U.S. patent application number 13/858820 was filed with the patent office on 2013-08-08 for transgenomic mitochondria, transmitochondrial cells and organisms, and methods of making and using.
The applicant listed for this patent is Michael D. Koob, Young G. Yoon. Invention is credited to Michael D. Koob, Young G. Yoon.
Application Number | 20130203160 13/858820 |
Document ID | / |
Family ID | 35197551 |
Filed Date | 2013-08-08 |
United States Patent
Application |
20130203160 |
Kind Code |
A1 |
Koob; Michael D. ; et
al. |
August 8, 2013 |
Transgenomic Mitochondria, Transmitochondrial Cells and Organisms,
and Methods of Making and Using
Abstract
This invention provides transgenomic mitochondria,
transmitochondrial cells and organisms, and the materials and
methods for making such mitochondria, cells and organisms.
Inventors: |
Koob; Michael D.;
(Roseville, MN) ; Yoon; Young G.; (Cottage Grove,
MN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Koob; Michael D.
Yoon; Young G. |
Roseville
Cottage Grove |
MN
MN |
US
US |
|
|
Family ID: |
35197551 |
Appl. No.: |
13/858820 |
Filed: |
April 8, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11578476 |
Oct 13, 2006 |
8420377 |
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PCT/US2005/012872 |
Apr 15, 2005 |
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13858820 |
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60562642 |
Apr 15, 2004 |
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Current U.S.
Class: |
435/325 ;
435/255.1; 435/317.1 |
Current CPC
Class: |
C12N 1/16 20130101; C12N
15/63 20130101; C12N 15/87 20130101; C12N 15/1027 20130101; C12N
15/81 20130101; C12N 15/8509 20130101; C12N 2800/90 20130101 |
Class at
Publication: |
435/325 ;
435/317.1; 435/255.1 |
International
Class: |
C12N 15/63 20060101
C12N015/63 |
Claims
1. A method for making a transgenomic mitochondrion incorporating
an exogenous mitochondrial genome comprising the steps of; a)
isolating a mitochondrial genome from a eukaryotic cell to yield an
exogenous mitochondrial genome; b) modifying said isolated
exogenous mitochondrial genome by insertion of one or more
selectable markers, screening markers, replication sequences, or
combinations thereof; c) introducing said modified isolated
exogenous mitochondrial genome into a host mitochondrion from a
second eukaryotic cell; d) removing said one or more selectable
markers, replication sequences, or combinations thereof, from said
modified isolated exogenous mitochondrial genome within said host
mitochondrion; and e) isolating said transgenomic mitochondrion
from said second eukaryotic cell.
2. The method of claim 1, wherein said host mitochondrion from said
second eukaryotic cell is a yeast mitochondrion.
3. The method of claim 2, wherein said yeast mitochondrion is
substantially free of endogenous mitochondrial DNA.
4. The method of claim 1, wherein said exogenous mitochondrial
genome is an isolated animal mitochondrial genome.
5. The method of claim 4, wherein said isolated animal
mitochondrial genome is modified prior to introduction into said
isolated yeast mitochondrion.
6. The method of claim 5, wherein said isolated animal
mitochondrial genome is modified by one or more selectable markers,
screening markers, replication sequences, or combinations thereof,
said selectable markers, screening markers, and replication
sequences having a prokaryotic origin.
7. The method of claim 5, wherein said isolated animal
mitochondrial genome is modified by one or more selectable markers,
screening markers, replication sequences, or combinations thereof,
said selectable markers, screening markers, and replication
sequences having a eukaryotic origin.
8. The method of claim 5, wherein said isolated animal
mitochondrial genome is modified by one or more selectable markers,
screening markers, replication sequences, or combinations thereof,
said selectable markers, screening markers, and replication
sequences having a synthetic origin.
9. The method of claim 5, wherein said isolated animal
mitochondrial genome constitutes at least 80% of naturally
occurring said isolated animal mitochondrial genome.
10. The method of claim 9, wherein said isolated animal
mitochondrial genome is introduced into said host yeast
mitochondrion.
11. The method of claim 10, wherein said introduction of said
isolated animal mitochondrial genome into said host yeast
mitochondrion is by particle bombardment.
12. A transgenomic mitochondrion comprising: a mitochondrion having
an outer membrane and an inner membrane; a matrix located inside
said inner membrane; and an exogenous mitochondrial genome that is
incorporated into and stably replicating within said matrix of said
mitochondrion.
13. The transgenomic mitochondrion of claim 12, wherein said
mitochondrion is an isolated yeast mitochondrion comprising an
exogenous mitochondrial genome.
14. The transgenomic mitochondrion of claim 12, wherein said
exogenous mitochondrial genome comprises an exogenous animal
mitochondrial genome.
15. The transgenomic mitochondrion of claim 14, wherein said animal
mitochondrial genome is selected from the group consisting of
avian, mammalian, murine, bovine, porcine, or human mitochondrial
genomes.
16. The transgenomic mitochondrion of claim 13, wherein said
mitochondrion is a yeast mitochondrion comprising an animal
mitochondrial genome.
17. The transgenomic mitochondrion of claim 15, wherein said animal
mitochondrial genome is a mouse mitochondrial genome.
18. The transgenomic mitochondrion of claim 15, wherein said animal
mitochondrial genome is a human mitochondrial genome.
19. The transgenomic mitochondrion of claim 13, wherein said
isolated yeast mitochondrion is substantially free of endogenous
yeast mitochondrial DNA.
20. A transmitochondrial cell comprising: a eukaryotic cell having
a plasma membrane, cytoplasm, a nucleus; and a transgenomic
mitochondrion incorporated into said cytoplasm of said eukaryotic
cell, said transgenomic mitochondrion having a modified exogenous
mitochondrial genome within said mitochondrion.
21. The transmitochondrial cell of claim 20, wherein said
transgenomic mitochondrion is not endogenous to said eukaryotic
cell.
22. The transmitochondrial cell of claim 20, wherein said modified
exogenous mitochondrial genome is stably replicating within said
transgenomic mitochondrion within said eukaryotic cell.
23. The transmitochondrial cell of claim 21, wherein said
transgenomic mitochondrion is a yeast mitochondrion.
24. The transmitochondrial cell of claim 22, wherein said modified
exogenous mitochondrial genome is an animal genome.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 11/578,476, filed 13 Oct. 2006, which was the national stage of
International Application No. PCT/US2005/012872, filed 15 Apr.
2005, which claims the benefit of U.S. Provisional Application No.
60/562,642, filed 15 Apr. 2004.
TECHNICAL FIELD
[0002] This invention generally relates to mitochondria and
mitochondrial DNA, and more particularly to transgenomic
mitochondria and transmitochondrial cells and organisms.
BACKGROUND OF THE INVENTION
[0003] The mammalian mitochondrial genome is a small, circular,
double-stranded molecule containing 37 genes. Of these, 24 (22
tRNAs and two rRNAs) are needed for mitochondrial DNA translation,
and 13 encode subunits of the respiratory chain complexes. Many
mitochondrial processes are strictly dependent on the interaction
between specific mitochondrial DNA sequences and the
nuclear-encoded mitochondrial polypeptides with which they have
co-evolved. Therefore, the majority of mitochondrial polypeptides
are nuclear encoded, translated in the cytoplasm, and then
transported across one or both of the mitochondrial membranes. It
is thought that mitochondria contain more than 1,000 different
proteins.
SUMMARY OF THE INVENTION
[0004] The invention discloses methods of transferring isolated
mitochondrial genomes into purified mitochondria or into
mitochondria within a cell. Mitochondria containing exogenous
mitochondrial genomes are "transgenomic mitochondria," while cells
or organisms containing exogenous mitochondrial genomes in the
mitochondria are "transmitochondrial cells" or "transmitochondrial
organisms." The invention provides such transgenomic mitochondria
and transmitochondrial cells and organisms.
[0005] Using the methods of the invention, a mitochondrial genome
can be manipulated prior to introducing it into mitochondria. In
addition, basic mitochondria-specific processes can be transferred
into transmitochondrial cells by introducing the appropriate
nuclear genes into the transmitochondrial cells. In one aspect, the
invention provides a transmitochondrial cell including an isolated
exogenous mitochondrial genome. Representative cells can include
yeast cells, mammalian cells, avian cells, bovine cells, and
porcine cells. Representative exogenous mitochondrial genomes
include mouse mitochondrial genomes and human mitochondrial
genomes.
[0006] In another aspect, the invention provides for a
transmitochondrial yeast cell that includes a transcriptionally
active mammalian mitochondrial genome. A representative mammalian
mitochondrial genome is a mouse mitochondrial genome. In still
another aspect, the invention provides a transmitochondrial yeast
cell that includes a yeast mitochondrion that is substantially free
of yeast mitochondrial DNA and that includes an isolated mammalian
mitochondrial genome. Such a transmitochondrial yeast cell also can
include an exogenous nuclear gene from the same species as the
mitochondrial genome. The exogenous nuclear gene can encode, for
example, a mitochondrial transcription factor or a polypeptide
involved in replication of the mitochondrial genome. In yet another
aspect, the invention provides a transgenomic yeast mitochondrion
that includes a mammalian mitochondrial genome. Representative
mammalian mitochondrial genomes include mouse mitochondrial genomes
and human mitochondrial genomes. In one embodiment, the
mitochondrion is substantially free of endogenous mitochondrial
DNA.
