G E N E T I C T R A N S C R I P T I O N IN ASPOROGENOUS MUTANTS OF BACILLUS
H. YAMAGISHI AND I. TAKAHASHI* Research Unit in Biochemistry, Biophysics and Molecular Biology and Department o/Biology, McMaster University, Hamilton (Canada)
(Received August 7th, 1967)
SUMMARY At I h (tl) and 3 h (ts) after the end of the log-phase, cells of a sporogenous and uracil-requiring strain of Bacillus subtilis were pulse labeled with [6-3Hluridine and RNA was extracted b y a phenol method. Hybrid competition experiments carried out with [3H]RNA fractionated b y sucrose gradient centrifugation indicated that messenger RNA (mRNA), which sedimented between 23-S and I6-S RNA's, was synthesized at very early stages and that an asporogenous m u t a n t (Sp-HI2-3) failed to produce this mRNA. The finding that the difference in competing capacity between unlabeled RNA's from a wild type strain and S p - H I 2 - 3 disappears when DNA from this m u t a n t is used in place of DNA from a wild type strain, supports our earlier conclusion drawn from genetic studies that the strain S p - H I 2 - 3 is a deletion mutant. Competition experiments with fractionated E3H]RNA (t3)revealed that early m R N A formed at tl was replaced b y another m R N A population which sedimented between I6-S and 4-S RNA's. Another asporogenous m u t a n t (Sp-N2-2) produced the early m R N A (tl) but not the late m R N A (t3). It is suggested that the strain Sp-N2-2 has a block at a later function for sporulation, whereas S p - H I 2 - 3 is blocked at a very early function.
INTRODUCTION An extensive turnover of RNA has been observed in sporulating Bacillus cultures, although the total amotmt of RNA remains constant during this period l-a. Hybrid competition experiments carried out by DoI 4 suggest that some of the messenger RNA (mRNA) made during this stage are specific for sporulation of Bacillus subtilis. A similar observation has been made with Bacillus cereus b y ARONSON5. Since genes concerning sporulation are numerous6a and they are randomly distributed along the chromosome 8, and since various spore components are synthesized at different stages 9, it is conceivable that sporulation m R N A ' s are heterogeneous with respect to the time of their appearance and their functions. * Correspondence should be addressed to I. TAKAHASHI. Biochim. Biophys. Acta, 155 (1968) 15o-158
m R N A FOR SPORULATION
We report here that a m R N A population is synthesized at very early stages of sporulation and this RNA is replaced b y another m R N A whose molecular size differs from that of the early mRNA. Hybrid competition experiments show in agreement with previous genetic studies that an asporogenous m u t a n t (Sp-HI2-3) m a y be a deletion m u t a n t and this m u t a n t is unable to synthesize the early mRNA, and that a point mutant, Sp-N2-2 is blocked at a later function.
MATERIALS AND METHODS
Bacterial cultures The following symbols were used to designate the genotypes of bacterial strains:
sp+, sporogenous; sp-, asporogenous. Other symbols were according to DEMEREC et al. 1°. Strains of B. subtilis used were: S B I 9 E (wild type), A26 (sp + ura), Sp-N2-2 (sp- ser) and S p - H I 2 - 3 (sp- phe). The wild type strain and sp- strains were described previously 7. Strain A26 was isolated from a wild type strain (W) b y ultraviolet irradiation. Cells were grown with aeration in a broth medium which contained the following ingredients: Difco Nutrient Broth, 8 g; KC1, I g; MgSO,, 50 mg; MnC12, 5 mg; CaC12, ioo mg; and FeC13, IO mg per 1 (pH 7.2). To obtain broth cultures of A26 the above medium was supplemented with uracil (io/zg/ml). To determine growth characteristics of various strains the broth medium was inoculated with cells grown overnight on Difco Tryptose Blood Agar Base (TB agar) and incubated for 2 h. The culture was diluted IO times with the same medium and bacterial growth was followed by means of the Klett-Summerson colorimeter. We designate various stages of sporulation as follows: t 0, the end of log phase; tn (tl, t2 etc.), n h after t 0. The number of spores was estimated b y heating cultures at 85 ° for IO min and plating on TB agar. Frequencies of sporulation in the broth medium after 24 h were 0.8 to I.O for S B I 9 E and A26, lO -7 to IO-s for Sp-N2-2, and < I O -10 for Sp-HI2-3. All incubations were at 37 °.
