Molec. gen. Genet. 137, 89--99 (1975) © by Springer-Verlag 1975

Lomofungin Inhibition of Allophanate Hydrolase Synthesis in Saccharomyces cerevisiae Robert P. Lawther, Stephen L. Phillips, and Terrance G. Cooper Department of Biochemistry, University of Pittsburgh, Pittsburgh, Pennsylvania Received January 20, 1975 Summary. The RNA polymerase inhibitor, lomofungin has been used to determine the half life of specific synthetic capacities (invertase and a-glucosidase) as well as that for gross protein synthesis. In both cases the studies conclude that cognate messenger RNAs decay with a half life of approximately 20 minutes. This antibiotic has been used to determine the half life of allophanate hydrolase specific synthetic capacity. We find that it decays with a half life of about three minutes; a value that agrees with the decay rates of allophanate hydrolase synthetic capacity following removal of inducer. These observations argue that mRNA may be metabolized by two separate routes in Saccharomyces.

Introduction Detailed studies of the molecular events involved in control of gene expression require availability of a specific inhibitor of RNA synthesis. To be of greatest value the inhibitor must stop RNA synthesis at the lowest possible concentration, and fail to significantly damage other cellular processes. Thus far, four compounds have been used successfully in this capacity to study RNA synthesis in yeast. Tonnesen and Freisen (1973) observed inhibition of RNA synthesis by both daunomycin and ethidium bromide. However, due to presumably poor penetration of cells by these compounds, massive quantities were required. Their investigations, designed to determine the half life of gross cellular messenger RNA, yielded mean values of 21 ~ 4 m i n u t e s . Fraser and Creanor (1974) observed that 8-hydroxyquinoline inhibited production of all RNA species at high concentration (50 ~g/ml), but at low concentration (10 fzg/ml) decreased ribosomal and polydisperse RNA by 50% without appreciably decreasing the amounts of 5S and tRNA. They suggest that 8-hydroxyquinoline inhibits RNA synthesis b y chelating the divalent metal ions known to be required for RNA synthesis. The inhibitor most widely used for studies of RNA synthesis in yeast is the phenazine antibiotic, lomofungin (Cannon et al., 1973; Cannon and Jimenez, 1974; Cano et al., 1973; Gottlieb and Nicolas, 1969; :Fraser etal., 1973). Lomofungin has been reported (Fraser and Creanor, 1974) to function in vivo analogously to 8-hydroxyquinoline. This finding has been largely verified by the preliminary report of Pavletich et al. (1974) using purified preparations of bacterial RNA and DNA polymerases. In addition to these studies concerning the effect of lomofungin on production of gross RNA, Kuo et al. (1973) have demonstrated that synthetic capacities to produce a-gluco7

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sidase a n d i n v e r t a s e are lost w i t h a half life of a b o u t 20 m i n u t e s following a d d i t i o n of t h e drug t o p r o t o p l a s t p r e p a r a t io n s . I n v i e w of t h e s e results we selected l o m o f u n g i n as a m e a n s of addressing questions concerning t h e m o l e c u l a r e v e n t s i n v o l v e d in a l l o p h a n a t e h y d r o l ase i n d u c t i o n in Saccharomyces cerevisiae. H o w e v e r , t h e g r e a t r an g e of drug c o n c e n t r a t i o n s e m p l o y e d in p r e v i o u s studies n e c e s s i t a t e d a t h o r o u g h e v a l u a t i o n of t h e conditions a n d l i m i t a t i o n s s u r r o u n d i n g t h e use of lomofungin. H e r e we r e p o r t a detailed c h a r a c t e r i z a t i o n of t h e conditions u n d e r w h i c h we used l o m o f u n g i n an d its effects u p o n p r o d u c t i o n of a l l o p h a n a t e hydrolase.

