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[CANCER RESEARCH 45, 1809-1814. April 1985] Nucleotide Sequence Preservation of Human Leukemic Mitochondria! DMA1 .... Raymond J. Monnat, Jr.,2 Clare L. Maxwell, and Lawrence A. Loeb The Joseph Gottstein Memorial Cancer Research Laboratory, Department of Pathology, University of Washington, Seattle, Washington 98195 ABSTRACT Nucleotide sequence variation in mitochondria! DNA isolated from human leukemic cells has been analyzed by recombinant DNA techniques. Three hundred eighty-seven independent recombinant DNA clones, each containing one of three defined segments of mitochondrial DNA isolated from the neoplastic cells of four leukemic patients, were analyzed. Partial nucleotide se quence determination of the 387 clones yielded a total of 81.7 kilobases of nucleotide sequence information. The only evidence of within-individual nucleotide sequence divergence consisted of three clones containing deletions of one or two nucleotides in one mitochondrial DNA region. These clones were three of 113 independent clones isolated from a patient with acute lymphocytic leukemia. The low level of nucleotide sequence divergence in the mitochondrial DNA population of neoplastic cells from individual leukemic patients suggests that a mechanism or mech anisms exist that limit the development of nucleotide sequence divergence in mammalian mitochondrial DNA. The results further suggest that this mechanism does not appear to be abrogated by neoplastic transformation in leukemic patients. INTRODUCTION Prior to the advent of recombinant DNA techniques, it was not possible to directly identify and characterize mutations occurring in the DNA of normal or neoplastic human cells. Now that individual cellular genes can be isolated, characterized, and reintroduced into mammalian cells, it has become possible to establish causal links between the presence of specific genetic alterations and either resistance to therapy or the development of métastases. We have applied recombinant DNA techniques to a specific question related to the phenomenon of neoplastic progression. Do mutations accumulate within human neoplastic cell popula tions? We have examined mtDNA3 isolated from neoplastic cells of patients with leukemia. We chose to study the mtDNA of human leukemic cells for 3 reasons: (a) mtDNA is well charac terized; its nucleotide sequence is known in entirety (3), and a great deal is known about between-individual nucleotide se quence differences; (b) mtDNA can be easily and reproducibly isolated from leukemic cells present in small amounts of periph eral blood; (c) Our previous work (29) demonstrated a low level of nucleotide sequence divergence within the mtDNA population of lymphocytes from individual normal donors. Thus, mtDNA mutations occurring in leukemic cells should be easily detected. 1This work was supported by grants from the NIH (CA-24845) and the United States Department of Energy (DE-AM06-76L02225). 1 Postdoctoral Fellow of the NIH (GM-07187). To whom requests for reprints should be addressed. 'The abbreviations used are: mtDNA, mitochondrial DNA; CO III, cytochrome oxidase subunit III; ALL, acute lymphocytic leukemia; CML/BC, chronic myelogenous leukemia in blast crisis; CLL, chronic lymphocytic leukemia. Received 9/5/84; revised, 12/11/84; accepted 1/10/85. CANCER RESEARCH In this paper, we report on the isolation and nucleotide sequence determination of 387 independently isolated recombinant clones containing mtDNA from the neoplastic cells of 4 patients with leukemia. MATERIALS AND METHODS Materials. Mononuclear cells were isolated from peripheral blood samples from 4 patients with clinical diagnoses of leukemia. The mononuclear cell fractions were isolated and stored at -70°C until confirmation of the clinical diagnosis. The cloning vector, M13mp11, and host, Escherichia coli strain JM103, were gifts of Dr. Joachim Messing, University of Minnesota. Protocols for growth of vectors and host strains are given in Ref. 27. Restriction endonucleases, T4 DNA ligase, and M13 