Preparation o[ DNA Crude DNA from exponentially growing cells was prepared as described previously s. H e a t treatment to stop DNA replication was omitted. The crude DNA was dissolved in I ×SSC (o.15 M N a C l + o . o I 5 M sodium citrate) and treated with boiled pancreatic ribonuclease (IO/~g/ml) for 2 h at 37 °. The DNA solution was then treated with self-digested pronase (50 #g/ml) for 2 h at 37 ° to destroy ribonuclease. The enzyme-treated DNA preparation was deproteinized again b y the sodium lauryl sulfate method s and purified further b y the isopropanol precipitation technique of MARMUR11. This step was repeated twice. The method for isolation of DNA from T 4 phage was described b y MANDELL AND HERSHEY12.
Preparation o / R N A Pulse-labeled RNA was prepared from cells of A26 b y the following technique. Cells at t 1 stage were labeled with [6-3Hluridine (20 #C/ml) for 3 min. As the rate of radioactive uridine incorporation was low, cells at t 3 were labeled for 15 min. The labeled cultures (15 ml) were poured into an equal volume of precooled buffer conBiochim. Biophys. Acta, 155 (1968) 15o-158
H. YAMAGISHI, I. TAKAHASHI
raining o.o2 M Tris, o.oi M MgCI~ and o.o2 M NaN 3 (pH 7-3). The labeled cells were collected b y centrifugation at 5 ° and resuspended in 2 ml of the above Tris buffer which was previously diluted twice with distilled water. The cell suspension was freeze-thawed three times in the presence of lysozyme (200 #g/ml) and deoxyribonuclease (20/~gfml). The concentration of lysozyme was raised to 500/~g/ml when cells from t~ were treated. The method for the extraction of RNA from freeze-thawed cells was described b y OKAMOTO, SUGINO AND NOMURA13. The final dialysis step was replaced b y an additional alcohol precipitation in our procedure. Unlabeled RNA from cells at various stages of growth was also prepared b y the above method. The concentration of RNA was estimated b y measuring the absorbance at 260 m~. An absorbance of 25 was taken as I m g of RNA per ml. The amount of DNA present in RNA preparations was less than I °/o as measured b y the fluorometric method '4. The pulse-labeled RNA was fractionated b y the following technique. Samples (0.2 ml) in o.02 M acetate buffer (pH 5.2) containing 0.02 M KC1, and o.oi M MgC12 were layered on the top of 4.7 ml of sucrose solution (5 % to 20 °/o linear gradient) containing o.oi M Tris (pH 7.4) and 0.05 M KC1 and centrifuged in the SW39 rotor at 37 ooo rev./min at 5 ° for 4 h. After the centrifugation the bottom of the tubes was punctured and every eight drops were collected in 2 ml of the Tris-KC1 buffer (o.oi M Tris; 0.5 M KC1, p H 7.3). After spotting Io-#1 samples on Toyoroshi No. 2 filter paper disks followed b y washing3 in cold 5 ~o trichloroacetic acid and in 95 9o ethanol, the radioactivity was determined b y means of a Nuclear-Chicago model 725 liquid scintillation counter.
Assay [or DNA-RNA hybrid [ormation The membrane-filter method described b y NYGAARD AND HALL15 was used with slight modifications. Heat-denatured DNA was prepared by keeping DNA samples (ioo/,g/ml) in I ×SSC in a boiling water b a t h for 15 min followed b y rapid cooling in ice water. Under our experimental conditions the absorption of heatdenatured DNA on membrane filters was complete as measured b y the fluorometric method 14. The amount of native DNA retained on membrane filters varied from o to 20 ~o. The typical annealing mixtures contained o.I ml of heat-denatured DNA (IOO/~g/ml) and 0. 9 ml of RNA samples in the Tris-KC1 buffer. The mixtures were incubated for 5 h at 67 ° and cooled slowly overnight to a temperature below 4 °° and chilled. Hybrids were collected by filtering through No. B6 filter membrane (Carl Schleicher and Schuell Co., Keene, New Hampshire) and washed with ioo ml of Tris-KC1 buffer. The filters were kept, without drying, in 5 ml of 2 × SSC containing 20/~,g/ml of boiled pancreatic ribonuclease for 60 min at room temperature as suggested b y GILLESPIE AND SPIEGELMAN 1~. The membranes were washed with IOO ml of 2 × SSC on each side before drying and counting.
