Volume 4 Number 5 1977
Nucleic Acids Research
A restriction endonuclease cleavage map of mouse mitochondrial DNA*
Kathleen Healy Mooret, Paul H.Johnson, Sarah E.W.Chandler and Lawrence l.Grossman
Department of Biochemistry, Wayne State University School of Medicine, Detroit, Ml 48201, USA
Received 22 February 1977
ABSTRACT A restriction endonuclease cleavage map is presented for mouse mitochondrial DNA. This map was constructed by electron microscopic measurements on partial digests containing fixed D-loops, and by electrophoretic analysis of partial and complete single enzyme digests, and of double digests. No map differences were detected between mitochondrial DNA from cultured LA9 cells and an inbred mouse line for the six endonucleases used. Three cleavage sites recognized by HpaI, five sites recognized by HincII, two sites recognized by PstI and four sites recognized by BwnI were located with respect to the origin of replication and the EcoRI and HinIII sites previously determined by others. No cleavages were produced by
KpnI
or
SalI.
The migration of linear DNA with a molecular weight greater than 1 x 106 was not a linear function of log molecular weight in 1% agarose gels run at 6.6 volts/cm. INTRODUCTION
Mitochondria from animal cells are a useful source of a well-defined eukaryotic DNA. Mitochondrial DNA (mtDNA) has been widely used for studies on DNA replication (1-10), complex DNA structure and formation (11-15) and genomic organization (16, 17), although its cellular function is poorly understood. In addition, its possible relationship to several human malignancies has been investigated (13, 15, 18-20). Restriction endonucleases have been used extensively for dissecting genomes in the size range of mtDNA and providing unique fractions for further study. We are interested in subdividing the mouse mtDNA genome for nucleotide sequence analysis of appropriate regions. We have expanded the initial cleavage map of mouse LA9 mtDNA constructed by Brown and Vinograd (16) using four ©) Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England
1273
Nucleic Acids Research additional enzymes. During the course of this work we have noted that restriction fragments with a molecular weight greater than 106 electrophoresed through 1% agarose gels at 6.6 volts/cm do not obey a linear relationship between mobility and log molecular weight.
MATERIALS AND METHODS
P&epaaution oj Mitochond&iat DNA. For most experiments, mtDNA was isolated from mouse LA9 cells grown in suspension culture (1) and harvested in log phase. MtDNA was purified according to method (i) of Smith et al. (21), except that the swelling buffer contained 1.5 mM EDTA in place of MgCl2 and ethidium bromide (EthBr) was used in the buoyant purification. In some cases, mitochondria were isolated from Swiss Webster mice, seventh generation inbred, kindly provided by Roger Acey, Wayne State University. Livers were minced in cold buffer (0.25 M sucrose, 10 mM Tris-HCl [pH 7.51, 10 mM KC1, 5 mM EDTA) (2 ml/g liver), dispersed briefly with a mechanical blender and homogenized with two strokes of a motordriven pestle. The crude homogenate was centrifuged at 2500 rev./min in an International centrifuge for 5 min. The supernatant was diluted with an equal volume of MS (14) buffer. The pellet was resuspended in an equal volume of MS and centrifuged as above. The supernatants were combined, centrifuged 2 to 3 times as above to remove contaminating nuclei, and the mitochondria collected at 12,000 rev./min, 15 min, in the Sorvall SS-34 rotor. Mitochondria were further purified by sedimentation through two-step sucrose gradients (21) and DNA isolated (21). OtheA DNA Ptepatations. Bacteriophage lambda DNA was purified as described (22). A wild-type lysogen was provided by D.A. Jackson, University of Michigan, and knin5cI857 was obtained from R.W. Davis, Stanford University. Bacteriophage PM2 DNA and qx174 RF DNA were purified as described (P.H. Johnson and L.I. Grossman, submitted for publication). Enzyma. EcoRI endonuclease was prepared as described (22) from 45 g of frozen cells (Escherichia coi DJ112 (R204), obtained from D.A. Jackson). HindIII was prepared by A. Nemanich of 1274