Materials and Methods

Culture Conditions Wild type strain M25 was grown in minimal medium as previously described (Cooper and Lawther, 1973). Cell density measurements were made using a Klett Summerson colorimeter (500-570 nm band pass filter). One hundred Klett units is approximately equivalent to 3 × l0 T cells per ml of culture. I t should be emphasized that the total metal ion concentration in the medium used here is significantly lower than in the medium used by others who have reported results obtained with lomofungin. In view of the chelation properties of lomofungin this difference is important.

Enzyme Assays Allophanate hydrolase was assayed as previously described by Whitney and Cooper (1972). Samples to be used for assay were, in all cases, transferred from the experimental culture to cold test tubes containing cyeloheximide (10 ~g/ml final concentration).

Assay o] Gross RNA and Protein Synthesis Cultures in which RNA or protein synthesis was monitored were incubated with 3It-uracil and 3H-leucine respectively. Samples were transferred from the experimental culture to cold test tubes containing 5 ml of cold 10% trichloroacetic acid and 1 mg/ml of non-radioactive uracil or leucine. Samples in which protein synthesis was monitored were placed in a boiling water bath for 15 to 20 minutes. All samples were collected on glass fiber filters and were washed four times using 5 ml of cold 5% trichloroacetic acid. After drying for 45 minutes at 80° the filters were transferred to scintillation vials for radioactivity determination.

Assay o/Poly A Containing RNA Cells were collected by centrifugation, washed with cold HeO and resuspended in 0.4 ml SDS buffer (0.1 M NaC1, 20 mM EDTA, 20 mM Tris, pH 7.0). Each sample was quick frozen and stored at --20 ° C. The pellet was thawed, brought t~ 0.7 ml with SDS buffer and frozen in the chamber of an Eaton press (1962) held on dry ice. The frozen cell suspension was forced through the 1.0 mm orifice of the chamber by continuous application of 10000 psi. The extract was brought to room temperature and cell debris was removed by centrifugation (5000 rpm for 5 minutes). RNA was isolated from the supernate using the hot SDS-phenol-ehloroform method described by Penman (1969). This method of extraction yields greater than 80 % of the total I~NA (unpublished results). Polymerization of TMP and attachment to cellulose in anhydrous pyridine was accomplished as described by Edmonds (1972). The I~NA was fractioned by affinity chromatography at room temperature in columns containing a bed volume of 1.5 ml of oligo (dT) cellulose equilibrated with I-IS buffer (0.1 M hTaC1, 5 mM EDTA, 50 mM Tris HCI, pH 7.5 and 0.2% SDS). RNA was dissolved in 0.4 ml HS buffer and layered on the column. The sample was permitted to flow into the column bed and held for 20 minutes. RNA

Lomofungin Inhibition in Saccharomyces

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which did not bind to oligo (dT) cellulose was removed by ehition with 14 ml HS buffer. The poly (A) containing RNA was recovered by elution with 14 ml LS buffer (10 mM Tris I-ICI, pH 7.5, 0.2 % SDS). Two ml fractions were collected and each fraction was precipitated at 4 ° C by the addition of 100 ~g of carrier RN:A and 0.5 ml of 50% TCA. Precipitates were collected on glass fiber filters, washed twice with three volumes of cold 5% TCA, once with cold 95% ethanol, then dried. Radioactivity retained on the filters was counted by scintillation spectrometry in 4.4 ml of scintillation solution (0.01 g POPOP, 6 g 1)1)0 per liter of toluene).