Chemicals The following chemicals were used: [6-3Hluridine (New England Nuclear Corp., Boston, Mass.); crystalline pancreatic deoxyribonuclease and ribonuclease (Worthington Biochemical Corp., Freehold, N. J.) ; pronase and lysozyme (California Corp. for Biochemical Research, Los Angeles, Calif.). Biochim. Biophys. Acta, 155 (1968) 15o-158
m R X A FOR SPORULATION
D N A - R N A hybrid [ormation Fig. i shows the growth curve of strain A26 from which pulse-labeled RNA was extracted. Various stages of growth are indicated b y the arrows.
FI-" LLJ J v
Fig. i. G r o w t h c u r v e of B. sublilis A26. V a r i o u s s t a g e s of g r o w t h are i n d i c a t e d b y t h e arrows. Fig. 2. E x t e n t of h y b r i d f o r m a t i o n a t v a r i o u s R N A / D N A ratios. R e a c t i o n m i x t u r e s (I ml) c o n t a i n e d v a r i o u s a m o u n t s of A26 (tl) [ 3 H I R N A (145 c o u n t s / m i n p e r /~g) a n d 2 0 p g of h e a t d e n a t u r e d S B I 9 E D N A ( O ) or T 4 D N A ( 0 ) .
Since it has been reported that the addition of actinomycin D to B. subtilis cultures in the presporulation stage (before t3) inhibits totally both the appearance of thermoresistant spores and the incorporation of [laCluraciP 7, we assume that m R N A for sporulation is being synthesized during that period. Arbitrarily pulselabeled RNA extracted at t 1 was taken as early m R N A and that extracted at t3 as late mRNA. To determine optimal conditions for D N A - R N A hybrid formation, the following experiments were carried out. When 20 #g of heat-denatured DNA (SBI9E) was annealed with various concentrations of pulse-labeled RNA, it was found that the amount of radioactivity fixed on membrane was proportional to the concentration of B. subtilis E3HIRNA present in the mixture (Fig. 2). Heat-denatured T 4 DNA, on the other hand, retained only a small amount of the added B. subfilis RNA and the amount of rSHJRNA hybridized remained almost constant at concentrations higher than 20/*g/ml. The experiments shown in Fig. 3 demonstrate that the use of D N A concentrations lower than I0 #g/ml results in considerable reduction in the hybridization efficiency and that the addition of carrier DNA does not improve the efficiency. Therefore, in our competition experiments the concentration of heat denatured DNA was maintained at I0/~g/ml and the addition of carrier DNA was omitted, although in some cases in Escherichia coli it has been reported that the addition of carrier DNA improved the hybridization efficiency at lower DNA concentrations 18.
Hybrid competition experiment with E3H]RNA extracted at tt The observation that actinomycin D strongly inhibits the spore formation when the antibiotic is added at early stages of sporulation 17 suggests that RNA Biochim. Biophys. Acta, 155 (1968) 15o-158
H. YAMAGISHI, I. TAKAHASHI
0 . 2 [ / /
~' ~' i '
DNA ADDED (I.llt)
I 5 FRACTION
Fig. 3. Effect of D N A c o n c e n t r a t i o n on h y b r i d formation. • : Reaction m i x t u r e s (I ml) contained 20 #g of A26 (tl) [SH]RNA (98o c o u n t s / r a i n per #g), various a m o u n t s of h e a t - d e n a t u r e d S B I 9 E D N A and carrier T 4 DNA. The total D N A concentration w a s 2o/~g/ml in all cases. × : Reaction m i x t u r e s (I ml) contained o. 5 #g of A26 (tl) [SH]RNA (20 ooo c o u n t s / m i n per/*g), various a m o u n t s of h e a t - d e n a t u r e d S B I 9 E D N A and carrier T 4 DNA. The total D N A c o n c e n t r a t i o n was 20 #g/ml. O : Reaction m i x t u r e s contained the same a m o u n t of A26 (q) [SH~RNA and S B I g E D N A as ( × ) b u t the addition of carrier T 4 D N A was omitted. Fig. 4. S e d i m e n t a t i o n profile of A26 [3H]RNA in sucrose gradients. • , Absorbance at 26o m/t; ©, c o u n t s i m i n p e r ml. (A): [3H]RNA f r o m t 1 cells; (B): [SH]RkNA f r o m t a cells.
