Proc. Natl. Acad. Sci. USA Vol. 76, No. 8, pp. 3947-3951, August 1979
Cell Biology
Isolation and characterization of germ line DNA from mouse sperm (genetic transmission/mammary tumor virus/leukemia virus/antibody diversity/testicular teratoma)
ROBERT SHIURBA AND S. NANDI Department of Zoology and Cancer Research Laboratory, University of California, Berkeley, California 94720
Communicated by Howard A. Bern, May 21, 1979
ABSTRACT Mouse germ line DNA was isolated from sperm by a physicochemical procedure that preferentially destroys contaminating somatic cell DNA. The use of reducing conditions and chelating agents in combination with phenol permitted extraction of high molecular weight DNA from mature sperm nuclei with approximately 80% efficiency. Less than 0.1% somatic cell DNA contamination remained in sperm DNA prepared by this method. Germ line DNA was characterized by determination of its ultraviolet absorbance spectrum, buoyant density in cesium chloride, and melting profile on a hydroxyapatite column. Contamination by mitochondrial DNA was assessed by cesium chloride/ethidium bromide gradient centrifugation. The significance of the mouse germ line DNA isolation procedure is discussed with respect to the possible genetic transmission of mammary tumor virus and leukemia virus, the origin of antibody diversity, and the origin of testicular teratomas. It is currently believed that mouse mammary tumor virus (MTV) is transmitted genetically via sperm and egg to successive generations in many inbred strains of mice. For example, in the case of the high-incidence mammary tumor strain, GR, both males and females transmit MTV infection to their offspring with equal facility (1). Furthermore, nucleic acid hybridization studies reveal a widespread distribution of MTVrelated nucleotide sequences in normal adult organ DNA of both high- and low-incidence strains (2, 3). However, germ line DNA from sperm or eggs of mouse strains with high incidence of mammary tumors has not been tested directly. We have isolated germ line DNA for the purpose of analyzing MTVrelated nucleotide sequences in the DNA of sperm and somatic cells of inbred mice. Our method is a modification of the procedure reported by Meistrich et al. (4), and it yields germ line DNA with a higher efficiency of extraction and purity. Germ line DNA from individual male mice also was isolated. We report here the details of the method, and we discuss the general significance of the use of germ line DNA in a number of major biological investigations. MATERIALS AND METHODS Animals. Donor mice were adult GR/A and BALB/c males 6-12 months of age from the Cancer Research Laboratory inbred mouse colony. Animals were sacrificed by cervical dislocation. Vasa deferentia and epididymides were removed surgically and stored at -70°C. The C3H-1 mouse embryo fibroblast cell line was a gift from A. Decleve (Stanford Uni-
versity) (5). Reagents. All chemicals were reagent grade. Standard saline citrate (NaCl/Cit) was 150 mM sodium chloride/15 mM sodium citrate, pH 7.0. Phosphate-buffered saline was 140 mM NaCl/3 mM KC1/8 mM Na2HPO4/1 mM KH2PO4, pH 7.4. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
[3HJThymidine Labeling of Somatic Cells. The C3H-1 cell line was grown in 100-mm petri dishes containing 10 ml of Dulbecco's modified Eagle's medium, 10% dialyzed fetal calf serum, 25 mM glucose, penicillin at 100 units/ml, and streptomycin at 100,g/ml. Cells were labeled continuously with [3H]thymidine at 5 ,uCi/ml for 72 hr with one change of label-containing medium at 41 hr (1 Ci = 3.7 X 1010 becquerels). Approximately 107 labeled C3H-1 cells were used for determination of the specific activity of the [3H]thymidine-labeled DNA. These cells were subjected to a modification of the Schmidt-Thannhauser procedure (6) for determination of DNA content similar to the one described in a subsequent section. Triplicate 100lOl samples from the final 1.5-ml acid hydrolysate were spotted and dried on Whatman GF/A filters for measurement of tritium radioactivity. Duplicate 500-Al samples were assayed with diphenylamine for deoxyribose by the method of Burton, with calf thymus DNA as a standard (7). Counts per 10 min were averaged and corrected for background and volume, and the specific activity of the [3H]DNA was estimated to be 56,000 cpm/tig. Isolation of Sperm Nuclei. The vasa deferentia and epididymides from 30 adult male GR/A or BALB/c mice (approximately 3 g of tissue) were minced finely, suspended in 10 ml of deionized double-distilled water, and homogenized at 4°C by shearing with a Brinkmann Polytron homogenizer at a setting of 6 (step 1). The homogenate was incubated at 37°C for 10 min and again was homogenized. Approximately 5 X 106 [3H]thymidine-labeled C3H-1 cells in 5 ml of medium were added, and afterwards the suspension was diluted to 17.5 ml with distilled water. Sonication was performed with a Branson S-75 Sonifier at the highest output (step 2). Six 15-sec intervals of sonication each were followed by 1-min cooling periods at 40C. The homogenate was transferred to a graduated cylinder, and the volume was adjusted to 20 ml with distilled water. Duplicate 1-ml samples were taken for analysis of contaminating [3H]DNA. The remainder of the homogenate was transferred to a siliconized 30-ml Corex tube and was centrifuged at 10,000 rpm (12,000 X g) for 15 min at 4°C in an SS-34 rotor in a Sorvall RC-2 centrifuge. The supernatant was decanted into a second Corex tube, and centrifugation was repeated. Each pellet was resuspended by pounding with a glass rod followed by Vortex mixing for 1 min in 5 ml of 1% (vol/vol) Triton X-100/1 mg of trypsin per ml/1 mM MgCl2/1 mM CaC12. The suspensions were pooled into a single tube, and the volume was adjusted to 20 ml with the same buffer. The tube was incubated at 370C for 60 min (step 3). The trypsinized homogenate was centrifuged as before, and the pellet was resuspended in 20 ml of 500 ,ug of soybean trypsin inhibitor per ml/10 mM EDTA (tetrasodium salt). The tube was incubated at 37°C for 10 min and was centrifuged. The pellet was resuspended in 20 ml of Abbreviations: MTV, mammary tumor virus; NaCl/Cit, standard saline citrate, pH 7.0.
