U.S. patent application number 14/276743 was filed with the patent office on 2015-04-02 for transformation of algal cells.
The applicant listed for this patent is Aurora Algae, Inc.. Invention is credited to Oliver Kilian, BERTRAND VICK.
Application Number | 20150093830 14/276743 |
Document ID | / |
Family ID | 41398588 |
Filed Date | 2015-04-02 |
United States Patent
Application |
20150093830 |
Kind Code |
A1 |
VICK; BERTRAND ; et
al. |
April 2, 2015 |
TRANSFORMATION OF ALGAL CELLS
Abstract
Exemplary methods include a method for transforming an algal
cell by preparing a transformation construct, preparing a particle
for bombarding the algal cell, adhering the transformation
construct to the particle, bombarding the algal cell with the
particle, and growing the algal cell into a colony. The
transformation construct is replicated within a nuclear genome of
the algal cell and the growing of the algal cell is in a nutrient
medium. Another exemplary method may include a method for
genetically modifying an algal cell, by adding nucleic acid to the
algal cell while the algal cell is suspended in a solution of low
conductivity, introducing the nucleic acid into the algal cell by
application of an electrical pulse resulting in a transformed algal
cell, and selecting a colony that includes the transformed algal
cell.
Inventors: |
VICK; BERTRAND; (Oakland,
CA) ; Kilian; Oliver; (Alameda, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Aurora Algae, Inc. |
Hayward |
CA |
US |
|
|
Family ID: |
41398588 |
Appl. No.: |
14/276743 |
Filed: |
May 13, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13372467 |
Feb 13, 2012 |
8759615 |
|
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14276743 |
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12480611 |
Jun 8, 2009 |
8119859 |
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13372467 |
|
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61059672 |
Jun 6, 2008 |
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Current U.S.
Class: |
435/446 |
Current CPC
Class: |
C12N 1/12 20130101; C12N
15/79 20130101; C12N 15/90 20130101; C12N 15/8207 20130101; C12N
15/8247 20130101; C12N 15/8237 20130101; C12N 15/8206 20130101 |
Class at
Publication: |
435/446 |
International
Class: |
C12N 15/90 20060101
C12N015/90 |
Claims
1. A method for genetically modifying an algal cell, the method
comprising: adding nucleic acid to the algal cell while the algal
cell is suspended in a solution of low conductivity, the nucleic
acid including a gene for a fatty acid; introducing the nucleic
acid into the algal cell by application of an electrical pulse
resulting in a transformed algal cell; and selecting a colony that
includes the transformed algal cell.
2. The method of claim 1, wherein the nucleic acid includes a
transformation construct.
3. The method of claim 1, wherein the electrical pulse is applied
with a field strength of higher than approximately 7500 V/cm.
4. The method of claim 1, wherein the electrical pulse is applied
with a field strength of higher than approximately 9000 V/cm.
5. The method of claim 1, wherein the electrical pulse is applied
with a field strength of higher than approximately 10,000 V/cm.
6. The method of claim 1, wherein the electrical pulse is applied
with a shunt resistor of greater than approximately 100 OHM.
7. The method of claim 1, wherein the algal cell is of genus
Nannochloropsis.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] The present application is a continuation of U.S.
Non-Provisional patent application Ser. No. 12/480,611 filed on
Jun. 8, 2009, titled "Transformation of Algal Cells," which claims
the benefit and priority of U.S. Provisional Patent Application
Ser. No. 61/059,672 filed on Jun. 6, 2008, titled "VCP-Based Vector
for Nannochloropsis Transformation," all of which are hereby
incorporated by reference.
[0002] The present application is related to U.S. Non-Provisional
patent application Ser. No. 12/480,635 filed on Jun. 8, 2009,
titled "VCP-Based Vectors for Algal Cell Transformation," which is
hereby incorporated by reference.
REFERENCE TO SEQUENCE LISTINGS
[0003] The present application is filed with sequence listing(s)
attached hereto and incorporated herein by reference.