extracted at this stage m a y contain m R N A specific for sporulation. Therefore it is expected that unlabeled RNA extracted at t 1 would compete with homologous ESHIRNA for loci on DNA more strongly than does RNA from log-phase cells or from asporogenous mutants. Experiments were performed b y adding increasing amounts of homologous or heterologous unlabeled RNA to a series of hybridization mixtures containing a constant amount of ESH~RNA (tl) and heat-denatured DNA. Contrary to the above expectation, all unlabeled RNA's showed almost identical competition pattern. The foregoing experiment suggests that hybrid competition due to spore-specific m R N A m a y be masked, because of the presence of a large excess of vegetative m R N A at t 1. Thus attempts were made to separate spore-specific m R N A from other RNA's b y centrifugation in a sucrose gradient. Figure 4 A shows the sedimentation profile of pulse-labeled RNA extracted at t 1. Fractions I, I I and I I I which had a relatively high specific activity were used in competition experiments. The amount of radioactive hybrid formed with Fractions I and I I I was reduced to about 35 ~o of the control value b y the addition of unlabeled homologous RNA, and RNA from log-phase cells and asporogenous mutants (Figs. 5 A and C). In contrast, when Fraction I I was used, RNA extracted from an asporogenous m u t a n t (Sp-HI2-3) competed much less than did RNA from other sources (Fig. 5B). Since the strain S p - H I 2 - 3 has been reported to be a deletion m u t a n t and has never produced any sp + revertant 7, the difference of IO °/o (average of three experiments) in the level of competition plateau between S p - H I 2 - 3 RNA and other RNA m a y represent an m R N A population which functions at very early stages of sporulation. This RNA sedimented between 23-S and I6-S RNA's. It was observed that RNA from another mutant, Sp-N2-2 which is a point m u t a n t 7 competed with Fraction I I at the same degree as homologous A26 RNA Biochim. Biophys. Acla, 155 (1968) 15o-158
m R N A FOR SPORULATION ,oot
m N ~
Fig. 5. Competition between fractionated A26 (4) [3H] R N A and various unlabeled R N A ' s on
DNA sites. (A) Reaction mixtures ( I ml) contained Io/zg of heat-denatured SB19E DNA. 0.24/*g of Fraction I RNA (90 ooo counts/min per/,g) and various amounts of unlabeled A26 (tl) RNA (©), A26 (log-phase) RNA (O), Sp-HI2-3 (4) RNA (×) and Sp-N2-2 (4) RNA (fN). (B) Reaction mixtures (I ml) contained io/2g of heat-denatured SBI9E DNA, o.45/~g of Fraction II RNA (27 ooo counts/min per/~g) and unlabeled RNA as indicated in (A). (C) Reaction mixtures (I ml) contained IO/zg of heat-denatured SBI9E DNA, o. 14/,g of Fraction III RNA (35o00 counts min per/zg) and unlabeled RNA as indicated in (A). (D) Same as (B) but io/zg of Sp-HI2- 3 DNA was used instead of SBI9E DNA. In the control mixtures, 9.9 % (A), 7.6 % (B), 9.8 % (C) and 8.8 % (D) of added [SH]RNA hybridized with heat-denatured DNA.