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Cell Biology: Shiurba and Nandi
phosphate-buffered saline/5 mM MgCl2, pH 7.4, and again was centrifuged. The resulting pellet was resuspended in 5 ml of phosphatebuffered saline/5 mM MgCl2, pH 7.4, and the volume was adjusted to 25 ml. DNase I was added to a final concentration of 150 ,gg/ml, and the tube was incubated at 370C for 90 min (step 4). DNase was inactivated by incubation at 750C for 5 min. The DNase I-treated pellet again was centrifuged. The DNase I-treated pellet was washed by sequential resuspension and centrifugation in 20 ml of each of the following solutions: 5% (vol/vol) Triton X-100/10 mM EDTA (step 5); 2 M NaCl/10 mM EDTA (step 6); 1% (wt/vol) sodium dodecyl sulfate/10 mM EDTA (step 7); 10 mM Tris-HCl/100 mM NaCl/10 mM EDTA, pH 8.0 (step 8). The final pellet again was resuspended and diluted to 20 ml with 10 mM Tris-HCI/100 mM NaCl/10 mM EDTA, pH 8.0. Duplicate 1-ml samples of the purified sperm nuclei were removed for analysis of somatic DNA contamination and electron microscopy. The remainder of the suspension was centrifuged, and the pellet was stored at -20°C for later DNA extraction. Determination of Somatic DNA Contamination. Samples of the original homogenates and final suspensions of purified sperm nuclei were prepared for tritium measurement by a modification of the procedure of Schmidt and Thannhauser (6). To each 1-ml sample from a total volume of 20 ml was added 1 ml of cold 1 M perchloric acid in a 3-ml Pyrex tube. The tubes were chilled for 30 min at 4°C. The resulting precipitates were centrifuged at 8000 rpm (7700 X g) for 10 min at 4°C in an SS-34 rotor. Precipitates were resuspended by Vortex mixing for 1 min and were washed three times in 2 ml of cold 0.5 perchloric acid by centrifuging in an identical manner. The final pellets were resuspended in 2 ml of 1 M NaOH and were incubated for 20 hr at room temperature followed by 4 hr at 370C. The alkali digests were neutralized with concentrated HCl, and 6 M perchloric acid was added to a final concentration of 0.5 M. The tubes again were chilled at 40C for 30 min. To each tube was added 300,ul of cold ether, and the tube was shaken vigorously for 1 min. The precipitates were pelleted and again were washed three times by centrifugation. Each pellet was resuspended in 1 ml of 0.5 M perchloric acid and was heated at 85°C for 30 min. The acid hydrolysates were cooled to room temperature and the tubes were centrifuged. Triplicate 100-pl samples were withdrawn from each hydrolysate and were spotted on Whatman GF/A filters. Filters were dried for 15 min at 100°C and were transferred to counting vials containing 5 ml of toluene/Omnifluor. Counts per 10 min were measured in a Packard model 3320 Tri-Carb scintillation spectrometer. Average input and output cpm were calculated and were corrected for background and volume. The total output/input cpm ratio was multiplied by 100 to estimate the percent of the original [3H]DNA contamination remaining in the final suspension of purified sperm nuclei. A summary of the results is shown in Table 1. Extraction of Sperm DNA. Sperm DNA was extracted by a modification of the p-aminosalicylate/phenol procedure of Kirby (8). Purified sperm nuclei from 30 mice were resuspended in 8 ml of 10 mM Tris-HCl/10 mM NaCl/10 mM EDTA, pH 8.0 (step 1). Dithiothreitol was added in 1 ml of the same buffer to a final concentration of 100 mM, and the mixture was incubated at 37°C for 30 min (step 2). Nuclease-free Pronase (self-digested 2 hr at 37°C) then was added in 1 ml of the same buffer to a final concentration of 500 utg/ml. Proteolytic digestion was performed by incubation for 3 hr at 370C
(step 3).
The digest was made 300 mM in sodium p-aminosalicylate,
Proc. Natl. Acad. Sci. USA 76 (1979) Table 1. Estimation of somatic DNA contamination of purified mouse sperm nuclei
Strain
GR/A
BALB/c
Total cpm Input Output 200 940,000 0 810,000 0 610,000 730,000 670,000
620,000
0 400 200
[3H]DNA
contamination, %
0.02 0.00 0.00 0.00 0.06 0.03
and it was shaken for 30 min at 125 rpm on a rotary shaker at room temperature (step 4). The solution was extracted similarly for 60 min with an equal volume of phenol, which was saturated with 10 mM Tris-HCI/10 mM NaCI/10 mM EDTA/300 mM sodium p-aminosalicylate at pH 8.0 (step 5). The phases were separated by centrifugation at room temperature, and the aqueous DNA phase was removed with a large-bore pipette. Sperm DNA was precipitated by adding 3 vol of cold 95% ethanol followed by incubating 18 hr at -20'C. Sperm DNA, which was collected by centrifugation at 15,000 X g for 15 min at 40C in an SS-34 rotor, was dissolved in NaCI/Cit. Alternatively, DNA was obtained by spooling from ethanol when sperm nuclei from 60 or more mice were prepared. The efficiency with which DNA may be extracted from mouse sperm nuclei also was estimated by a modification of the procedure of Schmidt and Thannhauser (6). Briefly, 1-ml samples from 20-ml suspensions of purified GR/A and BALB/c sperm nuclei were pretreated simultaneously in 100 mM dithiothreitol for 30 min at 370C. Nuclease-free Pronase was added to a final concentration of 500 ,g/ml, and the mixtures were digested for 3 hr at 370C. Each sample was made 0.5 M in perchloric acid and was incubated at 4°C for 15 min. The acid precipitable material was centrifuged at 10,000 rpm (12,000 X g) for 10 min at 4°C in an SS-34 rotor. The pellets were resuspended by Vortex mixing, and they were washed by centrifuging three times in 1 ml of cold 0.5 M perchloric acid. The washed pellets were resuspended in 1.1 ml of 0.5 M perchloric acid and were heated at 85°C for 30 min. The acid hydrolysates were cooled to room temperature and were centrifuged. The ultraviolet absorbance spectrum of a 1:10 dilution of each acid hydrolysate was measured in 5-nm increments between 220 and 290 nm with 0.5 M perchloric acid as a blank. The total DNA contents for GR/A and BALB/c sperm nuclei were calculated from the absorbances at 260 nm by assuming that mouse sperm DNA nucleotides have a molar extinction coefficient with respect to phosphorus of 8780 M-1 cm-1 (9). The corresponding amounts of pelleted DNA obtained by phenol extraction and ethanol precipitation as described were calculated from the absorbances at 260 nm in 5 ml of NaCl/Cit. After the data were corrected for volume, the efficiencies of extraction were estimated by dividing the yield of extracted DNA by the DNA content. Equilibrium Density Gradient Centrifugation. Preparative CsCl gradient (10 ml) and CsCl/ethidium bromide (250 Ag/ml) gradient centrifugation were performed simultaneously. Approximately 100 ug of native GR/A sperm DNA in NaCl/Cit was added to each solution. The solutions were centrifuged to equilibrium at 40,000 rpm (102,000 X g) for 65 hr at 17°C in a Beckman type 65 rotor. Six-drop fractions were collected. The refractive index of every other fraction was measured, and the corresponding density was determined from a table of values for CsCl (10). Density was plotted versus fraction number and
Proc. Natl. Acad. Sci. USA 76 (1979)
Cell Biology: Shiurba and Nandi line of best fit was calculated by the method of least squares. Each fraction then was diluted by adding 100,ul of NaCl/Cit, and its absorbance at 260 nm was determined. The guanine plus cytosine content was estimated by using the expression G+C = (p - 1.660)/0.098, in which p is the buoyant density and G+C equals the mole fraction of guanine plus cytosine (11). The CsCl/ethidium bromide gradient was examined and photographed in a darkroom with ultraviolet light from a mineral lamp. Melting Point Determination. Hydroxyapatite was prepared by the method of Miyazawa and Thomas (12). Determination of the melting temperature of native GR sperm DNA into single strands was performed on a 4-ml hydroxyapatite column according to the procedure of Drohan et al. (13). The temperature at which 50% of the DNA was eluted (tin) was estimated after plotting the percent DNA dissociated into single strands versus temperature. a
RESULTS The yield of high molecular weight mouse sperm DNA, obtained by spooling from ethanol as described, appeared to be strain dependent. We observed that GR/A mice yielded 1.6 mg/30 mice, whereas BALB/c mice gave 0.93 mg/30 mice, on the basis of measurements of the absorbances at 260 nm in NaCl/Cit. Because these results could not be explained on the basis of differences in age of the adult males used as donors, the efficiencies of DNA extraction were compared. The extraction procedure yielded DNA from GR/A and BALB/c sperm nuclei with efficiencies of 77% and 78%, respectively. Sperm counts of vasa deferential/epididymal homogenates also were measured with a hemocytometer. GR/A mice gave an average of 1.31 X 109 sperm per 30 mice whereas BALB/c mice gave an average of 9.26 X 108. Therefore, differences in the yields of sperm DNA from GR/A and BALB/c mice probably result from differences in the total number of sperm stored in the vasa deferentia and epididymides of these strains. High molecular weight mouse sperm DNA also was isolated from the vasa deferentia and epididymides of individual GR/A male mice. The microtechnique used was identical, except for volumes, to the sperm DNA isolation and extraction procedures described under.Materials and Methods. The yields of high molecular weight sperm DNA were in the range 50-100 jug per mouse. In Table 1 may be seen the results from six independent preparations of mouse sperm nuclei from 30 adult male donors. In each case, when [3H]thymidine-labeled C3H-1 cells were added to the original homogenate, less than 0.1% of the contaminating radioactive DNA remained in the final suspension of purified sperm nuclei. Furthermore, electron microscopy of the sperm nuclei showed no detectable contamination by whole cells, somatic cell nuclei, mitochondria, or sperm/midpiece tails. These findings are in agreement with those reported by Meistrich et al. (4), and they suggest that mouse straindependent differences do not exist with respect to the efficiency of the sperm nuclei purification procedure. In order to emphasize the differences between the sperm DNA extraction procedure described .here and that reported by Meistrich et al. (4), a parallel comparison using identical volumes was conducted. Sperm nuclei from 15 BALB/c males, which were purified as described under Materials and Methods, were used for extraction by each procedure. Yields were calculated from absorbances at 260 nm in NaCl/Cit. Our method gave 4545 jug of DNA per g wet weight, whereas only 20 Mg of DNA per g wet weight was obtained by the method of Meistrich et al. (4). This difference results from the fact that the procedure of Meistrich et al. (4) omits a critical step that requires that the phenol used for extraction be saturated with