BACKGROUND OF THE INVENTION
[0004] 1. Field of the Invention
[0005] This invention relates to molecular biology, and more
specifically to the transformation of algal cells.
[0006] 2. Description of Related Art
[0007] Transformed algae cells may be useful in aquaculture
production. The transformation of small algal cells with strong
cell walls, however, is difficult to achieve. Accordingly, there is
a need for improved methods of algal cell transformation.
SUMMARY OF INVENTION
[0008] Exemplary methods include a method for transforming an algal
cell by preparing a transformation construct, preparing a particle
for bombarding the algal cell, adhering the transformation
construct to the particle, bombarding the algal cell with the
particle, and growing the algal cell into a colony. The
transformation construct is replicated within a nuclear genome of
the algal cell and the growing of the algal cell is in a nutrient
medium.
[0009] Another exemplary method may include a method for
genetically modifying an algal cell, by adding nucleic acid to the
algal cell while the algal cell is suspended in a solution of low
conductivity, introducing the nucleic acid into the algal cell by
application of an electrical pulse resulting in a transformed algal
cell, and selecting a colony that includes the transformed algal
cell.
BRIEF DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1 shows a sequence of an exemplary transformation
construct.
[0011] FIG. 2 illustrates an exemplary method for transforming an
algal cell with a particle gun.
[0012] FIG. 3 shows an exemplary polymerase chain reaction result
obtained using ble primers as described in connection with Example
One.
[0013] FIG. 4 shows the exemplary polymerase chain reaction result
obtained as described in connection with Example Two.
[0014] FIG. 5 shows the number of algal cell transformants obtained
based on the field strength of electroporation applied to perform
the transformation as described in connection with Example
Three.
[0015] FIG. 6 shows the number of transformants obtained under
exemplary optimal electroporation conditions, while varying the
amount of transformation construct DNA used as described in
connection with Example Four.
[0016] FIG. 7 illustrates an exemplary method for transforming an
algal cell with electroporation.
DETAILED DESCRIPTION OF THE INVENTION
[0017] Provided herein are exemplary methods for transforming algal
cells. Transformed algae cells may be useful in aquaculture
production. The transformation of small algal cells with strong
cell walls, however, is difficult to achieve. Various exemplary
embodiments of the present invention are useful in the efficient
transformation of Nannochloropsis, a microalga of about 3-5
micrometers in size.
[0018] Typically, gold particles having a size of about 0.6
micrometers are used in biolistic transformation of algae smaller
than about 5 micrometers. A transformation vector or transformation
construct is precipitated onto the gold particles, and the gold
particles carrying the transformation construct are propelled
through the cell walls of the algae. The transformation efficiency
of Nannochloropsis with this method is very low.
[0019] According to the various exemplary methods provided herein,
aggregates of gold particles are broken into smaller fragments to
increase transformation efficiency. Using a 1510 Branson Bath
Sonicator, 0.6 micrometer gold particles are sonicated for one hour
just prior to precipitating a transformation construct onto the
gold particles. Using the sonicated gold particles or beads,
fourteen micrograms of transformation construct may produce
approximately twenty zeocine resistant transformants. Thus, this
exemplary embodiment provides about a twenty-fold increase in
transformation efficiency over currently available methods.
[0020] An alternative exemplary embodiment uses an improved
electroporation method to transform algal cells. In
electroporation, algal cells are subjected to an electric current
that opens transient pores in the membranes of the algal cells. A
transformation vector may pass through an open pore, eventually
leading to the successful transformation of the algal cell. Using a
Biorad Genpulser I electroporator set to the exponential protocol,
the following electroporation conditions were used: 2200V,
500.OMEGA. shunt resistor, 50 .mu.F capacity, 2 mm gap cuvette.
When 10 9 cells have been used in iso-osmotic non electrolyte
buffer (resistance higher than 1100.OMEGA.) in a volume of 100
microliters, typical time constants .tau. of 20-24 ms have been
reported by the device. This approach produced hundreds of zeocine
resistant transformants per microgram of transformation construct
used.
