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[172] Assay of M12 phage RNA infectivity in spheroplasts
880
BIOLOGICAL PROPERTIES OF NUCLEIC ACIDS
[172]
recipient bacteriaJ 2 £ ' C helper, for example, is less than 3% as effective as X. K helper on K12 recipients. Competence is lost if helper-infected bacteria are warmed to 30 ° or above, even in a medium lacking a source of carbon. The decay of competence is approximately exponential, falling to e -1 in 10 minutes at 30 °. Competence can be reestablished if the bacteria are reinfected with helper phage. Uptake o] D N A . The uptake of D N A by helper-infected bacteria requires divalent cations: a mixture of Mg 2÷ and Ca 2+, each at 0.01 M, was found to be most effective. Na ÷ at 0.1 M or 0.01 M, Ba ~÷, Zn 2÷, Cu :÷, Ni 2+, or Fe ~+ at 0.01 M had less than 10% the activity of Ca 2÷ and Mg 2÷. M n 2÷ at 0.01 M had 30% the activity of Ca 2÷ and Mg 2÷. Inorganic orthophosphate inhibited uptake: 0.01 M phosphate added during uptake inhibited D N A infection by 95%. Uptake, as measured by the transition of infectious centers to a pancreatic D N a s e resistant state is faster at 37 ° than at 30 °, but the maximal number of DNA-infected bacteria is the same at both temperatures. The rate of D N A uptake follows the curve of competence decay in the absence of D N A suggesting that uptake is limited by the decay of competence. ~D. Dussoix and W. Arber, J. Mol. Biol. 11, 238 (1965).
[172] Assay of M12 Phage
RNA
Infectivity in Spheroplasts
B y P. H. HOFSCHNEIDER and H. DELIUS
Introduction All methods for the assay of infectious phage R N A 1-1° of the f~ phage 1~ type (f2, fr, MS2, R17, M12, and others) are based on the same general procedure. Escherichia coli cells are harvested in the late loga1 j . E. Davis, J. H. Strauss, and R. L. Sinsheimer, Science 134, 1427 (1961).
2 p. Knolle and F. Kaudewitz, Biochem. Biophys. Res. Commun. 9, 208 (1962). 3 j. Fouace and J. ttuppert, Compt. Rend. Acad. Sci. 254, 4387 (1962). ' W. Paranchych, Biochem. Biophys. Res. Commun. 11, 28 (1963). J. E. Davis, D. Pfeifer, and R. L. Sinsheimer, J. Mol. Biol. 10, 1 (1964). 8 j. H. Strauss, J. Mol. Biol. 10, 422 (1964). ' D. L. Engelhardt and N. D. Zinder, Virology 23, 582 (1964). SA. Taketo, M. Ono, and It. Shibuya, J. Biochem. 57, 488 (1965). It. Delius, Thesis, Munich 1966. lo R. Benzinger, H. Delius, R. Jaenisch, and P. H. Hofschneider, manuscript submitted. "T. Loeb and N. D. Ziader, Proc. Natl. Acad. Sci. U.8. 47, 282 (1961).