3949
buffer and that the pH be made alkaline. When buffer-saturated phenol was used, the yields that we observed with the procedure of Meistrich et al. (4) were 795 Aug of DNA per g wet weight at pH 8.0 and 2976 Aig of DNA per g wet weight at pH 9.0. These DNAs exhibited a 260/280 absorbance ratio of approximately 1.79, whereas DNA obtained by our procedure showed a 260/280 ratio of 1.85. The ultraviolet absorbance spectrum of a solution of native GR/A sperm DNA in NaCl/Cit also was measured (Fig. 1). In addition to showing a maximum absorbance at 260 nmn and a minimum absorbance at 230 nm, which are characteristics of nucleic acids, the curve exhibited a 260/280 ratio of 1.87 and a 260/230 ratio of 2.40. These values indicated that the sperm DNA was sufficiently pure to permit further analysis by nucleic acid hybridization, analytical ultracentrifugation, or genetic mapping with restriction enzymes. Equilibrium density gradient centrifugation of native GR/A sperm DNA in a preparative CsCl gradient (Fig. 2) showed a
buoyant density of approximately 1.700 g/ml, which is in agreement with the value of 1.699 g/ml for mouse DNA obtained by analytical ultracentrifugation (14). The presence of satellite DNA was not revealed under these conditions, because the DNA was not sheared. By using the relationship of Schildkraut et al. (11), which expresses the dependence of DNA density on G+C content, a value of 41% G+C was calculated. When native GR/A sperm DNA was melted on a hydroxyapatite column, a tm of 870C was observed (Fig. 3). Possible contamination of sperm DNA by covalently closed, circular mitochondrial DNA was assessed by CsCl/ethidium bromide gradient centrifugation (15). Only a single band of intense ultraviolet fluorescence was observed in the middle of the gradient after 100 Ag of native GR/A sperm DNA was centrifuged to equilibrium. This result suggests that contamination by mitochondrial DNA was negligible.
0.1 -
0.4
a)
.0 o0
n0.
0.2
0. 0l
220
240
260
Wavelength,
280
300
nm
FIG. 1. Ultraviolet absorbance spectrum of native GR/A sperm DNA in NaCl/Cit.
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Cell Biology: Shiurba and Nandi
Proc. Natt. Acad. Sci. USA 76 (1979)
1aio
o0r
a
E
.)
C
C5 co
0 .0
CU 0) u
C
.0
.0
0
.0 4:
2 20~
I6
60o
10
20
30
40
50
Fraction
FIG. 2. Preparative CsCl equilibrium density gradient centrifugation of native high molecular weight GR/A sperm-DNA. The arrow indicates the position of density 1.700 g/ml as determined by measurement of refractive index.
~
~
701 70
80 80 Temperature, 0C
9010 90 1 00
FIG. 3. Melting profile of native GR/A sperm DNA in 120 mM sodium phosphate buffer, pH 6.8, on a hydroxyapatite column. tm = 870C.
is isolated may not be representative of the germ line, leaving
the results of analysis obtained from such material
open
to
criticism.
DISCUSSION The present paper reports a method for isolation of germ line DNA from mouse sperm with high purity. The procedure takes advantage of the relatively greater resistance of mature sperm nuclei to physicochemical degradation as compared to somatic cell nuclei. Mature sperm nuclei were isolated from homogenates of the vasa deferentia and epididymides of adult male GR/A and BALB/c mice by a modification of the procedure of Meistrich et al. (4). Somatic cell DNA contamination was measured by addition of [3H]thymidine-labeled somatic cells from the C3H-1 cell line at the beginning of the procedure to a control preparation. Counts per minute in [3H]DNA of the original homogenate then were compared to those cpm observed in the final -suspension of purified sperm nuclei. In contrast to mammalian somatic cells, it is well known that mammalian sperm are highly refractory to conventional procedures for extraction of DNA (16). However, high molecular weight mouse sperm DNA was phenol extracted with about 80% efficiency when dithiothreitol was employed to reduce disulfide crosslinkages between the proteins of condensed sperm chromatin (17), and sodium p-aminosalicylate was used to chelate transition metals, which may facilitate protein binding to DNA (8). Previously, several authors have reported the isolation of mouse sperm DNA for purposes of molecular hybridization (18-20) and for the study of chemical mutagenesis (21). These studies provide no assessment of contamination by somatic cell DNA. In our experience procedures that omit the use of sodium p-aminosalicylate have low efficiencies of extraction of sperm DNA, a property that may result in selective extraction of DNA sequences. Therefore, the sperm DNA that
The principle conclusions of this investigation may be summarized as follows: (i) High molecular weight germ line DNA was isolated from mouse sperm nuclei with less than 0.1% contamination by somatic cell DNA and without significant contamination by mitochondrial DNA. (ii) The efficiency with which sperm DNA was extracted approached 80% when dithiothreitol and sodium p-aminosalicylate were used in combination with buffer-saturated phenol at pH 8.0. (iii) Yields of sperm DNA were in the range 1-3 mg per 30 adult male mice, depending upon the mouse strain employed. (iv) Native sperm DNA exhibited characteristics of ultraviolet absorbance, buoyant density, and melting temperature which are in agreement with known values for mouse DNA (14, 22). (v) The method had a 227-fold higher DNA yield than that reported by Meistrich et al. (4). DNA purity also was increased. (vi) Germ line DNA was isolated from individual GR/A male mice by a microtechnique which was identical, except for volumes, to the procedure described under Materials and Methods. The yields were in the range of 50-100 /ig per mouse. By starting with this purification technique, it should be possible to distinguish between genetic transmission of MTV and congenital MTV infection of developing embryos in utero. If MTV is integrated in the mouse germ line, virus-related nucleotide sequences should be demonstrable in the DNA of sperm and eggs of high-incidence strains such as GR/A in which both males and females transmit MTV infection to offspring with equal facility. The high molecular weight mouse sperm DNA from high and low mammary tumor strains can be assayed for MTV-related nucleotide sequences by DNA-RNA hybridization with '25I-labeled GR MTV RNA. The genetic transmission of mouse leukemia virus may be investigated in
Cell Biology: Shiurba and Nandi similar manner. The possibility that the complete viral geis transmitted via the germ line may be tested by transfection of uninfected mammalian somatic cells with sperm DNA from' high-incidence males. An analogous approach also may be taken with respect to -the problem of the origin of antibody diversity. Theories that may account for the observed variable and constant regions within the light chains and heavy chains of antibody molecules can be classified as germ line or somatic mutational in emphasis (23). The germ line hypothesis predicts the existence of many genes that code for variable regions, whereas explanations that involve somatic mutation predict few such genes. Direct measurement of the number and organization of variable region genes in germ line DNA is required to resolve these opposing views. 'Not only is it possible to analyze mouse germ line DNA for MTV- or antibody gene-related nucleotide sequences by DNA-RNA hybridization, but also the relative location and organization of such sequences within the genome may be investigated by genetic mapping with restriction enzymes. The sites of specific cleavage of DNA by restriction enzymes may be used as fixed points of reference for locating particular gene sequences (24). Analysis of germ line DNA with restriction enzymes may provide evidence for the possible origin of MTV proviral DNA from retrovirus-infected germ cells of a vertebrate ancestor of the mouse (25) or from evolution of normal cellular gene sequences (26). Germ line DNA from sperm also may provide a base line for detecting possible translocation, or other rearrangement, of genes during normal development of antibody-producing cells (27) or during neoplastic transfora nome
mation.
