275
Biochimica et Biophysica Acta, 519 (1978) 275--278 © Elsevier/North-Holland Biomedical Press
BBA
Report
BBA 91470
STABILITY OF MESSENGER RNA FOR NITRATE REDUCTASE IN NEUROSPORA CRASSA
RAMASWAMY
PREMAKUMAR,
GEORGE
J. S O R G E R and D I N S D A L E G O O D E N
Department of Biology,McMaster University,Hamilton, OntarioL8S 4Kl (Canada) (Received January 31st, 1978)
Summary A m e t h o d has been developed to study the synthesis and decay of the messenger RNA for nitrate reductase in Neurospora crassa. Glutamine prevents the synthesis of the mRNA which appears to have a half-life of approximately 8.5 min. Nitrate reductase from Neurospora catalyzes the firststep in the assimilation of nitrate, is a 230 000 dalton, heteromultimeric enzyme and contains flavin, cytochrome b-557 and molybdenum (Mo) as components of its electron transport system [1--3]. Nitrate is believed to be required for the induced formation of nitrate reductase in Neurospora [4--6]. The effect of nitrate is to stimulate transcription [7, 8] and to partially protect nitrate reductase from decay [9, 10]. The stability of the mRNA of an enzyme is an important aspect of its regulation. Actinomycin D is widely used to block transcription in studies of mRNA stability [11]. Neurospora is relatively impermeable to the drug and the mycelia must, therefore, be pretreated with EDTA for the inhibitor to be effective [10, 12]. Even with this pretreatment, actinomycin D does not block RNA synthesis completely at the customarily used concentrations [12]. Another way of inhibiting the synthesis of a particular mRNA is to use a repressor. Glutamine represses nitrate reductase in Neurospora [13] (our unpublished results). We have studied the stability of the mRNA of nitrate reductase by making use of this finding and of the observation that tungsten (W), an analogue of Mo, can inhibit the development qf nitrate reductase, by being incorporated into nitrate reductase instead of the functional Mo [9]. A cirnilar procedure has been employed successfully in a recent study of the synthesis of nitrate reductase mRNA in tobacco cell lines [14]. Neurospora crassa, strain 3-1a (Stock No. 988, Fungal Stock Center, Humboldt) was grown in a basic medium [15] containing ammonium tar-
276 trate (4 g/l) and sucrose (20 g/l), until late log phase (39 h) at 27°C in standing culture. The mycelial pads were then transferred to fresh basic medium containing sodium nitrate (0.5 mM) as nitrogen source and sodium tungstate (6 mM} ("transcription medium"), and shaken at 27°C for the required time. Under these conditions one would expect the formation of the capacity to make nitrate reductase (presumably mRNA) and also of inactive tungsten-containing nitrate reductase. The mycelia, induced in this manner, were washed and transferred into a medium containing sodium nitrate (0.5 mM), sodium molybdate (6 mM) and glutamine (5 mM) ("translation medium"), and shaken for different time intervals at 27°C. The expectation here was that the accumulated mRNA would be translated into active nitrate reductase in this medium, and that no new mRNA would be made. When the stability of mRNA was examined, the mycelia were incubated in the transcription medium described above, with shaking, at 27°C, for 80 min, to form the mRNA. The mold was then transferred into a medium containing sodium nitrate (0.5 mM), sodium tungstate (6 mM) and glutamine (5 mM) ("decay medium"), and was shaken for different time intervals. Under these conditions, no new messenger RNA would be formed, the mRNA which had accumulated might decay, and no new active nitrate reductase would be formed. The mycelia, treated in this manner, were then transferred to the translation medium, and shaken for 20 min. Any mRNA remaining would be translated into active nitrate reductase. Extracts were prepared by grinding mycelia with an equal weight of Silica and 0.1 M potassium phosphate buffer, pH 7.0, containing 1 mM EDTA (2 ml buffer per 300 mg blotted weight of pads). Nitrate reductase was