Download Nucleotide Sequence Preservation of Human

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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.
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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
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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
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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
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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.
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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
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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
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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
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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. Meth
ods Enzymol., 97: 426-434, 1983.
39. Wallace, D. C. Assignment of the chloramphenicol resistance gene to mito
chondrial deoxyribonucleic acid and analysis of its expression in cultured
human cells. Mol. Cell. Biol., 7: 697-710, 1981.
40. Wheldon, T. E., and Kirk, J. An error cascade mechanism for tumor progres
sion. J. Theor. Biol., 42: 107-111,1973.
protooncogenes and those genes whose products play essential
roles in cell growth and division (8, 28).
ACKNOWLEDGMENTS
We thank Dr. Bernard Poiesz and Dr. Marshall Kadin for providing leukemic
cells and R. Martene Koplitz for help with DNA sequence determinations.
REFERENCES
1. Allen, J. A., and Coombs, M. M. Covalent binding of polycyclic aromatic
compounds to mitochondrial and nuclear DNA. Nature (Lond.), 287: 244-245,
1980.
2. Anderson, S. Shotgun DNA sequencing using cloned DNase l-generated
fragments. Nucleic Acids Res., 9: 3015-3027,1981.
3. Anderson, S., Bankier, A. T., Barrell, B. G., de Bruijn, M. H. L., Coulson, A.
R., Drouin, J., Eperon, I. C., Nierlich, D. P., Roe, B. A., Sanger, F., Schreier,
P. H., Smith, A. J. H., Staden, R., and Young, I. G. Sequence and organization
of the human mitochondrial genome. Nature (Lond.), 290: 457-465, 1981.
4. Anderson, S., de Bruijn, M. H. L., Coulson, A. R., Eperon, I. C., Sanger, F.,
and Young, I. G. Complete sequence of bovine mitochondrial DNA. Conserved
features of the mammalian mitochondrial genome. J. Mol. Biol., 756:683-717,
1982.
5. Aquadro, C. F., and Greenberg, B. D. Human mitochondrial DNA variation and
evolution: analysis of nucleotide sequences from seven individuals. Genetics,
703. 287-312, 1983.
6. Backer, J. M., and Weinstein, I. B. Mitochondrial DNA is a major cellular target
for a dihydrodiol-epoxide derivative of benzo[a]pyrene. Science (Wash. DC),
209:297-299,
1980.
7. Bibb, M. J.. Van Etten, R. A., Wright, C. T., Walberg. M. W., and Clayton, D.
A. Sequence and gene organization of mouse mitochondrial DNA. Cell, 26:
167-180,1981.
8. Bishop, J. M. Cellular oncogenes and retroviruses. Annu. Rev. Biochem., 52:
301-354, 1983.
9 Bogenhagen, D., and Clayton, D. A. The number of mitochondrial deoxyribo
nucleic acid genomes in mouse L and human HeLa cells. J. Biol. Chem., 249:
7991-7995, 1974.
10. Boyum, A. Separation of leucocytes from blood and bone marrow. IV. Isolation
of mononuclear cells and granulocytes from human blood. Scand. J. Clin. Lab.
Invest., 27(Suppl. 97): 77-89, 1968.
11. Brown, W. M. Polymorphism in mitochondrial DNA of humans as revealed by
restriction endonuclease analysis. Proc. Nati. Acad. Sci. USA, 77: 3605-3609,
1980.
12. Brown, W. M. Evolution of animal mitochondrial DNA. In: M. Nei and R. K.
Koehn (eds.), Evolution of Genes and Proteins, pp. 62-88. Sunderiand, MA:
Sinauer Associates, Inc., 1983.
13. Cann, R. L., Brown, W. M., and Wilson, A. C. Polymorphic sites and the
mechanism of evolution of human mitochondrial DNA. Genetics, 706: 479499, 1984.
14. Chaconas, G., and van de Sande, J. 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
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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
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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.
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