Nucleic Acids Research this laboratory using a modification of the method of Smith and Wilcox (23). BomI was prepared (24) from BacilZus omnyoZiquifaciens H (RUB 500, obtained from. G. Wilson, University of Rochester), or was obtained from New England BioLabs (Beverly, MA). HincII, PstI and KpnI were obtained from the above supplier;HindII and SaII were purchased from Bethesda Research Laboratories (Bethesda, MD); HpaI was obtained from both commercial sources. DNA polymerase I from E. coli was a gift of A. Kornberg, Stanford University; E. coli exonuclease III was provided by C. W. Schmid, University of California, Davis. ChemicatA. SeaKem agarose was purchased from Marine Colloids (Rockland, ME). EthBr was from Calbiochem. a-[32P]nucleoside triphosphates were from New England Nuclear. CsCl (99.9%) was from Reliable Chemical Co. (St. Louis, MO). All other chemicals were reagent grade. Enzymatic Rfet'izction o6 DNA. Restriction reactions, generally using 0.1 to 1 Wg of DNA, were carried out in volumes of 40 to 100 il for 1 to 2 hr at 37°C. The following reaction mixtures were used: EcoRI: 100 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 50 mM NaCl, 100 pg/ml gelatin; HpaI: 20 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 6 mM KC1, 5 mM 2-mercaptoethanol (2-SH); HinIl: 10 mM Tris-HCl (pH 8.0), 6.6 mM MgCl2, 60 mM NaCl, 6 mM 2-SH; HinIII: 10 mM Tris-HCl (pH 7.5), 7 mM MgCl2, 60 mM NaCl; PstI: 6 mM Tris-HCl (pH 7.5), 6 mM MgCl2, 50 mM NaCl, 6 mM 2-SH, 100 pg gelatin/ml; BamI: 6 mM Tris-HCl (pH 7.5), 6 mM MgCl2, 6 mM 2SH; KpnI: 6 mM Tris-HCl (pH 7.5), 6 mM MgC12, 50 mM NaCl, 6 mM 2-SH; SaII: 6 mM Tris-HCl (pH 7.9), 6 mM MgC12, 100 mM NaCl, 6 mM 2-SH.
Get EiectAophote,6". 1% solutions of agarose were prepared in running buffer (40 mM Tris base, 5 mM sodium acetate, 1 mM EDTA [pH 8.2]) by heating agarose suspensions in a boiling Evaporative losses were replaced by adding hot water bath. Slabs (15.5 cm high x agarose solution. the heated water to 13.5 cm x 0.25 cm) were poured between glass plates prewarmed The rear plate was sandblasted to improve adhesion to 60°C. and the front plate coated occasionally with dichlorodimethylsilane (Aldrich Chemical Co.). The electrophoresis apparatus 1275
Nucleic Acids Research was purchased from the Aquebogue Machine Shop (Aquebogue, NY). Samples (
Nucleic Acids Research
A restriction endonuclease cleavage map of mouse mitochondrial DNA*
Kathleen Healy Mooret, Paul H.Johnson, Sarah E.W.Chandler and Lawrence l.Grossman
Department of Biochemistry, Wayne State University School of Medicine, Detroit, Ml 48201, USA
Received 22 February 1977
ABSTRACT A restriction endonuclease cleavage map is presented for mouse mitochondrial DNA. This map was constructed by electron microscopic measurements on partial digests containing fixed D-loops, and by electrophoretic analysis of partial and complete single enzyme digests, and of double digests. No map differences were detected between mitochondrial DNA from cultured LA9 cells and an inbred mouse line for the six endonucleases used. Three cleavage sites recognized by HpaI, five sites recognized by HincII, two sites recognized by PstI and four sites recognized by BwnI were located with respect to the origin of replication and the EcoRI and HinIII sites previously determined by others. No cleavages were produced by
KpnI
or
SalI.
The migration of linear DNA with a molecular weight greater than 1 x 106 was not a linear function of log molecular weight in 1% agarose gels run at 6.6 volts/cm. INTRODUCTION
Mitochondria from animal cells are a useful source of a well-defined eukaryotic DNA. Mitochondrial DNA (mtDNA) has been widely used for studies on DNA replication (1-10), complex DNA structure and formation (11-15) and genomic organization (16, 17), although its cellular function is poorly understood. In addition, its possible relationship to several human malignancies has been investigated (13, 15, 18-20). Restriction endonucleases have been used extensively for dissecting genomes in the size range of mtDNA and providing unique fractions for further study. We are interested in subdividing the mouse mtDNA genome for nucleotide sequence analysis of appropriate regions. We have expanded the initial cleavage map of mouse LA9 mtDNA constructed by Brown and Vinograd (16) using four ©) Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England
1273
Nucleic Acids Research additional enzymes. During the course of this work we have noted that restriction fragments with a molecular weight greater than 106 electrophoresed through 1% agarose gels at 6.6 volts/cm do not obey a linear relationship between mobility and log molecular weight.