[0021] FIG. 1 shows the sequence of an exemplary transformation
construct. The exemplary transformation construct 100 includes a
promoter sequence 102, DNA encoding a transcribed but untranslated
5' region ("UTR") 104, an intron 106, a ble gene 108 and DNA
encoding a 3' UTR 110. The transformation construct may comprise
any number of promoters, genes, and/or other nucleic acid polymers
(naturally occurring or synthetic) and/or their analogs, or other
compounds that do not interfere with the ability of the
transformation construct to enter the algal cell or the algal
genome, or to function. In some embodiments, additional nucleotides
may appear in the transformation construct to facilitate or direct
the insertion of the construct (or any part thereof) into a desired
location in the genome.
[0022] The various exemplary methods described herein may be
employed using the exemplary transformation vectors or
transformation constructs PL90, H8 and B9 as described in U.S.
Non-Provisional patent application Ser. No. 12/480,635 filed on
Jun. 8, 2009, titled "VCP-Based Vectors for Algal Cell
Transformation," which is hereby incorporated by reference. The
difference between the three exemplary vector constructs is the
type of selection marker gene (SG) used: the sh ble gene (PL90),
the hygromycin B phosphotransferase gene (H8), or the blastocidin S
deaminase (B9) gene.
[0023] FIG. 2 illustrates an exemplary method for transforming an
algal cell with a particle gun.
[0024] At step 205, a desired transformation construct is prepared.
According to one exemplary embodiment, the transformation construct
may be either the PL90 transformation vector, the H8 transformation
vector, or the B9 transformation vector.
[0025] At step 210, small DNA-binding particles are prepared.
According to one exemplary embodiment, 0.6 .mu.m gold particles are
sonicated in buffer for one hour.
[0026] At step 215, the transformation construct is adhered to the
small DNA-binding particles. In one exemplary method,
transformation constructs are precipitated onto the gold
particles.
[0027] At step 220, algal cells are bombarded with the gold
particles having the transformation constructs precipitated onto
them. Nannochloropsis cells, according to one exemplary method, are
bombarded with fourteen micrograms of transformation construct DNA
that has been precipitated onto 0.42 mg of the 0.6 .mu.m gold
particles.
[0028] At step 225, the algal cells are grown into colonies on
selective medium. According to one exemplary embodiment, if the
transformed cells produce a large quantity of a desired gene
product, the cells may be further processed to collect the desired
product. One having ordinary skill in the art will recognize that
many appropriate buffers, media, and/or methods of product
collection may be used.
Example One
[0029] Nannochloropsis oceanica cells were grown in low light (85
.mu.E/(m2*s)) to mid log phase in F2 media, 50% seawater (See e.g.,
Guillard, R. R. L. and Ryther, J. H. 1962. Studies of marine
planktonic diatoms. I. Cyclotella nana Hustedt and Detonula
confervacea Cleve. Can. J. Microbiol. 8: 229-239). Nannochloropsis
cells were then pelleted at 2500 g for 15 min. The pellet was then
resuspended in fresh media. 5*10 9 resuspended cells were spread on
petri dishes (F2 media, 50% seawater, 1% Bactoagar.TM. from DIFCO)
and allowed to dry. 0.6 .mu.m gold particles (Biorad) were coated
essentially as recommended by Biorad with transformation vector
PL90. The plated cells were then bombarded with microparticles
(coated with transformation construct DNA as recommended by Biorad)
by a particle gun (Biolistic PDS-1000/He particle gun Bio-Rad) at
vacuum pressure of 29 mm Hg utilizing the heptameric adapter from
Biorad. All different stages were used in combination with 3
different rupture discs (1100 PSI, 1350 PSI, 1550 PSI) provided by
Biorad. Cells were subsequently resuspended in 10 ml fresh F2
Medium (half salinity) and allowed to recover overnight under low
light (.about.10-25 .mu.E/(m2*s)). 0.5*10 7 cells were then plated
on agar plates (0.8% Bactoagar.TM. from DIFCO) containing the
selection agent (2 .mu.g/ml zeocine, .mu.g/ml 300 hygromycin or 50
.mu.g/ml blasticidin, depending on the transformation construct
used) and incubated at 25.degree. C. at .about.50 .mu.E/(m2*sec).