[0007] The invention also provides for a transmitochondrial
mammalian cell that contains such a transgenomic yeast
mitochondrion. In another aspect, the invention provides a
transgenomic mitochondrion that includes an exogenous mitochondrial
genome. A representative transgenomic mitochondrion is a human
mitochondrion. In still another aspect, the invention provides for
a vector that includes a mammalian mitochondria genome and a
prokaryotic low-copy number origin of replication. Such a vector
also can include a prokaryotic selectable marker or a prokaryotic
screening marker, and/or an eukaryotic selectable marker or an
eukaryotic screening marker.
[0008] In yet another aspect, the invention provides a mammalian
mitochondria genome that includes a prokaryotic low-copy number
origin of replication. Such a genome also can include a prokaryotic
selectable marker or a prokaryotic screening marker, and/or an
eukaryotic selectable marker or an eukaryotic screening marker. In
one aspect, the invention provides a method for introducing an
isolated mammalian mitochondrial genome into a yeast mitochondrion.
Such a method includes providing a yeast cell that includes
mitochondria that are substantially free of endogenous DNA; and
introducing isolated mammalian mitochondrial genomes into the
mitochondria. In an embodiment of the invention, the mammalian
mitochondrial genomes are introduced into the yeast mitochondria by
particle bombardment. In one aspect, the invention provides a
method for introducing an isolated exogenous mammalian
mitochondrial genome into a mammalian mitochondrion. Such a method
includes providing a mammalian cell that includes mitochondria that
are substantially free of endogenous DNA; and introducing the
exogenous isolated mammalian mitochondrial genomes into the
mitochondria.
[0009] In an embodiment of the invention, the exogenous isolated
mammalian mitochondrial genomes are introduced into the
mitochondria by particle bombardment. In another aspect, the
invention provides a method for introducing an isolated exogenous
mammalian mitochondrial genome into a mammalian mitochondrion. Such
a method includes providing purified transgenomic yeast
mitochondria that contains an isolated mammalian mitochondrial
genome; injecting the purified transgenomic yeast mitochondria into
mammalian cells having mammalian mitochondria. In still another
aspect, the invention provides a transmitochondrial mammal that has
mitochondria containing exogenous isolated mammalian mitochondrial
genomes. Representative mammals can include mice, rats, or monkeys.
In yet another aspect, the invention provides a transmitochondrial
plant that has mitochondria containing exogenous isolated
mitochondrial genomes.
[0010] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. Although
methods and materials similar or equivalent to those described
herein can be used in the practice or testing of the present
invention, suitable methods and materials are described below. In
addition, the materials, methods, and examples are illustrative
only and not intended to be limiting. All publications, patent
applications, patents, and other references mentioned herein are
incorporated by reference in their entirety. In case of conflict,
the present specification, including definitions, will control. The
details of one or more embodiments of the invention are set forth
in the accompanying drawings and the description below. Other
features, objects, and advantages of the invention will be apparent
from the drawings and detailed description, and from the
claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0011] FIG. 1 is a schematic showing three mouse mitochondrial DNA
clones generated by an in vitro transposition reaction.
[0012] FIG. 2 shows the identification of mitochondrial DNA
fragments that serve as transcriptional promoters in E. coli. FIG.
2A is a schematic of the approach used to clone fragments from the
mouse mitochondrial genome with promoter activity. FIG. 2B is a
comparison of CAT activities generated from mitochondrial DNA
promoter fragments at low and high copy numbers. The black and gray
bars shown for each clone indicate the relative CAT activities from
low copy and high copy strains, respectively, with the value of the
low copy vector-only control arbitrarily set to 1.
[0013] FIG. 3 is a schematic representation of p2C.gamma.SSB, a
control plasmid used to examine DNA transcription assay by in
organello RNA synthesis.
[0014] FIG. 4 is a schematic showing transformation of S.
cerevisiae mitochondria in the cell by particle bombardment.
[0015] FIG. 5 shows a map of a construct containing the mouse
mitochondrial genome.
[0016] FIG. 6 shows the gene replacement strategy used to generate
single gene replacement yeast strains.
[0017] FIG. 7 shows a construct for examining in vivo transcription
initiation at mitochondrial promoters.
[0018] Like reference symbols in the various drawings indicate like
elements.
DETAILED DESCRIPTION
[0019] Saccharomyces cerevisiae yeast strains were generated that
contain entire mouse mitochondrial genomes in their mitochondria
rather than their own yeast mitochondrial DNA. Any mitochondrial
genome can be manipulated using routine recombinant techniques and
propagated using standard prokaryotic strains. Therefore, a new in
vivo mammalian mitochondrial model system has been created in which
the mitochondrial sequences can be genetically engineered. By
adding the appropriate mammalian genes for nuclear-encoded
mitochondrial proteins to these yeast strains, such
species-specific mitochondrial processes as transcription and
replication are recapitulated in these transmitochondrial yeast
strains. This methodology can be used to introduce any isolated
exogenous mitochondrial genomes into the mitochondria of any
eukaryotic cell.
Mitochondria, Mitochondrial DNA, and Eukaryotic Cells
[0020] The present invention provides transgenomic mitochondria and
transmitochondrial cells and organisms. Transgenomic mitochondria
can be generated by introducing isolated exogenous mitochondrial
genomes into purified mitochondria. Transmitochondrial cells and
organisms can be generated by introducing isolated exogenous
mitochondrial genomes into mitochondria within a cell, or by
introducing transgenomic mitochondria into a cell or organism.
[0021] Mitochondria can be purified using any of a number of
different gradient methods. (Sambrook et al., 1989, Molecular
Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y.; and Cavelier et al.,
2000, Exp. Cell Res., 259: 79-85). Mitochondria useful in the
invention are substantially free of DNA. As used herein,
"substantially free" is intended to mean mitochondria that are
nearly or completely void of DNA (i.e., mitochondrial DNA is
present in the mitochondria in small amounts or is present in only
a small number of mitochondria).
[0022] Mitochondrial DNA can be removed from mitochondria using a
variety of methods. In yeast, cells lacking DNA in their
mitochondria can be obtained by spontaneous mutation or by
treatment with a DNA-targeting drug such as acriflavin. These yeast
cells have been referred to as "petite mutants" because they are
deficient in respiration and therefore, smaller in size than
wild-type yeast cells. See, for example, Chen & Clark-Walker,
2000, Int. Rev. Cytol., 194: 197-238. Alternatively, cells can be
treated with a DNA intercalator such as rhodamine 6G or ethidium
bromide. When eukaryotic cells are exposed to a DNA intercalator,
replication in the circular genomes of mitochondria is blocked, and
the mitochondrial genomic DNA is eventually lost. See, for example,
Williams et al., 1999, Human Mol. Genetics, 8: 1691-7 and
Desjardins et al., 1985, Mol. Cell. Biol., 5: 1163-9.
[0023] As used herein, "exogenous" is generally defined as
"originating or derived externally." "Externally" typically means
from a different source. For example, mitochondrial genomes are
exogenous to host cells or host mitochondria when the mitochondrial
genomes originate from different cell types or different species
than the host cells or host mitochondria. In addition, "exogenous"
can also refer to mitochondrial genomes that are removed from
mitochondria, manipulated, and returned to the same mitochondria.
As used herein, "endogenous" is generally defined as "originating
or derived internally." For example, endogenous mitochondria are
mitochondria that are native to a cell.
[0024] A mitochondrial genome refers to the circular nucleic acid
present in a mitochondrion. Mitochondrial genomes can be obtained
from any number of organisms including mammalian organisms such as,
without limitation, mice and humans. As used herein, mitochondrial
"genome" refers to the complete mitochondrial genome (i.e., 100%)
or at least 80% (e.g., at least 85%, 90%, 95%, or 99%) of a
naturally occurring mitochondrial genome.
[0025] Generally, mitochondrial genomes are isolated so that the
genomes can be manipulated in any number of ways prior to
introduction into the mitochondria of the same or a different
organism. Methods of isolating mitochondrial genomes are known in
the art. See, for example, Hudson et al., 1969, PNAS USA, 62:
813-20; Ausenda & Chomyn, 1996, Methods Enzymol., 264: 122-128;
and Beckman et al., 1993, Promega Notes Magazine, 43: 10. As used
herein, "isolated" refers to mitochondrial genomes that are
separated from other nucleic acids usually associated with
mitochondrial genomes in their natural state. In addition, isolated
mitochondrial genomes can be genetically engineered, recombinant,
and/or synthetic mitochondrial genomes. Mitochondrial genomes or
fragments thereof existing among hundreds to millions of other
nucleic acids within, for example, a nucleic acid library (e.g., a
cDNA, or genomic library) or a portion of a gel (e.g., agarose, or
polyacrylamine) containing restriction-digested DNA are not to be
considered isolated mitochondrial genomes.
