Effects of electric fields and currents on living cells and their potential use in biotechnology: a survey

133

EioeIecrrachemisf~ and Bioenergetics, 20 (1988) 133-142 of J. Efectroanal. Chem.. and constituting Vol. 254 (19BB) Elsevicr Sequoia SA., Lausanne - Printed in The Nethertar&

A section

Effects of electric fiekls and currents on living cells and their potent&$use in biotechnology: a survey * J. Teissi6 Centre de Biochimie et de G&n&tique CeJhdaires du C.N.RS., 31062 Touloure Cedex (France)

Ii& Route de Nurbonne,

(Received in revised form 22 January 1988)

The behaviour of cells can be altered when they are submitted to electric fields. In this paper, the physical background which is needed to understand the effects of eIectr;c fields on living cells is described. The difierent biotechnological applications of those effects are then reported. The growth of micrwr8anisms and the differentiation of plant callus can be increased when cells are submitted to low intensity currents. Electrogenic metabolic activities can be modulated by external fields. Strong field pulses of short duration induce a new transient but fully reversible state of the plasma membrane which is then permeabilized and fusogenic. As the cell viabiity is not affected, this “electropermeabilization” is used for the direct transfer of genetic information into the cytoplasm and for the production of viable hybrids.

iNTROWJCTlON

The akm of this paper is to show to what extent, in 1987, the gap between basic research and applied biotechnology was bridged in the case of the use of electric fields and current. In other words, was the technological transfer present? One should not forget that in such a “partnership” the philosophy of the two partners is drastically different: scientists in basic research want to know why the results occur in SiiCh or such a way; engineerS in tile appk43 G&i jusi w;ru~i ‘*r~ld_y to use-’ procedures. Biotechnology is the industrial use of micro-organisms (such as bacteria, yeast, mammalian cells, plants) for the production of high value compounds. It is indeed a very old technology. Fermentation has been used for years and nowadays is still a

* Presented at the 9th International (Hungary), l-5 Septem&r 1987. 0302459B/BB,‘$O3.50

Symposium

on Bioelectrochemistry

0 1988 Elsevier Sequoia S.A.

and Bioenergetics,

Szeged

134

major part of biotechnology for the production of drugs, foods, antibiotics, etc. The improvements a new technique can give are either an increase in the production or a reduction in its costs. This can be done by several approaches which can be summarized by the following points: (aj an increase in the growth of the micro-organisms; (b) an increase in the metabolic activity of the micro-organisms; (c) the creation of “better” micro-organisms (i) by hybridation and (ii) by genetic engineering. As will be described briefly in this paper, electric fields *~u~d/or currents, when used in the proper way, can play a beneficial role at all levels. Numerous results have been described on academic systems but this paper will focus on works where the results can be used directly in the biotechnology industry. APPLICATIONSIN

BKO~C~~L~~Y

Effects of electric fkd& on cells

(1) A current is associated with the field that is applied to the cell suspension. This induces Joule heating of the sample. In most experiments, this effect should be kept as small as possible in order to avoid toxic consequences. (2) Chemical reactions involving charged species can be affected by external fields [l]. But it showuldbe noted that the range of field intensities which should be used is much larger than that for biotechnological purposes. (3) Zlectrid reacti~~a occur at the level of the electrodes inducing the degradation of the electrodes (formation of alumina) or the presence of highly reactive compounds. These effects remain local&d close to the electrodes. (4) Due to charge separation in the components of the membrane, dipoles will be induced. (5) The external field interacts with the cell’s permanent and induced dipoles and this leads to reorientation of the cells. (6) Cells will move electrophoretically. (7) Charged species present in the membrane will move electrophoretically along the ceJl smface [2]. This results in the clustering of these species at one pole of the cell facing the relevant electrode. (8) Due to the dielectric character of the eeli membrane, the electric field lines are distorted. Alteration of the txeabrane potential is associated with this perturbation 131. As will be described, points (6), (7) and (8) can be used to manipulate the behaviour of cells. Most applications t&e advantage of point (8). Cell membranes are considered to be impermeable or selectively permeable, but this is true only as long as the membrane potential remains within a limited range. Increasing the potential above a threshold induces “e1ectropermeabiion” of the membrane, This is easily obtained by the use of suitable external fields. After pulsing the cells, their membranes are permeable and exogenous molecules can cross them. But a clever choice of the electric pulse parameters (intensity, duration, number) can lead