In inbred strains of mice, for which linkage group maps have been constructed, nucleic acid hybridization and restriction enzyme analyses of germ line DNA may be correlated directly with genetic studies in vivo. The fact that high molecular weight sperm DNA may be isolated from individual male mice makes possible the study of Mendelian segregation and recombination of particular germ line sequences, such as those that are related to MTV, in male progeny of crosses between high-and low-incidence tumor strains. However, in contrast to classical Mendelian analysis in which genotype is inferred from phenotype, the genotype of the germ line DNA can be identified directly by nucleic acid hybridization and by mapping with restriction enzymes.
Germ line genes, such as those that specify immunoglobulins and protein hormones, may be cloned, and the regulation of their expression may be compared to that of corresponding genes from differentiated somatic cells. Finally, because germ line DNA represents the zero-time of the organism with respect to any permanent or reversible biochemical changes that may occur in the organization of the genome during differentiation and aging of somatic cells, the regulation of genetic expression in inbred mice may be analyzed from a unique frame of reference. Of particular interest in this regard may be the mouse testicular teratoma (28), whose origin from primordial germ cells or from somatic cells has been the subject of much discussion. DNAs from cloned teratoma cell lines, which may exhibit distinct characteristics of differentiation and loss of
Proc. Natl. Acad. Sci. USA 76 (1979)
3951
tumorigenicity, may be compared to the germ line DNA from which they were derived. These studies may provide evidence for the view that some malignancies do not result from irreversible genetic changes. We gratefully acknowledge Dr. K. DeOme for his interest and support. We thank S. Hamamoto for electron microscopy. We acknowledge P. Thompson for technical illustration, J. Underhill for photography, B. Hayes and J. Kop for technical assistance, M. Webb for providing mice, and S. Castillo for typing the manuscript. This investigation was supported by Fellowship 5 F22 CA03162, awarded to R.S. by the National Cancer Institute, U.S. Department of Health, Education, and Welfare, and by Grant CA05388, awarded by the National Cancer Institute. 1. Muhlbock, 0. (1965) Eur. J. Cancer 1, 123-124. 2. Morris, V. L., Medeiros, E., Ringold, G. M., Bishop, J. M. & Varmus, H. E. (1977) J. Mol. Biol. 114,73-91. 3. Schlom, J., Colcher, D., Drohan, W. & Kettmann, R. (1978) Prog. Exp. Tumor Res. 21, 140-158. 4. Meistrich, M. L., Reid, B. 0. & Barcellona, W. J. (1975) J. Cell Biol. 64,211-222. 5. Decleve, A., Niwa, O., Hilgers, J. & Kaplan, H. S. (1974) Virology 57,491-502. 6. Schmidt, G. & Thannhauser, S. J. (1945) J. Biol. Chem. 161, 83-89. 7. Burton, K. (1968) Methods Enzymol. eds. Grossman, L. & Moldave, K. (Academic, New York), Vol. 12, pp. 163-166. 8. Kirby, K. S. (1957) Biochem. J. 66, 495-504. 9. Ogur, M. & Rosen, G. (1950) Arch. Biochem. 25,262-276. 10. Handbook of Biochemistry (1968) ed. Sober, H. A. (CRC, West Palm Beach, FL), pp. J252-256. 11. Schildkraut, C. L., Marmur, J. & Doty, P. (1962) J. Mol. Biol. 4, 430-443. 12. Miyazawa, Y. & Thomas, C. A., Jr. (1965) J. Mol. Biol. 11, 223-237. 13. Drohan, W., Kettmann, R., Colcher, D. & Schlom, J. (1977) J. Virol. 21, 986-994. 14. Cech, T. R., Rosenfeld, A. & Hearst, J. E. (1973) J. Mol. Biol. 81, 299-325. 15. Radloff, R., Bauer, W. & Vinograd, J. (1967) Proc. Natl. Acad. Sci. USA 57, 1514-1521. 16. Chargaff, E. (1955) The Nucleic Acids, eds. Chargaff, E. & Davidson, J. N. (Academic, New York), Vol. 1, pp. 307-371. 17. Borenfreund, E., Fitt, E. & Bendich, A. (1961) Nature (London) 191, 1375-1377. 18. Morrison, M. R., Paul, J. & Williamson, R. (1972) Eur. J. Biochem. 27, 1-9. 19. Harrison, P. R., Birnie, G. D., Hall, A., Humphries, S., Young, B. D. & Paul, J. (1974) J. Mol. Biol. 84,539-554. 20. Lueders, K. K. & Kuff, E. L. (1977) Cell 12, 963-972. 21. Sega, G. A., Cumming, R. B. & Walton, M. F. (1974) Mutation Res. 24, 317-333. 22. Marmur, J. & Doty, P. (1962) J. Mol. Biol. 5, 109-118. 23. Williamson, A. R. (1976) Annu. Rev. Biochem. 45, 467-500. 24. Danna, K. & Nathans, D. (1971) Proc. Natl. Acad. Sci. USA 68, 2913-2917. 25. Todaro, G. J. & Huebner, R. J. (1972) Proc. Natl. Acad. Sci. USA 69, 1009-1015. 26. Temin, H. M. (1971) J. Natl. Cancer Inst. 46, iii-vii. 27. Gally, J. A. & Edelman, G. M. (1970) Nature (London) 227, 341-348. 28. Stevens, L. C. (1967) Adv. Morphol. 6, 1-31.