assayed as described previously [9], after a preiucubation of the extract for 30 min, at 30°C, with all substrates except NADPH. This procedure has been found in our laboratory to result in maximal increase of nitrate reductase activity (unpublished observations). One unit of activity is defined as the production of one nmole of nitrite per min at 30°C. Specific activity is defined in terms of units of activity per mg protein. Protein was determined by the biuret method [16]. The capacity to synthesize nitrate reductase accumulated after a lag of 20 min in mycelia exposed to transcription medium (Fig. 1). The capacity to synthesize nitrate reductase is attributed here to the formation of messenger RNA for nitrate reductase. The presence of glutamine in the transcription medium prevented the accumulation of mRNA. The above increase in activity is not merely due to the activation of pre-existing tungsten-containing enzyme, because it occurred only minimally in the presence of cycloheximide (Fig. 1). Mycelia that were exposed to the transcription medium for 80 min, began to synthesize active nitrate reductase immediately upon transfer to the translation medium, whereas mycelia that had not previously been exposed to the transcription medium displayed a lag of more than 20 min before active nitrate reductase was synthesized (Fig. 2). This observation is further evidence that the mRNA accumulates during the mold's incubation in transcription medium. The presence of cycloheximide in the translation medium prevented the synthesis of active nitrate reductase, suggesting again that this increase in activity is not due to the activation of pre-existing, inactive, tungsten-containing, enzyme.
277
2O~
,,
/
i
/ L.
41
~
40
80
120 160 Time (mln)
2~
10
20
3O nO 50 Time (rain)
60
Fig. 1. Effect o f t i m e o f i n c u b a t i o n in transcription m e d i u m on the m y c e l i a l a c c u m u l a t i o n o f the capacity to synthesize nitrate reductase. Neurompora mycetia were grown as described in the t e xt , transferred to ~ t i o n m e d i u m Coasic m e d i u m containing nitrate and t u n g r t a t e ) and ~ - ~ e n at 27°C for the indicated times, w a s h e d and finally i n c u b a t e d for 20 m i n a t 27°C, w i t h s bs ki ng in trln*1.tion mediu m (bas/c m e d i u m contmln4nf nitrate, m o l y b d a t e and glutamine). The myc e l l a were t he n washed and extr acted w i t h 0 . I M p h o s p h a t e buffer, pH 7.0, containing I mM EDTA (2 ml per 300 mg b l o t t e d weight of myceila). --', i n c u b a t i o n for 20 m/n in tr-n-IAtion m e d i u m after expoeure for the i ndi c a t e d tim es to tran scription m e d i u m ; o-----¢, i n c u b a t i o n in transcription m e d i u m for the t/rues s how n b u t no expose to tzanalation m e d i u m ; ~ ~, i n c u b a t i o n in tro nal-t i on m e d i u m for 20 rain in t h e p z ~ e n e e o f cycloheximid e (1 , g / m l ) after exposure to tzanacflption m e d i u m for the tlmes.shown; ± ±, i n c u b a t i o n in tr anslation m e d i u m for 20 rain after exposure to transcription m e d i u m containing 5 mM glutamine for the times shown.
Fig. 2. Effect of time of i n c u b a t i o n in translation m e d i u m on the mycelinl a c c u m u l a t i o n of active nitr ate reductase. Neurospora w a s g r o w n as described. One set o f m y c e l i a w a s transferred to basic m e d i u m containin~ 0.~ mM nitrate (c~----Q) foz the ~ m e s IDd_!cated. Myceital pads from the ot he r set were incub a t e d in tran scription m e d i u m (basic m e d i u m eontsi~inB nitra t e and tungmate) either for SO m i n (o o) or alternatively for 8 0 rain ( e - - - - 4 ) and s u b s e q u e n t l y e x p o ~ d to t ra ns l a t i on m e d i u m (basic m e d i u m containing_ nitrate, m o l y b d a t e and glutamine) in the absence (-" "-) or presence (~ ~ ) of l ~ g / m l c y c l o h e x i m i d e for the times shown. Extracts of the myceUa were t h e n made in 0.1 M phos pha t e buffer, pH 7 . 0 , eont~tning 1 mM E D T A and asaayed.