135

to a very interesting situation. This permeability can be transient and the membrane impermeability can be recovered spontaneously. As the cell viability is not affected, such electrical treatments allow manipulation of the cytoplasmic content, The basic principles of this electropermeabiition were described in the 1970s by Neumann and Rosenbeck [4], Zimmermann et al, [5] and Kinosita and Tsong [6], but their practical uses for biotechnology are more recent. Another useful feature of this electropermeabilization is the associated induction of a “fusogenic” state of the cell surface. In other words, cell fusion can be obtained with the production of viable and stable hybrids in high yield. Technical aspects

Electric fields and currents are generated by applyiug a voltage to two electrodes either in direct contact with the cell suspension or capacitively coupled 173. In the case of small currents, the electrodes are simply wires and consequently the current density is not uniform throughout the sample. In the case of high intensity pulsed fields, two designs are used: (A) &.. capacitor is loaded to z high voltage and then discharged in the cell suspension. The field is then decaying exponentially with time. Two parameters are characteristic of the pulse [Sj: (1) the initial intensity E, and (2) the decay time T, which is a function of the capacitor C and the resistance of the sample, i.e. of its ionic content. (B) An electronically wntrolled high-power voltage generator delivers the voltage during a pre-selected duration, The field intensity dependence on time is then a squure waue. Two parameters are under control [9]: (I) the field intensity, which is constant, and (2) the pulse duration, which is electronically selected. Flat and parallel electrodes must be selected in order to obtain a uniform field intensity all over the sample. Other shapes would give problems in the control of the experiments but are proposed for the induction of dielectrophoresis [lG]. Neverthe!;tss, th5 appears not to be necessary Ill]. The nature of the electrodes should be chosen to obey the two requirements: (a) electrical conductivity and (b) absence of electrochemical reactions. For these reasons, aluminium foils must be avoided, the non-conductive aluminium oxide b&g eiectrochemically generated. The experimental conditions must be observed on. line. For this purpose, the shape of the e!ectric pulse must be observed with au oscilloscope [8,9]. Other aspects of the eq:erimental conditions are dependent on the cellular system that we are dealing with. The general rules of cellular biology must be observed LS far as viability and sterility are corxerned. Working under a laminar flow hood is a technical advantage [9]. Use of small currents Endogenous electric currents are known to be present in tissues 1121. They appear to control the polarity of cells. This vectorial property is associated with microlocali-

136

zation of ionic pumps at GI:Zend of the cell. As described in the Introduction, such microlocalization can be induced by an external field through the electrophoretic effect. This is indeed of ~~XHIIKII~practical use. In the case of neme cells in culture, neurite outgrowth was observed to increase by a factor of two when a very minute electric current (4 nA/cm*) was applied 1131. This treatment was apparently as effective as that obtained when the nerve growth factor is added at physiological concentration. But the most directly usable effects are those in the plant biotechnology field. One of the major problems is linked to the use of protoplasts (wall-free cells obtained by enzymatic treatment) and the associated regeneration problems. In most cases, protoplasts are able to resynthesize their wall and to divide up to a callus. A 70% increase in growth was observed with tobacco when a negative electrode was impaled in the calli and a 1 PA current was applied, auxin being present in the culture medium. A major advantage of this electrical stimulation was an increase in the shoot formation in the case of wheat 1141. More recently, plant ambryogenesis has been observed without direct contact between one electrode and the callus [15]. All these results on plant systems (increase in growth rate, induction of differentiation) are direct evidence that electrical stimulation can in many cases be a great improvement in the regeneration of protoplasts to the plant level. Ektrkal