[172]
M12 RNA INFECTIVITY
881
rithmic phase, washed, resuspended in hypertonic medium and converted to spheroplasts by the action of lysozyme and E D T A . 12 Then, after attaining competence for phage R N A infection, the spheroplasts are mixed with the R N A to be tested. After the adsorption period either of two methods are followed: (a) the adsorption mixture is directly plated with agar and the number of infectious complexes formed is counted ~-4,~-1° or (b) the adsorption mixture is diluted into liquid medium and the number of phage produced by the infectious complexes is determined. 1,2,(~ In the latter case a bacterial strain resistant to phage infection is required in order to avoid additional infection cycles initiated by the progeny phages. The titer of infectious RNA in a sample is calculated from the number of infectious complexes or mature phages produced, divided by the efficiency of the assay. The efficiency is defined as the number of plaques resulting per phage R N A equivalent (infectious or physical) of a "standard R N A " preparation 12a (e.g., if 10,000 phage RNA equivalents yield one plaque, the efficiency would be 10-4). The spheroplast assay has already been studied in detail for the determination of infectious ~X174 DNA, 1°,1~-~6 which is similar to f~ RNA both with respect to molecular weight and single-strandedness. Efficiencies of up to 10-3 are reproducibly obtained. However, when this assay is adapted to infectious phage RNA, efficiencies of only 10-; to 10 -~ are obtained. The following method °,~° is a modification of those described earlier. Efiiciencies of 10 G-10-~ are reproducibly obtained. Materials MEDIA FOR PREPARATION OF SPHEROBLASTS
G r o w t h m e d i u m I is similar to 3XD medium 17 and contains per
liter 1: R. Repaske, Biochim. Biophys. Acta 22, 189 (1956). 12aThe "standard RNA" preparation is an RNA solution prepared from a phage solution with known infectious titer or an RNA solution with known concentration of phage RNA molecules as calculated from the optical density of the solution and the molar extinction coefficient of phage RNA. 13 G. D. Guthrie and R. L. Sinsheimer, J. Mol. Biol. 2, 297 (1960). " G. D. Guthrie and It. L. Sinsheimer, Biochim. Biophys. Acta 72, 290 (1963). 15 It. Wahl, J. Huppert, and L. Emerique-Blum, Compt. Rend. Acad. Sci. 250, 4227 (1960). ~ M. Sekiguctii, A. Taketo, and Y. Takagi, Biochim. Biophys. Acta 45, 199 (1960). " D. Fraser and E. A. Jerrel, J. Biol. Chem. 205, 291 (1953).
882
[172]
BIOLOGICAL PROPERTIES OF NUCLEIC ACIDS
16 g Bacto technical casamino acids (Difco Laboratories, Detroit, Michigan) 1Ta 5 g glucose 150 ml Tris-HC1, 0.1 M, pH 8.5 100 ml of a salt solution containing per liter: NH4C1, 10.0 g; MgS04, 3.0 g; and CaC12, 0.33 g 100 ml phosphate buffer containing per liter: Na2HPO4"2 H20, 105.0 g; and KH2P04, 45.0 g (autoclave separately) Growth medium H contains per liter 10.0 g Bacto nutrient broth (Difco) or 10.0 g meat peptone (E. Merck, Darmstadt) 5.0 g NaC1 Conversion medium contains 70 ml sucrose, 0.5M (Saccharose, E. Merck or sucrose M.A. special enzyme grade, Mann Research Laboratories, New York) 20 ml Tris-HC1, 0.1 M, pH 8.1 4 ml EDTA, 4% (Disodium salt, adjusted to pH 8.1 with NaOH) 2 ml lysozyme, 0.4% (3X crystallized, Sigma Chem. Co., St. Louis, Missouri) Dilution medium I (PAM medium 13,14) contains per liter 10 g Bacto nutrient broth (Difco) 10 g Bacto technical casamino acids (Difco) 1 g glucose 100 g sucrose 1 g MgSO~ (autoclaved separately) Before use a 30% solution of bovine serum albumin (BSA, Hyland Laboratories, Los Angeles, California or albumin "Povite," BiotestSerum Institut, Frankfurt/Main) was added to give a final concentration of 2% BSA. Dilution medium I I is the same as dilution medium I, but without albumin / SOLUTIONS FOR THE PREPARATION OF INFECTIOUS PHAGE
RNA
Cell suspension .medium containing NaC1, 0.3 M and EDTA, 0.003 M (disodium salt, adjusted to pH 7.0) Sodium dodecylsulfate, 10% Water-saturated phenol Ethanol, 96% EDTA, 0.003 M (disodium salt, adjusted to pH 7.0) '~aFiltration of concentrated casamino acid solutions through filter paper and nitrocellulose filters (group 50, Membranfiltergesellschaft, GSttingen) 3 times each, increased the reproducibility of the method.