The decay of mRNA is shown in Fig. 3. The observed half-life of the mRNA appeared to be 8.5 rain. The short half-life of the nitrate reductase mRNA may not be unusual. The reported half-lives of the capacity to synthesize ailophanate hydrolase, omithine carbamoyl transferase and arginase are 3 rain [17--19], 5--7 rain [20] and 4--5 rain [21], respectively, in yeast. In Aspergillus nidulans, the half-life of the synthetic capacity for arginase was determined to be 2.5 min [22]. Lampen et al. [23] and Creanor et al. [24] found that the synthetic capacities for constitutive and derepressible acid phosphatase decay with half-lives of 25 rain and 3--5 rain, respectively. Two classes of polydisperse RNA have been reported to occur in cultured animal and insect cells [25, 26]. The most labile species possessed a half-life of 1.2 h whereas the most stable class decayed with a half-life of 20 h. Stiles et al. [27] observed that, in animal cells, the synthetic capacities to produce alanine amino transfemse and tyrosine amino transfemse decayed with half-lives of 12-14 h and 2 h, respectively. It would seem from the foregoing that relatively
278
>, >
20
o'~16 o ' $.,,-' ',z ¢, a l , ~
~E oN8
Sg
Z
2b
4'o 6'o Time (rain)
Fig. 3. Decay of the m R N A for nitrate reductasc. Neurospora was grown as described and the pads incubated w i t h Jdl=king at 27°C in transcription m e d i u m (basic m e d i u m covt=inlng ni t ra t e and tungstate) for 80 rain. The i n d u c e d pads were then transferred to decay m e d i u m (basic m e d i u m cont=intnw nitrate, t u n p t a t e and g/utamine). After incubating at 27°C with shaking for the i ndi c a t e d times, the myceltal pads were exposed to tr anslation m e d i u m (baltic m e d i u m containing nitrate, m o l y b d a t e and gl ut a mi ne ) for 20 rain, at 27°C w i t h shaking. E x t r a c t s of the m y c e l l s were made and the nitrate reductase c o n t e n t assayed in the usual manner.
unstable mRNAs are not an unusual feature of the regulation of eukaryotic enzymes with a rapid rate of turnover, as is the case with nitrate reductase [9] This work was supported by N.R.C. grant No. A-3649.
References I 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 28 24 25 26 27
Pan, S.S. and N u o n , A. (1976) Fed. Proc. 35, 887 Pan, S.S., Erlckson, R.H. and N u o n , A. (1976) Fed. Proc. 3 4 , 6 8 2 Garrett, R.H. and Nason, A. (1969) J. Biol. Chem. 244, 2 8 7 0 - - 2 8 8 2 Kinsky, S.C. (1961) J. Bacteriol. 82, 898--904 Nason, A. and Evans, H.J. (1956) Methods in Enzymol . 2, 4 1 1 - - 4 1 5 Sorger, G.J. (1965) Biochim. Biophys. Acta 99, 284--245 Sorger, G.J. and Davies, J. (1973) Biochem. J. 134, 6 7 3 - - 6 8 5 Subramanian, K.N. and Sorger, G.J. (1972) J. Bacteriol. 110, 536--546 Sorger, G.J., Debanne, M.T. and Davies, J. (1974) Biochem. J. 140, 395--403 Subramanian, K.N. and Sorger, G.J. (1972) J. Bacterlol. 110, 547--553 Reich, E. and Goldberg, I.H. (1964) in Progress of Nucleic Acid Research and Molecular Biology (Davidson, J.N. and Cohn, W.E., eds.), 3 pp. 183--234 Urey, J.C. and Horowitz. N.H. (1967) Biochim. Biophys. Acta 132, 300--309 Subra~snIRn, K.N., P a d m a n a b a n , G. and Sarma, P.S. (1968) Biochim. Biophys. Acta 151, 20-32 He/mar, Y.M., Ben-Huz, E. and Rik}Is~ E. (1977) Nature 268, 170--171 Sorger, G.J. and Giles, N.H. (1965) Genetics 52, 777--788 Gornall, A.G., Bardawill, C.S. and David, M.M. (1949) J. Biol. Chem. 177,751--756 Bomdnger, J. and Cooper, T.G. (1976) J. Bacteriol. 