stimulation

of metabolic activities

Cellular activities which are under the control of the membrane potential (electrogenic) can be stimulated by an external field. (I) Ionic pumping across red blood ceils membranes K+ (or Rb+) ions are pumped into erytknzytes when they are submitted to ac fields [16]. This effect is inhibited by the physiological inhibitor of Na+/K+ ATPase - ouabain. A systematic investigation of this process showed that it was under the control of the field intensity and the frequency [17]. A detailed description of their theoretical aspects has recently been proposed [18]. (2) ATP synthesis in c?zergy transducing membranes The electrical part of the protomotive force which is supposed to trigger ATP mAby external fields. This was shown to synthesis in membranes [19] can be modulatUu be effective in generating ATP in thylakoids [20,21]> SMP 1221,mitochondria [23] and bacteria [24). In the case of Escherichia coli, it was shown that this effect was linked to activation of the FIFO ATP synthase. (3) Electrical stimulation of metabolism in bone ceils in culture By using capacitively coupled electrodes, Korenstein et al. were able tc show that after stimulating a cell culture during a short period they were able to induce a

137

cascade of events affecting the metabolism [7]. Immediate biochemical changes were electric field intensity dependent. The cyclic AMP level was modified. The cytoplasmic Ca*+ content was increased and the organization of the cytoskeleton was affected. The activity of omithine decarboxylase @DC), a regulating enzyme in the biosynthesis of polyamines, was observed to be increased (up to three-fold) 4 h after the stimulation. DNA synthesis was observed to be enhanced by the electrical stimulation. As proposed by the authors, these effect are linked to each other, the long-term effects (ODC activity, DN.4 synthesis) being induced by the increase in the CAMP level. Another interesting feature cf this electricai stimulation is its cellular specificity. In other words, when working on a mixed population, the electrical parameters can be selected in order to stimulate only one strain, leaving the other one unaffected. (4) Elect&Cal stimuiatiun of antibody-producing hybridoma cells The continuous direct stimulation of hybridoma cells over 48 h has been shown to increase the production of lactic acid by 30% 1251.The cell concentration was observed to increase over ‘ihe control (unstimulated cells) and a concomitant increase in the antibody concentration was detected. These observations indicated that electrical stimulation, when used under suitable conditions, wcadld improve the hybridoma antibody productivity by increasing the growth rate of the cells, the slow rate observed in most cases being a bottleneck in the large-scale production of monoclonal antibodies. Electric field mediated direct gene transfer: “electrolransformation ”

As described above, the cell plasma membrane is reversibly permeabilized when pulsed by a suitably calibrated electric field.In 1982 plasmids were shown to be able to permeate across the “electropermeabilizecl” membrane and then to be expressed [is]. The methodology which was then given in this reference work has been proved to be valid for any kind of cells or protopiasts. The experimental scheme is described in Table 1. A large amount of work has been published since then dealing with mammalian cells. The major conclusions were as follows: (1) The number of transformants increased linearly with the DNX concentration. (2) Cotransformation was occurring. (3) Linearized plasmids were apparently more effective. (4) In the case of exponentially decreasing fields;, the electrical “window” (intensity, decay time) was apparently narrow. As a conclusion, square-wave conditions were easier to handle for optimization. (5) The inherited genetic character is stable. These conclusions appeared to be valid with any cell strain (even primary culture) and were extended to other cellular systems. Micro-organisms of biotechnological relevance were shown to be easily electrotransformed. In the case of bacteria, the group in Jena showed that the PEG

TABLE 1 Experimental proosdure?or ‘*elcctrotfamfommion” 1. 2. 3. 4. 5. 6. 7. 8. 9.