Cell Biology
Isolation and characterization of germ line DNA from mouse sperm (genetic transmission/mammary tumor virus/leukemia virus/antibody diversity/testicular teratoma)
ROBERT SHIURBA AND S. NANDI Department of Zoology and Cancer Research Laboratory, University of California, Berkeley, California 94720
Communicated by Howard A. Bern, May 21, 1979
ABSTRACT Mouse germ line DNA was isolated from sperm by a physicochemical procedure that preferentially destroys contaminating somatic cell DNA. The use of reducing conditions and chelating agents in combination with phenol permitted extraction of high molecular weight DNA from mature sperm nuclei with approximately 80% efficiency. Less than 0.1% somatic cell DNA contamination remained in sperm DNA prepared by this method. Germ line DNA was characterized by determination of its ultraviolet absorbance spectrum, buoyant density in cesium chloride, and melting profile on a hydroxyapatite column. Contamination by mitochondrial DNA was assessed by cesium chloride/ethidium bromide gradient centrifugation. The significance of the mouse germ line DNA isolation procedure is discussed with respect to the possible genetic transmission of mammary tumor virus and leukemia virus, the origin of antibody diversity, and the origin of testicular teratomas. It is currently believed that mouse mammary tumor virus (MTV) is transmitted genetically via sperm and egg to successive generations in many inbred strains of mice. For example, in the case of the high-incidence mammary tumor strain, GR, both males and females transmit MTV infection to their offspring with equal facility (1). Furthermore, nucleic acid hybridization studies reveal a widespread distribution of MTVrelated nucleotide sequences in normal adult organ DNA of both high- and low-incidence strains (2, 3). However, germ line DNA from sperm or eggs of mouse strains with high incidence of mammary tumors has not been tested directly. We have isolated germ line DNA for the purpose of analyzing MTVrelated nucleotide sequences in the DNA of sperm and somatic cells of inbred mice. Our method is a modification of the procedure reported by Meistrich et al. (4), and it yields germ line DNA with a higher efficiency of extraction and purity. Germ line DNA from individual male mice also was isolated. We report here the details of the method, and we discuss the general significance of the use of germ line DNA in a number of major biological investigations. MATERIALS AND METHODS Animals. Donor mice were adult GR/A and BALB/c males 6-12 months of age from the Cancer Research Laboratory inbred mouse colony. Animals were sacrificed by cervical dislocation. Vasa deferentia and epididymides were removed surgically and stored at -70°C. The C3H-1 mouse embryo fibroblast cell line was a gift from A. Decleve (Stanford Uni-
versity) (5). Reagents. All chemicals were reagent grade. Standard saline citrate (NaCl/Cit) was 150 mM sodium chloride/15 mM sodium citrate, pH 7.0. Phosphate-buffered saline was 140 mM NaCl/3 mM KC1/8 mM Na2HPO4/1 mM KH2PO4, pH 7.4. The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
[3HJThymidine Labeling of Somatic Cells. The C3H-1 cell line was grown in 100-mm petri dishes containing 10 ml of Dulbecco's modified Eagle's medium, 10% dialyzed fetal calf serum, 25 mM glucose, penicillin at 100 units/ml, and streptomycin at 100,g/ml. Cells were labeled continuously with [3H]thymidine at 5 ,uCi/ml for 72 hr with one change of label-containing medium at 41 hr (1 Ci = 3.7 X 1010 becquerels). Approximately 107 labeled C3H-1 cells were used for determination of the specific activity of the [3H]thymidine-labeled DNA. These cells were subjected to a modification of the Schmidt-Thannhauser procedure (6) for determination of DNA content similar to the one described in a subsequent section. Triplicate 100lOl samples from the final 1.5-ml acid hydrolysate were spotted and dried on Whatman GF/A filters for measurement of tritium radioactivity. Duplicate 500-Al samples were assayed with diphenylamine for deoxyribose by the method of Burton, with calf thymus DNA as a standard (7). Counts per 10 min were averaged and corrected for background and volume, and the specific activity of the [3H]DNA was estimated to be 56,000 cpm/tig. Isolation of Sperm Nuclei. The vasa deferentia and epididymides from 30 adult male GR/A or BALB/c mice (approximately 3 g of tissue) were minced finely, suspended in 10 ml of deionized double-distilled water, and homogenized at 4°C by shearing with a Brinkmann Polytron homogenizer at a setting of 6 (step 1). The homogenate was incubated at 37°C for 10 min and again was homogenized. Approximately 5 X 106 [3H]thymidine-labeled C3H-1 cells in 5 ml of medium were added, and afterwards the suspension was diluted to 17.5 ml with distilled water. Sonication was performed with a Branson S-75 Sonifier at the highest output (step 2). Six 15-sec intervals of sonication each were followed by 1-min cooling periods at 40C. The homogenate was transferred to a graduated cylinder, and the volume was adjusted to 20 ml with distilled water. Duplicate 1-ml samples were taken for analysis of contaminating [3H]DNA. The remainder of the homogenate was transferred to a siliconized 30-ml Corex tube and was centrifuged at 10,000 rpm (12,000 X g) for 15 min at 4°C in an SS-34 rotor in a Sorvall RC-2 centrifuge. The supernatant was decanted into a second Corex tube, and centrifugation was repeated. Each pellet was resuspended by pounding with a glass rod followed by Vortex mixing for 1 min in 5 ml of 1% (vol/vol) Triton X-100/1 mg of trypsin per ml/1 mM MgCl2/1 mM CaC12. The suspensions were pooled into a single tube, and the volume was adjusted to 20 ml with the same buffer. The tube was incubated at 370C for 60 min (step 3). The trypsinized homogenate was centrifuged as before, and the pellet was resuspended in 20 ml of 500 ,ug of soybean trypsin inhibitor per ml/10 mM EDTA (tetrasodium salt). The tube was incubated at 37°C for 10 min and was centrifuged. The pellet was resuspended in 20 ml of Abbreviations: MTV, mammary tumor virus; NaCl/Cit, standard saline citrate, pH 7.0.