126, 198--204 Lawther, R ~ . and Cooper, T.G. (1973) Binchem. Biophys. Res. C o m m u n . 5b, 1 1 0 0 - - 1 1 0 4 Lawther, R.P. and Cooper, T.G. (1975) J. Bacteriol. 121, 1064--1073 M e u e n g u y , F. and Cooper, T.G. (1977) J. Bactedol. 130, 1208--1261 Bouinger, J. and Cooper, T.G. (1977) J. Bacteriol. 1 8 1 , 1 6 8 - - 1 7 3 Cybts, J. and Weglemdd, P. (1972) Eur. J. Blochem. 30, 262--265 L a m p e n , J.O., Kuo, S.C. and Cano, F.R. (1973) in Yeast, Mould and Plant P r o t o p l ~ t s (VLLIanueva, J.R., Gareia-Acha, I., Granson, S. and Uruburu, F., eds.), pp. 143--156, A c a de mi c Press Inc., New Y o r k Ckeanor, J., May, J,W. and Mitchln~on° J.M. (1975) Eur. J. Biochem. 60, 467--493 Perry, R.P. and Kelley, D.E. (1973) J. Mol. Biol. 7 9 , 6 8 1 - - 6 9 6 Spradilng, A., Hull, H. and Penman, S. (1976) Cell 4, 181--137 Stiles, C.D., Lee, K. and Kenney, F.T. (1976) Proc. Natl. Acad. Sci. U.S. 73, 2 6 3 4 - - 2 6 3 8
Biochimica et Biophysica Acta, 519 (1978) 275--278 © Elsevier/North-Holland Biomedical Press
BBA
Report
BBA 91470
STABILITY OF MESSENGER RNA FOR NITRATE REDUCTASE IN NEUROSPORA CRASSA
RAMASWAMY
PREMAKUMAR,
GEORGE
J. S O R G E R and D I N S D A L E G O O D E N
Department of Biology,McMaster University,Hamilton, OntarioL8S 4Kl (Canada) (Received January 31st, 1978)
Summary A m e t h o d has been developed to study the synthesis and decay of the messenger RNA for nitrate reductase in Neurospora crassa. Glutamine prevents the synthesis of the mRNA which appears to have a half-life of approximately 8.5 min. Nitrate reductase from Neurospora catalyzes the firststep in the assimilation of nitrate, is a 230 000 dalton, heteromultimeric enzyme and contains flavin, cytochrome b-557 and molybdenum (Mo) as components of its electron transport system [1--3]. Nitrate is believed to be required for the induced formation of nitrate reductase in Neurospora [4--6]. The effect of nitrate is to stimulate transcription [7, 8] and to partially protect nitrate reductase from decay [9, 10]. The stability of the mRNA of an enzyme is an important aspect of its regulation. Actinomycin D is widely used to block transcription in studies of mRNA stability [11]. Neurospora is relatively impermeable to the drug and the mycelia must, therefore, be pretreated with EDTA for the inhibitor to be effective [10, 12]. Even with this pretreatment, actinomycin D does not block RNA synthesis completely at the customarily used concentrations [12]. Another way of inhibiting the synthesis of a particular mRNA is to use a repressor. Glutamine represses nitrate reductase in Neurospora [13] (our unpublished results). We have studied the stability of the mRNA of nitrate reductase by making use of this finding and of the observation that tungsten (W), an analogue of Mo, can inhibit the development qf nitrate reductase, by being incorporated into nitrate reductase instead of the functional Mo [9]. A cirnilar procedure has been employed successfully in a recent study of the synthesis of nitrate reductase mRNA in tobacco cell lines [14]. Neurospora crassa, strain 3-1a (Stock No. 988, Fungal Stock Center, Humboldt) was grown in a basic medium [15] containing ammonium tar-