Mix DNA and cells in a Ca2+, Mg2+-free physiological buffer Coolat4°Cfr10min Pulse (field intensity, duration and shape are a function of the strain) Keep the pulsed sample at 4O C for ten more min Incubate at room temperature or at 37 OC Wash the plasmid out Let the cells grow for 24-48 h (this growth period is longer in the experknents with protoplasts) Plate the cells (proroplasts) on a selective medium In the case of plant proloplasts, when selected calli have been isolated, a regeneration step is undertaken in order to obtain plants

mediated transformation of Batiks cereup spheroplasts vzas improved when the mixture was treated with very strong short pulses [26]. In the case of Streptomyces iividans spheroplasts, it was indeed shown that transformation was obtzined by pulsing in a PEG-free solution [273. As we are faced with the problem of regeneration when spheroplasts are used, the direct transformation of intact bacteria appears to be advantageous. This has recently been shown to be possible with two strains of industrial relevance: StreptococcusthermopJCh.s(281 and Lactobuciliuscase7 [29] by the use of pukes with a decay time in the ms time rage. Yeasts were electrotransformed either by treating spheroplasts with very short pulses (ps) [30] or by pulsing intact cells with longer pulses, PEG being present in the solution [31j. Nevertheless, it should be noted that in this second case the yield was low. PIznt genetic manipulation is one of the biggest challenges in biotechnology nowadays. The aim is to obtain plants capable of resisting herbicides and pesticides. The approach is to transfer a plasmid where a suitable gene is present in the plant protoplast and then to regenerate the resistant plant. This. has been shown with several Nicotianu strains [32-361 and very recently with Brassica [37]. Here again, two approaches were described, one dealing with decay times in tile ms time range 136,373 and the second with mu+ shorter pulsing periods [32-351. It should be noted ths< as more systematic investigations were performed with this second approach, it apparently provided more reproducible results and a much higher yield in stable transformants. EiectricfieId mediatedcellfurion: “electrofusion” In the early 198&z, results from different laboratories showed that cells in tight contact were observed to fuse when pulsed under suitable conditions [IO]. 1s~fact, it has recently been shown that electropermeabilized cells are fusogenic [38,393. Electrofusion can then be obtained by two procedures (see Table 2, A and B).

139 TABLE 2 Experimental procedure for “electrofusion”

(A) 1.

2. 3. 4. 5. 6. 7.

Mix the cell populations in a suitable buffer Create contact betw8xn the cells Pulse under well-defimed electrical conditions which are a function of the cells that are being de& with Incubate the sample (at 37OC for mammalian cells) Let the cells grow Run the selection assay In the case of plant protoplasts, when hybrid calli have been isolated, a regeneration step is undertaken h or&r to obtain hybrid plants

(B) 1. Pulse each parent cell population under its most suitable electrical conditions (intensity, duration or decay, number, delay) 2, Mix the pulsed populations 3. Create contact between the cells 4. Then aII the other steps are as in (A) above

In both cases, the basic req.tir~~~* ~~~~ is to obtain contact between the plasma membranes of the two cells to be fused and as such electrofusioncan been obtained only between protoplasts in the case of walled celJs (bacteria, yeasts, plants). Contact can be obtained by many approaches (Table 3). The composition of the buffer was chosen to be iso-osmotic but, owing to technologicalreasons connectedwith the power of the generator or with the use of dielectrophoresis(Joule effects), its ionic content was in mosi cases rather low. The major use of mammalian cell hybrids in biotechnology is the obtention of antibodies producing hybridoma. Electrofusion has been shown to be a very efficient tool for this purpose. Two different philosophies have been proposed: (I) an increasein the yield of variable hybrids between lymphocytesand myeloma &Is followed by selectionof the specific antibodies producing hybridoma [40-441; and (2) a specific targeting of the antibodies producing lymphocytes with the myeloma

TABLE 3 Proposed methods to obtain cell contact in “electrofusion” 1. Natural [&6]

2. 3. 4. 5. 6. 7.