sequencing primer were obtained from New England Biolabs or Bethesda Research Labo ratories, Inc. Calf intestinal alkaline phosphatase was obtained from Boehringer-Mannheim. E. coli DNA polymerase I large fragment was prepared by digestion of homogeneous E. coli DNA polymerase I (22) with Bacillus subtilis subtilisin, followed by a separation on Sephadex G100 (23), or obtained from Bethesda Research Laboratories. FicolhHypaque was obtained from Pharmacia Fine Chemicals. Deoxy- and dideoxynucleoside triphosphates and ATP were obtained from Pharma cia P-L Biochemicals. [«-32P]dCTPand [«-32P]dTTPwere obtained from New England Nuclear. Methods. Peripheral blood mononuclear cells were isolated from 4 patients with leukemia by gradient centrifugation in FicolhHypaque (10). The resulting mononuclear cell fractions were washed twice in phos phate-buffered saline, pelleted, and frozen at -70°C. mtDNA was iso lated from the frozen cell pellets by a modification of the "no gradient" technique of Bogenhagen and Clayton (9) [Tapper ef al. (38)]. The resulting mtDNA was resuspended in 50 M!of 50 rriM Tris (pH 7.5):1 HIM EDTA. Prior to cloning, each mtDNA preparation was digested sequen tially with the restriction endonucleases Sacl and Xbal, extracted with phenol, and precipitated with ethanol. To test for complete digestion, 5 ti\ (1/10 volume) of each restriction digest containing approximately 10 ng of mtDNA were end-labeled with 2 MCiof [«-32P]dCTP(3000 Ci/mmol) (17). The labeled DNA was precipitated with ethanol at -70°C and washed with cold 70% ethanol to remove unincorporated dCTP. The labeled DNA fragments were separated on a 0.8% neutral agarose gel by electrophoresis in Trisiborate buffer [90 rtiM Tris-HCI:90 rriM boric acid:1 mM EDTA, pH 8.2 (26)]. The gel was transferred to Whatman No. 3MM chromatography paper and dried at 80°Cfor 30 min under vacuum. Autoradiography was performed with an enhancing temperature (22°C)for 12 to 24 h. screen at room The cloning vector, M13mp11, was prepared by cutting the viral replicative form sequentially with the restriction endonuclease Sacl and Xbal. The 2 resulting fragments were dephosphorylated with calf intes tinal alkaline phosphatase (14), extracted with phenol, and precipitated with ethanol. Ligation reactions were performed in a volume of 30 ^l of 50 ÕTIM Tris-HCI (pH 7.5):10 mM MgCI2:10 mw dithiothreitoM mM ATP containing 100 ng of dephosphorylated M13 DNA, approximately 50 ng of mtDNA, and 0.1 to 0.5 units of T4 DNA ligase. After ligation at 18°C for 24 h, aliquots of the mixture were used to transform the E. coli host strain JM103 (27). DNA Sequence Analysis. Single-stranded M13 DNA containing mi tochondrial inserts was prepared from individual plaque-purified colonies VOL. 45 APRIL 1985 1809 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1985 American Association for Cancer Research. HUMAN LEUKEMIA by virus precipitation with polyethylene glycol and extraction with phenol (27). DMA sequence determination was by a modification of the dideoxy chain termination method of Sanger ef al. (36). Single-stranded recom binant DNA template (0.5 Mg) was hybridized wtih a 2-fold molar excess of a synthetic oligonucleotide sequencing primer (New England Biolabs; 17-mer) in 13 n\ of 12 mw Tris-HCI (pH 7.9):70 mM NaCI:7.5 HIM MgCI2; annealing was at 55°C for 1 h. Each base-specific chain termination reaction contained in 6 ^l of 20 mw Tris-HCI (pH 7.5): 67.5 mw NaCI: 7.5 mM MgCI2: 10 mw dithiothreitol: 1.67 ^M [a-^PJdTTP (150 Ci/mmol), mtDNA ments 4, 5, or 7 was used as a template for nucleotide sequence determination by the dideoxy chain termination method of Sanger ef a/. (36). The nucleotide sequences of the light (L)-strand of Fragment 4 and of the H-strand of Fragment 7 were determined from the Sacl site that divides the CO III gene into halves with a cut between L-strand nucleotides 9647 and 9648. The H-strand sequence of Fragment 5 was determined from the Sacl site that cuts between H-strand nucleotides 36 and 37 (Chart 1). Patient information and a summary of the