276 trate (4 g/l) and sucrose (20 g/l), until late log phase (39 h) at 27°C in standing culture. The mycelial pads were then transferred to fresh basic medium containing sodium nitrate (0.5 mM) as nitrogen source and sodium tungstate (6 mM} ("transcription medium"), and shaken at 27°C for the required time. Under these conditions one would expect the formation of the capacity to make nitrate reductase (presumably mRNA) and also of inactive tungsten-containing nitrate reductase. The mycelia, induced in this manner, were washed and transferred into a medium containing sodium nitrate (0.5 mM), sodium molybdate (6 mM) and glutamine (5 mM) ("translation medium"), and shaken for different time intervals at 27°C. The expectation here was that the accumulated mRNA would be translated into active nitrate reductase in this medium, and that no new mRNA would be made. When the stability of mRNA was examined, the mycelia were incubated in the transcription medium described above, with shaking, at 27°C, for 80 min, to form the mRNA. The mold was then transferred into a medium containing sodium nitrate (0.5 mM), sodium tungstate (6 mM) and glutamine (5 mM) ("decay medium"), and was shaken for different time intervals. Under these conditions, no new messenger RNA would be formed, the mRNA which had accumulated might decay, and no new active nitrate reductase would be formed. The mycelia, treated in this manner, were then transferred to the translation medium, and shaken for 20 min. Any mRNA remaining would be translated into active nitrate reductase. Extracts were prepared by grinding mycelia with an equal weight of Silica and 0.1 M potassium phosphate buffer, pH 7.0, containing 1 mM EDTA (2 ml buffer per 300 mg blotted weight of pads). Nitrate reductase was assayed as described previously [9], after a preiucubation of the extract for 30 min, at 30°C, with all substrates except NADPH. This procedure has been found in our laboratory to result in maximal increase of nitrate reductase activity (unpublished observations). One unit of activity is defined as the production of one nmole of nitrite per min at 30°C. Specific activity is defined in terms of units of activity per mg protein. Protein was determined by the biuret method [16]. The capacity to synthesize nitrate reductase accumulated after a lag of 20 min in mycelia exposed to transcription medium (Fig. 1). The capacity to synthesize nitrate reductase is attributed here to the formation of messenger RNA for nitrate reductase. The presence of glutamine in the transcription medium prevented the accumulation of mRNA. The above increase in activity is not merely due to the activation of pre-existing tungsten-containing enzyme, because it occurred only minimally in the presence of cycloheximide (Fig. 1). Mycelia that were exposed to the transcription medium for 80 min, began to synthesize active nitrate reductase immediately upon transfer to the translation medium, whereas mycelia that had not previously been exposed to the transcription medium displayed a lag of more than 20 min before active nitrate reductase was synthesized (Fig. 2). This observation is further evidence that the mRNA accumulates during the mold's incubation in transcription medium. The presence of cycloheximide in the translation medium prevented the synthesis of active nitrate reductase, suggesting again that this increase in activity is not due to the activation of pre-existing, inactive, tungsten-containing, enzyme.
277
2O~
,,
/
i
/ L.