[172]
M12 RNA INFECTIVITY
883
SOLUTIONS AND AGARS FOR THE ASSAY
DNase solution 1 m g / m l (Deoxyribonuclease I, electrophoretically purified, Worthington Biochemical Corp., Freehold, New Jersey)
Dilution medium I I I is the same as dilution medium I, but without albumin and MgS04 E D T A , 0.003 M (disodium salt, adjusted to p H 7.0) Top layer agar contains per liter: 7 g agar (E. Merck) l0 g peptone (E. Merck) or Bacto nutrient broth (Difco) 5 g NaC1 After autoclaving 125 ml sucrose, 1 M and 125 ml phosphate buffer (containing N a : H P Q . 2 H20, 0.7%, NaC1, 0.4%, and KH~P04, 0.3~,) arc added
Bottam layer agar contains per liter 15 g agar (E. Merck) l0 g peptone (E. Merck) 5 g NaCl T h e p H is adjusted to 7 after autoclaving with N a O H P r e p a r a t i o n of Spheroplasts Eight milliliters of a fresh E. coli K12 ll2()~)F +18 overnight culture are added to 2 liters of growth medium I or II. The culture is shaken in a 5-liter bottle at 20°. 19 When a titer of 4 X l0 s cells/mF ° is reached 200 ml portions of the culture are centrifuged for 10 minutes at 5000 g. Each pellet is immediately t a k e n up in 5 ml of the conversion medium. After a 3 minute incubation at room t e m p e r a t u r e the cells begin to appear spherical by phase-contrast microscopy and after 10 minutes 80-95% of the rod-shaped bacterial cells have been converted to spheroplasts. The lysozyme action is stopped at this time b y the addition of 20 ml of dilution medium I. 21 I~E. coIi K12 112 ( h ) F ÷ was kindly given b y Dr. D. Pratt, Madison, Wisconsin. 19Bacteria grown u n d e r these conditions were as good for spheroplast preparation as
aerated bacterial cultures grown at 37° , but at 20° infection by contaminating phages was inhibited. The efficiency of the spheroplasts varied by a factor of 5 when cell cultures were harvested at different cell concentrations between 1 × 10~ to 6 × 10~/ml. The optimal cell concentration may be influenced by the bacterial strain used, strength of aeration etc. :1Some BSA lots used in the dilution medium I yielded efficiencies three times as high as those observed without albumin. However, other BSA preparations lowered the efficiency of the spheroplasts. The difference was probably due to different concentrations of contaminating RNase. Infectious M12 phage RNA in 0.003 M
884
BIOLOGICAL PROPERTIES OF NUCLEIC ACIDS
[172]
Incubation is continued for another 20 minutes. Then 200 ml of dilution medium I I are added and the spheroplasts are sedimented for 15 minutes at 2500 g. Each pellet is carefully resuspended in 10 ml of dilution medium II using a large-bore pipette. Storage of Spheroplast Preparations
When the spheroplasts are stored at 4 ° their efficiency reaches a maximum the day after their preparation and decreases to less than 10% within two weeks. The efficiency of poor spheroplast preparations (lower than 10 -6) decreases from the time of preparation. Spheroplasts which are stored frozen give a lower but a more constant efficiency. One-tenth volume of dimethylsulfoxide is added to the spheroplasts which are divided into portions of suitable size and sedimented at 4 ° (15 minutes at 2500 g). The supernatants are discarded and the pellets stored at --20 ° . The spheroplast pellets are thawed by resuspension in the original volume of prewarmed dilution medium II. The efficiency of the thawed preparations is generally only 10% of the efficiency of the same preparation before freezing but is unchanged for several months. Preparation of Infectious P h a g e R N A
The following method can be applied to purified phage, crude lysates, or phage-infected bacteria (up to 101° cells/ml, washed in cell suspension medium). To the solution containing phage, lysate, or infected bacteria 1/10 volume of 10% dodecyl sulfate and a few drops of chloroform are added. Phage or lysate are shaken for 1 minute; infected cells are shaken for 1-5 minutes until lysis of cells has occurred. The mixture is now deproteinized by shaking with one volume of water-saturated phenol at 4 °, centrifuging for 5 minutes at 2000 g and discarding the phenol layer and the interface. Fresh phenol is added and, the protein extraction is repeated once. The R N A contained in the aqueous phase can be stored in two ways: (a) the R N A is kept in solution at 4 ° over a phenol phase. In order to reduce the concentration of phenol the R N A is diluted at least 100-fold for the assay. (b) The R N A is precipitated by adding 3 volumes of ethanol at 4 ° . After storage at --20 ° for 2-3 hours the precipitate is EDTA was inactivated 10-fold in 25 minutes at 37° by a final concentration of 0.3% "good" albumin. "Bad" albumin caused more than a 100-fold inactivation under the same conditions. When no "good" albumin was available the lysozyme treatment was stopped after 5 minutes by the addition of dilution medium II and the spheroplasts were immediately sedimented.
