J. Inst. Brew., May-June, 1985, Vol. 9\,pp. 169-173
169
DIFFERENTIATION OF BREWERY YEAST STRAINS BY RESTRICTION ENDONUCLEASE ANALYSIS
OF THEIR MITOCHONDRIAL DNA
By Sun Y. Lee, Finn B. Knudsen
(Research and Development Department, Adolph Coors Company, Golden, Colorado 80401)
and Robert O. Poyton
Department of Molecular, Cellular and Developmental Biology, University of Colorado at Boulder, Campus Box 347
Boulder, Colorado 80309
Received 24 July 1984
Mitochondrial DNA (mtDNA) was isolated from different strains of brewery yeast and digested with
various restriction endonucleases. The digestion products were separated by electrophoresis in
agarose gels. Of the twenty restriction endonucleases used, only two—Aval and Haelll—produced
different restriction fragment patterns when applied to the mtDNA from two strains of Saccharomyces
uvarum. The restriction fragment patterns produced by the other eighteen enzymes were identical.
Analysis of mtDNA from a strain of Saccharomyces cerevisiae with the same twenty restriction
endonucleases revealed several differences with respect to Saccharomyces uvarum. Taken together,
these results indicate that restriction endonuclease fragmentation patterns of mtDNA are useful as
diagnostic tools for distinguishing strains of ale and larger yeast.
Key words: Saccharomyces uvarum, Saccharomyces
cerevisiae, mitochondrial DNA; Electrophoresis.
Introduction
The differentiation of the yeasts relies on a number of
criteria including: modes of sexual and asexual reproduc
tion, carbohydrate utilization, cell wall composition and
serology,
and
the
nucleotide composition
of nuclear
DNA. -7 As noted previously,7 each of these criteria
frequently suffers from a lack of sensitivity and specificity.
In brewing, fermentation characteristics are often used as
the criteria for distinguishing different yeast strains from
one another. However, these criteria are also often not
specific enough to differentiate one production strain from
another.
Recently, restriction endonuclease fingerprinting of yeast
genomic DNA (i.e., both nuclear and mitochondrial DNA)
has proven effective in differentiating some yeast strains.4
For example, with this method it has been possible to
identify different groups of species within the genus
Saccharomyces. In view of the rapid rate of evolution
of mitochondrial DNA, relative to nuclear DNA,2pl° the
application of restriction endonuclease fingerprinting to
mtDNA has the potential for being far more sensitive in
yeast strain differentiation than its application to total
genomic DNA. Indeed, previous studies with mtDNA
restriction endonuclease fingerprints have already revealed
differences between several wild type laboratory strains
of Saccharomyces which had no detectable differences
otherwise.1'6*8 In the study reported here we have used
endonuclease fingerprinting of mtDNA to differentiate
important production strains of Saccharomyces uvarum
from one another and from Saccharomyces cerevisiae.
at 30°C for 7 to 8 hours on a rotary shaker (200 rpm). The
preculture was then seeded into a liter of YPD broth and
grown overnight at 30cC in an environmental shaker (New
Brunswick) 200 rpm. Cell growth was monitored turbidometrically with a Klett colorimeter (No. 54 green filter).
Preparation of Mitochondrial DNA.—Mitochondrial
DNA was prepared from yeast sphaeroplasts and isolated
by cesium chloride density jradient centrifugation as fol
lows (Fig. 1). Approximately 5-5 g (wet weight) of cells, in
the late lag to early stationary phase were harvested by
centrifugation at 4,000 xg for 5 min. The pelleted cells were
washed once with 100 ml of sterile distilled water, sus
pended in 01 M Tris base-2-5mM dithiothreitol at 0-2 g
(wet weight) per ml, and incubated at 30°C for 20 min on a
rotary shaker at 200 rpm. Cells were pelleted by centrifuga
tion at 4,000 xg for 5 min and then suspended at 0-5 g (wet
weight) per ml of 1-35 M sorbitol-01 M Na2 EDTA, pH
7-0. Zymolyase 60,000 (Mile Laboratories) was then added
at 05 mg/ml and the cells were converted to sphaeroplasts
by incubation at 30°C on a rotary shaker at 100 rpm.
