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[99] The isolation of nucleic acids from bacterial spores
[99]
99
NUCLEIC ACIDS FROM BACTERIAL SPORES
MoL~s BAs~ PER 100 MOLES
Fraction
Guanine
Adenine
Cytosine
Thymine
1
24.9
25.3
24.9
24.9
2 3 4
26.0 21.5 16.3
28..8 30.8 30.8
20.8 18.7 20.5
24.4 29.0 32.4
[ 9 9 ] T h e I s o l a t i o n of N u c l e i c A c i d s f r o m B a c t e r i a l S p o r e s
By I. TAKAHASHI Treatment by reagents known to rupture protein disulfide bonds sensitizes spores of many Bacillus species to the action of lysozyme.1 This article Will describe the method for the isolation of nucleic acids from spores of Bacillus subtilis by sensitization with 2-mercaptoethanol or thioglycolic acid followed by lysozyme lysis.
Reagents Buffered saline, 0.1 M NaC1 plus 0.01 M phosphate, pH 7.0 Lysozyme (Worthington Biochemical Corp., Freehold, New Jersey). Pancreatic DNase (Worthington Biochemical Corp., Freehold, New Jersey). 2-Mercaptoethanol, 10% (v/v) in 8 M urea, pH 3.0 with HC1 Thiogtycolic acid, 2 5 ~ (v/v) in 8 M urea Penassay Broth (Difco Laboratories, Detroit, Michigan) Sporulation agar contains dehydrated nutrient broth (Difco), 8 g; MgSO~.7 H20, 0.25 g; KC1, 1 g; MnC12, 1.25 mg; and agar, 15 g per liter. The pH of the medium is adjusted to 7.0 with 1 N NaOH. To the medium autoclaved for 15 minutes, FeSO4 and Ca(NOs)2 solutions, sterilized separately, are added to at the concentrations of 10-~ M and 10-s M, respectively.
Procedure. Cells of B. su/~t///s SB 19 grown in Penassay Broth for 4 hours with shaking are plated on sporulation agar (0.2 ml/plate) and incubated for 24 hours (spores incubated more than 2 days are not sensitized to the action of lysozyme2). Spores are collected, washed once, and resuspended in buffered saline. To purify spores the spore suspension 1G. W. Gould and A. D. Hitchins, Nature 197, 622 (1963). 2 I. Takahashi, J. Bacterial. 87, 1499 (1964).
100
ISOLATION OF TOTAL NUCLEIC ACIDS
[100]
is treated with lysozyme (100 #g/ml) for 30 minutes, then with pancreatic DNase in the presence of MgSO, (0.01 M) for 20 minutes. Spores are collected by centrifugation (3000 g, 20 minutes) and washed twice in buffered saline. The washed spores are resuspended in the mercaptoethanol reagent or thioglycolie acid reagent and incubated for 60 minutes. Mercaptoethanol or thioglyeolic acid is removed by five cycles of washings and resuspended in buffered saline. The sensitized spores are treated with lysozyme (500 #g/ml) for 60 minutes or longer. The resulting viscous suspension is centrifuged at 12,000 g for 20 minutes and supernatant fluid is used as starting material for the isolation of nucleic acids. All incubations and treatments are carried ou* at 37 °, except centrifugations which are at 4 °. The yield of spores and nucleic acids in this method varies considerably from one preparation to another.
[100] I s o l a t i o n a n d P u r i f i c a t i o n of P l a n t N u c l e i c A c i d s f r o m Whole Tissues and from Isolated Nuclei
By
HERBERT STERN
The procedures here described may require modifications in detail according to the nature of the material used. Three factors, especially, must be taken into account: (1) Hardness of tissue. Tissues which are soft pose no problems in mechanical breakage (meristematic regions, for example), but if the investigator is interested in other types of tissue, care must be taken to assure breakage of cells. Specific methods for some types of tissue will be given below, but the point must be emphasized that besides being refractory to breakage, plant cell walls are formidable barriers to the diffusion of large molecules. Incomplete cell breakage may result not only in low yields of nucleic acid, but what is more important, preferential breakage of young cells results in selective extractions. (2) pH. In a number of plant species vacuolar pH is low and in such cases, macromolecules may be denatured upon cell injury. Vacuolar membranes are likely to be disrupted before cells are broken and Tris or phosphate buffers may not penetrate rapidly enough to offset vacuolar acidity. If this appears to be the case, and if preprocessing with organic solvents is not desired (see Section II,A,1), prior infiltration of tissue pieces with dilute ammonium hydroxide (e.g., 0.001 M) should be tried. This procedure has not been adequately tested in our laboratory. (3) Nucleic acid content. Nucleic acids may be isolated and purified from most types of plant tissues, but special procedures are required for tissues that con-
99
NUCLEIC ACIDS FROM BACTERIAL SPORES
MoL~s BAs~ PER 100 MOLES
Fraction
Guanine
Adenine
Cytosine
Thymine
1
24.9
25.3
24.9
24.9
2 3 4
26.0 21.5 16.3
28..8 30.8 30.8
20.8 18.7 20.5
24.4 29.0 32.4
[ 9 9 ] T h e I s o l a t i o n of N u c l e i c A c i d s f r o m B a c t e r i a l S p o r e s
By I. TAKAHASHI Treatment by reagents known to rupture protein disulfide bonds sensitizes spores of many Bacillus species to the action of lysozyme.1 This article Will describe the method for the isolation of nucleic acids from spores of Bacillus subtilis by sensitization with 2-mercaptoethanol or thioglycolic acid followed by lysozyme lysis.