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phosphate-buffered saline/5 mM MgCl2, pH 7.4, and again was centrifuged. The resulting pellet was resuspended in 5 ml of phosphatebuffered saline/5 mM MgCl2, pH 7.4, and the volume was adjusted to 25 ml. DNase I was added to a final concentration of 150 ,gg/ml, and the tube was incubated at 370C for 90 min (step 4). DNase was inactivated by incubation at 750C for 5 min. The DNase I-treated pellet again was centrifuged. The DNase I-treated pellet was washed by sequential resuspension and centrifugation in 20 ml of each of the following solutions: 5% (vol/vol) Triton X-100/10 mM EDTA (step 5); 2 M NaCl/10 mM EDTA (step 6); 1% (wt/vol) sodium dodecyl sulfate/10 mM EDTA (step 7); 10 mM Tris-HCl/100 mM NaCl/10 mM EDTA, pH 8.0 (step 8). The final pellet again was resuspended and diluted to 20 ml with 10 mM Tris-HCI/100 mM NaCl/10 mM EDTA, pH 8.0. Duplicate 1-ml samples of the purified sperm nuclei were removed for analysis of somatic DNA contamination and electron microscopy. The remainder of the suspension was centrifuged, and the pellet was stored at -20°C for later DNA extraction. Determination of Somatic DNA Contamination. Samples of the original homogenates and final suspensions of purified sperm nuclei were prepared for tritium measurement by a modification of the procedure of Schmidt and Thannhauser (6). To each 1-ml sample from a total volume of 20 ml was added 1 ml of cold 1 M perchloric acid in a 3-ml Pyrex tube. The tubes were chilled for 30 min at 4°C. The resulting precipitates were centrifuged at 8000 rpm (7700 X g) for 10 min at 4°C in an SS-34 rotor. Precipitates were resuspended by Vortex mixing for 1 min and were washed three times in 2 ml of cold 0.5 perchloric acid by centrifuging in an identical manner. The final pellets were resuspended in 2 ml of 1 M NaOH and were incubated for 20 hr at room temperature followed by 4 hr at 370C. The alkali digests were neutralized with concentrated HCl, and 6 M perchloric acid was added to a final concentration of 0.5 M. The tubes again were chilled at 40C for 30 min. To each tube was added 300,ul of cold ether, and the tube was shaken vigorously for 1 min. The precipitates were pelleted and again were washed three times by centrifugation. Each pellet was resuspended in 1 ml of 0.5 M perchloric acid and was heated at 85°C for 30 min. The acid hydrolysates were cooled to room temperature and the tubes were centrifuged. Triplicate 100-pl samples were withdrawn from each hydrolysate and were spotted on Whatman GF/A filters. Filters were dried for 15 min at 100°C and were transferred to counting vials containing 5 ml of toluene/Omnifluor. Counts per 10 min were measured in a Packard model 3320 Tri-Carb scintillation spectrometer. Average input and output cpm were calculated and were corrected for background and volume. The total output/input cpm ratio was multiplied by 100 to estimate the percent of the original [3H]DNA contamination remaining in the final suspension of purified sperm nuclei. A summary of the results is shown in Table 1. Extraction of Sperm DNA. Sperm DNA was extracted by a modification of the p-aminosalicylate/phenol procedure of Kirby (8). Purified sperm nuclei from 30 mice were resuspended in 8 ml of 10 mM Tris-HCl/10 mM NaCl/10 mM EDTA, pH 8.0 (step 1). Dithiothreitol was added in 1 ml of the same buffer to a final concentration of 100 mM, and the mixture was incubated at 37°C for 30 min (step 2). Nuclease-free Pronase (self-digested 2 hr at 37°C) then was added in 1 ml of the same buffer to a final concentration of 500 utg/ml. Proteolytic digestion was performed by incubation for 3 hr at 370C
(step 3).
The digest was made 300 mM in sodium p-aminosalicylate,
Proc. Natl. Acad. Sci. USA 76 (1979) Table 1. Estimation of somatic DNA contamination of purified mouse sperm nuclei
Strain
GR/A
BALB/c
Total cpm Input Output 200 940,000 0 810,000 0 610,000 730,000 670,000
620,000
0 400 200
[3H]DNA
contamination, %
0.02 0.00 0.00 0.00 0.06 0.03
and it was shaken for 30 min at 125 rpm on a rotary shaker at room temperature (step 4). The solution was extracted similarly for 60 min with an equal volume of phenol, which was saturated with 10 mM Tris-HCI/10 mM NaCI/10 mM EDTA/300 mM sodium p-aminosalicylate at pH 8.0 (step 5). The phases were separated by centrifugation at room temperature, and the aqueous DNA phase was removed with a large-bore pipette. Sperm DNA was precipitated by adding 3 vol of cold 95% ethanol followed by incubating 18 hr at -20'C. Sperm DNA, which was collected by centrifugation at 15,000 X g for 15 min at 40C in an SS-34 rotor, was dissolved in NaCI/Cit. Alternatively, DNA was obtained by spooling from ethanol when sperm nuclei from 60 or more mice were prepared. The efficiency with which DNA may be extracted from mouse sperm nuclei also was estimated by a modification of the procedure of Schmidt and Thannhauser (6). Briefly, 1-ml samples from 20-ml suspensions of purified GR/A and BALB/c sperm nuclei were pretreated simultaneously in 100 mM dithiothreitol for 30 min at 370C. Nuclease-free Pronase was added to a final concentration of 500 ,g/ml, and the mixtures were digested for 3 hr at 370C. Each sample was made 0.5 M in perchloric acid and was incubated at 4°C for 15 min. The acid precipitable material was centrifuged at 10,000 rpm (12,000 X g) for 10 min at 4°C in an SS-34 rotor. The pellets were resuspended by Vortex mixing, and they were washed by centrifuging three times in 1 ml of cold 0.5 M perchloric acid. The washed pellets were resuspended in 1.1 ml of 0.5 M perchloric acid and were heated at 85°C for 30 min. The acid hydrolysates were cooled to room temperature and were centrifuged. The ultraviolet absorbance spectrum of a 1:10 dilution of each acid hydrolysate was measured in 5-nm increments between 220 and 290 nm with 0.5 M perchloric acid as a blank. The total DNA contents for GR/A and BALB/c sperm nuclei were calculated from the absorbances at 260 nm by assuming that mouse sperm DNA nucleotides have a molar extinction coefficient with respect to phosphorus of 8780 M-1 cm-1 (9). The corresponding amounts of pelleted DNA obtained by phenol extraction and ethanol precipitation as described were calculated from the absorbances at 260 nm in 5 ml of NaCl/Cit. After the data were corrected for volume, the efficiencies of extraction were estimated by dividing the yield of extracted DNA by the DNA content. Equilibrium Density Gradient Centrifugation. Preparative CsCl gradient (10 ml) and CsCl/ethidium bromide (250 Ag/ml) gradient centrifugation were performed simultaneously. Approximately 100 ug of native GR/A sperm DNA in NaCl/Cit was added to each solution. The solutions were centrifuged to equilibrium at 40,000 rpm (102,000 X g) for 65 hr at 17°C in a Beckman type 65 rotor. Six-drop fractions were collected. The refractive index of every other fraction was measured, and the corresponding density was determined from a table of values for CsCl (10). Density was plotted versus fraction number and
Proc. Natl. Acad. Sci. USA 76 (1979)
Cell Biology: Shiurba and Nandi line of best fit was calculated by the method of least squares. Each fraction then was diluted by adding 100,ul of NaCl/Cit, and its absorbance at 260 nm was determined. The guanine plus cytosine content was estimated by using the expression G+C = (p - 1.660)/0.098, in which p is the buoyant density and G+C equals the mole fraction of guanine plus cytosine (11). The CsCl/ethidium bromide gradient was examined and photographed in a darkroom with ultraviolet light from a mineral lamp. Melting Point Determination. Hydroxyapatite was prepared by the method of Miyazawa and Thomas (12). Determination of the melting temperature of native GR sperm DNA into single strands was performed on a 4-ml hydroxyapatite column according to the procedure of Drohan et al. (13). The temperature at which 50% of the DNA was eluted (tin) was estimated after plotting the percent DNA dissociated into single strands versus temperature. a
RESULTS The yield of high molecular weight mouse sperm DNA, obtained by spooling from ethanol as described, appeared to be strain dependent. We observed that GR/A mice yielded 1.6 mg/30 mice, whereas BALB/c mice gave 0.93 mg/30 mice, on the basis of measurements of the absorbances at 260 nm in NaCl/Cit. Because these results could not be explained on the basis of differences in age of the adult males used as donors, the efficiencies of DNA extraction were compared. The extraction procedure yielded DNA from GR/A and BALB/c sperm nuclei with efficiencies of 77% and 78%, respectively. Sperm counts of vasa deferential/epididymal homogenates also were measured with a hemocytometer. GR/A mice gave an average of 1.31 X 109 sperm per 30 mice whereas BALB/c mice gave an average of 9.26 X 108. Therefore, differences in the yields of sperm DNA from GR/A and BALB/c mice probably result from differences in the total number of sperm stored in the vasa deferentia and epididymides of these strains. High molecular weight mouse sperm DNA also was isolated from the vasa deferentia and epididymides of individual GR/A male mice. The microtechnique used was identical, except for volumes, to the sperm DNA isolation and extraction procedures described under.Materials and Methods. The yields of high molecular weight sperm DNA were in the range 50-100 jug per mouse. In Table 1 may be seen the results from six independent preparations of mouse sperm nuclei from 30 adult male donors. In each case, when [3H]thymidine-labeled C3H-1 cells were added to the original homogenate, less than 0.1% of the contaminating radioactive DNA remained in the final suspension of purified sperm nuclei. Furthermore, electron microscopy of the sperm nuclei showed no detectable contamination by whole cells, somatic cell nuclei, mitochondria, or sperm/midpiece tails. These findings are in agreement with those reported by Meistrich et al. (4), and they suggest that mouse straindependent differences do not exist with respect to the efficiency of the sperm nuclei purification procedure. In order to emphasize the differences between the sperm DNA extraction procedure described .here and that reported by Meistrich et al. (4), a parallel comparison using identical volumes was conducted. Sperm nuclei from 15 BALB/c males, which were purified as described under Materials and Methods, were used for extraction by each procedure. Yields were calculated from absorbances at 260 nm in NaCl/Cit. Our method gave 4545 jug of DNA per g wet weight, whereas only 20 Mg of DNA per g wet weight was obtained by the method of Meistrich et al. (4). This difference results from the fact that the procedure of Meistrich et al. (4) omits a critical step that requires that the phenol used for extraction be saturated with