[0026] Isolated mitochondrial genomes can be obtained using
isolation techniques routine in the art such as those exemplified
herein. In addition, isolated mitochondrial genomes within the
scope of the invention can be obtained using other methods
including, without limitation, recombinant nucleic acid technology,
and/or the polymerase chain reaction (PCR). General PCR techniques
are described, for example in PCR Primer: A Laboratory Manual,
Dieffenbach & Dveksler, Eds., Cold Spring Harbor Laboratory
Press, 1995; and U.S. Pat. Nos. 4,683,202, 4,683,195, 4,800,159,
and 4,965,188. Recombinant nucleic acid techniques include, for
example, restriction enzyme digestion and ligation. Isolated
nucleic acids of the invention also can be chemically synthesized,
either as a single nucleic acid molecule or as a series of
oligonucleotides. In addition, isolated nucleic acid molecules of
the invention also can be obtained by mutagenesis.
[0027] Mitochondrial genomes can be, but do not necessarily need to
be, cloned into a vector. Mitochondrial genome sequences can be
produced and manipulated by recombinant DNA technology methods
routine in the art. Sequences that allow mitochondrial genomes to
be amendable to manipulation include, for example, origin of
replication sequences and additional sequences such as those
encoding a selectable marker (e.g., an antibiotic resistance gene)
or a screenable marker (e.g., glucuronidase (GUS)).
[0028] An origin of replication sequence can include a prokaryotic
origin of replication, which initiates replication of nucleic acid
sequences in prokaryotes, or a eukaryotic origin of replication
(also referred to as an "autonomously replicating sequence" (ARS)),
which initiates replication of nucleic acid sequences in
eukaryotes. Origin of replication sequences can be generally
referred to as "low-copy number" origins and "high-copy number"
origins based upon the number of rounds of replication initiated.
Mitochondrial genomes are generally more stable with a low-copy
number origin of replication (e.g., .ltoreq.25 copies of
mitochondrial genomes/cell, e.g., 1 copy/cell, 5 copies/cell, 10
copies/cell, 15 copies/cell, 20 copies/cell, or 25
copies/cell).
[0029] Screenable and selectable markers are typically used to
identify and separate, respectively, those cells containing nucleic
acid encoding the marker from cells that don't contain the marker
nucleic acid. Screenable markers generally produce a colored or
fluorescent product when provided with the appropriate substrate
and include but are not limited to glucuronidase (GUS),
.beta.-galactosidase (.beta.-gal), luciferase, or green fluorescent
protein (GFP). Selectable markers can provide resistance to a
compound (e.g., an antibiotic or a herbicide) and include but are
not limited to NPTII (encoding resistance to kanamycin), and HPT
(encoding resistance to hygromycin).
TABLE-US-00001 TABLE 1 List of Selectable and Screenable Markers
Type or Source of Selection Marker Enzyme/Mode of Action
Agent/Substrate Reference A. Selectable markers I. Antibiotics
Neomycin Neomycin Neomycin/kanamycin Bevan et al., 1983,
phosphotransferse phosphotransferase antibiotic C418 Nature, 304:
184-7 II gene (neo) type II (NPT II), detoxification Hygromycin
Hygromycin Hygromycin van den Elzen et al., phosphotransferase
phosphotransferase 1985, Plant Mol. Biol., gene (hpt)
detoxification 5: 299-302 Bleomycin Binding protein,
Bleomycin/phleomycin Mazodier et al., 1985, resistance gene
detoxification Nuc. Acids Res., 13: 195-205; Gatignol et al., 1987,
Mol. Gen. Genet., 207: 342-8 Sulphonamide Dihydropteroate
Sulfadiazine/asulam Guerineau et al., 1990, resistance gene
synthase, detoxification Plasmid, 23: 35-41; (sull) Guerineau et
al., 1990, Plant Mol. Biol., 15: 127-36 II. Herbicides Csr1-1 gene
Acetolactate synthase, Chlorsulfuron Haughn &Somerville,
insensitive (sulphonylurease) 1986, Mol. Gen. Genet., 204: 430-4;
Haughn et al., 1988, Mol. Gen. Genet., 211: 266-271 aroA gene EPSP
synthase, sensitive Glyphosate Rogers et al., 1983, and insensitive
Appl. Environ. Microbiol., 46: 37-43; Stalker et al., 1985, J.
Biol. Chem., 260: 4724-8 psbA gene Q. protein, insensitive Atrazine
Goloubinoff et al., 1984, Nuc. Acids Res., 12: 9489-96; Sato et
al., 1988, Mol. Gen. Genet., 214: 358-60 bar gene Phosphinothricin
Phosphinothricin, Thompson et al., 1987, acetyltransferase,
bialaphos EMBO J., 9: 2519-23 detoxification bxn gene Nitrilase,
detoxification Bromoxynil Stalker et al., 1988, J. Biol. Chem.,
263: 6310-4 dehl gene Dehalogenase, Dalapon Busto et al., 1992,
Sci. detoxification Total Environ., 123-124: 267-77 tfd gene 2,4-
2,4-D Vedler et al., 2000, dichlorophenoxyacetate Gene, 245: 161-8
monoxygenase, detoxification III. Metabolic agents dhfr gene
Dihydrofolate Methotrexate Nakamura &Littlefield,
(bacterial/mouse) reductase, insensitive 1972, J. Biol. Chem., 247:
179-87; Eichholtz et al., 1987, Somat. Cell Mol. Genet., 13: 67-76
ocd gene Ornithine L-ornithine Schindler et al., 1989,
cyclodeaminase J. Bacteriol., 171: 847-54 B. Scorable/assayable
markers nos/ocs genes Nopaline/octopine Nopaline/octopine Yang et
al., 1987, Anal. synthase Biochem., 160: 342-5 lacZ gene
.THETA.-galactosidase (.THETA.-gal), X-gal Pardee et al., 1959, J.
color reaction Mol. Biol., 1: 165-78 uidA gene
.THETA.-glucuronidase (GUS), X-glu Jefferson et al., 1986, color
reaction PNAS USA, 83: 8447-51 luc gene Luciferase, light Luciferin
Wood &DeLuca, 1987, emission Anal. Biochem., 161: 501-7 lux
gene Luciferase, light Hydrocarbon Boivin et al., 1988, emission
compounds Mol. Gen. Genet., 213: 50-5 Streptomycin Streptomycin
Streptomycin Boronin et al., 1979, phosphotransferase
phosphotransferase, Antibiotiki, 24: 585-90 gene detoxification
Chloramphenicol Chloramphenicol Chloramphenicol Shaw & Brodsky,
1968, acetyltransferase acetyltransferase, J. Bacteriol., 95: 28-36
gene detoxification
[0030] The cloning procedure described herein for mtDNA takes
advantage of the fact that DNA must be circular in order to
replicate in E. coli and that the mtDNA genome is typically the
only circular DNA in most eukaryotic cells. Since the transposition
reaction does not circularize linear DNA, the genomic DNA fragments
and broken mtDNA fragments that invariably contaminate mtDNA
preparations are not cloned by this procedure and so the background
is very low. The replication ori and selectable marker are also
inserted at random locations and orientations throughout the
mitochondrial genome and so many different clones are generated in
the same experiment. The most stable of these resulting constructs
are readily identified during the initial plasmid analysis as those
that have faithfully replicated the mitochondrial genome in E.
coli.
[0031] Representative eukaryotic cells that can be made
transmitochondrial for exogenous mitochondrial genomes include
yeast cells, mammalian cells, avian cells, bovine cells, porcine
cells, and plant cells. Mammalian cells include, but are not
limited to, rodent cells, primate cells, and human cells. Methods
of culturing yeast cells, mammalian cells, avian cells, bovine
cells, and porcine cells are known in the art. See, for example,
Cell and Tissue Culture: Laboratory Procedures, Doyle et al. eds,
John Wiley & Sons, 1998; Harrison & Rae, General Techniques
of Cell Culture, Harris, ed., Cambridge University Press, 1997;
Basic Cell Culture: A Practical Approach, 2nd Ed., Davis, ed.,
Oxford Press, 2002; and Griffiths & Doyle, Mammalian Cell
Culture: Essential Techniques, John Wiley & Sons, 1997.
[0032] The invention further provides for cells containing viable
mitochondrial genomes. "Viable mitochondrial genomes" refer to
mitochondrial genomes that can be replicated (e.g., that are
maintained through multiple rounds of cell division). Maintenance
of exogenous mitochondrial genomes usually requires the presence of
one or more species-specific nuclear genes (e.g., encoding a
mitochondrial replication factor). "Transcriptionally active"
refers to a mitochondrial genome that, under the appropriate
conditions, can be partially or entirely transcribed. Transcription
of mitochondrial genomes generally requires the presence of one or
more species-specific nuclear genes (e.g., encoding a mitochondrial
transcription factor). "Species-specific" means that the nuclear
genes, whether for replication or transcription, originate from the
same species from which the mitochondrial genomes originated, or a
species very closely related thereto.
Transmitochondrial Cells and Organisms, and Methods of Making Such
Cells and Organisms
[0033] The invention provides for methods of introducing an
isolated mammalian mitochondrial genome into a mitochondrion. As
discussed above, the mitochondrial genome can be introduced into a
purified mitochondrion or into a mitochondrion within a cell.
Methods of introducing DNA into purified mitochondria are known in
the art and generally utilize electroporation. Methods of
introducing mitochondrial genomes into mitochondria in a cell have
proven much more difficult. The present invention describes methods
of introducing an exogenous mitochondrial genome into a
mitochondrion in a cell, as well as methods of replicating and
transcribing the exogenous mitochondrial genome.