Negative control DNA was linearized pJet1 vector DNA (Fermentas).
Growth of colonies could be observed after .about.2-3 weeks.
Colonies were then analyzed for the presence of the selection
marker gene. Colonies on selective media were never obtained in
experiments utilizing the control DNA. Only utilization of 1350 PSI
rupture disc, stage 1 (uppermost stage, just underneath heptameric
adapter) and 1550 PSI rupture disc stage 2 yielded a few
transformants under these conditions.
[0030] To proof for the presence of transformation construct within
the cells (and thus that the transformation experiment was a
success), a single colony was picked and restreaked on unselective
medium and allowed to grow for approximately one week. Cells were
then scraped (.about.10 .mu.g cells), washed 3 times in F2-50%
seawater, incubated with 30 units DNAse I in DNAse I buffer
(Fementas) 1 hour at 37.degree. C. Cells were then washed once in
F2-50% seawater and resuspended in 50 .mu.l F2-50% seawater. Cells
were pelleted and the supernatant collected; cells were resuspended
in 50 .mu.l A ddH20. Cell and supernatant fractions were incubated
7 minutes at 95.degree. C.; 5 .mu.l each fraction were subjected to
PCR (50 .mu.l reaction volume) with specific primers against the
ble gene (rev: 5'TTA GTC CTG CTC CTC GGC CAC GAA3', for: 5'ATG GCC
AAG TTG ACC AGT GCC GT3').
[0031] The PCR program was:
[0032] 1. 94 C, 3 min, 1 cycle;
[0033] 2. 94 C, 15 sec;
[0034] 3. 58 C, 30 sec;
[0035] 4. 72 C, 30 sec (steps 2-4 are amplification stage, 38
cycles);
[0036] 5. 72 C, 5 min; and
[0037] 6. 4 C, cool down and keep temperature.
[0038] FIG. 3 shows the exemplary polymerase chain reaction result
obtained using ble primers as described in connection with Example
One.
Example Two
[0039] We followed the procedures of Example One, except this time
utilizing a 1510 Branson Bath Sonicator, 0.6 .mu.m gold particles
were sonicated for 1-hour just prior to DNA precipitation onto
beads. Using the sonicated beads, 14 .mu.g DNA produced
approximately 20 zeocine-resistant transformants.
[0040] Detailed Procedure.
[0041] Growth and preparation of cells: the cells were grown in 2 L
flasks in F2 medium (containing seawater of half salinity) to early
log phase. The cells were then harvested by centrifugation for 10
min. at 2500 and gently resuspended in fresh F2 medium. 5*10 9
cells (in .about.400 .mu.l) were then plated on an agar plate
(containing 1% Bactoagar.TM. from DIFCO) and allowed to dry for
.about.20 minutes in a sterile hood.
[0042] Preparation of Particles.
[0043] Different sizes and materials of particles were used (all
obtained from Biorad), i.e. Tungsten particles 0.7 and 1 .mu.m
average diameter and gold particles 1 .mu.m or 0.6 .mu.m average
diameter. Prior to the precipitation of the DNA onto the particles
(the micro carrier), the particles were resuspended in ddH2O and
sonicated for 1 hour in a 1510 Branson Bath Sonicator. 3 mg
particles were coated with 100 .mu.g linearized transformation
construct DNA as described elsewhere (see user manual Biolistic
PDS-1000/He particle gun from Bio-Rad) and resuspended in 500 .mu.l
Ethanol after 2 washing steps in the same volume ethanol. 10 .mu.l
of this DNA coated particle solution (`the microcarrier`) was
pipetted onto each macrocarrier which was fitted subsequently into
the Hepta Adapter (optional component of the Biolistic.RTM.