[172]
M12 RNA INFECTIVITY
885
sedimented for 20 minutes at 12,000 g, The pellets are redissolved in E D T A , 0.003 M and stored frozen at - - 2 0 °. Under these conditions the R N A loses no more t h a n 10-20% of its biological activity within 6 months. T h e preparation and purification of M12 infectious replieative-form RNA22,53 is described in Vol. X I I , P a r t A, p. 613.
Assay for Single-Stranded Phage RNA The spheroplast suspension is incubated for 15 minutes at 37 ° with 10 v / m l of DNase. ~4 I t is then diluted with 3 volumes of dilution medium I I I at 20 °. Portions of 0.5 ml of these spheroplasts are mixed with 0.1 ml of the R N A sample (diluted in 0.003 M E D T A ) and left standing at room t e m p e r a t u r e for 30 minutesY 5 Then the tubes are placed into a 37 ° bath for about 30 seconds. After addition of 3 ml of top agar the mixtures are poured immediately on agar plates. No indicator bacteria have to be added, since about 10% of the spheroplasts can still form colonies. The plates are incubated for about 12 hours at 37 ° and the plaques counted. To obtain good reproducibility, care in cleaning test tubes and pipettes is required. All tests should be performed in triplicate.
Assay for M12 D o u b l e - S t r a n d e d R e p l i c a t i v e - F o r m R N A (Replicative I n t e r m e d i a t e ) Replicative-form R N A (RF) of phage M12 was infectious in this spheroplast assay only after heat denaturation. 22 For the assay, R F samples (diluted in 0.003 M E D T A ) are therefore incubated for 1 minute in a boiling-water bath and chilled rapidly in an ice bath. The assay for infectivity is performed on the denaturated mixture as described above. Yields T h e plaque yield in the assay was proportional to the concentration of infectious R N A from 10 -3 to one infectious phage R N A equivalents ~J. Ammann, H. Delius, and P. H. Hofschneider, J. Mol. Biol. 10, 557~(1964). ~3B. Francke and P. H. Hofschneider, J. Mol. Biol. 16, 544 (1966). ~ Treatment of spheroplasts with RNase-free DNase usually raised the efficiency of the spheroplasts 2 to 5-fold. A maximum increase of 40-fold was observed. ~Approximately 70% of the infectious complexes are formed within 30 seconds after addition of the RNA. A maximum is reached between 30 and 40 minutes. No mature phage could be detected at this time after lyzing the spheroplasts by dilution into 0.04 M phosphate buffer, pH 6.7. Plating in agar stops new formation of infectious complexes. Whenever a large number of assays were performed the mixing of RNA and spheroplasts was staggered by 30 seconds for pairs of tests and "standard RNA" was tested at the beginning and end of each experiment to check the constancy of the efficiency.
886
BIOLOGICAL PROPERTIES OF NIICL~.IC ACIDS
[172]
per spheroplast (at a spheroplast concentration of 2 X 10'~/ml and an efficiency of 4 X 10 -5).26 E v e n when n o n s a t u r a t i n g concentrations of infectious R N A were used the n u m b e r of infectious complexes was directly p r o p o r t i o n a l to the n u m b e r of spheroplasts between 5 X 105 and 5 X 10s/m177 T h e m a x i m u m efficiency of the a s s a y calculated on the basis of infectious complexes yielded per infectious p h a g e R N A equivalent was 2 X 10 -4 ( R N A was p r e p a r e d from purified M 1 2 phage; only one in ten phages was infectious as calculated from the optical density at 260 n m of the phage solution). M o s t of the spheroplast p r e p a r a t i o n s showed an efficiency of about lO-~.2s
~The mixtures of the less diluted RNA samples with spheroplasts were diluted subsequent to adsorption in dilution medium I I I before plating. '~ The data are in accord with the kinetics of a bimolecular reaction found in the ¢X174 DNA assay?4 ~Different lots of media components (casamino acids, nutrient broth, peptone, sucrose, and agar) and of enzymes sometimes changed the efficiencies of the assays appreciably. When one lot has to be replaced by another, both should be tested with standard RNA in parallel. Tests with frozen spheroplasts of constant competence are well suited to check whether the media for the preparation of spheroplasts or the components of the assay system are affected.