41
~
40
80
120 160 Time (mln)
2~
10
20
3O nO 50 Time (rain)
60
Fig. 1. Effect o f t i m e o f i n c u b a t i o n in transcription m e d i u m on the m y c e l i a l a c c u m u l a t i o n o f the capacity to synthesize nitrate reductase. Neurompora mycetia were grown as described in the t e xt , transferred to ~ t i o n m e d i u m Coasic m e d i u m containing nitrate and t u n g r t a t e ) and ~ - ~ e n at 27°C for the indicated times, w a s h e d and finally i n c u b a t e d for 20 m i n a t 27°C, w i t h s bs ki ng in trln*1.tion mediu m (bas/c m e d i u m contmln4nf nitrate, m o l y b d a t e and glutamine). The myc e l l a were t he n washed and extr acted w i t h 0 . I M p h o s p h a t e buffer, pH 7.0, containing I mM EDTA (2 ml per 300 mg b l o t t e d weight of myceila). --', i n c u b a t i o n for 20 m/n in tr-n-IAtion m e d i u m after expoeure for the i ndi c a t e d tim es to tran scription m e d i u m ; o-----¢, i n c u b a t i o n in transcription m e d i u m for the t/rues s how n b u t no expose to tzanalation m e d i u m ; ~ ~, i n c u b a t i o n in tro nal-t i on m e d i u m for 20 rain in t h e p z ~ e n e e o f cycloheximid e (1 , g / m l ) after exposure to tzanacflption m e d i u m for the tlmes.shown; ± ±, i n c u b a t i o n in tr anslation m e d i u m for 20 rain after exposure to transcription m e d i u m containing 5 mM glutamine for the times shown.
Fig. 2. Effect of time of i n c u b a t i o n in translation m e d i u m on the mycelinl a c c u m u l a t i o n of active nitr ate reductase. Neurospora w a s g r o w n as described. One set o f m y c e l i a w a s transferred to basic m e d i u m containin~ 0.~ mM nitrate (c~----Q) foz the ~ m e s IDd_!cated. Myceital pads from the ot he r set were incub a t e d in tran scription m e d i u m (basic m e d i u m eontsi~inB nitra t e and tungmate) either for SO m i n (o o) or alternatively for 8 0 rain ( e - - - - 4 ) and s u b s e q u e n t l y e x p o ~ d to t ra ns l a t i on m e d i u m (basic m e d i u m containing_ nitrate, m o l y b d a t e and glutamine) in the absence (-" "-) or presence (~ ~ ) of l ~ g / m l c y c l o h e x i m i d e for the times shown. Extracts of the myceUa were t h e n made in 0.1 M phos pha t e buffer, pH 7 . 0 , eont~tning 1 mM E D T A and asaayed.
The decay of mRNA is shown in Fig. 3. The observed half-life of the mRNA appeared to be 8.5 rain. The short half-life of the nitrate reductase mRNA may not be unusual. The reported half-lives of the capacity to synthesize ailophanate hydrolase, omithine carbamoyl transferase and arginase are 3 rain [17--19], 5--7 rain [20] and 4--5 rain [21], respectively, in yeast. In Aspergillus nidulans, the half-life of the synthetic capacity for arginase was determined to be 2.5 min [22]. Lampen et al. [23] and Creanor et al. [24] found that the synthetic capacities for constitutive and derepressible acid phosphatase decay with half-lives of 25 rain and 3--5 rain, respectively. Two classes of polydisperse RNA have been reported to occur in cultured animal and insect cells [25, 26]. The most labile species possessed a half-life of 1.2 h whereas the most stable class decayed with a half-life of 20 h. Stiles et al. [27] observed that, in animal cells, the synthetic capacities to produce alanine amino transfemse and tyrosine amino transfemse decayed with half-lives of 12-14 h and 2 h, respectively. It would seem from the foregoing that relatively
278
>, >
20
o'~16 o ' $.,,-' ',z ¢, a l , ~
~E oN8
Sg
Z
2b
4'o 6'o Time (rain)
Fig. 3. Decay of the m R N A for nitrate reductasc. Neurospora was grown as described and the pads incubated w i t h Jdl=king at 27°C in transcription m e d i u m (basic m e d i u m covt=inlng ni t ra t e and tungstate) for 80 rain. The i n d u c e d pads were then transferred to decay m e d i u m (basic m e d i u m cont=intnw nitrate, t u n p t a t e and g/utamine). After incubating at 27°C with shaking for the i ndi c a t e d times, the myceltal pads were exposed to tr anslation m e d i u m (baltic m e d i u m containing nitrate, m o l y b d a t e and gl ut a mi ne ) for 20 rain, at 27°C w i t h shaking. E x t r a c t s of the m y c e l l s were made and the nitrate reductase c o n t e n t assayed in the usual manner.
unstable mRNAs are not an unusual feature of the regulation of eukaryotic enzymes with a rapid rate of turnover, as is the case with nitrate reductase [9] This work was supported by N.R.C. grant No. A-3649.