Ccntrifugation in order IO obtain a cell pellet [39] Contact inhibition (for plated cells) [9] Dielectrophoresis (40,49,52,58] Agglutinating agents [47,48,50,6S] Targeting [45,46) Micromanipulation [62]

cells [45,46]. In case (I), cells were first treated with pronase and then brought into contact by dielectrophoresis. They were then pulsed for periods in the ps range. In case (2), after targeting, the celis were pulsed for periods in the us range. In this case, the yield in viable hybrids was low but all were specific producers and no selection was required. Hybrids between bacteria can be obtained by electrofusion. A preliminary step is the fornxtion of protoplasts and a regeneration step must follow the selection procedure. Another characteristic of electrofusion of bacterial protoplasts is that, due to their small size, very strong field intensities are needed. The PEG mediated fusion yield was increased ten-fold when a PEG -!-Bacilius thuringensis protoplast mixture was pulsed (20 kV/cm, r = 5 JS) [47J. In the case of Clostridium giutamicum, electrofusion of chemically aggregated protoplasts (7.5 kV/cm, t = 100 J.LS) gave a high yield of viable and stable hybrids (a lOO-fold increase as compared with the PEG method) [48]. The difficulty connected with the small size of the bacteria could be overcome by blocking their division during growth by using cephalexin. The “giant” protoplasts obtained by such meazs in the case of Escherichiu coli and Salmonella tiphymurium were agglutinated by dielectrophoresis and ekxtrofused (4 kV/cm, 15 ps) [49]. Protoplast formation is also required for yeast electrofusion. The PEG mediated fusion yield was increased up to go-fold by pulsing the suspension (decay time in the ps time range). Optimization of the field intensity was dependent on the yeast strain (Succharomyces iipolytica, - Lodderomyces eiongisporus, Saccharomyces cerevisiue) [50,51]. S. cerevisiae protoplasts can be brought into contact by dielectrophoiesis and then electrofused (10 kV/cm, 10 ps). The yield of the hybrids w2s apparently a function of the ionic content of the buffer [52-573. As fas as plant biotechnology is concerned, hybrid plants have been obtained by different ways in the case of Nicotiana. The differences were at the level of the methodology which was used to create the contact between the protoplasts to be fused: dielectrophoresis [58-611, micromanipulation 1623,sedimentation 163,641and sedimentation with added PEG 1651.The electrical conditions in all cases were rather similar (l-l.5 kV/cm, 50-100 ps). The hybrids are stable and the seeds keep the hybrid character as predicted by Mendelian laws. In the case of parents where multiple mutations were present, electrofusion gave somatic hybrids with a much greater genetic variation than the PEG method [65]. This should be an advantage in the obtention of the most suitable hybrid for biotechnology. CONCLUSlON

From this paper, it can be concluded that 2s far as growth or production is concerned, the use of small fields and current can help to improve the yield of production. As far as the creation of new species either by fusion or by gene transfer is concerned, the tool is ready but it is still not obvious how to use it. It is clear that no definite rule has been proposed for successful use of electric fields. It should be kept in mind that all the theoretical studies were done using a very naive model of the cell and forgetting all “physiological” aspects. The molecular

141

aspects of electropermeabilization and its associated cell fusogenicity are still poorly understood. At the present state of knowledge 1661, the costly method of empiricism must still be used. This would explain why experimental conditions looked so different from one laboratory to another (see, for example, the results on direct gene electrotransfer in plants). From my own experience, I would rather suggest the folIowing approach. For each strain to be used, one shouId (1) obtain the permeabilization vs. field intensity plot (by the use of a dye or radioactive compound); (2) obtain the survival ratio vs. field intensity plot, survival meaning aptitude to divide (not the use of viability dyes); and (3) check the dependence of (1) and (2) on other parameters (pulse duration, buffer content). From these results, the optimal conditions can be chosen. Thereafter, the problem will be the regeneration of intact systems, a very difficult one indeed where, as reported here, the use of small fields might be useful. ACKNOWLEDGEMENTS

Thanks are due to Drs. Berg, Emeis, Glassy, Goldsworthy, Karube, Korenstein, Krizaj, Mattanovich, Morikawa, Neumann, Siegemund and Steinbiss for providing preprints and reprints of their works in this field. This work was supported by grants from the CNRS and MRES. REFERENCES ‘: 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