BIOLOGICAL PROPERTIES OF NUCLEIC ACIDS
[172]
recipient bacteriaJ 2 £ ' C helper, for example, is less than 3% as effective as X. K helper on K12 recipients. Competence is lost if helper-infected bacteria are warmed to 30 ° or above, even in a medium lacking a source of carbon. The decay of competence is approximately exponential, falling to e -1 in 10 minutes at 30 °. Competence can be reestablished if the bacteria are reinfected with helper phage. Uptake o] D N A . The uptake of D N A by helper-infected bacteria requires divalent cations: a mixture of Mg 2÷ and Ca 2+, each at 0.01 M, was found to be most effective. Na ÷ at 0.1 M or 0.01 M, Ba ~÷, Zn 2÷, Cu :÷, Ni 2+, or Fe ~+ at 0.01 M had less than 10% the activity of Ca 2÷ and Mg 2÷. M n 2÷ at 0.01 M had 30% the activity of Ca 2÷ and Mg 2÷. Inorganic orthophosphate inhibited uptake: 0.01 M phosphate added during uptake inhibited D N A infection by 95%. Uptake, as measured by the transition of infectious centers to a pancreatic D N a s e resistant state is faster at 37 ° than at 30 °, but the maximal number of DNA-infected bacteria is the same at both temperatures. The rate of D N A uptake follows the curve of competence decay in the absence of D N A suggesting that uptake is limited by the decay of competence. ~D. Dussoix and W. Arber, J. Mol. Biol. 11, 238 (1965).
[172] Assay of M12 Phage
RNA
Infectivity in Spheroplasts
B y P. H. HOFSCHNEIDER and H. DELIUS
Introduction All methods for the assay of infectious phage R N A 1-1° of the f~ phage 1~ type (f2, fr, MS2, R17, M12, and others) are based on the same general procedure. Escherichia coli cells are harvested in the late loga1 j . E. Davis, J. H. Strauss, and R. L. Sinsheimer, Science 134, 1427 (1961).
2 p. Knolle and F. Kaudewitz, Biochem. Biophys. Res. Commun. 9, 208 (1962). 3 j. Fouace and J. ttuppert, Compt. Rend. Acad. Sci. 254, 4387 (1962). ' W. Paranchych, Biochem. Biophys. Res. Commun. 11, 28 (1963). J. E. Davis, D. Pfeifer, and R. L. Sinsheimer, J. Mol. Biol. 10, 1 (1964). 8 j. H. Strauss, J. Mol. Biol. 10, 422 (1964). ' D. L. Engelhardt and N. D. Zinder, Virology 23, 582 (1964). SA. Taketo, M. Ono, and It. Shibuya, J. Biochem. 57, 488 (1965). It. Delius, Thesis, Munich 1966. lo R. Benzinger, H. Delius, R. Jaenisch, and P. H. Hofschneider, manuscript submitted. "T. Loeb and N. D. Ziader, Proc. Natl. Acad. Sci. U.8. 47, 282 (1961).