nucleotide sequence information obtained are given in Table 1. A total of 81.7 kiloside triphosphates were 4.2 MMfor dATP, dCTP, and dGTP and 1.67 /¿M bases of sequence information, an average of 211 nucleotides per clone, was determined. Single-base substitutions were found for dTTP in the respective chain termination reactions. The dideoxy: deoxynucleoside triphosphate ratios used in individual termination reac approximately once every 230 nucleotides when patient mtDNA tions were 60:1 for adenosine, 30:1 for cytidine, 22.5:1 for guanosine, sequences were compared with the published human mtDNA and 50:1 for thymidine. The concentrations of nonlimiting deoxynucleo sequence. The published sequence was derived largely from side triphosphates were 42 ^M in adenosine-, cytidine-, and guanosinemtDNA isolated from a single placenta (3). A small portion of the specific base termination reactions and 31 ^M in thymidine-specific base published sequence (<5%) was derived from HeLa mtDNA or termination reactions. assumed to be identical to bovine mtDNA (3, 4). Single-base Chain termination reactions were performed at 37°Cfor 10 min. The substitutions were identifed in all patients and in all 3 mtDNA sequencing reactions were stopped with 5 ¡¡\ of 25 mw EDTA (pH 8), fragments studied (Table 1). Each between-individual substitu vacuum dried, and resuspended in 2 ¿tl of 99% deionized formamide, 10 0.125 ng of primed viral DNA template, and 0.2 unit of E. coli DNA polymerase I large fragment. The concentrations of limiting deoxynucleo- m«EDTA, 10 mM sodium hydroxide, and 0.3% each of xylene cyanol FF and bromphenol blue. After denaturation for 3 min at 95°C, 1.5-^1 aliquots from each reaction were loaded onto a 39- x 33- x 0.035-cm 8% polyacrylamide:7 M urea gel. The ratio of acrylamide to bisacrylamide was 19:1; the gel buffer was 135 mM Tris-HCI:45 mw boric acid:2.5 mM EDTA (pH 8.9) (2). After electrophoresis at 1500 V for 2 h, the gels were fixed for 2 min. in 10% acetic acid, transferred onto Whatman No. 3MM chromatography paper, and dried at 80°C for 30 min under vacuum. Autoradiography was performed at room temperature without an en hancing screen for 20 h. All nucleotide sequence differences from the published human mtDNA sequence were verified by repeating nucleotide sequence determination of the template. RESULTS mtDNA was isolated from the leukemic peripheral blood mononuclear cells of 4 patients. The 4 leukemic patients were unre lated; 3 of the 4 received no therapy, while one, CML/BC, received chemotherapy a year prior to isolation of the cells. Three different types of leukemia are represented: ALL; CLL; and CML/BC (Table 1). Purified mtDNA from each patient was cut into 7 fragments by sequential digestion with the restriction endonucleases Sacl and Xbal. Of the 4 potential fragments that could be ligated into the cloning vector, M13mp11 cut with Sacl and Xòal,3 (Nos. 4, 5, and 7) were recovered as recombinant molecules (Chart 1). Restriction endonuclease Fragments 4 and 7 span nucleotides 8,287 to 10,256 of the human mitochondrial genome (3). Each of these 2 fragments contains approximately one-half of the gene for CO III. In addition to CO III, Fragment 4 contains genes for ATPase 6, lysyl-tRNA, and an open reading frame (A6L). Restric tion endonuclease Fragment 7 contains the glutamyl-tRNA gene and a portion of open reading frame 3, in addition to one-half of the CO III gene. Restriction endonuclease Fragment 5 spans nucleotides 41 to 1,197 of the human mitochondrial genome. Fragment 5 contains the heavy (H) strand origin of mtDNA replication ("D-loop region"), promoters for mtDNA transcription, the phenylalanyl-tRNA gene, and a portion of the 12S rRNA gene (3). Single-stranded viral recombinant M13mp11 DNA prepared from 387 independently isolated clones containing mtDNA Frag CANCER RESEARCH tion in mtDNA Fragment 4, 5, or 7 was found in all independently isolated clones containing that fragment from the same patient. Short deletions of one or 2 nucleotides were identified in 3 of 81 clones containing mtDNA