[0034] The methods of the invention have been exemplified herein by
introducing mouse mitochondrial genomes into endogenous yeast
mitochondria, but the methods are applicable and amenable to other
eukaryotic organisms as well. For example, to make mammalian cells
transmitochondrial only requires appropriate tissue culture
conditions and appropriate selectable or screenable markers. Both
tissue culture conditions of mammalian cells and selectable and/or
screenable markers suitable for use in mammals (see Table 1 above)
are well known in the art.
[0035] According to the methods of the invention, exogenous
isolated mitochondrial genomes can be introduced into mitochondria
in a cell using particle bombardment or any comparable technique
that allows the exogenous mitochondrial genomes to pass through
both mitochondrial membranes. Particle bombardment conditions for
introducing DNA into endogenous mitochondria have been described
(see, for example, Johnston et al., 1988, Science, 240: 1538-1541;
Fox et al., 1988, PNAS USA, 85: 7288-7292), and differ little from
the particle bombardment conditions for introducing DNA into plant
cells.
[0036] Alternatively, exogenous mitochondrial genomes can be
introduced into endogenous mitochondria via fusion and/or
conversion of mitochondria by transferring the exogenous
transgenomic mitochondria containing the mitochondrial genome of
interest into eukaryotic cells (e.g., by microinjection of
transgenomic mitochondria). In the case of fusion, the two
membranes (inner and outer) of one mitochondria fuse with that of
another, forming long, thin, branched "mitochondrial networks" from
which mitochondria can be transiently split off (i.e., fission).
Fission and fusion create a dynamic mitochondrial network system in
the cell. In the case of conversion, the exogenous mitochondrial
structures are gradually converted to endogenous structures as
their protein and lipid components are replaced with those
generated by the host cell. Following fusion and/or conversion, the
exogenous mitochondrial genome of interest is maintained in
endogenous mitochondria. Therefore, the transgenomic mitochondria
containing the mitochondrial genomes of interest can be used
essentially as carriers or delivery vehicles, and their structure
in the newly-formed transmitochondrial cell is transient. Prior to
this invention, fusion or conversion could only be performed with
closely related species.
[0037] Irrespective of the mechanism of uptake (fusion or
conversion), transmitochondrial cells or organisms can be generated
using, for example, the transgenomic yeast mitochondria described
herein. Transgenomic yeast mitochondria can be generated using, for
example, particle bombardment, and purified using, for example, a
sucrose gradient, and introduced into non-yeast eukaryotic cells
(e.g., by microinjection). Yeast cells are particularly useful
carriers or delivery vehicles for exogenous mitochondrial genomes
because yeast cells have a fairly promiscuous replication system.
As discussed above, the mitochondrial proteins from the
transgenomic yeast mitochondria are exogenous to eukaryotic host
cells and are eventually replaced by mitochondrial proteins
endogenous to the eukaryotic cells. The methods of the invention,
therefore, provide several approaches for producing an eukaryotic
cell containing endogenous mitochondria that, themselves, contain
exogenous mitochondrial genomes.
[0038] There are many diseases caused by mutations in the
mitochondrial genome. See, for example, Dahl & Thorburn, 2001,
Am. J. Med. Genet., 106:1-3; Thorburn & Dahl, 2001, Am. J. Med.
Genet., 106: 102-14; and Dimauro & Schon, 2001, Am. J. Med.
Genet., 106: 18-26. Using the methods of the invention,
mitochondrial genomes containing a mutation can be introduced into
the mitochondria of an egg or an embryo using particle bombardment
as described herein. Alternatively, a mutant mitochondrial genome
can be introduced into mitochondria of an egg or an embryo by a
fusion-like process similar to that described above. If
mitochondrial genomes are introduced into mitochondria of an
embryo, the embryo is usually an early-stage embryo (e.g.,
generally 4-6 cells) so that the majority of cells carry
mitochondria containing the mutant mitochondrial genomes.
Techniques to fertilize an egg and grow the resulting embryo to an
organism, particularly with model organisms such as Drosophila,
Zebrafish (Danio rerio), mice, rats, and primates, as well as with
domestic animals, are well known in the art. See, for example,
McEvoy, 2003, Reprod. Domest. Anim., 38: 268-75; Hearn, 2001,
Reprod. Fertil. Dev., 13: 517-22; and Ishiwata et al., 2001, Hum.
Cell, 14: 283-91.
[0039] The invention provides for a transmitochondrial organism
such as a mammal that contains exogenous isolated mitochondrial
genomes. The mitochondrial genomes can be isolated from
mitochondria originating from an individual afflicted with a
disease, or mitochondrial genomes can be manipulated recombinantly
using standard techniques such as site directed mutagenesis,
saturation mutagenesis, or PCR mediated mutagenesis to contain
either a specific or a random mutation. Any of a number of mammals
can be used to generate a transmitochondrial mammal containing
exogenous mutant mitochondrial genomes. Useful animals include, but
are not limited to, mice, rat, or primates. The invention describes
methods of making model organisms having mitochondria-originating
diseases.
[0040] Methods of the invention also provide for a
transmitochondrial organism (e.g., an animal or a plant) containing
exogenous isolated mitochondrial genomes. For example, a
mitochondrial genome can be manipulated to include nucleic acids
encoding enzymes involved in the production of particular amino
acids and/or vitamins, for example, those that are not naturally
produced by an organism. A transmitochondrial organism containing
mitochondria that, in turn, contain the engineered mitochondrial
genome can make such amino acids and/or vitamins on their own. The
diet of such a transmitochondrial organism, for example, would not
need to be supplemented with such amino acids and/or vitamins.
[0041] Such transmitochondrial organisms are amenable to the
production of such compounds because of the high number of
mitochondria per cell, and because many if not all of the metabolic
intermediates required during synthesis reactions (e.g.,
co-factors) are already present in the mitochondria. Another
significant advantage of using a mitochondrial genome is that
multiple genes should be able to be added and co-regulated as a
group, like operons in bacteria, rather than needing to
individually regulate them with separate promoters as is the case
with mammalian nuclear genes.
[0042] With respect to plants, plant mitochondrial genomes are
generally larger than other eukaryotic mitochondrial genomes.
Therefore, a vector capable of allowing manipulation of large
nucleic acids such as a bacterial artificial chromosome (BAC)
vector can be used when manipulating plant mitochondrial genomes.
Particle bombardment techniques are well established in plants (see
references cited above), as are tissue culture methods and plant
regeneration methods (see, for example, Smith, Plant Tissue
Culture: Techniques and Experiments, 2nd Ed., Academic Press, 2000;
and Evans et al., Plant Cell Culture (The Basics), Bios Scientific
Pub. Ltd., 2003).
[0043] The invention will be further described in the following
examples, which do not limit the scope of the invention described
in the claims.
EXAMPLES
Example 1
Escherichia coli Strains, Cell Lines, Plasmids, and Culture
Media
[0044] Escherichia coli strains DH5.alpha. .lamda.att::pirwt,
DH5.alpha. .lamda.att::pir200 (Koob et al., 1994, Ann. N.Y. Acad.
Sci., 745: 1-3) and DH5.alpha. .lamda.att::pir116 were used for
maintaining plasmids containing the R6K .gamma. origin of
replication (.gamma.-ori). p2C.gamma.CmR (Koob et al., 1994, Ann.
N.Y. Acad. Sci., 745:1-3), which contains the .gamma.-ori and a
chloramphenicol resistance gene (CmR), was used as a template for
making a transposon DNA fragment (see below). The mouse cell line
LL/2 (ATCC CRL-1642) (Bertram & Janik, 1980, Cancer Lett., 11:
63-73) was grown in DMEM (Life Technologies, Rockville, Md.) in the
presence of heat-inactivated 10% fetal bovine serum at 37.degree.
C. in a humidified 10% CO.sub.2 incubator. The mtDNA-less .rho.0
LL/2 cell line described herein, a derivative of LL/2 cells, was
grown in DMEM supplemented with 10% fetal bovine serum, 50 .mu.g/ml
uridine and 0.1 mg/ml pyruvate.
Example 2
Isolation of mtDNA-less (.rho.0) LL/2 Cell Line
[0045] The mtDNA-less .rho.0 LL/2 cell line was isolated by a
modification of a method previously described (King & Attardi,
1989, Science, 246: 500-3; Bai & Attardi, 1998, EMBO J., 17:
4848-58), which involves treatment of LL/2 cells with high
concentrations of ethidium bromide. LL/2 cells were exposed to 5
.mu.g/ml ethidium bromide for 4 weeks in medium supplemented with
50 pg/ml uridine and 0.1 mg/ml pyruvate. After 4 weeks, clonal
.rho.0 LL/2 cell lines were obtained by diluting these ethidium
bromide-treated cells to single cells per well on 96-well plates.
The clonal cells were then cultured in normal medium supplemented
with 50 .mu.g/ml uridine and 0.1 mg/ml pyruvate. The .rho.0 state
of the cloned cells was verified by a PCR assay using L-strand
(5'-ACC CAA CGC GGC AAA CTA ACC-3' (SEQ ID NO:1)) and H-strand
(5'-TCT TGT TCG TCT GCC AGG CT-3' (SEQ ID NO:2)) primers and by
assaying for the unique growth requirements of the .rho.0 cells
(King & Attardi, 1996, Methods Enzymol., 264: 304-13).