E. Neumann, Prog. Biophys. Mol. Bial., 47 (1986) 197. M.M. Poe, Annu. Rev. Biophys. Bioeng.. 10 (1981) 245. J. Bernhard1 and H. Pauly. Biophysik (Berlin). 10 (1973) 89. E. Neumann and K. Rosenbeck. J. Membr. Biol., 14 (1973) 194. U. Zimmermann, G. Pilwat and F. Riemann, Biophys. J.. 14 (1974) 881. K. Kinosita and T-Y. Tsong, Proc. Natl. Acad. Sci. USA, 74 (1977) 1923. R. Korenstein, D. Somjcn, F. Laub. A. Danon, H. Fischler and I. Bindcrman in Balaban fEds.). Biological Structures and Coupled Flows, Academic Press; New E. Neumann, M. Schaeffer-Ridder, Y. Wong and P.H. Hoffschneider, EMBO J., C. Blangcro and J. Teissie, B&him. Biophys. Res. Commun., 114 (1983) 663. U. Zimmermann, B&him. Biophys. Acta, 694 (1982) 227. A. Zachrisson and C.H. Bomman. Physiol. Plant., 61 (1984) 314. L.F. Jaffe and R. Nuccitelli, Annu. Rev. Biophys. Bioeng., 6 (1977) 445. B.F. Sisken. B. McLeod and A.A. Pilla, J. bioelcctr., 3 (1984) 81. A. Goklsworthy, TIBTECH. 4 (1986) 327. M. Dijak, D-L. Smith, T-J. Wilson and DC.‘?:. Brown, Plant Cell Rep., 5 (1986) J. TeissiC and T.Y. Tsong, J. Phys. (Faris), 77 (1981) 1043. T.Y. Tsong, Biosci. Rep., 3 (1983) 487. R.D. Asrumian, P.B. Chock and T.Y. Tsong, Stud. Biophys., 119 (1987) 123. P.D. Bayer. B. Chance, L. Emster, P. Mitchell, E. Racker and E.C. Slater. Annu. (1977) 955. H.T. Witt. E. Schlodder and P. Grzber. FEBS Lett., 69 (1976) 272. C. Vinkler and R. Korenstein, Proc. Natl. Acad. Sci. USA, 79 (1982) 3183. J. Teissi& B.E. Knox, T.Y. Tsong and J. Wehrle. Proc. Natl. Acad. Sci. USA, 78

A. Oplatka and M. York, 1983. p. 401. 1 (1982) 841.

468.

Rev. B&hem..

(1981)

7473.