MATERIALS AND METHODS
P&epaaution oj Mitochond&iat DNA. For most experiments, mtDNA was isolated from mouse LA9 cells grown in suspension culture (1) and harvested in log phase. MtDNA was purified according to method (i) of Smith et al. (21), except that the swelling buffer contained 1.5 mM EDTA in place of MgCl2 and ethidium bromide (EthBr) was used in the buoyant purification. In some cases, mitochondria were isolated from Swiss Webster mice, seventh generation inbred, kindly provided by Roger Acey, Wayne State University. Livers were minced in cold buffer (0.25 M sucrose, 10 mM Tris-HCl [pH 7.51, 10 mM KC1, 5 mM EDTA) (2 ml/g liver), dispersed briefly with a mechanical blender and homogenized with two strokes of a motordriven pestle. The crude homogenate was centrifuged at 2500 rev./min in an International centrifuge for 5 min. The supernatant was diluted with an equal volume of MS (14) buffer. The pellet was resuspended in an equal volume of MS and centrifuged as above. The supernatants were combined, centrifuged 2 to 3 times as above to remove contaminating nuclei, and the mitochondria collected at 12,000 rev./min, 15 min, in the Sorvall SS-34 rotor. Mitochondria were further purified by sedimentation through two-step sucrose gradients (21) and DNA isolated (21). OtheA DNA Ptepatations. Bacteriophage lambda DNA was purified as described (22). A wild-type lysogen was provided by D.A. Jackson, University of Michigan, and knin5cI857 was obtained from R.W. Davis, Stanford University. Bacteriophage PM2 DNA and qx174 RF DNA were purified as described (P.H. Johnson and L.I. Grossman, submitted for publication). Enzyma. EcoRI endonuclease was prepared as described (22) from 45 g of frozen cells (Escherichia coi DJ112 (R204), obtained from D.A. Jackson). HindIII was prepared by A. Nemanich of 1274
Nucleic Acids Research this laboratory using a modification of the method of Smith and Wilcox (23). BomI was prepared (24) from BacilZus omnyoZiquifaciens H (RUB 500, obtained from. G. Wilson, University of Rochester), or was obtained from New England BioLabs (Beverly, MA). HincII, PstI and KpnI were obtained from the above supplier;HindII and SaII were purchased from Bethesda Research Laboratories (Bethesda, MD); HpaI was obtained from both commercial sources. DNA polymerase I from E. coli was a gift of A. Kornberg, Stanford University; E. coli exonuclease III was provided by C. W. Schmid, University of California, Davis. ChemicatA. SeaKem agarose was purchased from Marine Colloids (Rockland, ME). EthBr was from Calbiochem. a-[32P]nucleoside triphosphates were from New England Nuclear. CsCl (99.9%) was from Reliable Chemical Co. (St. Louis, MO). All other chemicals were reagent grade. Enzymatic Rfet'izction o6 DNA. Restriction reactions, generally using 0.1 to 1 Wg of DNA, were carried out in volumes of 40 to 100 il for 1 to 2 hr at 37°C. The following reaction mixtures were used: EcoRI: 100 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 50 mM NaCl, 100 pg/ml gelatin; HpaI: 20 mM Tris-HCl (pH 7.5), 10 mM MgCl2, 6 mM KC1, 5 mM 2-mercaptoethanol (2-SH); HinIl: 10 mM Tris-HCl (pH 8.0), 6.6 mM MgCl2, 60 mM NaCl, 6 mM 2-SH; HinIII: 10 mM Tris-HCl (pH 7.5), 7 mM MgCl2, 60 mM NaCl; PstI: 6 mM Tris-HCl (pH 7.5), 6 mM MgCl2, 50 mM NaCl, 6 mM 2-SH, 100 pg gelatin/ml; BamI: 6 mM Tris-HCl (pH 7.5), 6 mM MgCl2, 6 mM 2SH; KpnI: 6 mM Tris-HCl (pH 7.5), 6 mM MgC12, 50 mM NaCl, 6 mM 2-SH; SaII: 6 mM Tris-HCl (pH 7.9), 6 mM MgC12, 100 mM NaCl, 6 mM 2-SH.
Get EiectAophote,6". 1% solutions of agarose were prepared in running buffer (40 mM Tris base, 5 mM sodium acetate, 1 mM EDTA [pH 8.2]) by heating agarose suspensions in a boiling Evaporative losses were replaced by adding hot water bath. Slabs (15.5 cm high x agarose solution. the heated water to 13.5 cm x 0.25 cm) were poured between glass plates prewarmed The rear plate was sandblasted to improve adhesion to 60°C. and the front plate coated occasionally with dichlorodimethylsilane (Aldrich Chemical Co.). The electrophoresis apparatus 1275
Nucleic Acids Research was purchased from the Aquebogue Machine Shop (Aquebogue, NY). Samples (