Example 3
DNA Manipulation
[0046] Mini-scale preparations of plasmid DNA were prepared by the
alkaline lysis method (Sambrook et al., 1989, Molecular Cloning: A
Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press,
Cold Spring Harbor, N.Y.) and large quantities of plasmid DNA were
prepared by the PEG precipitation method and other recombinant DNA
techniques were performed essentially as previously described
(Sambrook et al., supra). Restriction enzymes and T4 DNA ligase
were purchased from New England Biolabs (Beverly, Mass.) and used
as recommended by the manufacturer. AmpliTaq DNA polymerase was
purchased from Applied Biosystems (Branchburg, N.J.) and
deoxyoligonucleotides (oligos) were synthesized by Life
Technologies (Rockville, Md.) or IDT (Coralville, Iowa). Ampicillin
(Amp), tetracycline and chloramphenicol (Cm) were used at
concentrations of 50, 12.5 and 12.5 .mu.g/ml, respectively.
Example 4
Isolation of mtDNA From Mouse Liver
[0047] The liver of a freshly killed mouse was removed and placed
in cold isolation medium (0.32 M sucrose, 1 mM potassium EDTA, 10
mM Tris-HCl pH 7.4). All subsequent steps were performed at
4.degree. C. or on ice. The tissue was minced with scissors and
rinsed several times with the same medium. The chopped tissue was
suspended in isolation medium (4 ml/g liver) and homogenized in a
Potter-Elvehjem tissue grinder (Kontes Glass Co., Vineland, N.J.).
The homogenate was transferred to 50 ml centrifuge tubes and
centrifuged at low speed (1000 g, 5 min). The supernatant was then
centrifuged at high speed (13 000 g, 20 min). The resultant
supernatant was discarded, the pellet was resuspended in 5 ml of
DNase I buffer (210 mM mannitol, 70 mM sucrose, 5 mM Tris-HCl pH
7.4, 10 mM MgCl2) containing 0.5 mM phenylmethylsulfonyl fluoride.
The suspension was incubated with 4000 Kunitz units of DNase I
(Sigma) at 37.degree. C. for 1 hour. After incubation, the
suspension was washed three times in 10 vol of washing buffer (210
mM mannitol, 70 mM sucrose, 5 mM Tris-HCl pH 7.4, 10 mM EDTA) by
centrifugation at 13,000 g for 20 min. The washed pellet was
resuspended in 5 ml of sucrose-TE buffer (20% sucrose, 50 mM
Tris-HCl pH 7.4, 10 mM EDTA) and loaded on top of sucrose layers
consisting of 15 ml of 1.5 M sucrose (lower layer) and 15 ml of 1.0
M sucrose (upper layer), both containing 10 mM Tris-HCl pH 7.4, 5
mM EDTA. After centrifugation at 25,000 rpm for 30 min in a Beckman
SW28 rotor, the interface fraction (red-brown color) between the
1.0 and 1.5 M sucrose gradients was collected and washed twice with
4 vol of washing buffer by centrifugation at 18,000 g for 20 min.
The mitochondrial pellet was suspended in 3 ml of STE buffer (100
mM NaCl, 10 mM EDTA, 50 mM Tris-HCl pH 7.4) and incubated with 330
pl of 10% SDS and 400 .mu.l of proteinase K (10 mg/ml) at
50.degree. C. for 3 hours. After incubation, the digest was
extracted with 3 ml of phenol/chloroform saturated with TE (10 mM
Tris-HCl pH 7.4, 1 mM EDTA) by shaking gently for 10 minutes and
the extraction was repeated with 3 ml of chloroform. The DNA in the
aqueous phase was precipitated by the addition of 370 pl of 3 M
sodium acetate and 6 ml of absolute ethanol (30 minutes at
-20.degree. C.). The DNA was collected by centrifugation, washed
with 3 ml of 70% ethanol, dried, and resuspended in TE.
Example 5
In vitro Transposon Insertion Reaction
[0048] A synthetic transposon in which the .gamma.-ori and a
chloramphenicol resistance gene are flanked by Tn5 mosaic ends (ME)
was generated by a PCR using BamHI-linearized p2C.gamma.CmR as
template and the primers MESalCmR (5'-CTG TCT CTT ATA CAC ATC TGT
CGA CAG AAG CCA CTG GAG CA-3' (SEQ ID NO:3); ME sequence
italicized, SaII restriction site underlined) and MESma .gamma. ori
(5'-CTG TCT CTT ATA CAC ATC TCC CGG GCT MT TCT GTC AGC CGT T-3'
(SEQ ID NO:4); Smal restriction site underlined). A total of 30
amplification cycles were performed: 1 minute at 95.degree. C., 1
minute at 55.degree. C., followed by 90 s at 72.degree. C. PCR
products were isolated from low melting point agarose gels (FMC,
Rockland, Me.) using AgarACE.TM. (Promega, Madison, Wis.). For the
in vitro transposon insertion reaction, a purified hyperactive Tn5
transposase (EZ::TN; Epicentre, Madison, Wis.) was used in a
reaction consisting of 1 U transposase, 200 ng mouse mitochondrial
DNA and 10 ng PCR-amplified transposon in the buffer provided by
the supplier. After incubating the reaction mixture for 2 hours at
37.degree. C., the reaction was terminated by adding 1 .mu.l of the
supplied 10x stop solution and by heating at 70.degree. C. for 10
minutes. An aliquot of 1 .mu.l of the in vitro transposon insertion
reaction mixture was used for electrotransformation of DH5.alpha.
.lamda.att::pirwt.
Example 6
Cloning of mtDNA Fragments With Promoter Activity in E. coli and
the Chloramphenicol Acetyltransferase (CAT) Assay
[0049] Mouse mtDNA digested with AIuI, HaeIII and Sau3AI was
ligated into the HincII and BgIII sites 5' of the promoter-less
chloramphenicol acetyltransferase gene (CAT, CmR) of the `promoter
trapping` vector pANTS.gamma.-Cm (FIG. 2A). The ligation mixture
was transformed into E. coli strain DH5.alpha. .lamda.att::pir200
and recombinant clones were selected on Luria broth (LB)-Cm plates
(12.5 .mu.g/ml Cm). DNA isolated from these colonies was sequenced
and retransformed into DH5.alpha. .lamda.att::pirwt (LB-Amp
selection). The CAT activity from the same recombinant clones was
assayed in both the pir200 and pirwt strains grown in LB-Amp medium
to an optical density of .about.0.6 at 600 nm. CAT activity was
assayed using the FAST CAT.RTM. Green (deoxy) Chloramphenicol
Acetyltransferase Assay Kit (Molecular Probes Inc., Eugene, Oreg.)
according to the manufacturer's instructions. A Phosphorlmager
Storm 840 (Molecular Dynamics, Sunnyvale, Calif.) system was used
to measure band intensity of the acetylated products after
fractionating on a thin layer chromatography plate (Merck KGaA,
Darmstadt, Germany). Values of the intensities were calculated
relative to the CAT activity of the vector-only low copy control
sample.
Example 7
Electroporation of Mitochondria
[0050] Exponentially growing LL/2 cells were harvested by
centrifugation, washed twice with 1 mM Tris-HCl pH 7.0, 0.13 M
NaCl, 5 mM KCl and 7.5 mM MgCl.sub.2. The cell pellet was
resuspended in half the cell volume with 1/10.times. IB (4 mM
Tris-HCl pH 7.4, 2.5 mM NaCl, 0.5 mM MgCl.sub.2) and the cells were
broken using a Pellet Pestle tissue grinder (Kontes Glass Co.). The
homogenate was mixed with a one-ninth volume of the packed cell
volume of 10X IB (400 mM Tris-HCl pH 7.4, 250 mM NaCl, 50 mM
MgCl.sub.2) resulting in a buffer concentration of roughly 1.times.
IB (40 mM Tris-HCl pH 7.4, 25 mM NaCl, 5 mM MgCl.sub.2). The
unbroken cells and nuclei were removed by two consecutive low speed
centrifugations (2000 rpm for 5 min). The supernatant was placed
into new 1.5 ml Eppendorf tubes and centrifuged at full speed for
10 minutes to obtain a crude mitochondrial pellet. Mitochondria
were rinsed once with 500 .mu.l of 1.times. IB, centrifuged again
at full speed for 10 min and resuspended into 0.33 M sucrose/10%
glycerol (at a concentration of 100 mg mitochondrial protein/ml)
for electroporation. Electroporations were performed essentially as
described (Collombet et al., 1997, J. Biol. Chem., 272: 5342-7). An
aliquot of 10 .mu.g of cloned mouse mtDNA was added to 50 .mu.l of
100 mg/ml mitochondrial suspension and the mixture was transferred
into a cold electroporation cuvette (0.1 cm gap cuvette; Bio-Rad).
Electroporation was carried out using a Bio-Rad Gene Pulser at a
capacitance of 25 .mu.F, a resistance of 400 .OMEGA. and a field
strength of 10-16 kV/cm. After electroporation, 1 ml of incubation
buffer (40 mM Tris-HCl pH 7.4, 25 mM NaCl, 5 mM MgCl.sub.2, 10%
glycerol) was added to the electroporation cuvette. The
electroporated mitochondria were rapidly mixed by pipetting,
transferred to a new Eppendorf tube and washed three times with
incubation buffer by centrifugation.