Fragment 7 from one patient (Patient ALL; Table 1). Two of the 3 deletions consisted of a deleted cytidine at L-strand position 9731 or 9732. From which of the 2 positions the cytidine was deleted cannot be determined from the nucleotide sequence of the deletion-containing clones. The third deletion consisted of a deletion of thymidine at L-strand position 9730 and a cytidine from L-strand positions 9727-9 or 9731-2. Again, which cytidine was deleted could not be estab lished unambiguously (Fig. 1). No additional examples of muta tions were identified that involved only a portion of independently isolated clones containing the same mtDNA fragment from a single patient. DISCUSSION We have used recombinant DNA techniques to identify mtDNA mutations in human leukemic cells. With the exception of 3 clones, we found no evidence of mtDNA nucleotide sequence divergence in 387 independently isolated mtDNA clones isolated from the leukemic cells of 4 patients. The small amount of nucleotide sequence divergence identified in the mtDNAs of 4 leukemic patients has several implications for the biology of human mtDNA and for the pathogenesis of leukemia. Mitochondrial Nucleotide Sequence Differences between Leukemic Patients. Nucleotide sequence differences in the mtDNAs of unrelated normal humans and other mammals have been documented by restriction endonuclease mapping and by limited DNA-sequencing studies (5, 7, 11-13, 21, 33). In this study, using DNA sequencing, single-base substitutions were identified in each of the 4 patients and in each of the 3 mtDNA fragments studied. Several identical nucleotide substitutions were found in different patients. For example, a substitution of guanosine for adenosine at L-strand position 263 was identified in both of the patients with CLL. This substitution occurs in the noncoding "D-loop" region, near the heavy strand origin of mtDNA replication; thus, it does not alter or disrupt a known mitochondrial gene product. This and several other nucleotide VOL. 45 APRIL 1985 1810 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1985 American Association for Cancer Research. HUMAN LEUKEMIA mtDNA Table 1 Nucleotìdesequencing summary Summary of human leukemic mtDNA nucleotide sequencing. Three hundred eighty-seven independently isolated recombinant clones containing one of 3 mtDNA restriction fragments were partially sequenced. The number of clones containing each mtDNA fragment, the number of nucleotides sequenced, and the position and type of nucleotide sequence alterations identified are indicated for each of the patients studied. The nucleotide positions are from, and changes are given with respect to, the published L-strand sequence of human mtDNA (3). differencePatient8 Nucleotide sequence frag- (yr)ALL Age of clones nucleotides of position9,5599,731-9,7329,727-9,7329.559731521992042072502639,55926 studied32814314214821396622Total sequenced6,36917,5279,8813,1393,81010,1114,2607,85013,6005,177L-strand clonesAll21AllAlAllAlAlAlAllAlAl Sex mentM 47M 15CLL C— CTG-^:A-43T-£T-.CT— 457F 57CLL Patient 1 CG—AT— CA— GNoneG-*CA-GNoneG-*CT— 457M 80CML/BC patient 2 16mtDNA 47No. °Patient ALL. Age 15, male, with testicular mass, lymphadenopathy, CT— CNo. and leukemia. Peripheral WBC count, 250 x 10* I"', with a95% blasts. A minority of leukemic blasts expressed cell surface la antigen, detected by an la (null) heteroantiserum. No surface immunoglobulin, T-cell differentiation antigens, or sheep erythrocyte, complement, or immunoglobulin Fc receptors could be demonstrated. The leukemic blasts were Sudan black, phosphatase. and nonspecific esterase negative. Impression: acute lymphoblastic leukemia, FAB L-2, null cell type. Patient CLL-1. Age 57, male, with diagnosed though untreated CLL of 4-year duration. Peripheral WBC differential count revealed 100% lymphocytes. Patient CLL-2. Age 80, female, with diagnosed though untreated CLL. Patient CML/BC. Age 16. male, with CML/BC diagnosed previously, treated with Adriamycin and splenectomy 13 months prior to admission. Peripheral WBC count. 