10 U. All permeabilization treatments required mixing during the assay for optimum conditions. Table I summarizes the effect of treatment on intracellular ß-galactosidase activity detected. The untreated (control) sample had 4.0 U of intracellular ß-galactosidase activity. Ten min of agitation in T:E was optimal, showing the highest 12.4 U activity. Agitation of 5 or 15 min yielded an enzyme activity value 10.5 U. The 15 min T:E agitation treatment reduced enzyme activity. If these values are considered in conjunction with Fig. 1, 15 min agitation in T:E reduces the enzymatic activity, whereas 5 min of agitation does not permeabilize the BS sufficiently and less substrate enters the BS, resulting in reduced enzyme activity. This illustrates the importance of assessing ß-galactosidase activities in both pellet and supernatant fractions. The ß-galactosidase activity in the supernatant fraction had significantly less enzyme activity than the pellet fraction for all treatments. This indicates the treatments have a marginal effect on the release of ß-galactosidase through permeabilized BS membranes, i.e., 2.0 versus 3.0 U. The activity of the ß-galactosidase detected in the pellet fraction is highest (9.1 U) after the 10 min permeabilization treatment, indicating a 4.5x increase in intracellular enzyme activity in the assay results when compared to no treatment. The BS permeabilization ability of CTAB, Triton X-100 + CTAB or 5 min agitation in T:E was equal. T:E agitation is more effective than heating of the BS in the presence of T:E (9.0 U).

View larger version (15K): [in this window] [in a new window]   FIG. 1. Relationship between duration of permeabilization with 10% (v/v) T:E and ß-galactosidase activity (U) in pellet (BS) and supernatant fractions. Enzyme activities present in each fraction were used to determine effectiveness of permeabilization. Beauveria bassiana BS were agitated during permeabilization at 1400 rpm. The value at 0 min represents untreated BS

Permeabilization protocol was tested for dTTP incorporation. Permeabilization is absolutely required for uptake of the ³H-dTTP (Fig. 2). A period of 10 min agitation with 10% (v/v) T:E was most optimal (results not shown). Radioisotope uptake reaches a maximum after 10 min and remains constant thereafter. The addition of 5% (w/v) glucose to the permeabilization buffer increased the rate of uptake. To ensure that radioisotope incorporation reflected DNA synthesis, we used the protocol of a standard isotope dilution technique, which determines the effect of diluting the ³H-dTTP with unlabeled dTTP. In such an experiment, the isotope-labeled precursor is kept at a fixed, small quantity and nonradioactive species at increasing concentrations. The uptake of labeled species is measured in relation to its dilution with the non-labeled form. As a result, while the total radioactivity per volume of reaction remains fixed the CPM or the amount of the compound incorporated will change due to the specific activity (sum of labeled + unlabeled precursor). As the amounts of unlabeled dTTP is increased, the uptake of ³H-dTTP (in CPM) would decrease, according to the dilution ratios used (Fig. 3). We conclude that the incorporation truly reflects incorporation and is specific and saturable. Therefore, the uptake of the nucleotide is not nonspecific or due to entrapment but is DNA being synthesized.

View larger version (18K): [in this window] [in a new window]   FIG. 2. Percent uptake of 3H-dTTP in permeabilized and nonpermeabilized BS. The BS were permeabilized by agitating with 10% (v/v) T:E for 10 min at 1400 rpm

View larger version (18K): [in this window] [in a new window]   FIG. 3. Relationship between 3H-dTTP uptake and increasing dilution by unlabeled dTTP. Reaction tube conditions were: incubation in the presence of an equal volume of 5% (w/v) TCA with 30 min TCA precipitation (A), 30 min TCA precipitation (B) and TCA precipitation for 3 h (C)

Examining the DNA extracted from permeabilized cells, which had incorporated ³H-dTTP for 10 min showed the presence of the nucleotide. The B. bassiana DNA in our incorporation experiment contained 4 x 10¹³ molecules ³H-dTTP/µg BS DNA. Extraction of DNA showed that uptake of ³H-dTTP ended in the DNA as opposed to the ³H-dTTP being retained within the soluble cytoplasmic pool or associated with the BS membranes and BS walls. Study of kinetics of nucleotide uptake and role of TCA washing procedure also indicates that the described experimental conditions prevented unincorporated ³H-dTTP to remain BS bound and can be excluded from the radioactivity analyses of DNA synthesis.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
In B. bassiana, and other filamentous fungi that produce BS or other single cell units, permeabilization is advantageous to whole cells or cell-free lysates for enzyme activity measurement. When compared with BS, mycelia have added structural and compartmental complexity, making biosynthetic activities, such as DNA replication in terms of cell units, difficult to assess. When compared with whole cells, cell-free extracts are tedious to prepare. The permeabilization method for whole-cell, or BS in this case, extracts is a preferred over cell-free extracts in the in situ DNA synthesis method. The advantage of the former is that (i) the DNA replicating enzymes, cofactors and other nucleotide triphosphates' concentration remain intact inside the permeabilized cells (Serrano et al 1973, Choudary and Rao 1976, Choudary 1984), and (ii) permeabilization removes the need to stringently control environmental conditions to maintain enzyme activity (Basabe et al 1979, Ram et al 1983).