PDS-1000/He Particle Delivery system). Thus, each shot with the
particle gun provided 7.times.2 .mu.g=14 .mu.g DNA precipitated on
micro carrier. Coated particles were used for biolistic
transformation within an hour.
[0044] Delivery of Particles to Cells.
[0045] The petri dish containing the cells was placed on the
corresponding stage within the particle delivery system (stage 1
being the nearest to the heptamer adapter, stage 3 being the one
most far away). One of three different rupture discs (Biorad) was
used: 1100 psi, 1350 psi, 1550 psi. Vacuum was generally applied to
29 mm Hg (obtained within .about.20 sec.) and the He pressure was
increased at max speed in order to provide the He shock wave after
rupture of the rupture disc. The pressure in the delivery chamber
was instantly increased at maximal rate (less than 10 sec until
atmospheric pressure was reached) and the petri dish with the
bombarded cells was recovered.
[0046] Post Delivery Treatment of Cells.
[0047] The cells were gently scraped off the plate resuspended in
10 ml of F2 medium (half salinity compared to seawater) and allowed
to recover in low light (.about.14 .mu.E/(m2*s)) over night. Next
day, 0.5*10 7 cells were plated on F2 agar plates (petri dish, F2
medium with half salinitiy compared to seawater and 0.8%
Bactoagar.TM. from DIFCO) and the respective selection agent.
Plates were incubated at 25.degree. C. at 50 .mu.E/(m2*sec).
Colonies were visible after .about.2-3 weeks.
[0048] Results.
[0049] 1. From all the particles tested, only the 0.6 .mu.m gold
particles produced transformants.
[0050] 2. We tested all different combinations of stage position
(within the particle delivery system) and rupture disc.
[0051] 3. A major difference was observed when we pretreated the
gold particles with ultrasound.
[0052] The following table reflects the quantity of transformants
observed when using different experimental parameters. The vector
used in these experiments was PL90 conferring resistance against
the drug zeocine.
TABLE-US-00001 Gold particles pretreatment.sup.1 no no no yes yes
yes Rupture 1100 1350 1550 1100 1350 1550 disc psi psi psi psi psi
psi Stage 1 0 1 0 11 4 77 2 0 0 2 25 4 22 3 0 0 0 8 4 5 .sup.1gold
particles were washed and then disrupted by 1 h ultra sound
treatment
[0053] These results indicate that:
[0054] 1. Pretreatment of the 0.6 .mu.m gold particles by ultra
sound dramatically increases the transformation efficiency.
[0055] 2. That the transformation efficiency is highest at 1100 Psi
pressure (rupture disc) and if the petri dish containing the cells
is localized on stage 2.
[0056] 3. The vector PL90 may be used for the nuclear
transformation of Nannochloropsis by conferring resistance against
the drug zeocine.
[0057] FIG. 4 shows the exemplary polymerase chain reaction result
obtained as described in connection with Example Two.
Example Three
Transformation Via Electroporation
[0058] Detailed Procedure.
[0059] Growth and Preparation of Cells.
[0060] Nannochloropsis oceanica was grown in 2 L flasks in F2
medium (containing seawater of half salinity) to mid log phase. The
cells were then harvested by centrifugation for 10 min. at 2500 and
gently resuspended in 375 mM sorbitol. The cells were washed
several times in 375 mM sorbitol solution in order to minimize
conductivity of remaining medium. The cells were finally
resuspended to a final concentration of 10 10 cells/ml and used for
transformation within an hour.
[0061] Electroporation.
[0062] Electroporation was performed in a Biorad GenPulser I
Electroporator utilizing 2 mm cuvettes. 100 .mu.l cells at 10 10
cells/ml were pipetted into a 2 mm cuvette and varying amounts of
transformation construct in <5 .mu.l volume ddH2O added. The
cuvette containing the DNA-cell mixture was gently snipped for
mixing and then placed into the electroporation chamber.