[172]
M12 RNA INFECTIVITY
881
rithmic phase, washed, resuspended in hypertonic medium and converted to spheroplasts by the action of lysozyme and E D T A . 12 Then, after attaining competence for phage R N A infection, the spheroplasts are mixed with the R N A to be tested. After the adsorption period either of two methods are followed: (a) the adsorption mixture is directly plated with agar and the number of infectious complexes formed is counted ~-4,~-1° or (b) the adsorption mixture is diluted into liquid medium and the number of phage produced by the infectious complexes is determined. 1,2,(~ In the latter case a bacterial strain resistant to phage infection is required in order to avoid additional infection cycles initiated by the progeny phages. The titer of infectious RNA in a sample is calculated from the number of infectious complexes or mature phages produced, divided by the efficiency of the assay. The efficiency is defined as the number of plaques resulting per phage R N A equivalent (infectious or physical) of a "standard R N A " preparation 12a (e.g., if 10,000 phage RNA equivalents yield one plaque, the efficiency would be 10-4). The spheroplast assay has already been studied in detail for the determination of infectious ~X174 DNA, 1°,1~-~6 which is similar to f~ RNA both with respect to molecular weight and single-strandedness. Efficiencies of up to 10-3 are reproducibly obtained. However, when this assay is adapted to infectious phage RNA, efficiencies of only 10-; to 10 -~ are obtained. The following method °,~° is a modification of those described earlier. Efiiciencies of 10 G-10-~ are reproducibly obtained. Materials MEDIA FOR PREPARATION OF SPHEROBLASTS
G r o w t h m e d i u m I is similar to 3XD medium 17 and contains per
liter 1: R. Repaske, Biochim. Biophys. Acta 22, 189 (1956). 12aThe "standard RNA" preparation is an RNA solution prepared from a phage solution with known infectious titer or an RNA solution with known concentration of phage RNA molecules as calculated from the optical density of the solution and the molar extinction coefficient of phage RNA. 13 G. D. Guthrie and R. L. Sinsheimer, J. Mol. Biol. 2, 297 (1960). " G. D. Guthrie and It. L. Sinsheimer, Biochim. Biophys. Acta 72, 290 (1963). 15 It. Wahl, J. Huppert, and L. Emerique-Blum, Compt. Rend. Acad. Sci. 250, 4227 (1960). ~ M. Sekiguctii, A. Taketo, and Y. Takagi, Biochim. Biophys. Acta 45, 199 (1960). " D. Fraser and E. A. Jerrel, J. Biol. Chem. 205, 291 (1953).
882
[172]
BIOLOGICAL PROPERTIES OF NUCLEIC ACIDS
16 g Bacto technical casamino acids (Difco Laboratories, Detroit, Michigan) 1Ta 5 g glucose 150 ml Tris-HC1, 0.1 M, pH 8.5 100 ml of a salt solution containing per liter: NH4C1, 10.0 g; MgS04, 3.0 g; and CaC12, 0.33 g 100 ml phosphate buffer containing per liter: Na2HPO4"2 H20, 105.0 g; and KH2P04, 45.0 g (autoclave separately) Growth medium H contains per liter 10.0 g Bacto nutrient broth (Difco) or 10.0 g meat peptone (E. Merck, Darmstadt) 5.0 g NaC1 Conversion medium contains 70 ml sucrose, 0.5M (Saccharose, E. Merck or sucrose M.A. special enzyme grade, Mann Research Laboratories, New York) 20 ml Tris-HC1, 0.1 M, pH 8.1 4 ml EDTA, 4% (Disodium salt, adjusted to pH 8.1 with NaOH) 2 ml lysozyme, 0.4% (3X crystallized, Sigma Chem. Co., St. Louis, Missouri) Dilution medium I (PAM medium 13,14) contains per liter 10 g Bacto nutrient broth (Difco) 10 g Bacto technical casamino acids (Difco) 1 g glucose 100 g sucrose 1 g MgSO~ (autoclaved separately) Before use a 30% solution of bovine serum albumin (BSA, Hyland Laboratories, Los Angeles, California or albumin "Povite," BiotestSerum Institut, Frankfurt/Main) was added to give a final concentration of 2% BSA. Dilution medium I I is the same as dilution medium I, but without albumin / SOLUTIONS FOR THE PREPARATION OF INFECTIOUS PHAGE
RNA
Cell suspension .medium containing NaC1, 0.3 M and EDTA, 0.003 M (disodium salt, adjusted to pH 7.0) Sodium dodecylsulfate, 10% Water-saturated phenol Ethanol, 96% EDTA, 0.003 M (disodium salt, adjusted to pH 7.0) '~aFiltration of concentrated casamino acid solutions through filter paper and nitrocellulose filters (group 50, Membranfiltergesellschaft, GSttingen) 3 times each, increased the reproducibility of the method.