3949
buffer and that the pH be made alkaline. When buffer-saturated phenol was used, the yields that we observed with the procedure of Meistrich et al. (4) were 795 Aug of DNA per g wet weight at pH 8.0 and 2976 Aig of DNA per g wet weight at pH 9.0. These DNAs exhibited a 260/280 absorbance ratio of approximately 1.79, whereas DNA obtained by our procedure showed a 260/280 ratio of 1.85. The ultraviolet absorbance spectrum of a solution of native GR/A sperm DNA in NaCl/Cit also was measured (Fig. 1). In addition to showing a maximum absorbance at 260 nmn and a minimum absorbance at 230 nm, which are characteristics of nucleic acids, the curve exhibited a 260/280 ratio of 1.87 and a 260/230 ratio of 2.40. These values indicated that the sperm DNA was sufficiently pure to permit further analysis by nucleic acid hybridization, analytical ultracentrifugation, or genetic mapping with restriction enzymes. Equilibrium density gradient centrifugation of native GR/A sperm DNA in a preparative CsCl gradient (Fig. 2) showed a
buoyant density of approximately 1.700 g/ml, which is in agreement with the value of 1.699 g/ml for mouse DNA obtained by analytical ultracentrifugation (14). The presence of satellite DNA was not revealed under these conditions, because the DNA was not sheared. By using the relationship of Schildkraut et al. (11), which expresses the dependence of DNA density on G+C content, a value of 41% G+C was calculated. When native GR/A sperm DNA was melted on a hydroxyapatite column, a tm of 870C was observed (Fig. 3). Possible contamination of sperm DNA by covalently closed, circular mitochondrial DNA was assessed by CsCl/ethidium bromide gradient centrifugation (15). Only a single band of intense ultraviolet fluorescence was observed in the middle of the gradient after 100 Ag of native GR/A sperm DNA was centrifuged to equilibrium. This result suggests that contamination by mitochondrial DNA was negligible.
0.1 -
0.4
a)
.0 o0
n0.
0.2
0. 0l
220
240
260
Wavelength,
280
300
nm
FIG. 1. Ultraviolet absorbance spectrum of native GR/A sperm DNA in NaCl/Cit.
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Cell Biology: Shiurba and Nandi
Proc. Natt. Acad. Sci. USA 76 (1979)
1aio
o0r
a
E
.)
C
C5 co
0 .0
CU 0) u
C
.0
.0
0
.0 4:
2 20~
I6
60o
10
20
30
40
50
Fraction
FIG. 2. Preparative CsCl equilibrium density gradient centrifugation of native high molecular weight GR/A sperm-DNA. The arrow indicates the position of density 1.700 g/ml as determined by measurement of refractive index.
~
~
701 70
80 80 Temperature, 0C
9010 90 1 00
FIG. 3. Melting profile of native GR/A sperm DNA in 120 mM sodium phosphate buffer, pH 6.8, on a hydroxyapatite column. tm = 870C.
is isolated may not be representative of the germ line, leaving
the results of analysis obtained from such material
open
to
criticism.
DISCUSSION The present paper reports a method for isolation of germ line DNA from mouse sperm with high purity. The procedure takes advantage of the relatively greater resistance of mature sperm nuclei to physicochemical degradation as compared to somatic cell nuclei. Mature sperm nuclei were isolated from homogenates of the vasa deferentia and epididymides of adult male GR/A and BALB/c mice by a modification of the procedure of Meistrich et al. (4). Somatic cell DNA contamination was measured by addition of [3H]thymidine-labeled somatic cells from the C3H-1 cell line at the beginning of the procedure to a control preparation. Counts per minute in [3H]DNA of the original homogenate then were compared to those cpm observed in the final -suspension of purified sperm nuclei. In contrast to mammalian somatic cells, it is well known that mammalian sperm are highly refractory to conventional procedures for extraction of DNA (16). However, high molecular weight mouse sperm DNA was phenol extracted with about 80% efficiency when dithiothreitol was employed to reduce disulfide crosslinkages between the proteins of condensed sperm chromatin (17), and sodium p-aminosalicylate was used to chelate transition metals, which may facilitate protein binding to DNA (8). Previously, several authors have reported the isolation of mouse sperm DNA for purposes of molecular hybridization (18-20) and for the study of chemical mutagenesis (21). These studies provide no assessment of contamination by somatic cell DNA. In our experience procedures that omit the use of sodium p-aminosalicylate have low efficiencies of extraction of sperm DNA, a property that may result in selective extraction of DNA sequences. Therefore, the sperm DNA that
The principle conclusions of this investigation may be summarized as follows: (i) High molecular weight germ line DNA was isolated from mouse sperm nuclei with less than 0.1% contamination by somatic cell DNA and without significant contamination by mitochondrial DNA. (ii) The efficiency with which sperm DNA was extracted approached 80% when dithiothreitol and sodium p-aminosalicylate were used in combination with buffer-saturated phenol at pH 8.0. (iii) Yields of sperm DNA were in the range 1-3 mg per 30 adult male mice, depending upon the mouse strain employed. (iv) Native sperm DNA exhibited characteristics of ultraviolet absorbance, buoyant density, and melting temperature which are in agreement with known values for mouse DNA (14, 22). (v) The method had a 227-fold higher DNA yield than that reported by Meistrich et al. (4). DNA purity also was increased. (vi) Germ line DNA was isolated from individual GR/A male mice by a microtechnique which was identical, except for volumes, to the procedure described under Materials and Methods. The yields were in the range of 50-100 /ig per mouse. By starting with this purification technique, it should be possible to distinguish between genetic transmission of MTV and congenital MTV infection of developing embryos in utero. If MTV is integrated in the mouse germ line, virus-related nucleotide sequences should be demonstrable in the DNA of sperm and eggs of high-incidence strains such as GR/A in which both males and females transmit MTV infection to offspring with equal facility. The high molecular weight mouse sperm DNA from high and low mammary tumor strains can be assayed for MTV-related nucleotide sequences by DNA-RNA hybridization with '25I-labeled GR MTV RNA. The genetic transmission of mouse leukemia virus may be investigated in
Cell Biology: Shiurba and Nandi similar manner. The possibility that the complete viral geis transmitted via the germ line may be tested by transfection of uninfected mammalian somatic cells with sperm DNA from' high-incidence males. An analogous approach also may be taken with respect to -the problem of the origin of antibody diversity. Theories that may account for the observed variable and constant regions within the light chains and heavy chains of antibody molecules can be classified as germ line or somatic mutational in emphasis (23). The germ line hypothesis predicts the existence of many genes that code for variable regions, whereas explanations that involve somatic mutation predict few such genes. Direct measurement of the number and organization of variable region genes in germ line DNA is required to resolve these opposing views. 'Not only is it possible to analyze mouse germ line DNA for MTV- or antibody gene-related nucleotide sequences by DNA-RNA hybridization, but also the relative location and organization of such sequences within the genome may be investigated by genetic mapping with restriction enzymes. The sites of specific cleavage of DNA by restriction enzymes may be used as fixed points of reference for locating particular gene sequences (24). Analysis of germ line DNA with restriction enzymes may provide evidence for the possible origin of MTV proviral DNA from retrovirus-infected germ cells of a vertebrate ancestor of the mouse (25) or from evolution of normal cellular gene sequences (26). Germ line DNA from sperm also may provide a base line for detecting possible translocation, or other rearrangement, of genes during normal development of antibody-producing cells (27) or during neoplastic transfora nome
mation.