[0063] Device settings were exponential decay protocol with 500 Ohm
Shunt resistance and 50 .mu.F capacity, 2 mm Gap
[0064] Post Delivery Treatment of Cells.
[0065] After electroporation the cells were allowed to stay in the
cuvette for 3 minutes before they were recovered and resuspended in
10 ml F2 medium (half salinity). After an overnight incubation in
low light (.about.14 .mu.E/(m2*s), 0.5*10 7 cells were plated on F2
agar plates and the respective selection agent. Plates were
incubated at 25.degree. C. at 50 .mu.E/(m2*sec). Colonies were
visible after .about.2-3 weeks.
[0066] Results.
[0067] In initial experiments, we used 0.5 .mu.g DNA/100 .mu.l cell
suspension and varied the voltage. We routinely measured electrical
resistance of the cell-DNA mixture with the respective option in
the Biorad GenPulser I Electroporator to ensure that resistance was
>1100 Ohm. The actual electroporation usually returned
exponential decay times .tau. of 20-24 ms. Initially, when
performing these experiments with varying field strengths we got a
single transformant (again we used the PL90 vector linearized and
performed selection on agar plates containing zeocine) at the
highest field strength initially tried (which was 1000V, =5 kV/cm).
5 kV/cm is a very high field strength and we did not expect that we
might obtain transformants at all at such a high field strength.
Since we obtained a single transformant at this high voltage we
performed electroporations in additional experiments at very high
voltages.
[0068] FIG. 5 shows the number of algal cell transformants obtained
based on the field strength of electroporation applied to perform
the transformation as described in connection with Example Three.
The result of this experiment as shown in FIG. 5 was very
surprising: transformation was successful at field strengths equal
or higher than 8 kV/cm and efficiency peaked at 10.5 kV/cm. Note
that applied field strengths for the electroporation of algae are
typically factor 10 lower. Higher Field strengths than 12000V/cm
were not possible to apply (cuvettes arced).
[0069] We conclude that we can transform Nannochloropsis with the
vector PL90 if we treat the cells as indicated above.
Example Four
Ideal Electroporation
[0070] Parameters are (within Biorad GenPulser I Electroporator):
10 9 cells in 100 .mu.l 375 mM sorbitol (washed several times), 500
Ohm shunt resistor, 2 mm gap cuvette, 10500 volt/cm (=2100V/2 mm),
exponential decay program.
[0071] We then tested the efficiency of transformation by adding
varying amounts of DNA to the electroporation cuvette (again
containing 10 9 cells in 100 .mu.l).
[0072] FIG. 6 shows the number of transformants obtained under
exemplary optimal electroporation conditions, while varying the
amount of transformation construct DNA used as described in
connection with Example Four. We observed a linear increase in the
number of transformants obtained based on increasing the amount of
transformation construct DNA used. For example, 3 .mu.g of
transformation construct DNA added during a single electroporation
experiment yielded approximately 9000 transformants, 1.5 .mu.g of
transformation construct DNA yielded approximately 4500
transformants. This result indicates that no saturation with DNA
occurred and that the transformation method via electroporation
which we established is extremely efficient.
[0073] FIG. 7 illustrates an exemplary method for transforming an
algal cell with electroporation.
[0074] At step 705, a desired transformation construct is prepared.
According to one exemplary embodiment, the transformation construct
may be either the PL90 transformation vector, the H8 transformation
vector, or the B9 transformation vector.
[0075] At step 710, the transformation construct is added to algal
cells, while the algal cells are suspended in a solution of low
conductivity. In various exemplary embodiments, the solution is a
sorbitol solution.
[0076] At step 715, the transformation construct is introduced into
the algal cells by application of an electrical pulse, resulting in
transformation of the algal cells. According to some exemplary
embodiments, the electrical pulse is applied with a field strength
of higher than approximately 10,000 V/cm, and with a shunt resistor
of greater than approximately 100 OHM.