[172]
M12 RNA INFECTIVITY
883
SOLUTIONS AND AGARS FOR THE ASSAY
DNase solution 1 m g / m l (Deoxyribonuclease I, electrophoretically purified, Worthington Biochemical Corp., Freehold, New Jersey)
Dilution medium I I I is the same as dilution medium I, but without albumin and MgS04 E D T A , 0.003 M (disodium salt, adjusted to p H 7.0) Top layer agar contains per liter: 7 g agar (E. Merck) l0 g peptone (E. Merck) or Bacto nutrient broth (Difco) 5 g NaC1 After autoclaving 125 ml sucrose, 1 M and 125 ml phosphate buffer (containing N a : H P Q . 2 H20, 0.7%, NaC1, 0.4%, and KH~P04, 0.3~,) arc added
Bottam layer agar contains per liter 15 g agar (E. Merck) l0 g peptone (E. Merck) 5 g NaCl T h e p H is adjusted to 7 after autoclaving with N a O H P r e p a r a t i o n of Spheroplasts Eight milliliters of a fresh E. coli K12 ll2()~)F +18 overnight culture are added to 2 liters of growth medium I or II. The culture is shaken in a 5-liter bottle at 20°. 19 When a titer of 4 X l0 s cells/mF ° is reached 200 ml portions of the culture are centrifuged for 10 minutes at 5000 g. Each pellet is immediately t a k e n up in 5 ml of the conversion medium. After a 3 minute incubation at room t e m p e r a t u r e the cells begin to appear spherical by phase-contrast microscopy and after 10 minutes 80-95% of the rod-shaped bacterial cells have been converted to spheroplasts. The lysozyme action is stopped at this time b y the addition of 20 ml of dilution medium I. 21 I~E. coIi K12 112 ( h ) F ÷ was kindly given b y Dr. D. Pratt, Madison, Wisconsin. 19Bacteria grown u n d e r these conditions were as good for spheroplast preparation as
aerated bacterial cultures grown at 37° , but at 20° infection by contaminating phages was inhibited. The efficiency of the spheroplasts varied by a factor of 5 when cell cultures were harvested at different cell concentrations between 1 × 10~ to 6 × 10~/ml. The optimal cell concentration may be influenced by the bacterial strain used, strength of aeration etc. :1Some BSA lots used in the dilution medium I yielded efficiencies three times as high as those observed without albumin. However, other BSA preparations lowered the efficiency of the spheroplasts. The difference was probably due to different concentrations of contaminating RNase. Infectious M12 phage RNA in 0.003 M
884
BIOLOGICAL PROPERTIES OF NUCLEIC ACIDS
[172]
Incubation is continued for another 20 minutes. Then 200 ml of dilution medium I I are added and the spheroplasts are sedimented for 15 minutes at 2500 g. Each pellet is carefully resuspended in 10 ml of dilution medium II using a large-bore pipette. Storage of Spheroplast Preparations
When the spheroplasts are stored at 4 ° their efficiency reaches a maximum the day after their preparation and decreases to less than 10% within two weeks. The efficiency of poor spheroplast preparations (lower than 10 -6) decreases from the time of preparation. Spheroplasts which are stored frozen give a lower but a more constant efficiency. One-tenth volume of dimethylsulfoxide is added to the spheroplasts which are divided into portions of suitable size and sedimented at 4 ° (15 minutes at 2500 g). The supernatants are discarded and the pellets stored at --20 ° . The spheroplast pellets are thawed by resuspension in the original volume of prewarmed dilution medium II. The efficiency of the thawed preparations is generally only 10% of the efficiency of the same preparation before freezing but is unchanged for several months. Preparation of Infectious P h a g e R N A
The following method can be applied to purified phage, crude lysates, or phage-infected bacteria (up to 101° cells/ml, washed in cell suspension medium). To the solution containing phage, lysate, or infected bacteria 1/10 volume of 10% dodecyl sulfate and a few drops of chloroform are added. Phage or lysate are shaken for 1 minute; infected cells are shaken for 1-5 minutes until lysis of cells has occurred. The mixture is now deproteinized by shaking with one volume of water-saturated phenol at 4 °, centrifuging for 5 minutes at 2000 g and discarding the phenol layer and the interface. Fresh phenol is added and, the protein extraction is repeated once. The R N A contained in the aqueous phase can be stored in two ways: (a) the R N A is kept in solution at 4 ° over a phenol phase. In order to reduce the concentration of phenol the R N A is diluted at least 100-fold for the assay. (b) The R N A is precipitated by adding 3 volumes of ethanol at 4 ° . After storage at --20 ° for 2-3 hours the precipitate is EDTA was inactivated 10-fold in 25 minutes at 37° by a final concentration of 0.3% "good" albumin. "Bad" albumin caused more than a 100-fold inactivation under the same conditions. When no "good" albumin was available the lysozyme treatment was stopped after 5 minutes by the addition of dilution medium II and the spheroplasts were immediately sedimented.