Preparation and Use o/Lomo/ungin Preparation of lomofungin for experimental use was found to be of utmost importance if the drug was to be effective at very low concentrations. All preparations used in subsequent experiments were prepared by dissolving lomofungin in fresh dimethyl sulfoxide to a final concentration of 100 ~g/ml. As shown in Fig. 1 A, h'eshly prepared lomofungin exhibited only one peak of absorption in the visible region. I-Iowever, as the drug aged this peak was obscured by appearance of at least four others. A simultaneous loss of drug potency accompanied this spectral shift. A similar shift in lomofungin spectral characteristics is immediately observed if the drug is dissolved in dimethyl sulfoxide that has been opened for a month or two as compared to a freshly opened bottle (Fig. 1B). We also observed that lomofungin reacts with rubber cap liners prompting us thereafter to dissolve the drug in acid washed test tubes in place of screw-capped vials. The drug was always added to growing cultures of cells; never to medium devoid of cells. Addition of lomofungin to minimal culture medium followed by addition of the ceils resulted in almost total loss of the drug's effectiveness.

Results

Effects o/Lomo/ungin upon Gross R N A and Protein Synthesis I n preparation for studies of the effect of lomofungin upon allophanate hydrolase synthesis, we monitored its influence u p o n gross production of R N A and protein. As shown in Fig. 2, the range of concentrations over which R N A synthesis is blocked and protein synthesis remains essentially intact is r e m a r k a b l y small (0.25-0.6 ~g/ml). Also, macromolecular synthesis was very sensitive to the drug; only 1.5 9g/ml were required for 80% loss of both R N A and protein synthesizing ability. These observations are in striking contrast to those of K u o et al. (1973), who reported t h a t : (1) lomofungin at a concentration of 40 ~g/ml was required to decrease R N A synthesis 84 % and (2) over a broad concentration range (0-40 ~g/ml) protein synthesis fell only 12%. I n view of these results we ascertained the time course of drug induced losses of macromolecular synthetic ability. As shown in Fig. 3, the kinetic response of R N A and protein synthesis to lomofungin addition are different in two respects. The action of lomofungin u p o n R N A synthesis is immediate, i.e. the rate of synthesis observed within 10 to 30 seconds of drug addition is identical to t h a t observed 50 minutes later. I n contrast the rate of protein synthesis decreases with time after addition of lomofungin at concentrations up to 1 ~zg/ml. A t 2.5 ~zg/ml, however, protein synthesis is immediately terminated. The d a t a in Fig. 2 demonstrate t h a t at 0.25 ~zg/ml, lomofungin inhibits gross R N A synthesis 50% whereas protein synthesis decreases only 10%. This argues t h a t at this concentration, lomofungin is inhibiting ribosomal and messenger Rb~A synthesis differentially. This a r g u m e n t was verified (Table 1) b y direct measurements of p o l y A containing R N A (presumably m R N A ) and R N A t h a t does not 7*

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bind to oligo (dT) cellulose (principally ribosomal R N A ; Groner, Hynes and Phillips, manuscript in press) synthesized at various drug concentrations. The rate of poly A containing R N A synthesis is essentially uneffected at 0.25 ~g/ml lomofungin. However, the rate of synthesis of I~NA that is unable to bind oligo (dT) cellulose is reduced by about one half.

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Fig. 2. Effect of lomofungin on gross protein and RNA synthesis. A series of flasks were prepared containing an amount of ]omofungin sufficient to yield the final concentration indicated in the figure after cell culture was added. At zero time 1.0 ml of wild type strain M25 (cell density, 25 KIett units) were added to the lomofungin. Ten minutes later either all-uracil (6 ~g/ml, specific activity, 185 ~c/~Mole) or 3H-leueine (1O ~zg/m], specific activity, 532 ~zc/ ~Mole) were added. After 30 minutes incubation in the presence of the radiaetive metabolite a 0.5 m] sample was removed from each flask for assay as described in Materials and Methods

Table 1. Oligo (dT) cellulose fractionation of RNA synthesized in the presence of different concentrations of lomofungin Lomofungin concentration (~zg/ml)

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a Values in parenthesis are percentages of control values observed in the absence of drug. Six m] of a culture of strain M25 were added to each of four flasks containing an amount of Iomofungin sufficient to yield final concentrations of 0, 0.25, 1.0 and 2.5 ~zg/ml respectively. After three minutes at 30 ° C, all-uracil (0.5 (zg/ml, specific activity, 2.25 me/izMole) was added. After three minutes incubation in the presence of radioactive uracil ceils were harvested and processed as described in Materials and Methods.