In inbred strains of mice, for which linkage group maps have been constructed, nucleic acid hybridization and restriction enzyme analyses of germ line DNA may be correlated directly with genetic studies in vivo. The fact that high molecular weight sperm DNA may be isolated from individual male mice makes possible the study of Mendelian segregation and recombination of particular germ line sequences, such as those that are related to MTV, in male progeny of crosses between high-and low-incidence tumor strains. However, in contrast to classical Mendelian analysis in which genotype is inferred from phenotype, the genotype of the germ line DNA can be identified directly by nucleic acid hybridization and by mapping with restriction enzymes.
Germ line genes, such as those that specify immunoglobulins and protein hormones, may be cloned, and the regulation of their expression may be compared to that of corresponding genes from differentiated somatic cells. Finally, because germ line DNA represents the zero-time of the organism with respect to any permanent or reversible biochemical changes that may occur in the organization of the genome during differentiation and aging of somatic cells, the regulation of genetic expression in inbred mice may be analyzed from a unique frame of reference. Of particular interest in this regard may be the mouse testicular teratoma (28), whose origin from primordial germ cells or from somatic cells has been the subject of much discussion. DNAs from cloned teratoma cell lines, which may exhibit distinct characteristics of differentiation and loss of
Proc. Natl. Acad. Sci. USA 76 (1979)
3951
tumorigenicity, may be compared to the germ line DNA from which they were derived. These studies may provide evidence for the view that some malignancies do not result from irreversible genetic changes. We gratefully acknowledge Dr. K. DeOme for his interest and support. We thank S. Hamamoto for electron microscopy. We acknowledge P. Thompson for technical illustration, J. Underhill for photography, B. Hayes and J. Kop for technical assistance, M. Webb for providing mice, and S. Castillo for typing the manuscript. This investigation was supported by Fellowship 5 F22 CA03162, awarded to R.S. by the National Cancer Institute, U.S. Department of Health, Education, and Welfare, and by Grant CA05388, awarded by the National Cancer Institute. 1. Muhlbock, 0. (1965) Eur. J. Cancer 1, 123-124. 2. Morris, V. L., Medeiros, E., Ringold, G. M., Bishop, J. M. & Varmus, H. E. (1977) J. Mol. Biol. 114,73-91. 3. Schlom, J., Colcher, D., Drohan, W. & Kettmann, R. (1978) Prog. Exp. Tumor Res. 21, 140-158. 4. Meistrich, M. L., Reid, B. 0. & Barcellona, W. J. (1975) J. Cell Biol. 64,211-222. 5. Decleve, A., Niwa, O., Hilgers, J. & Kaplan, H. S. (1974) Virology 57,491-502. 6. Schmidt, G. & Thannhauser, S. J. (1945) J. Biol. Chem. 161, 83-89. 7. Burton, K. (1968) Methods Enzymol. eds. Grossman, L. & Moldave, K. (Academic, New York), Vol. 12, pp. 163-166. 8. Kirby, K. S. (1957) Biochem. J. 66, 495-504. 9. Ogur, M. & Rosen, G. (1950) Arch. Biochem. 25,262-276. 10. Handbook of Biochemistry (1968) ed. Sober, H. A. (CRC, West Palm Beach, FL), pp. J252-256. 11. Schildkraut, C. L., Marmur, J. & Doty, P. (1962) J. Mol. Biol. 4, 430-443. 12. Miyazawa, Y. & Thomas, C. A., Jr. (1965) J. Mol. Biol. 11, 223-237. 13. Drohan, W., Kettmann, R., Colcher, D. & Schlom, J. (1977) J. Virol. 21, 986-994. 14. Cech, T. R., Rosenfeld, A. & Hearst, J. E. (1973) J. Mol. Biol. 81, 299-325. 15. Radloff, R., Bauer, W. & Vinograd, J. (1967) Proc. Natl. Acad. Sci. USA 57, 1514-1521. 16. Chargaff, E. (1955) The Nucleic Acids, eds. Chargaff, E. & Davidson, J. N. (Academic, New York), Vol. 1, pp. 307-371. 17. Borenfreund, E., Fitt, E. & Bendich, A. (1961) Nature (London) 191, 1375-1377. 18. Morrison, M. R., Paul, J. & Williamson, R. (1972) Eur. J. Biochem. 27, 1-9. 19. Harrison, P. R., Birnie, G. D., Hall, A., Humphries, S., Young, B. D. & Paul, J. (1974) J. Mol. Biol. 84,539-554. 20. Lueders, K. K. & Kuff, E. L. (1977) Cell 12, 963-972. 21. Sega, G. A., Cumming, R. B. & Walton, M. F. (1974) Mutation Res. 24, 317-333. 22. Marmur, J. & Doty, P. (1962) J. Mol. Biol. 5, 109-118. 23. Williamson, A. R. (1976) Annu. Rev. Biochem. 45, 467-500. 24. Danna, K. & Nathans, D. (1971) Proc. Natl. Acad. Sci. USA 68, 2913-2917. 25. Todaro, G. J. & Huebner, R. J. (1972) Proc. Natl. Acad. Sci. USA 69, 1009-1015. 26. Temin, H. M. (1971) J. Natl. Cancer Inst. 46, iii-vii. 27. Gally, J. A. & Edelman, G. M. (1970) Nature (London) 227, 341-348. 28. Stevens, L. C. (1967) Adv. Morphol. 6, 1-31.