45

142 23 M. Rogner, K. Ohno. T. Hamamoto. N. !&me and Y. Kagawa, B&hem. Biophys. I&s_ Commun., 91 (1979) 362. 24 J. Teissi&, Biochemistry, 25 (1986) 369. 2S M, Suzuki, E, Tamiya, H. Matsuoko, M. Sugi and I. Karube. Biochim. Biophys. Acta, 889 (1986) 149. 26 N. Shivarova, W. F&ster, HE Jacob and R. Grigorova 2, Allg. Mikrobiol., 23 (1983) 595. 27 D.J. McNeil, FEMS Lett., 42 (1987; 239. 28 G. Somkuti, TIBTECH. S (1987) 186. 29 B. Chassy and J.L. Flickinger, FEMS Lett., 44 (1987) 173. 30 I. Kanrbe, E. Tamiya and H. Matsuoka, FEBS Lett., 182 (198s) 90. 31 H. Hashimoto, H. Morikawa, Y. Yamada and A. Kimura, Appt. Microbial. Biotechnol., 21 (1985) 336. 32 R.D. Shitlito, M.W. Saul, J. Paszkowski, M. Multer and I. Potrykus, Biotechnology. 3 (1985) 1099. 33 M. Pietrzak, R.D. Shiltito. T. Hahn and 1. Potrykus, Nucleic Acid. Res., 14 (1986) 5857, 34 R.J. Schochter, R.D. Shillito, M.W. Saul. J. Paszkowski and I. Potrykus, Biotechnology, 4 (1986) 1093. 35 C.D. Riggs and G.W. Bates, Proc. Natl. Acaci. Sci. ‘U’S& 83 (1986) 5602. 36 M. North, I. Negrutiu, A_ Bumy, M. Van Montagu atld L. Herrera-Estretla, EMBO J., 6 (1987) 2525. 37 P. Guerche, M. Charbonnier, L. Jouanin, C. Toumeur. J. Paszkowski and G. Pelletier. Plant Sci., 52 (1987) 11. 38 A.E. Sowers, 3. Cell Biot., 102 (1986) 1358. 39 J. Teissie and M.P. RoIs, B&hem. Biophys. Res. Ccmmun., 140 (1986) 258. 40 J. Vienken and U. Zimmcrmann, FEBS Lett.. 182 (1985) 278. 41 U. Karslen, G. Papsdorf, G. Roloff, P. Stoltey, H. Ate& I. Watther and H. Weiss. Eur. J. Cancer Clin. Onwl., 21 (1985) 733. 42 M.C. Glassy and G. Hoffmann, Hybridoma. 4 (1985) 61. 43 S.M. Brown, Q-F. Akhong, A.D. Sage and J.A. Lucy. B&hem_ Sot. Trans., 14 (1986) 297. 44 K. Oh&hi, J. Chiba, Y. Goto and T. Tokunaga, J. Immunol. Methods, 100 (1987) 181. 45 M.M.S. ‘Lo, T.Y. Tsong, M.K. Conrad, S.D.M. Stritlmatter, L.D. Hester and S.H. Snyder, Nature, 310 (1984) 792. 46 D.M. Wojchowski and A.J. Sytkowski, 3. Immunol. Methods, 90 (1986) 173. 47 N. Shivarova, R. Grigorova, W. Farster, H-E. Jacob and Ii. Berg. Bioelectrochem. Bioenerg., 11 (1983) 181. 48 M.P. Rots, P. Bandiera, G. Laneetle and J. Teissih, Stud. Biophys., 119 (1987) 37. 49 H.J. Ruthe and J. Adler, Biochim. Biophys. Acta. 819 (1985) 105. SO H. Weber, W. Farster, H.E. Jacob and H. Berg, 2. Altg. Mikrobiol,, 21 (1981) 555. 51 H. Weber, W. Fi%ter, H. Berg and H.E. Jacob, Curr. Genet.. 4 (1981) 165. 52 H.J. Halfmann, W. ROcken, CC. Emeis and U. Zimmermann, Curr. Genet.. 6 (1982) 25. 53 H.J. Halfmann, C.C. Emeis and U. Zimmermann, Arch. Xicrobiol, 134 (1983) 1. 54 R. Schnettttr, U. Zimmermann and CC. Emeis. FEMS Lett., 24 (1984) 81. SS R. Schnettter and U. Zimmennann, FEMS Lett., 27 (1985) 195. 56 H.G. Broda, R. S&nettler and U. Zimmermann, B&him. Biophys. Acta, 899 (1987) 25. 57 E. Forster and C.C. Em&, FEMS Lett., 34 (1986) 69. 58 K.J. Polite. ?_ Van Wiksetaar and H. Verhoevcn, Plant Ccl1 Rep., 4 (1985) 274. 59 H. Kohn, R. Schieder and 0. Schieder, Plant Sci., 38 (1985) 121. 60 G.W. Bates and CA. Hascnkampf, Theor. Appt. Genet., 70 (1985) 227. 61 S.E. De Vries, E. Jacobsen, M.G.K. Jones, A.E.H.M. Loonen, M.J. Tempelaar, J. Wijbrandi atad W.J. Feenstra, Plant Sci., 51 (1987) 105. 62 H.U. Koop and H-G. Schweiger, Eur. J. Cell. Biol.. 39 (1985) 46. 63 H. Morikawa, K. Sugino, Y. Hayashi, J. Takeda, M. Senda, A. Hiril’i and Y_ Yamada, Biotechnology, 4 (1986) 57. 64 H. Morikawa, T. Kumashiro, K. Kusakari, A. Iida, A. Hirai’ and Y. Yamada, Theor. Appt. Genet.. 75 (1988) 1. 65 M-H. MO&n&, G. Atibert and J. TeissiC, Stud. Biophys., i19 (1987) 89. 66 H. Berp, Stud. Biophys., 119 (1987) 17.