[0077] At step 720, the algal cells are grown into colonies on
selective medium. According to one exemplary embodiment, if the
transformed cells produce a large quantity of a desired gene
product, the cells may be further processed to collect the desired
product. One having ordinary skill in the art will recognize that
many appropriate buffers, media, and/or methods of product
collection may be used.
[0078] While various embodiments have been described above, it
should be understood that they have been presented by way of
example only, and not limitation. Thus, the breadth and scope of a
preferred embodiment should not be limited by any of the
above-described exemplary embodiments.
Sequence CWU 1
1
111820DNANannochloropsis 1ggcggtcttt tgtcctttcc tctatagccc
acccgtctag agggcacacg cgatgatctt 60tatatctctt catgtgtctt tgttttaact
aggatactgc cgggtgaatg cccatcggac 120aagaggccaa actctatcta
cacccttttg acttctgttg tggtcgtagt gtgtgcttgc 180atgccctgaa
agtccaggca tcccacttgt gctctaaccc cattcaaaac agcagaagtg
240cttaattaag atatagattc atgatctcct gtcccctcct tcttaccttt
tcacaaacct 300cacacagaag tctccactct tcgcctctaa aacctctttt
taaattatgg taagttcgtg 360cggcagtggg ttttcggatc tatatttgtc
aagatccagt tcaaggtcag ggatgtagat 420taagtacaga aggagaagca
caagcgcgcc agttcgcccc tcacggcctg gagcagggca 480tttaatccct
ctatcttacc agaaccatac tatacaacca atcctgttgg catcgctctg
540tctatttgtc gtgcgtgcat gtgtccatgg tgtggtgggg ggcaggggtt
ttcggggttg 600cggttgaagg caccttatca gaaagatgcc ctcagagata
gaggtagccc cctccccccg 660atcttcgacc agtcctgtca ggcgaacact
ttcacccgtc gttcacctcg ttacacacaa 720ggagtagacc tctgaagttc
taattgtcat aaatgcccct cccccctccc tctttccctt 780gatcttcccc
tccgagcaga ttatggccaa gttgaccagt gccgttccgg tgctcaccgc
840gcgcgacgtc gccggagcgg tcgagttctg gaccgaccgg ctcgggttct
cccgggactt 900cgtggaggac gacttcgccg gtgtggtccg ggacgacgtg
accctgttca tcagcgcggt 960ccaggaccag gtggtgccgg acaacaccct
ggcctgggtg tgggtgcgcg gcctggacga 1020gctgtacgcc gagtggtcgg
aggtcgtgtc cacgaacttc cgggacgcct ccgggccggc 1080catgaccgag
atcggcgagc agccgtgggg gcgggagttc gccctgcgcg acccggccgg
1140caactgcgtg cacttcgtgg ccgaggagca ggactaagct tctgtggaag
agccagtggt 1200agtagcagta gcagcagcag tagcagccgc agcactcagt
gttggcgcga gagattgtcc 1260atcccttctt aacctaccgg aagagaaata
aggcctttct cccgtagctg tcttcgtttg 1320tttgtgctga ttgcttgata
tgagagtgtt gaattcctgc atcatgtttt tctctgtagt 1380cctttcctac
ccccgtcatt ttcttttctc cctggttctt cttttgtcac ccttatttta
1440cataaaattt tctttgttta tagtgagagg aaggtagaga ggggaaaaca
agaacaacga 1500acgcaagcgt gtgaaaggag ggcgagtaga agagaaacag
atctgttgag cattgagagt 1560ggagccgggg gaaaggcttg tgtgttgtct
ttgaaaaagt tgtttaaatc acgaatccgt 1620tagttctcat gtgtacctct
ttcactacat gtgatggaga aaacaaaagt gtgaggatta 1680attgaagaaa
aagaagagtt cgacacgtca aaccgcccaa aagacgtcac aaagagaact
1740tgattctctt tgccgtgttg atcctgtctt ttcccccagc ttttcttgcc
acccgtggca 1800cacgagatgg acaagatcag 1820
* * * * *