E//ects o/ Lomo/ungin upon Synthesis o/ Allophanate Hydrolase A l l s u b s e q u e n t e x p e r i m e n t s were p e r f o r m e d using lomofungin a t a final conc e n t r a t i o n of 1 Ezg/ml. This c o n c e n t r a t i o n was selected because lomofungin a d d i t i o n to this level r e s u l t e d in loss of gross p r o t e i n synthesis w i t h a half life of t w e n t y m i n u t e s (see Fig. 3B). As shown in Fig. 4, if lomofungin is a d d e d to a log p h a s e c u l t u r e of Saccharomyces j u s t p r i o r to i n d u c e r (urea) no increase in a U o p h a n a t e h y d r o l a s e a c t i v i t y occurs. These d a t a a n d those in T a b l e I i n d i c a t e t h a t a t a ]omofungin c o n c e n t r a t i o n sufficient to t o t a l l y block p r o d u c t i o n of a l l o p h a n a t e h y d r o -

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lase, p o l y A c o n t a i n i n g R N A a n d gross p r o t e i n a p p e a r a t 55% of t h e i r u n i n h i b i t e d levels. If, indeed, lomofungin is p r e v e n t i n g p r o d u c t i o n of a l l o p h a n a t e h y d r o l a s e b y i n h i b i t i n g s y n t h e s i s of its cognate messenger R N A , t h e f u n c t i o n a l half life of this s y n t h e t i c c a p a c i t y m a y be a s c e r t a i n e d b y a d d i n g t h e d r u g to a f u l l y i n d u c e d c u l t u r e a n d d e t e r m i n i n g t h e t i m e r e q u i r e d for e n z y m e p r o d u c t i o n t o cease. As shown Fig. 5A, e i g h t t o t e n m i n u t e s elapse b e t w e e n a d d i t i o n of lomofungin a n d

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Fig. 4. Induction of allophanate hydrolase in the presence and absence of lomofungin. A culture of strain M25 was grown to a cell density of 35 Klett units and divided into two portions. One portion received urea (10 mM final concentration) and the other received lomofungin (1 ~zg/ ml final concentration) followed one minute later by addition of urea (10 mM final concentration). Zero time in this figure represents the time of urea addition to both cultures. At the indicated times, 10 ml samples were removed from both cultures and assayed for allophanate hydrolase activity using standard procedures

cessation of hydrolase production. This is consistent with losing hydrolase specific syntheitc capcity with a half life of about three minutes. On the other hand, 90 minutes elapse before gross protein synthesis stops (Fig. 5B) ; a value consistent with a messenger I~NA half life of about 20 minutes. The fact t h a t hydrolase levels continue to slowly decline rather than remaining constant after reaching a plateau (Fig. 5A closed circles after 20 minutes) is likely a secondary effect of lomofungin upon the enzyme or its production. This loss of hydrolase activity proceeds with a half life of about 90 minutes even in the absence of protein synthesis (Fig. 6A). T h a t this effect is not general is documented in Fig. 6 B which depicts results of monitoring gross protein synthesis in the above experiment. Although the reason behind this activity loss is unknown, the same result is observed when daunomycin is used in place of lomofungin (Lawther and Cooper, unpublished observations).