Example 8
In Organello RNA Synthesis of Electroporated Mitochondria and
RT-PCR Analysis
[0051] For in organello RNA synthesis (Gaines, 1996, Methods
Enzymol., 264: 43-9), the final mitochondrial pellet was
resuspended in 50 pl of incubation buffer (10% glycerol, 40 mM
Tris-HCl pH 7.4, 25 mM NaCl, 5 mM MgCl2, 1 mM pyruvate, 1 mM ATP
and 1 mg/ml BSA) and incubated at 37.degree. C. for 2 hours. After
incubation, the mitochondrial suspension was pelleted at full speed
in a microcentrifuge for 10 min and washed twice with incubation
buffer. The pellets were suspended in 200 .mu.l of DNase I buffer
(10% glycerol, 10 mM Tris-HCl pH 8.0, 1 mM MgCl2) and incubated
with 200 Kunitz units of DNase I (Sigma) at 37.degree. C. for 30
min. After incubation, the mitochondria were pelleted and washed
twice with washing buffer (10% glycerol, 10 mM Tris-HCl pH 7.4, 1
mM EDTA) to inactivate and remove the nuclease. The mitochondrial
samples were lysed with 300 pl of lysis buffer (0.5% SDS, 10 mM
Tris-HCl pH 7.4, 150 mM NaCl, 1 mM EDTA) and incubated with 100 pg
proteinase K for 15 min at 37.degree. C. Phenol extraction was then
performed, total mitochondrial nucleic acids were collected by
ethanol precipitation and residual DNA contaminants were removed
using RNase-free DNase I (Promega) as directed by the manufacturer.
RT-PCR was carried out using 16S rRNA-specific or ND6-specific
primers. RT-PCR analysis was carried out using a SuperScript
First-Strand Synthesis system (Life Technologies) and the following
primers: CmR-F (5'-GTA CCT ATA ACC AGA CCG TTC AGC-3' (SEQ ID
NO:5)), CmR-R (5'-CAG CGG CAT CAG CAC CTT GTC-3' (SEQ ID NO:6)),
16S rRNA-F (5'-GCA GCC ACC AAT AAA GAA AG-3' (SEQ ID NO:7)), 16S
rRNA-R (5'-GGA CCC TCG TIT AGC CGT TC-3' (SEQ ID NO:8)),
MusMt-MIuIA (5'-AGG TGG ATT ATT TAT AGT GTG ATT ATT GCC-3' (SEQ ID
NO:9)), ND6-F (5'-GGA GAT CTT GAT GTA TGA GGT TGA TGA TGT TGG A-3'
(SEQ ID NO:10)) and ND6-R (5'-CCC GCA MC AAA GAT CAC CC-3' (SEQ ID
NO:11)). The following PCR cycle parameters were used for each of
the RT-PCR reactions: 95.degree. C. for 5 minutes, followed by 30
cycles of 95.degree. C. for 30 s, 60.degree. C. for 45 s and
72.degree. C. for 30 s, and finally 72.degree. C. for 10 minutes
16S rRNA-R and ND6-R primers were used as gene-specific primers for
the first strand synthesis of 16S rRNA and ND6 transcription,
respectively, and the 16S rRNA-F and MusMt-MIuIA primer set or
ND6-F and ND6-R primer set was used for subsequent PCR
amplification, respectively. Expected band sizes of the RT-PCR
products were 232 by for 16S rRNA and 123 by for ND6,
respectively.
Example 9
Cloning the Mouse Mitochondrial Genome in E. coli
[0052] A scheme was devised for cloning complete mitochondrial
genomes in E. coli that uses an in vitro transposition reaction to
insert an E. coli origin of DNA replication (ori) and selectable
marker at random locations into circular mtDNA. To make a synthetic
transposon containing the y-ori from the R6K plasmid (Kolter &
Helinski, 1982, J. Mol. Biol., 161: 45-56; Shafferman et al., 1982,
J. Mol. Biol., 161: 57-76) and the CmR gene, a PCR amplification
was performed using linearized plasmid p2C.gamma.CmR as template
and the PCR primers MESalCmR and MESma .gamma. ori. These primers
contain the 19 by Tn5 transposase recognition sequences (mosaic end
or ME) and the unique restriction sites for SaII and SmaI,
respectively, at their 5' ends and plasmid-specific sequences at
their 3' ends. The 1.5 kb PCR-amplified transposon, therefore,
consisted of the CmR marker and the .gamma.-ori flanked by inverted
Tn5 ME sequences at each of its ends. To perform the in vitro
transposition reaction, this linear synthetic transposon was
incubated with hyperactive Tn5 transposase and isolated, circular
mouse mtDNA. The products from this transposition reaction were
electroporated into an E. coli strain containing a chromosomal copy
of the R6K pir gene, which encodes the replication initiator
protein needed for .gamma.-ori replication (Kolter & Helinski,
supra; Shafferman et al., supra), and transformants were selected
on Cm plates.
[0053] Three transposon-inserted mouse mtDNA clones obtained using
this cloning strategy were characterized by restriction enzyme
mapping and sequencing. FIG. 1 shows the schematic representation
of the mouse mtDNA clones. All of the transposons were inserted
into the mouse mtDNA in the same orientation. When the transposon
junctions of these clones were sequenced using vector-specific
sequencing primers, it was found that the transposons were inserted
into the ND1, COXII and ND5 genes on the mouse mtDNA at nucleotides
3347, 7688 and 13371, respectively. Tn5 transposition reactions
typically generate a 9 by target duplication immediately flanking
the transposon insertion site and these sequence duplications were
found in each of the mouse mtDNA clones. Extensive sequencing of
these clones identified several sequence polymorphisms present in
the mtDNA of the mouse strain used as the source of the mtDNA but
further confirmed the overall sequence integrity of the
constructs.
Example 10
Comparison of the Stability of Mouse mtDNA Clones in E. coli at Low
and High Copy Number
[0054] To examine the stability of the cloned mouse mtDNA in E.
coli, the three mapped mouse mtDNA clones were transformed into one
of two E. coli strains. One of these strains contained the
wild-type pir gene (pirwt) in the chromosome and replicated the
mtDNA clones at a moderately low number of copies/cell (-10-15
copies/cell), and the other strain contained a mutant pir gene
(pir116) (Greener et al., 1990, Mol. Gen. Genet., 224: 24-32) that
replicated the clones at a relatively high number of copies/cell
(200 copies).
[0055] After transforming the mtDNA plasmids into the pirwt strain,
cultures were grown in LB medium at 30 or 37.degree. C., plasmid
DNA was isolated at regular time intervals and the restriction
patterns of these DNA preparations were compared. No deletions or
rearrangements were observed in the mouse mtDNA fragments even
after several days of culture at either slow growth (30.degree. C.)
or rapid growth (37.degree. C.) temperatures. When the mouse mtDNA
clones were transformed into the pir116 strain, however, the
transformation efficiency using the same standard chemical
transformation method was dramatically lower than that seen with
the pirwt strain (Table 2). The mtDNA clones were electroporated
into the pir116 strain in order to obtain more transformants, and
restriction analyses of plasmid DNA isolated from the few colonies
that were formed was performed. It was found that almost all of
these colonies contained plasmids in which all or most of the mouse
mtDNA sequence was deleted.
TABLE-US-00002 TABLE 2 Transformation efficiency of the mouse mtDNA
clones in two different E. coli strains Transformation efficiency
DH5.alpha. Colonies/.mu.g DNA DH5.alpha. Plasmid
.lamda.att::pirwt.sup.a .lamda.att::pir116.sup.a P2C.gamma.Cm.sup.R
1.2 .times. 10.sup.5 1.3 .times. 10.sup.5 pMusMtTN-ND1 7.5 .times.
10.sup.3 1 pMusMtTN-COXII 9.0 .times. 10.sup.3 2 pMusMtTN-ND5 8.5
.times. 10.sup.3 1
Example 11
Identification of mtDNA Fragments with Transcriptional Promoter
Activity in E. coli
[0056] It was hypothesized that transcription of mtDNA in E. coli
was a likely underlying cause of the apparent toxicity of the mtDNA
sequences at high copy number. To test this hypothesis, mtDNA
fragments were cloned into a `promoter-trap` vector designed to
directly select recombinant clones in which a promoter-less CmR
(CAT) gene is transcribed only if an inserted DNA fragment has
promoter activity (FIG. 2A). AIuI, HaeIII and Sau3AI were used to
digest mouse mtDNA into relatively small fragments, which were
cloned into the HincII or BgIII sites upstream of the CAT gene in
pANTS .gamma.-Cm. Recombinant clones with inserts that had promoter
activity were selected by transforming them into an E. coli strain
that replicated the plasmids at high copy number (pir200) and
plated on LB-Cm plates. The mouse mtDNA fragments identified in
this screen are listed in Table 3. The plasmids containing these
inserts were transformed into the low copy E. coli strain (pirwt)
and the CAT activity generated from each clone was measured at both
low and high copy numbers (FIG. 2B). For each of the mtDNA fragment
clones, the CAT activity generated in the high copy strain was
significantly higher than that generated in the low copy strain,
with many of the fragments only producing background levels of CAT
activity at low copy numbers.