560 x 10" \'\ with a95% blasts. The blasts were terminal deoxynucleotidyl transferase negative and contained Ph1 (Philadelphia) chromosome. Blast morphology and cytochemistry were as follows. Acid phosphatase: undifferentiated, negative; promyelocytic, positive (needle-like). Periodic acid-Schiff: undifferentiated, negative; promyelocytic, negative. Peroxidase: undifferentiated, negative; promyelocytic, positive. Sudan Black B: undifferentiated, positive (granular); promyelocytic, positive. Naphthol-AS-D-chloroacetate esterase: undifferentiated.positive (14% of blasts); promyelocytic, weakly positive (fluoride inhibitable). Impression: CML/BC (myelomonocytic), treated previously. 6 G. guanosine; C. cytidine; T, thymidine; A, adenosine. substitutions identified in Patient CLL-1 have been identified in sequence comparisons of the D-loop region of unrelated normal humans (5, 21). Three additional between-individual differences outside the Dloop region of mtDNA were identified in this study. Substitution of cytidine for thymidine at L-strand positions 9698 and 9725 was found in all clones containing these regions from Patient CML/BC. Both of these base substitutions occur in codon third positions in the CO III gene; neither produces an amino acid substitution. A third between-individual difference identified in all 4 patients studied was substitution of cytidine for guanosine at L-strand position 9559. This nucleotide substitution was identified by comparing the nucleotide sequence of each patient's mtDNA Fragment 4 with the published human mtDNA sequence. A substitution of cytidine for guanosine at L-strand position 9559 results in a substitution of proline for arginine at CO III amino acid 118. We identified this base substitution previously in mtDNAs of 5 normal unrelated individuals (29) and have subse quently identified the same base substitution in the mtDNAs of 6 additional unrelated normal or leukemic individuals.4 Thus, it is likely that the guanosine at L-strand position 9559 of the pub lished human mtDNA sequence is an error and/or that the original sequence was from an individual with an uncommon base sub4 R. J. Monnat, Jr., C. L. Maxwell, and L. A. Loeb, unpublished results. CANCER RESEARCH stitution at this site. Mitochondria! Sequence Divergence within Individual Leu kemic Patients. A major goal of this study was to identify and characterize mtDNA mutations in the neoplastic cells of individual leukemic patients. In a previous study of mtDNAs from lympho cytes of 5 normal unrelated donors, we found that sequence divergence between normal individuals was as great as 0.9%; yet, there was no evidence of within-individual mitochondrial nucleotide sequence divergence at a frequency of greater than 1 in 49,000 nucleotides (0.002%) (29). Against this low back ground of spontaneous mtDNA mutation, we anticipated that any increase in mutation associated with neoplastic transforma tion would be easy to detect. We expected nucleotide sequence differences to accumulate in the mtDNA population of individual leukemic patients for several reasons: (a) a high susceptibility of mtDNA to modification and damage by mutagens and carcino gens (1,6,31) and by altered cellular nucleotide pools (16); (b) transformed cells appear to have higher mutation rates than do corresponding normal cells (15, 30); and (c) human leukemic cells appear to replicate DNA less faithfully than do normal human lymphocytes (37). How much nucleotide sequence divergence could arise in the mtDNA population of an expanding neoplastic cell population? Many human leukemias appear to be of monoclonal origin, i.e., arise from one or a small number of cells (18). If a neoplasm is of monoclonal origin, the one or small number of neoplastic cells VOL. 45 APRIL 1985 1811 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1985 American Association for Cancer Research. HUMAN LEUKEMIA mtDNA least 3 ways. They could represent (a) preexisting mtDNA vari ation that was fortuitously amplified with the neoplastic cell population; (b) mutations that originated in the neoplastic