Discussion I n agreement with others, we have shown lomofungin can be used to specifically inhibit I~NA synthesis. I n contrast to previous reports we find the drug effective at 10-40 fold smaller concentrations and specific to RNA synthesis over a much narrower, two fold range (0.25-0.6 ~zg/ml). Two sources likely contribute to these discrepancies; the way in which we handle the drug and the strains we use. These observations, however, emphasize the caution required if use of lomofungin is to avoid the confusion t h a t surrounded early experiments in eucaryotie systems using an analogous drug, actinomycin D. I n addition to differential

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effects o n t r a n s c r i p t i o n a n d t r a n s l a t i o n , Miteheson (Fraser et al., 1973) has reported differential effects on synthesis of various R N A species; 5S a n d transfer R N A being refraetfle to l o m o f u n g i n c o n c e n t r a t i o n s capable of i n h i b i t i n g ribosomal a n d polydisperse R N A . R e f i n i n g their observations, we find t h a t ribosomal R N A synthesis is m u c h more sensitive to l o m o f u n g i n i n h i b i t i o n t h a n is the synthesis of

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p o l y (A) containing R N A . A t 0.25 ~zg/ml of lomofungin p o l y (A) c o n t a i n i n g R N A is p r o d u c e d a t t h e control levels while r i b o s o m a l R N A has decreased a t l e a s t 5 0 % . T h r o u g h use of lomofungin we h a v e shown t h a t R N A synthesis is r e q u i r e d for p r o d u c t i o n of a l l o p h a n a t e hydrolase. W h e t h e r failure of a l l o p h a n a t e h y d r o l a s e a c t i v i t y to a p p e a r d u r i n g i n h i b i t i o n of R N A synthesis derives f r o m failure to p r o d u c e t h e specific h y d r o l a s e messenger or a n a c t i v a t o r molecule can o n l y be c o n v i n c i n g l y distinguished when these e x p e r i m e n t s are r e p e a t e d a n d either i m m u n o - p r e e i p i t a b l e h y d r o l a s e p r o t e i n or h y d r o l a s e specific m R N A is m o n i t o r e d .

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R.P. Lawther et at.

Kuo et al. (1973) reported t h a t the synthetic capacity to produce invertase decays with a half life of 20 minutes as does poly (A) containing R N A (Hynes and Phillips, unpublished observations) and gross protein synthetic capacity (Hutchison et al., 1943). If R N A metabolism in eucaryotes follows the pattern established in bacteria, all synthetic capacities would be expected to decay with the same rate. This is not the case. We observed loss of allophanate hydrolase synthetic capacity with a half life of approximately 3 minutes (Lawther and Cooper, 1973). This is in agreement with the studies of Singer and P e n m a n (1973), who observed t h a t decay of m R N A from t t e L a occurs at two very different rates. Caution is needed, however, in the use of lomofungin to determine the half life of a macromolecular synthetic process. As shown in Fig. 3 B the kinetic response of protein synthesis to lomofungin addition (concentrations between 0.25 and 2.5 tzg/ml) implies t h a t the time required for cessation of synthesis to occur is a function of the drug concnetration used and m a y involve effects on both R N A and protein synthesis. We arbitrarily selected a lomofungin concentration of 1 ~zg/ml because at t h a t concentration the capacity for gross protein synthesis decays at a rate previously reported for mRNA. This was used as a standard against which to compare the decay rate of allophanate hydrolase synthetic capacity. While these facts preclude assigning great significance to absolute decay rates of synthetic capacity obtained using lomofungin, it can be concluded t h a t the synthetic capacity to produce hydrolase decays quite differently from t h a t to produce gross cellular proteins. If, indeed, synthetic capacity to produce an enzyme is a reflection of the presence of its mRI~A, the existence of different rates for loss of hydrolase and invertase or gross protein synthetic capacity suggests two paths of metabolism for their respective messenger RNAs. Acknowledgement. The authors express their sincere gratitude to Dr. G. B. Whiffield of Upjohn Co. for providing the samples of lomofungin used in these experiments. This work was supported by Public Health Service Grants GM-19386 and GM-20693 from the National Institute of General Medical Sciences and Grant Q-52 from the ttealth Research Services Foundation of Pittsburgh. Robert Lawther was supported by an Andrew Mellon Predoctoral Fellowship Award.