(Fig. 5B). Therefore a t t I this m u t a n t m a y be still s y n t h e s i z i n g m R N A p o p u l a t i o n s similar to those of sp ÷ b a c t e r i a . The b e h a v i o u r of u n l a b e l e d R N A from cells in t h e log phase is u n e x p e c t e d . T h e log-phase R N A c o m p e t e d a g a i n s t [ 3 H ] R N A (tl) to t h e s a m e e x t e n t as d i d homologous t 1 R N A . This o b s e r v a t i o n a p p a r e n t l y indicates t h a t cells in t h e log phase are s y n t h e s i z i n g the e a r l y m R N A . A l t e r n a t e l y it is also possible t h a t this strong comp e t i t i o n is due to the fact t h a t F r a c t i o n I I R N A still contains a r e l a t i v e l y large a m o u n t of m R N A for v e g e t a t i v e functions as well as the e a r l y m R N A . E x p e r i m e n t s are being carried o u t to t e s t the a b o v e possibilities. The foregoing e x p e r i m e n t s were carried o u t w i t h h e a t - d e n a t u r e d D N A from a wild t y p e s t r a i n ( S B I 9 E ) . H o w e v e r , when similar e x p e r i m e n t s were m a d e w i t h D N A from S p - H I 2 - 3 , t h e difference in c o m p e t i t i o n p a t t e r n b e t w e e n S p - H I 2 - 3 R N A a n d R N A from o t h e r sources d i s a p p e a r e d (Fig. 5D). I n t e r p r e t a t i o n of this result m a y be t h a t sites c o r r e s p o n d i n g to t h e e a r l y m R N A are d e l e t e d in t h e D N A of S p - H I 2 - 3 .
Competition experiments with [3H]RNA extracted at t 3 To c o m p a r e e a r l y m R N A (tl) w i t h m R N A m a d e at l a t e r stages, cells of A26 were pulse l a b e l e d a t t 3 a n d R N A was e x t r a c t e d from these cells. The pulse-labeled R N A was t h e n f r a c t i o n a t e d b y sucrose g r a d i e n t c e n t r i f u g a t i o n . F i g u r e 4 B i l l u s t r a t e s the d i s t r i b u t i o n of R N A a n d its r a d i o a c t i v i t y . F r a c t i o n s I a n d I I which showed higher specific a c t i v i t y were selected a n d used in h y b r i d c o m p e t i t i o n e x p e r i m e n t s . F r a c tion I s e d i m e n t e d b e t w e e n I6-S a n d 4-S R N A ' s a n d F r a c t i o n I I b e t w e e n 23-S a n d I6-S Biochim. Biophys. Acta, 155 (1968) 15o-158
H. YAMAGISHI, I. TAKAHASHI (A)
Fig. 6. Competition between fractionated A26 (l~) [3H]RNA and various unlabeled RNA's on DNA sites. Reaction mixtures (I ml) contained io/~g of heat-denatured SBI9E DNA, (A) Fraction I RNA (o.31/~g, 53 7°0 counts/min per pg) or (B) Fraction II RNA (o.56 #g, 18 600 counts• min p e r / , g ) and various amounts of unlabeled A26 (t3) RNA ((2)), A26 (log-phase) RNA ( 1 ) , Sp-HI2-3 (t3) RNA ( × ) and Sp-N2-2 (t3) RNA ([3). In the control mixture, lO. 5 % (A), 8.3 % (B) of added [3H]RNA hybridized with heat-denatured DNA.
RNA's. Fig. 6 shows results of competition experiments carried out with these RNA fractions. When Fraction I was used, it was found that homologous A26 (ta) RNA reduced the amount of radioactive hybrid by 8o % and heterologous RNA's; log-phase RNA, Sp-HI2-3 RNA and Sp-N2-2 RNA by 70 %, 65 % and 67 %, respectively (Fig. 6A). These figures are the average of three independent experiments. The difference in competing capacity between t3 RNA and other RNA's would probably represent mRNA for sporulation synthesized at t3. The results also show that strain Sp-N2-2, which is able to synthesize the early mRNA, is unable to form sporulation mRNA at t~. When Fraction II was used, there was practically no difference in competing ability among various non-radioactive RNA preparations tested (Fig. 6B). This would suggest that the early mRNA which sediments between 23-S and I6-S RNA's is no longer synthesized at the t~ stage. DISCUSSION
Using the hybrid competition technique, Do# has shown that sporulating B. subtilis cells contain both mRNA for sporulation and mRNA for vegetative functions. ARONSON5 has reported that 13. cereus cells synthesize, during the transition period,
an mRNA which persists throughout a large part of the sporulation process as well as mRNA of a short half-life which are required for metabolic events. From our experimental data it is not possible to determine whether mRNA for sporulation in B. subtilis are stable or whether they resemble those of vegetative Biochim. Biophys. Acta, I55 (1968) 15o-158