TABLE-US-00003 TABLE 3 Functional E. coli promoter-like sequences
identified in mouse mtDNA Clone No. Restriction Fragment Position
in mouse mtDNA.sup.a 1 HaeIII 2216-2857 2 HaeIII 2857-3336 3 Sau3AI
4276-5887 4 HaeIII 4671-6622 5 Sau3AI 5884-5991 6 AluI 6336-6717 7
AluI 7709-8254 8 Sau3AI 8165-8940 9 AluI 8491-9045 10 HaeIII
8812-9757 11 AluI 9045-9138 12 AluI 10978-11083 13 HaeIII
11119-11193 14 Sau3AI 12028-13403 15 AluI 13844-14913 16 Sau3AI
14749-15333 17 HaeIII 15190-15740
Example 12
Modification of pMusMtTN Plasmids in E. coli
[0057] Numerous modifications were made to the pMusMtTN plasmids
using standard cloning techniques. It was determined that these
plasmids are amenable to most alterations as long as the
recombinant clones were propagated in pirwt E. coli strains. To
determine if any segments of the mouse mtDNA genome contain
sequences that might make modifying or subcloning these segments
difficult, four segments spanning the entire mtDNA sequence were
each isolated and subcloned. The appropriate clones were digested
with the restriction enzyme pairs BgIII/MIuI, MIuI/BspEI,
BspEI/SphI, and SphII/BgIII (see FIG. 1), the smaller DNA segments
between these restriction sites were isolated and cloned into a
plasmid with a replication on from pBR322. The expected subclones
were obtained from each of these segments. These subcloned
fragments were then recloned back into the full pMusMtTN
constructs. No problems were experienced with any of these segments
except the SphI-BgIII fragment, from which only a small number of
recombinant clones were obtained. To further localize the source of
this cloning difficulty, this mtDNA segment was subdivided into
SphI-XhoI and XhoI-BgIII fragments. It was found that the SphI-XhoI
segment could be cloned with a normal level of efficiency whereas
the number of clones obtained from the XhoI-BgIII segment was once
again noticeably reduced. The XhoI-BgIII segment in the clones was
sequenced and found to still contain the full, unaltered mtDNA
sequence.
Example 13
In Organello RNA Synthesis from Electroporated Mitochondria Using
Cloned Mouse mtDNA
[0058] p2C .gamma.SSB (FIG. 3) was constructed as a control
template to develop a RT-PCR assay for transcription of foreign DNA
introduced into purified mitochondria. This plasmid was designed so
that the CAT gene of the plasmid would be transcribed from the
mouse mitochondrial H-strand promoter (HSP) once the construct was
introduced into physiologically active mitochondria. p2C .gamma.SSB
also contain the D-loop region and L-strand promoter (LSP)
sequences from the mouse mitochondrial genome.
[0059] Electroporation was used to introduce p2C.gamma.SSB into
wild-type and mtDNA-less (.rho.0) mitochondria purified from mouse
LL/2 and .rho.0 LL/2 cell lines, respectively. In order to optimize
the electroporation reaction, multiple electroporation reactions
were performed with field strengths ranging from 10 to 16 kV/cm (25
pF capacitance, 400 resistance). The electroporated mitochondria
were incubated in a buffer suitable for in organello RNA synthesis,
total RNA was isolated from these mitochondria, and RT-PCR analysis
was performed to identify the RNA transcribed from the
electroporated DNA. For these experiments, the RNA transcribed
across the CAT gene by the HSP mitochondrial promoter was analyzed
using the CAT-specific primers CmR-R and CmR-F. Mitochondrial RNA
transcripts were clearly detected from DNA electroporated into both
wild-type and .rho.0 mitochondria. Positive signals were detected
for both 12 and 14 kV/cm electroporation conditions. No signal was
detected in the control reactions without reverse transcriptase
(-RT) or in the no electroporation controls, in which plasmid
p2C.gamma.SSB was mixed with purified mitochondria without
electroporation.
[0060] Once robust electroporation and transcription assay
conditions had been established, the mouse mtDNA clone
pMusMtTN-COXII was electroporated into purified mouse .rho.0
mitochondria and in organello RNA synthesis was performed as
described herein. In this experiment, RT-PCR was used to assay for
the expression of the 16S rRNA and ND6 genes, which are transcribed
from HSP and LSP, respectively. RNA transcripts were clearly
detected from each strand of the modified mitochondrial genome
electroporated into the .rho.0 mitochondria. These experiments were
performed using a variety of electroporation conditions. Results
determined that using 16 kV/cm for the electroporation reaction
most often resulted in the most efficient transfer of the 17.8 kb
mouse mtDNA construct into transcriptionally active .rho.0
mitochondria.
Example 14
Full Genomic Constructs are Replicated with a High Degree of
Fidelity in Yeast Mitochondria
[0061] FIG. 4 shows a schematic of the methods used to make
transmitochondrial yeast cells. The DNA that is targeted to the
yeast mitochondria carries a wild-type copy of the yeast
mitochondrial COX2 gene and is mixed with a second plasmid
designated pRS426 that carries a nuclear genetic marker (URA3) and
a yeast DNA replication origin (ARS) (Christianson et al., 1992,
Gene, 110: 119-22; and GenBank Accession No. UO3451). This DNA
mixture is then combined with inert tungsten carrier particles and
is introduced into a lawn of .rho.0 yeast cells (MCC109.rho.0
cells) plated on uracil deficient selection plates using a
biolistic transformation apparatus (PDS-1000/He; BioRad) using a
previously described procedure (Fox et al., 1988, PNAS USA, 85:
7288-92 and Bonnefoy & Fox, 2002, Methods Enzymol., 350:
97-111).
[0062] Yeast colonies that grew on uracil deficient selection
plates after bombardment were a result of nuclear transformation
with the URA3 gene. In order to find the fraction of cells
(.about.0.1%) that have incorporated exogenous DNA into their
mitochondria as well as into their nucleus, the colonies from the
bombardment plate were replica plated onto a lawn of a "tester"
yeast strain. This tester strain (MCC124) is of the opposite mating
type than the transformed cells and has a full yeast mitochondrial
genome with a point mutation in the mitochondrial COX2 gene
(.rho.+cox2). The mitochondrial networks of the two cell types fuse
after mating, and those bombarded cells that have a functional COX2
gene in their mitochondria complement the cox2 mutation of the
tester strain. These mated cells are themselves replica plated onto
non-fermentable growth media on which only those mating progeny
with full mitochondrial function can grow.
[0063] In initial experiments using the above-described
mitochondrial transformation procedure, several different COX2
fragments were tested to determine the optimal marker. mtDNA
fragments that included both the COX2 promoter and full coding
frame were the easiest to score in the screening assay. Therefore,
the full-length COX2 was used for most of the subsequent
experiments. As noted by Fox et al. (1988, PNAS USA, 85:
7288-7292), the transcribed COX2 genes apparently complement the
cox2 mutation in trans without requiring recombination between the
two mitochondrial genomes. The resulting increased complementation
efficiency has the added benefit of allowing the use of a color
selection on the original mating plate to determine which colonies
contain functional mitochondria. The .rho.0 strain used in the
transformations described herein has an ade2 mutation that leads to
accumulation of an intermediate in the adenine biosynthesis
pathway. In those cells that have fully functional mitochondria,
this intermediate is converted to a red pigment and the cells turn
pink. The cells that do not have mitochondrial function cannot
oxidize this intermediate and the cells remain white.
[0064] To make a clone containing the full mouse mitochondrial
genome without any gene disruptions, the transposon insertion in
the plasmid pMusMtEZTN-ND5 was moved to regenerate the complete ND5
and ND6 genes. The plasmid pMusMtEZTN-ND5 was double-digested with
XhoI and SaII, and a synthetic XhoI-SaII linker containing the 3'
end-sequences of the ND6 gene and an additional FspI site was
cloned between the XhoI and SaII sites. This cloning regenerated
the entire ND6 gene. To regenerate an uninterrupted ND5 gene, the
wild type mouse XhoI/SphI mtDNA fragment from the plasmid
pMusMtEZTN-ND1 was cloned between the XhoI and SphI sites of
p2C.gamma.ApR-Mt. This vector contained a modified multiple cloning
sites (MCS) that included the 3' end sequences of the ND5 gene from
the mtDNA XhoI site to the stop codon of the ND5 gene. The
NotI/SphI fragment from this construct, which contained the
.gamma.-ori and mtDNA sequences from the end of ND5 to the SphI
site, was isolated and was used to replace the NotI/SphI fragment
of the ND6-linker modified pMusMtEZTN-ND6 construct. A SmaI/KpnI
DNA fragment containing the complete COX2 gene from yeast mtDNA was
generated by PCR and introduced into the SmaI/KpnI of this plasmid
clone to generate the mouse mtDNA construct used in yeast
bombardment experiments (FIG. 5).
[0065] Modification of the pMusMtEZTN-ND5 results in two XhoI sites
flanking the E. coli vector sequences (.gamma.-ori+CmR) and the
yeast screening marker (COX2). Digesting the vector with XhoI to
remove the E. coli vector sequences and self-ligating the
mitochondrial DNA sequences would allow intact and entire mouse
mitochondrial genome to be recovered from this vector.