References Cannon, M., Davies, J. E., Jimenez, A. : Inhibition by lomofungin of nucleic acid and protein synthesis in Saccharomyces cerevisiae. FEBS Letters 82, 277-280 (1973) Cannon, M., Jimenez, A.: Lomofungin as an inhibitor of nucleic acid synthesis in Saccharomyces cerevisiae. Biochen. J. 142, 457-463 (1974) Cano, F. R., Kuo, S.-C., Lampen, J. 0.: Lomofungin, an inhibitor of deoxyribonucleic aciddependent ribonucleic acid polymerascs. Antimicrob. Ag. Chemther. 8, 723-728 (1973) Cooper, T. G., Lawther, R. P. : Induction of allantoin degradative enzymes in Saccharomyces cerevisiae by the last intermediate of the pathway. Proc. nat. Acad. Sci. (Wash.) 10, 23402344 (1973) Eaton, N. R. : New press for disruption of micro-organisms. J. Bact. 83, 1359-1360 (1962) Edmonds, M. : In: Procedures in nucleic acid research (Cantoni and Davies, eds.), vol. 2, p. 629. New York: Harper & Row 1972 Fraser, R. S. S., Creanor, J. : Rapid and selective inhibition of RNA synthesis in yeast by 8-hydroxyquinoline. Europ. J. Biochem. 46, 67-73 (1974) Fraser, R. S. S., Creanor, J., Mitchison, J. M. : Rapid and selective inhibition of the synthesis of high molecular weight RNA in yeast by lomofungin. Nature (Lond.) 244, 222-224 (1973)

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Gottlieb, D., Nicolas, G. : Mode of action of lomofungin. Appl. Microbiol. 18, 3540 (1969) Hutchison, H. T., Hartwell, L. H., MacLaughlin, C. S. : Temperature-sensitive yeast mutant defective in ribonucleic acid production. J. Bact. 99, 807-814 (1969) Kuo, S.-C., Cano, F. R., Lampcn, J. O. : Lomofungin, an inhibitor of ribonucleic acid synthesis in yeast protoplasts: Its effect on enzyme formation. Antimicrob. Ag. Chemther. 3, 716-722 (1973) Lawther, R. P., Cooper, T. G.: Effects of inducer addition and removal upon the level of allophanate hydrolase in Saccharomyces cerevisiae. Biochem. biophys. Res. Commun. 55, 1100-1104 (1973) Pavletich, K., Kuo, S.-C., Lampen, J. O. : Chelation of divalent cations by ]omofungin: Role in inhibition of nucleic acid synthesis. Biochem. biophys. Res. Commun. 69, 934-941 (1974) Penman, S. : In: Fundamental techniques in virology (Habel and Sulzman, eds.), p. 35. New York: Academic Press 1969 Singer, R. H., Penman, S. : Messenger RNA in tieLa Cells kinetics of formation and decay. J. molec. Biol. 18, 321-324 (1973) Tonnesen, T., Friesen, J. D. : Inhibitors of ribonucleie acid synthesis in Saccharomyce~ cerevisiae: Decay rate of messenger ribonucleic acid. J. Bact. 115, 889-896 (1973) Whitney, P. A., Cooper, T. G. : Urea carboxylase and allophanate hydrolase. Two components of adenosine triphosphate: urea amido-lyase in Saccharomyces cerevisiae. J. biol. Chem. 247, 1349-1353 (1972) C o m m u n i c a t e d b y W. Maas Robert P. Lawther Stephen L. Phillips Terrance G. Cooper Department of Biochemistry Faculty of Arts and Sciences University of Pittsburgh Pittsburgh, Pennsylvania 15261 USA

Lomofungin inhibition of allophanate hydrolase synthesis in Saccharomyces cerevisiae.

The RNA polymerase inhibitor, lomofungin has been used to determine the half life of specific synthetic capacities (invertase and alpha-glucosidase) a...
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