mRNA FOR SPORULATION
functions. Our results, however, show that m R N A ' s for sporulation are heterogeneous in molecular size and the time of their appearance. This observation seems to be compatible with the facts that genes concerning sporulation are numerous 6,7 and they are distributed randomly along the chromosome 8, and that various spore components are synthesized at different stages 9. I t is conceivable that these genes form multi-operons and they become functional at various stages of sporulation. It should be noted, however, that none of the products of sporulation genes has been identified so far. Therefore m R N A ' s discussed in this paper merely represent RNA populations which are rapidly labeled with radioactive uridine during the post log-phase at which time actinomycin D can prevent the development of mature spores. Furthermore, correct interpretation of results obtained from hybrid competition experiments could be somewhat hindered b y the fact that B. subtilis cultures used in our experiments are not completely synchronized for sporulation. Nevertheless, the difference between RNA's extracted from two types of asporogenous mutants is striking. Competition pattern of S p - H I 2 - 3 RNA at tl indicates that this strain is unable to synthesize the early mRNA. In addition, the difference in competing ability between S p - H I 2 - 3 RNA and RNA from a wild type strain (SBI9E) disappears when DNA from S p - H I 2 - 3 is used in place of DNA from SBI9E. This finding m a y be explained if competing sites for the early m R N A were deleted in the S p - H I 2 - 3 DNA. From previous genetic studies, the strain S p - H I 2 - 3 has been reported to be a deletion mutantL Since no sporulation m R N A has been detected in S p - H I 2 - 3 throughout the presporulation period, this m u t a n t might have a block at a very early function. It is tempting to postulate that this function m a y be related to the activation of an early gene whose product triggers the sequential induction of other genes. Further studies are being conducted in our laboratory to verify this hypothesis. The behaviour of RNA from another s p - mutant, Sp-N2-2 is quite different from that of S p - H I 2 - 3 RNA. The strain Sp-N2-2 produces spontaneous sp + revertants at a frequency of 10 -7 to IO-8, is transducible to sp + and its mutation is located near the terminus of B. subtilis chromosome 7,8. Results shown in Fig. 5B indicate that Sp-N2-2 is capable of synthesizing the early m R N A (tl) which is absent Jn Sp-HI2-3. However, at a later stage (4) this strain is unable to produce sporulation m R N A (Fig. 6). Thus strain Sp-N2-2 m a y have a block at a later function which presumably is the synthesis of some spore constituents. ACKNOWLEDGEMENTS
This work was supported b y a grant from the National Research Council of Canada. One of us (H.Y.) was a Postdoctoral Fellow of the National Research Council of Canada.
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5 A. I. ARONSON, J. Mol. Biol., i i (1965) 576. 6 P. SCHAEFFER, H. IONESCO, A. RYTER AND G. BALASSA, Mdchanismes de rdgulation des activit~s cellulaires chez microorganismes, Centre N a t i o n a l de la R e c h e r c h e Scientifique, Paris, 1963, P. 553. 7 I. TAKAHASHI, dr. Bacteriol., 89 (1965) 294. 8 I. TAKAHASHI, dr. Bacteriol., 89 (1965) lO65. 9 H. O. HALVORSON, Syrup. Soc. Gen. Microbiol., 15 (1965) 343. lO M. DEMEREC, E. A. AOELBERG, A. J. CLARK AND P. E. HARTMAN, Genetics, 54 (1966) 61. i i J. MARMUR, J. Mol. Biol., 3 (1961) 2o8. 12 J. D. MANDELL AND A. D. HERSHEY, Anal. Biochem., I (196o) 66. I3 K. OKAMOTO, Y. SUGINO AND M. NOMURA, J. Mol. Biol., 5 (1962) 527. 14 J. M. KISSANE AND E. ROBINS, J. Biol. Chem., 233 (1958) 184. 15 A. P. NYGAARD AND B. D. HALL, Biochem. Biophys. Res. Commun., 12 (1963) 98. 16 D. GILLESPIE AND S. SPIEGELMAN, J. Mol. Biol., 12 (1965) 829. 17 J. SZULMAJSTER, R. E. CANFIELD AND J. BLICHARSKA, Compt. Rend., 256 (1963) 2057. 18 F. IMAMOTO, N. MORIKAWA, K. SATO, S. NISHIMA, T. NISHIMURA AND A. MATSUSHIRO, J. Mol. Biol., 13 (1965) 157.
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