Reagents Buffered saline, 0.1 M NaC1 plus 0.01 M phosphate, pH 7.0 Lysozyme (Worthington Biochemical Corp., Freehold, New Jersey). Pancreatic DNase (Worthington Biochemical Corp., Freehold, New Jersey). 2-Mercaptoethanol, 10% (v/v) in 8 M urea, pH 3.0 with HC1 Thiogtycolic acid, 2 5 ~ (v/v) in 8 M urea Penassay Broth (Difco Laboratories, Detroit, Michigan) Sporulation agar contains dehydrated nutrient broth (Difco), 8 g; MgSO~.7 H20, 0.25 g; KC1, 1 g; MnC12, 1.25 mg; and agar, 15 g per liter. The pH of the medium is adjusted to 7.0 with 1 N NaOH. To the medium autoclaved for 15 minutes, FeSO4 and Ca(NOs)2 solutions, sterilized separately, are added to at the concentrations of 10-~ M and 10-s M, respectively.
Procedure. Cells of B. su/~t///s SB 19 grown in Penassay Broth for 4 hours with shaking are plated on sporulation agar (0.2 ml/plate) and incubated for 24 hours (spores incubated more than 2 days are not sensitized to the action of lysozyme2). Spores are collected, washed once, and resuspended in buffered saline. To purify spores the spore suspension 1G. W. Gould and A. D. Hitchins, Nature 197, 622 (1963). 2 I. Takahashi, J. Bacterial. 87, 1499 (1964).
100
ISOLATION OF TOTAL NUCLEIC ACIDS
[100]
is treated with lysozyme (100 #g/ml) for 30 minutes, then with pancreatic DNase in the presence of MgSO, (0.01 M) for 20 minutes. Spores are collected by centrifugation (3000 g, 20 minutes) and washed twice in buffered saline. The washed spores are resuspended in the mercaptoethanol reagent or thioglycolie acid reagent and incubated for 60 minutes. Mercaptoethanol or thioglyeolic acid is removed by five cycles of washings and resuspended in buffered saline. The sensitized spores are treated with lysozyme (500 #g/ml) for 60 minutes or longer. The resulting viscous suspension is centrifuged at 12,000 g for 20 minutes and supernatant fluid is used as starting material for the isolation of nucleic acids. All incubations and treatments are carried ou* at 37 °, except centrifugations which are at 4 °. The yield of spores and nucleic acids in this method varies considerably from one preparation to another.
[100] I s o l a t i o n a n d P u r i f i c a t i o n of P l a n t N u c l e i c A c i d s f r o m Whole Tissues and from Isolated Nuclei
By
HERBERT STERN
The procedures here described may require modifications in detail according to the nature of the material used. Three factors, especially, must be taken into account: (1) Hardness of tissue. Tissues which are soft pose no problems in mechanical breakage (meristematic regions, for example), but if the investigator is interested in other types of tissue, care must be taken to assure breakage of cells. Specific methods for some types of tissue will be given below, but the point must be emphasized that besides being refractory to breakage, plant cell walls are formidable barriers to the diffusion of large molecules. Incomplete cell breakage may result not only in low yields of nucleic acid, but what is more important, preferential breakage of young cells results in selective extractions. (2) pH. In a number of plant species vacuolar pH is low and in such cases, macromolecules may be denatured upon cell injury. Vacuolar membranes are likely to be disrupted before cells are broken and Tris or phosphate buffers may not penetrate rapidly enough to offset vacuolar acidity. If this appears to be the case, and if preprocessing with organic solvents is not desired (see Section II,A,1), prior infiltration of tissue pieces with dilute ammonium hydroxide (e.g., 0.001 M) should be tried. This procedure has not been adequately tested in our laboratory. (3) Nucleic acid content. Nucleic acids may be isolated and purified from most types of plant tissues, but special procedures are required for tissues that con-