[0066] A Southern analysis of two transmitochondrial clones grown
to stationary phase five times sequentially on YPD media was
performed. Although a separate analysis of individual colonies
isolated during this culturing process indicated that some cells do
lose the mouse mitochondrial genome, no significant rearrangements
or deletions were detected in the mouse genomic constructs.
Although mtDNA replication in mouse mitochondria is strictly
dependent on transcription, these results confirm that replication
of the mouse mitochondrial genome in yeast is completely
independent of transcription from the mouse mitochondrial
promoters. By transferring this genome into yeast, therefore, an in
vivo experimental system has been generated in which most aspects
of mammalian mitochondrial transcription can be tested without
jeopardizing either mitochondrial DNA integrity or cell
viability.
Example 15
Species-specific Control of Mitochondrial Transcription
[0067] The gene replacement strategy shown in FIG. 6 was used to
generate single gene replacement yeast strains in which the mouse
POLRMT, TFB2M, or TFAM genes were inserted in-frame immediately 3'
of the mitochondrial targeting sequence of their respective
homologous yeast genes. A yeast strain was also generated in which
the TFB2M gene was inserted into the yeast COX4 gene. This strain
was generated because the yeast homolog of TFB2M, MTF1, does not
have a known mitochondrial targeting sequence, while COX4 does. The
chromosomal location of each of these insertions was confirmed by
PCR, and the precise recombination junctions and integrity of the
mammalian transcription genes were confirmed by sequencing.
Insertion strains in which the GFP gene is fused in-frame at the 3'
of each of these genes were generated to confirm that the mouse
transcription proteins are properly expressed and targeted to the
mitochondria.
[0068] The .rho.0 haploid strains were mated with a haploid strain
of opposite mating type that contains a synthetic mitochondrial DNA
construct (.rho.-) containing both a yeast and mouse mitochondrial
promoter to determine the effect of these gene insertions on
transcription from both mouse and yeast mitochondrial promoters.
The E. coli plasmid used to generate this .rho.-strain contains
both the HSP and LSP and full D-loop region of the mouse
mitochondrial genome as well as the yeast mitochondrial COX2 gene
and promoter (FIG. 7).
[0069] An oligonucleotide complementary to an internal site in
yeast mitochondrial C''OX2 (COX2PrEx2, 5'-ATC ATG TM TTC TM MT
AC-3' (SEQ ID NO:12)) or mouse mitochondrial D-loop region
(LSPrEx2, 5'-TCA TGC TTG TTA GAO ATA M-3' (SEQ ID NO:13); and
T7PRO2, 5'-CCT ATA GTG AGT CGT ATT A-3' (SEQ ID NO:14)) was
radiolabeled with [.alpha.-32P]ATP (3000 Ci/mmol; PerkinElmer) and
T4 polynucleotide kinase (Epicenter). The labeled oligonucleotide
was used as a primer in reverse transcription reactions using total
yeast RNA. The labeling reaction conditions are shown below in
Table 4. The components were combined, incubated at 37.degree. C.
for 30 minutes, and then incubated at 65.degree. C. for 5 minutes
to inactivate the T4 polynucleotide kinase.
TABLE-US-00004 TABLE 4 Labeling Reaction Conditions Component
Amount (.mu.l) DEPC-treated water 12.0 10X Kinase buffer 2.5 Primer
(20 .mu.M) 2.5 [.alpha.32P]ATP 6.0 T4 polynucleotide kinase (10
U/.mu.l) 2.0 Total 25.0
[0070] Total RNAs from yeast cultures were collected by isopropyl
alcohol precipitation and residual DNA contaminants were removed
using RNase-free DNase I (Promega) (Table 5). The components were
combined, and incubated at 37.degree. C. for 1 hour. 1.5 .mu.l of
stop solution was THEN added, and the reaction was incubated at
65.degree. C. for 10 minutes to inactivate the RNase-free DNase
I.
TABLE-US-00005 TABLE 5 Elimination of DNA Contamination from RNA
Preparations Component Amount (.mu.l) Total RNA (~50 .mu.g) 10.0
10X DNase I buffer 1.5 RNase-free DNase I (1 U/.mu.l) 1.0
DEPC-treated water 2.5 Total 15.0
[0071] Primer extension reactions were then carried out using a
SuperScript first-strand cDNA synthesis system (Life Technologies).
The reaction components of the primer extension reactions are shown
below in Table 6. After the reaction components were combined, the
reaction was slow cooled to anneal the primers by reducing the
temperature from 70.degree. C. to less than 37.degree. C. over a
15- to 20-minute interval. The reaction was put on ice for 1 minute
and the following reagents were added: 4.0 .mu.l of 10.times.
reverse transcriptase buffer, 4.0 pl of 0.1 M DTT, 1.0 .mu.l of
RNaseOut ribonuclease inhibitor (40 U/.mu.l), and 5.0 .mu.l of
DEPC-treated water. The reaction was then incubated at 42.degree.
C. for 2 minutes, 1.0 .mu.l of SuperScript II reverse transcriptase
(200 U/.mu.l; Invitrogen) was added, and the reaction was incubated
at 42.degree. C. for 50 minutes. The reaction was then terminated
by heating to 70.degree. C. for 15 minutes.
TABLE-US-00006 TABLE 6 Reaction Mix for Primer Extensions Component
Amount (.mu.l) DNase I-treated RNA 16.5 10 mM dNTPs 2.0 2 .mu.M
labeled primer 2.5 DEPC-treated water 4.0 Total 25.0
[0072] One-tenth volume (.about.4 .mu.l) of 3 M sodium acetate and
three volumes (120 .mu.l) of ethanol were added into each sample
and the samples were chilled at -70.degree. C. for 10 minutes. The
primer extension products were then precipitated by centrifugation
(13,200 rpm for 30 minutes) and washed with 80% ethanol for 5
minutes. The products were dried and resuspended with TE containing
RNase A (40 .mu.g/ml).
[0073] Each sample was heated to 92.degree. C. for 5 minutes to
denature the primer extension products after adding 10.times.
formamide buffer (23.75 ml formamide, 12.5 mg bromophenol blue,
12.5 mg xylene cyanol FF, 1 ml 0.5 M EDTA (pH 8.0), and 0.25 ml
sterile water). The samples were loaded on an 8% polyacrylamine-8 M
urea-TBE gel to fractionate the radiolabeled primer extension
products. The gel was then dried and exposed on an X-ray film for
autoradiography.
[0074] The level of COX2 transcript was measured by primer
extension experiments in the control haploid strains (.rho.+,
.rho.0, or synthetic .rho.-) and in diploid cells derived from
mating the .rho.- cells to the strains containing the mouse TFB2M
gene inserted into either the COX4 or the MTF1 gene. A similar
experiment was performed in which the level of the HSP transcript
was measured in the same RNA samples (i.e., the COX2 assay was
repeated on two samples). These results demonstrated that mouse
mitochondrial promoters are not functional in yeast, and that a
single mouse mitochondrial transcription protein is not sufficient
to activate mouse mitochondrial promoters.
[0075] Extension products of the size predicted for HSP
transcription initiation were detected when all three mouse
mitochondrial transcription genes were present in the strain,
whereas no HSP extension products were detected in control
reactions using RNA isolated from a similar strain containing only
two of the mouse transcription genes. The shorter HSP extension
products may result from RNA processing in the yeast mitochondria,
alternative transcription start sites, secondary structure in the
mouse tRNAphe sequence that leads to premature termination of the
reverse transcriptase extension product, or any combination
thereof.
OTHER EMBODIMENTS
[0076] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not limit the
scope of the invention, which is defined by the scope of the
appended claims. Other aspects, advantages, and modifications are
within the scope of the following claims.
Sequence CWU 1
1
14121DNAArtificial SequenceOligonucleotide 1acccaacgcg gcaaactaac c
21220DNAArtificial SequenceOligonucleotide 2tcttgttcgt ctgccaggct
20341DNAArtificial SequenceOligonucleotide 3ctgtctctta tacacatctg
tcgacagaag ccactggagc a 41443DNAArtificial SequenceOligonucleotide
4ctgtctctta tacacatctc ccgggctaat tctgtcagcc gtt 43524DNAArtificial
SequenceOligonucleotide 5gtacctataa ccagaccgtt cagc
24621DNAArtificial SequenceOligonucleotide 6cagcggcatc agcaccttgt c
21720DNAArtificial SequenceOligonucleotide 7gcagccacca ataaagaaag
20820DNAArtificial SequenceOligonucleotide 8ggaccctcgt ttagccgttc
20930DNAArtificial SequenceOligonucleotide 9aggtggatta tttatagtgt
gattattgcc 301034DNAArtificial SequenceOligonucleotide 10ggagatcttg
atgtatgagg ttgatgatgt tgga 341120DNAartificial
sequenceoligonucleotide 11cccgcaaaca aagatcaccc 201220DNAArtificial
SequenceOligonucleotide 12atcatgtaat tctaaaatac 201320DNAArtificial
Sequenceoligonucleotide 13tcatgcttgt tagacataaa 201419DNAArtificial
Sequenceoligonucleotide 14cctatagtga gtcgtatta 19
* * * * *