The permeabilization process should alter the cell cytoplasmic membrane, and leave the outer membrane intact (Felix 1982). We used ß-galactosidase activity to determine the optimal conditions for achieving permeabilization for uptake of nucleotide molecules, without inducing the leakage of intracellular enzymes (Fig. 1). Most ß-galactosidase activity detected was contained in the BS (pellet) fraction rather than the supernatant fraction. Results to the contrary would have indicated disintegration of BS membranes rather than their permeabilization. Our method of treatment enabled the entry of dTTP, an impermeable substrate, into the BS.

Labeling B. bassiana DNA during growth in the presence of exogenous thymine and thymidine, and applying the usual approach with prokaryotic cells is not possible (results not shown). Fungi lack thymidine kinase for the production of nucleotide triphosphate pools (Grivel and Jackson 1968, Hatzfeld 1973, Oertel and Goulian 1979, Kornberg and Baker 1992). The incorporation of dTTP (M_r = 242 Da) is within the molecular-weight range of other compounds taken up by fungal transport systems (Burgstaller 1997). However, B. bassiana cannot transport dTTP into the BS (Fig. 2). The uptake of molecules that in general are impermeable, due to structure, size or lack of a transport system, is facilitated by permeabilization. The permeabilized system enables dTTP uptake (Fig. 2), direct incorporation into the DNA without concern to equilibration of cellular nucleotide triphosphate pools. The permeabilized system represents genuine uptake and incorporation of the nucleotide as demonstrated by decreased ³H-dTTP incorporation when dilution with unlabeled dTTP is increased (Fig. 3). We conclude that this method of permeabilization can be used independently or in conjunction with in vitro methods to study enzyme kinetics and DNA synthesis processes in BS of B. bassiana.

	ACKNOWLEDGMENTS

 
Natural Sciences and Engineering Research Council of Canada Research Grant No. 493 (GG Khachatourians) and a postgraduate scholarship (L Chelico) supported this research.

	FOOTNOTES

 
¹ Corresponding author, Email: lic131{at}mail.usask.ca

Accepted for publication February 17, 2003.

	LITERATURE CITED

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Basabe JR, Lee CA, Weiss RL., 1979 Enzyme assays using permeabilized cells of Neurospora. Anal Biochem 92:356-360[Medline]

Burgstaller W., 1997 Transport of small ions and molecules through the plasma membrane of filamentous fungi. Crit Rev Microbiol 23:1-46[Medline]

Choudary VP., 1984 A simple micromethod for rapid kinetic analyses of yeast enzymes in situ. Anal Biochem 138:425-429[Medline]

Choudary VP., Rao R., 1976 Regulatory properties of yeast nitrate reductase in situ. Can J Microbiol 22:35-42[Medline]

Dien BS, Srienc F., 1991 Bromodeoxyuridine labeling and flow cytometric identification of replicating Saccharomyces cerevisiae cells: lengths of cell cycle phases and population variability at specific cell cycle positions. Biotechnol Prog 7:291-298[Medline]

Felix H., 1982 Permeabilized cells. Anal Biochem 120:211-234[Medline]

Feng MG, Poprawski TJ, Khachatourians GG., 1994 Production, formulation and application of the entomopathogenic fungus Beauveria bassiana for insect control worldwide. Biocont Sc Tech 4:3-34

Förster C, Marienfeld S, Wilhelm R, Krämer R., 1998 Organelle purification and selective permeabilisation of the plasma membrane: two different approaches to study vacuoles of the filamentous fungus Ashbya gossypii. FEMS Microbiol Lett 167:209-214[Medline]

Grivel AR, Jackson JF., 1968 Thymidine kinase: evidence for its absence from Neurospora crassa and some other microorganisms, and the relevance of this to the specific labeling of DNA. J Gen Microbiol 54:307-317[Medline]

Hatzfeld J., 1973 DNA labeling and its assay in yeast. Biochim Biophys Acta 299:34-42[Medline]

Hegedus DD, Bidochka MJ, Khachatourians GG., 1990 Beauveria bassiana submerged conidia production in a defined medium containing chitin, two hexosamines or glucose. Appl Microbiol Biotechnol 33:641-647

Hegedus DD, Khachatourians GG., 1995 The impact of biotechnology on hyphomycetous fungal insect biocontrol agents. Biotech Adv 13:455-490