References I 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 28 24 25 26 27
Pan, S.S. and N u o n , A. (1976) Fed. Proc. 35, 887 Pan, S.S., Erlckson, R.H. and N u o n , A. (1976) Fed. Proc. 3 4 , 6 8 2 Garrett, R.H. and Nason, A. (1969) J. Biol. Chem. 244, 2 8 7 0 - - 2 8 8 2 Kinsky, S.C. (1961) J. Bacteriol. 82, 898--904 Nason, A. and Evans, H.J. (1956) Methods in Enzymol . 2, 4 1 1 - - 4 1 5 Sorger, G.J. (1965) Biochim. Biophys. Acta 99, 284--245 Sorger, G.J. and Davies, J. (1973) Biochem. J. 134, 6 7 3 - - 6 8 5 Subramanian, K.N. and Sorger, G.J. (1972) J. Bacteriol. 110, 536--546 Sorger, G.J., Debanne, M.T. and Davies, J. (1974) Biochem. J. 140, 395--403 Subramanian, K.N. and Sorger, G.J. (1972) J. Bacterlol. 110, 547--553 Reich, E. and Goldberg, I.H. (1964) in Progress of Nucleic Acid Research and Molecular Biology (Davidson, J.N. and Cohn, W.E., eds.), 3 pp. 183--234 Urey, J.C. and Horowitz. N.H. (1967) Biochim. Biophys. Acta 132, 300--309 Subra~snIRn, K.N., P a d m a n a b a n , G. and Sarma, P.S. (1968) Biochim. Biophys. Acta 151, 20-32 He/mar, Y.M., Ben-Huz, E. and Rik}Is~ E. (1977) Nature 268, 170--171 Sorger, G.J. and Giles, N.H. (1965) Genetics 52, 777--788 Gornall, A.G., Bardawill, C.S. and David, M.M. (1949) J. Biol. Chem. 177,751--756 Bomdnger, J. and Cooper, T.G. (1976) J. Bacteriol. 126, 198--204 Lawther, R ~ . and Cooper, T.G. (1973) Binchem. Biophys. Res. C o m m u n . 5b, 1 1 0 0 - - 1 1 0 4 Lawther, R.P. and Cooper, T.G. (1975) J. Bacteriol. 121, 1064--1073 M e u e n g u y , F. and Cooper, T.G. (1977) J. Bactedol. 130, 1208--1261 Bouinger, J. and Cooper, T.G. (1977) J. Bacteriol. 1 8 1 , 1 6 8 - - 1 7 3 Cybts, J. and Weglemdd, P. (1972) Eur. J. Blochem. 30, 262--265 L a m p e n , J.O., Kuo, S.C. and Cano, F.R. (1973) in Yeast, Mould and Plant P r o t o p l ~ t s (VLLIanueva, J.R., Gareia-Acha, I., Granson, S. and Uruburu, F., eds.), pp. 143--156, A c a de mi c Press Inc., New Y o r k Ckeanor, J., May, J,W. and Mitchln~on° J.M. (1975) Eur. J. Biochem. 60, 467--493 Perry, R.P. and Kelley, D.E. (1973) J. Mol. Biol. 7 9 , 6 8 1 - - 6 9 6 Spradilng, A., Hull, H. and Penman, S. (1976) Cell 4, 181--137 Stiles, C.D., Lee, K. and Kenney, F.T. (1976) Proc. Natl. Acad. Sci. U.S. 73, 2 6 3 4 - - 2 6 3 8