cell population; or (c) mutations that arose during mtDNA DNA cloning and/or nucleotide sequence determination. We consider the third possibility unlikely. A previous study of mitochondrial nucleotide sequence divergence in normal individuals, which utilized 78 independently isolated mtDNA clones containing the same region of human mtDNA, failed to reveal either deletioninsertion or nucleotide substitution mutations at positions 9727- Chart 1. Structure, cloning, and nucleotide sequence determination of human mtDNA. mtDNA isolated from the neoplastic cells of leukemic patients was digested with the restriction endonucleases Sacl (A) and Xtoal (A). The resulting fragments were ligated into M13mp11. prepared by sequential digestion with Sacl and Xöal, and dephosphorylation Three classes of recombinant molecules were recovered, containing insertion of mitochondrial Fragments 4, 5, or 7. Arrows indicate the origin and direction of nucleotide sequence determination in these fragments. Cross-hatched segments of the mtDNA molecule contain the mitochondrial tRNA genes. Nucleotide sequences of the H-strand of Fragment 5-bearing clones were determined from the Sacl site that cleaves between H-strand nucleotides 36 and 37. The nucleotide sequences of the L-strand of Fragment 4-bearing clones and of the H-strand of Fragment 7-bearing clones were determined from the Sacl site that cleaves between L-strand nucleotides 9647 and 9648. Gene designations and nucleotide numbering are from Ref 3. URF, unidentified or open reading frame; Cyt b, cytochrome b. from which it originates must double at least 40 times before a sufficiently large number of neoplastic cells accumulates to pro duce the clinical problems by which we recognize leukemia. If the initial neoplastic cell(s) began with a homogeneous mtDNA population, and if the error rate for mtDNA replication is 10~4 (estimated on the basis of fidelity measurements with mtDNA polymerase) (24), then one nucleotide in 250 should be mutated in each mitochondrial molecule in leukemic cells at the time of diagnosis. These mitochondrial mutations would be in addition to mutations that accumulated during the >45 cell population doublings required to convert the zygote into a fully formed organism. Most importantly, a progressive increase in base substitutions would be expected if tumor progression in leukemic cells proceeded by an error-prone DNA replication mechanism that was able to affect mtDNA (25). In this study, only 3 mitochondrial mutations were identified in 81,700 nucleotides of DNA sequence information obtained from the neoplastic cells of 4 leukemic patients. All 3 of the withinindividual differences consisted of deletion of one or 2 nucleo tides in the one patient with ALL. These clones were 3 of a total of 81 containing Fragment 7 that were isolated as independent clones from the patient with ALL. The single-base deletions of cytidine in 2 of the clones produce a -1 frame shift in the CO III gene and new chain termination codons 24, 48, and 70 down stream from the deletion site. The deletion of a cytidine and thymidine from the L-strand nucleotide region 9727-9732 of the third clone produces a -2 frame-shift mutation in the CO III gene and new chain termination codons 4,26, 70, and 84 downstream from the 2 nucleotide deletion sites. These within-individual mutations could have originated in at CANCER RESEARCH 9732 (29). Thus, there does not appear to be preferential muta tion of these nucleotide positions when human mtDNA is cloned in M13 and the resulting clones used for nucleotide sequence determination by the dideoxy chain termination method. Concerted Preservation of Human Leukemic mtDNA. Our results strongly suggest that nucleotide sequence divergence is not present in the mtDNA population of neoplastic cells of leukemic patients at a level of greater than one nucleotide substitution per molecule. Two implications of this finding are: (a) that a mechanism (or mechanisms) exist to restrict the de velopment of mitochondrial nucleotide sequence divergence; and (b) that this mechanism does not appear to be abrogated by neoplastic transformation. With only 3 exceptions, this study revealed an invariant mtDNA population in neoplastic cells of individual leukemic patients. The low level of mitochondrial nucleotide sequence divergence in both normal (29) and neoplastic human cells suggests the exist ence of a mechanism to limit the accumulation of mutations in the mtDNA population derived from the zygote. How such a mechanism might work is not clear. The most direct mechanism, a passive loss of altered or damaged mtDNA molecules, fails to explain the absence of all nucleotide substitutions, regardless of their coding effects, from human mtDNA. A second mechanism of mitochondrial nucleotide sequence preservation could be more accurate mtDNA replication or mtDNA repair. For example, a hundredfold increase in the accuracy of mtDNA replication, pre dicted from measurements of the fidelity of the suspected mtDNA polymerase, 7, in vitro (24) would be sufficient to limit the development of mitochondrial nucleotide sequence divergence during development. However, neither more faithful mtDNA rep lication nor mtDNA repair appears adequate to limit mutation accumulation during mtDNA turnover in normal somatic cells following completion of development (35) or in an expanding neoplastic cell population. A third possible mechanism that could explain the absence of nucleotide substitutions in the mtDNA population of an individual is the repeated correction of all mtDNA molecules in a cell against a master copy. This mechanism in its simplest form is unlikely to be correct, as genetically distinct mtDNA molecules appear to be able to persist in single human cells (39). In this study, the nucleotide sequence of mtDNA isolated from the neoplastic cells of individual leukemic patients was found to be highly conserved. Thus, critical tests of a somatic mutational origin of tumor cell heterogeneity and tumor progression (19, 20, 25, 32, 34, 40) must focus on defined nuclear genes. The methods and approach developed in the course of this investi gation can be used to identify and characterize nucleotide se quence divergence in specific nuclear genes from individual tumor cell populations. Two families of nuclear genes that will be particularly important to analyze in this manner are cellular VOL. 45 APRIL 1985 1812 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1985 American Association for Cancer Research. HUMAN LEUKEMIA 17. Drouin, J. Cloning of human mitochondrial DNA in Escherichia coli. J. Mol. Biol., 740: 15-34, 1980. 18. Fialkow, P. J. Cell lineages in hematopoietic neoplasia studied with glucose-6phosphate dehydrogenase cell markers. J. Cell. Physiol., (Suppl. 1): 37-43, 1982. 19. Fidler, I. J., and Hart, I. R. Biological diversity in metastatic neoplasms: origins and implications. Science (Wash. DC), 277: 998-1003, 1982. 20. Foulds, L. Tumour progression and neoplastic development. In: P. Emmelot and O. Muhlbock (eds.), Cellular Control Mechanism and Cancer, pp. 242258. New York: Elsevier Publishing Company, 1964. 21. Greenberg, B. D., Newbold, J. E., and Sugino, A. Intraspecific nucleotide sequence variability surrounding the origin of replication in human mitochondrial DNA. Gene, 27: 33-49, 1983. 22. Jovin, T. M., Englund, P. T., and Bertsch, L. L. Enzymatic synthesis of deoxyribonucleic acid. XXVI. Physical and chemical studies of a homogeneous deoxyribonucleic acid polymerase. J. Biol. Chem., 244: 2996-3008, 1969. 23. Klenow, H., and Henningsen, I. Selective elimination of the exonuclease activity of the deoxyribonucleic acid polymerase from Escherichia coli B by limited proteolysis. Proc. Nati. Acad. Sci. USA, 65: 168-175, 1970. 24. Kunkel, T. A., and Loeb, L. A. Fidelity of mammalian DNA polymerases. Science (Wash. DC), 273: 765-767,1981. 25. Loeb, L. A., Springgate, C. F., and Battula, N. Errors in DNA replication as a basis of malignant changes. Cancer Res., 34: 2311 -2321, 1974. 26. Maniatis, T., Fritsch, E. F., and Sambrook, J. Molecular Cloning. A Laboratory Manual, pp. 156-162. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1982. 27. Messing, J. New M13 vectors for cloning. Methods Enzymol., 707: 20-78, 1983. 28. Monnat, R. J., Jr., and Loeb, L. A. Mechanisms of neoplastic transformation. Cancer Invest., 7: 175-183, 1983. 29. Monnat, R. J., Jr., and Loeb, L. A. Nucleotide sequence preservation of human mitochondrial DNA. Proc. Nati. Acad. Sci. USA, in press, 1985. 30. Nichols, W. W. Viral interactions with the mammalian genome relevant to neoplasia. In: J. German (ed.). Chromosome Mutation and Neoplasia, pp. 317332. New York: Alan R. Liss, 1983. 31. Niranjan, B. G., Bhat, N. K., and Avadhani, N. G. Preferential attack of mitochondrial DNA by aflatoxin B, during hepatocarcinogenesis. Science (Wash. DC), 275: 73-75,1982. 32. Nowell, P. C. The clonal evolution of tumor cell populations. Science (Wash. DC), 794: 23-28, 1976. 33. Olivo, P. D., Van de Walle, M. J., Laipis, P. J., and Hauswirth, W. W. Nucleotide sequence evidence for rapid genotypic shifts in the bovine mitochondrial DNA D-loop. Nature (Lond.). 306: 400-402, 1983. 34. Poste, G., and Greig, R. The experimental and clinical implications of cellular heterogeneity in malignant tumors. J. Cancer Res. Clin. Oncol., 706:159-170, 1983. 35. Rabinowitz, M., and Swift, H. Mitochondrial nucleic acids and their relation to the biogenesis of mitochondria. Physiol. Rev., 50: 376-427, 1970. 36. Sanger, F., Nicklen, S., and Coulson, A. R. DNA sequencing with chainterminating inhibitors. Proc. Nati. Acad. Sci. USA, 74: 5463-5467, 1977. 37. Springgate, C. F., and Loeb, L. A. Mutagenic DNA polymerase in human leukemic cells. Proc. Nati. Acad. Sci. USA, 70: 245-249,1973. 38. Tapper, D. P., Van Etten, R. A., and Clayton, D. A. Isolation of mammalian mitochondrial DNA and RNA and cloning of the mitochondrial genome. 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H. 5'-MP labeling of RNA and DNA restriction fragments. Methods Enzymol., 65: 75-85, 1980. 15. Cifone, M. A., and Fidler, I. J. Increasing metastatic potential is associated with increasing genetic instability of clones isolated from murine neoplasms. Proc. Nati. Acad. Sci. USA, 78: 6949-6952, 1981. 16. Dimnik, L. S., and Hoar, D. I. Thymidine deprivation is mutagenic to the mitochondrial genome. Genetics, 97 (Suppl. 1).• s31,1981. CANCER RESEARCH mtDNA VOL. 45 APRIL 1985 1813 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1985 American Association for Cancer Research. NO. OF CLONES: DELETION(S): POSITIONS*: 78/81 — — 2/81 -C 9731 or 32 1/81 -C,T 9727-32 GTACGTACGT 9735 Fig. 1. Nucleotide deletions identified in mtDNA Fragment 7 of Patient ALL. The published L-strand sequence of nucleotide positions 9721 to 9738 of human mtDNA is given (left). Seventy-eight of 81 clones containing Fragment 7 from Patient ALL had a nucleotide sequence identical to that of the published sequence. The nucleotide sequencing gel autoradiogram of one of these 78 clones is shown (Lanes 1 to 4). Two of 81 clones contained a deleted cytidine at position 9731 or 9732. The nucleotide sequencing gel autoradiogram of one of these 2 clones is shown (Lanes 5 to 8). One of 81 clones contained a deletion of the thymidine at position 9730 and of a cytidine from positions 9727 to 9729 or 9731 to 9732. The nucleotide sequencing gel autoradiogram of this one double-deletion clone is shown (Lanes 9 to 72). •, it is not possible to assign the deleted cytidines unambiguously to positions 9727 to 9729 or 9731 to 9732. CANCER RESEARCH VOL. 45 APRIL 1985 1814 Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1985 American Association for Cancer Research. Nucleotide Sequence Preservation of Human Leukemic Mitochondrial DNA Raymond J. Monnat, Jr., Clare L. Maxwell and Lawrence A. Loeb Cancer Res 1985;45:1809-1814. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/45/4/1809 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected]. To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected]. Downloaded from cancerres.aacrjournals.org on June 15, 2017. © 1985 American Association for Cancer Research.