Khachatourians GG., 1996 Biochemistry and molecular biology of entomopathogenic fungi. In: Howard DH, Miller JD, eds. The mycota. Vol. VI: Animal and human relationships. Berlin, Germany: Springer Verlag. p 331–363

Khachatourians GG., Valencia E, Miranpuri GS., 2002 Beauveria bassiana and other entomopathogenic fungi in the management of insect pests. In: Koul O, Dhaliwal GS, eds. Microbial Biopesticides. Vol. 2. Reading, United Kingdom: Harwood Academic Publishers. p 239–275

Kippert G., 1995 A rapid permeabilization method procedure for accurate quantitative determination of ß-galactosidase activity in yeast cells. FEMS Microbiol Lett 128:201-206[Medline]

Kornberg A, Baker TA., 1992 Biosynthesis of DNA precursors. In: Kornberg A, Baker TA, eds. DNA Replication. 2nd ed. New York, New York: W.H. Freeman. p 75–89

Laouar L, Mulligan BJ, Lowe KC., 1992 Yeast permeabilization with surfactants. Biotechnol Lett 14:719-720

Liu Y, Hama H, Fujita Y, Kondo A, Inoue Y, Kimura A, Fukuda H., 1999 Production of S-lactoylglutathione by high activity whole cell biocatalysts prepared by permeabilization of recombinant Saccharomyces cerevisiae with alcohols. Biotechnol Bioeng 64:54-60[Medline]

Mac Pherson JM, Khachatourians GG., 1991 Partial purification and characterization of ß-galactosidase produced by Beauveria bassiana. Biotechnol Appl Biochem 13:217-230

Maggesse MC, Galvagno MA, Cantore ML, Passeron S., 1982 In situ measurement of cAMP related enzymes in the dimorphic fungus Mucor rouxii. Biol Int Rep 6:1101-1108

Miozzari BF, Niederberger P, Hunter R., 1978 Permeabilization of microorganisms by Triton X-100. Anal Biochem 90:220-223[Medline]

Oertel W, Goulian M., 1979 Deoxyribonucleic acid synthesis in Saccharomyces cerevisiae permeabilized cells with ether. J Bacteriol 140:333-341[Abstract/Free Full Text]

Oertel W, 1977 Deoxyribonucleic acid synthesis in permeabilized spheroplasts of Saccharomyces cerevisiae. J Bacteriol 132:233-246[Abstract/Free Full Text]

Oliveira DE, Lucia AC, Neto S, Panek AD., 1981 Permeabilization of yeast for in situ determination of -glucosidase. Anal Biochem 113:188-192[Medline]

Quigley DR, Jabri E, Selitrennikoff CP., 1987 Permeabilization of Neurospora crassa hyphae with toluene-ethanol and filipin. Curr Microbiol 14:269-274

Ram SP, Sullivan PA, Shepherd MG., 1983 The in situ assay of Candida albicans enzymes during yeast growth and germ–tube formation. J Gen Microbiol 129:2367-2378[Medline]

Sachs MS, Selker EU, Lin B, Roberts CJ, Luo Z, Vaught-Alexander D, Margolin BS., 1977 Expression of herpes virus thymidine kinase in Neurospora crassa. Nucleic Acids Res 25:2389-2395

Sambrook J, Fritsch EF, Maniatis T., 1989 Commonly used techniques in molecular cloning. In: Molecular Cloning: A Laboratory Manual. Vol. 3. 2nd ed. Plainview, New York: Cold Spring Harbor Laboratory Press. p E.3

Seip JE, Di Cosimo R., 1992 Optimization of accessible catalase activity in polyacrylamide gel–immobilized Saccharomyces cerevisiae. Biotechnol Bioeng 40:638-642

Serrano R, Gancedo JM, Gancedo C., 1973 Assay of yeast enzymes in situ: a potential tool in regulation studies. Eur J Biochem 34:479-482[Medline]

esták S, Farka V., 2001 In situ assays of fungal enzymes in cells permeabilized by osmotic shock. Anal Biochem 292:34-39[Medline]

Shailasree S, Nagajyothi B, Bhat SG, Sekhar S, Bhat N., 1999 Preparation of detergent permeabilized bakers' yeast whole cell catalase. Process Biochem 34:349-354

Sorol MR, Pereyra E, Mizyrycki C, Silvia Rossi S, Moreno S., 2001 Protein kinase A activity in permeabilized cells as an approximation to in vivo activity. Exp Cell Res 271:337-343[Medline]

Wellman AM, Pendyala L., 1979 Permeability changes in membranes of Neurospora crassa after freezing and thawing. Cryobiol 16:184-195

This Article Abstract Full Text (PDF) Services Similar articles in this journal Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Chelico, L. Articles by Khachatourians, G. G. Search for Related Content PubMed Articles by Chelico, L. Articles by Khachatourians, G. G. Agricola Articles by Chelico, L. Articles by Khachatourians, G. G.