[172]
M12 RNA INFECTIVITY
885
sedimented for 20 minutes at 12,000 g, The pellets are redissolved in E D T A , 0.003 M and stored frozen at - - 2 0 °. Under these conditions the R N A loses no more t h a n 10-20% of its biological activity within 6 months. T h e preparation and purification of M12 infectious replieative-form RNA22,53 is described in Vol. X I I , P a r t A, p. 613.
Assay for Single-Stranded Phage RNA The spheroplast suspension is incubated for 15 minutes at 37 ° with 10 v / m l of DNase. ~4 I t is then diluted with 3 volumes of dilution medium I I I at 20 °. Portions of 0.5 ml of these spheroplasts are mixed with 0.1 ml of the R N A sample (diluted in 0.003 M E D T A ) and left standing at room t e m p e r a t u r e for 30 minutesY 5 Then the tubes are placed into a 37 ° bath for about 30 seconds. After addition of 3 ml of top agar the mixtures are poured immediately on agar plates. No indicator bacteria have to be added, since about 10% of the spheroplasts can still form colonies. The plates are incubated for about 12 hours at 37 ° and the plaques counted. To obtain good reproducibility, care in cleaning test tubes and pipettes is required. All tests should be performed in triplicate.
Assay for M12 D o u b l e - S t r a n d e d R e p l i c a t i v e - F o r m R N A (Replicative I n t e r m e d i a t e ) Replicative-form R N A (RF) of phage M12 was infectious in this spheroplast assay only after heat denaturation. 22 For the assay, R F samples (diluted in 0.003 M E D T A ) are therefore incubated for 1 minute in a boiling-water bath and chilled rapidly in an ice bath. The assay for infectivity is performed on the denaturated mixture as described above. Yields T h e plaque yield in the assay was proportional to the concentration of infectious R N A from 10 -3 to one infectious phage R N A equivalents ~J. Ammann, H. Delius, and P. H. Hofschneider, J. Mol. Biol. 10, 557~(1964). ~3B. Francke and P. H. Hofschneider, J. Mol. Biol. 16, 544 (1966). ~ Treatment of spheroplasts with RNase-free DNase usually raised the efficiency of the spheroplasts 2 to 5-fold. A maximum increase of 40-fold was observed. ~Approximately 70% of the infectious complexes are formed within 30 seconds after addition of the RNA. A maximum is reached between 30 and 40 minutes. No mature phage could be detected at this time after lyzing the spheroplasts by dilution into 0.04 M phosphate buffer, pH 6.7. Plating in agar stops new formation of infectious complexes. Whenever a large number of assays were performed the mixing of RNA and spheroplasts was staggered by 30 seconds for pairs of tests and "standard RNA" was tested at the beginning and end of each experiment to check the constancy of the efficiency.
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per spheroplast (at a spheroplast concentration of 2 X 10'~/ml and an efficiency of 4 X 10 -5).26 E v e n when n o n s a t u r a t i n g concentrations of infectious R N A were used the n u m b e r of infectious complexes was directly p r o p o r t i o n a l to the n u m b e r of spheroplasts between 5 X 105 and 5 X 10s/m177 T h e m a x i m u m efficiency of the a s s a y calculated on the basis of infectious complexes yielded per infectious p h a g e R N A equivalent was 2 X 10 -4 ( R N A was p r e p a r e d from purified M 1 2 phage; only one in ten phages was infectious as calculated from the optical density at 260 n m of the phage solution). M o s t of the spheroplast p r e p a r a t i o n s showed an efficiency of about lO-~.2s
~The mixtures of the less diluted RNA samples with spheroplasts were diluted subsequent to adsorption in dilution medium I I I before plating. '~ The data are in accord with the kinetics of a bimolecular reaction found in the ¢X174 DNA assay?4 ~Different lots of media components (casamino acids, nutrient broth, peptone, sucrose, and agar) and of enzymes sometimes changed the efficiencies of the assays appreciably. When one lot has to be replaced by another, both should be tested with standard RNA in parallel. Tests with frozen spheroplasts of constant competence are well suited to check whether the media for the preparation of spheroplasts or the components of the assay system are affected.