YEAST CELLS
♦
SPHAEROPLASTS
LYSATE
Cs Cl GRADIENT CENTRIFUGATION
IN PRESENCE OF DAPI
NUCLEAR DNA
(Lower Band)
MITOCHONDRIAL DNA
(Upper Band)
DISCAR D
R EMOVAL OF DAPI WITH
ISOAMYLALCOHOL
Experimental methods
Yeast Strains.—Two production strains of Saccharo
myces uvarum, LA and LC, were obtained from the Coors
culture
collection.
Saccharomyces
cerevisiae
strain
D273-10B (Mat, ATCC #24657) was obtained from the
American Type Culture Collection.
Media and Cell Growth.—Yeast strains were grown in
liquid YPD medium,9 containing (per liter): 10 g of yeast
extract (Difco), 20 g of peptone (Difco), and 20 g of
dextrose. Solid medium was prepared by incorporating 2%
agar (20 g/1) into YPD liquid medium. Yeast strains used
for the preparation of mitochondrial DNA were precultured by inoculating a freshly grown colony from a YPD
agar plate into 10 ml of YPD liquid medium and incubating
DIALYSIS
PRECIPITATION OF MITOCHONDRIAL
DNA WITH ETHANOL
RE-EXTRACTION OF mtDNA WITH
PHENOL-CHLOROFORM-ISOAMYLALCOHOL
(24:24:1)ANDCHLOROFORMISOAMYLALCOHOL (24:1)
ETHANOL PRECIPITATION
♦
PELLET ( = Purified Mitochondrial DNA)
Fig. I. Isolation scheme Tor yeast mtDNA.
170
[J. Inst. Brew.
lee etal: differentiation of yeast strains
The conversion of cells to sphaeroplasts was followed
spectrophotometrically by measuring their optical density
at 650 nm. Cells were completely sphaeroplasted after
incubation for 30 to 60 min. Sphaeroplasts were pelleted by
centrifugation at 3500 xg for 5 min and resuspended in ice
cold 1 -35 M sorbitol at 3 g (wet weight) per ml. Ten volumes
the conditions recommended by Bethesda Research Labor
atories. Restriction endonuclease digests were subjected to
of TE buffer (0-05 M Tris-005 M Na2 EDTA, ph 80) was
recognize either four or six nucleotide bases (Table I) were
added and the mixture was adjusted to 2% sarkosyl by the
gradual addition of a 20% sarkosyl solution. This mixture
was kept on ice for 10 min to produce a sphaeroplast lysate.
This lysate was clarified by centrifugation at 27,000 xg for
10 min. The clear supernatant was transferred to 30 ml
Corex (Corning) tubes, containing solid CsCl (8 g of CsCI
per 7-9 ml of lysate). After the CsCl had dissolved, a solu
tion (10mg/ml) of either bisbenzimide (Hoechst 33258)
or DAPI (4'6-Diamidino-2-phenylindole) was added to a
ratio of 0-2 ml/7-9 ml of clear lysate. This mixture was
transferred to cellulose nitrate centrifuge tubes and centrifuged at 120,000 xg for 2 days in a Beckman Ti50 rotor. After
centrifugation, mtDNA (top band) was harvested, under a
U V lamp, by puncturing each centrifuge tube with a syringe
needle and drawing it up into a syringe. Either dye was
extracted by mixing the mtDNA fraction with isoamylalcohol saturated with an equal volume of IM NaCl. This ex
traction was repeated 4 to 5 times. The CsCl was removed
by extensive dialysis first against a buffer containing IM
NaCl-002M NaPO4, 6mM EDTA, pH 7-2 at 4°C over
night and then against 2 changes of 0-03 M NaCl-2mM
Tris-0-2mM Na2 EDTA, pH 80 over a 48hr period.
Mitochondria! DNA was precipitated at — 20°C overnight
or - 70°C for 1 hr after addition of 1/10 volume of 2 M Na2
acetate, followed by 2 to 2-5 volumes of cold absolute
ethanol. The mtDNA precipitate was purified further by
extracting first in phenol: chloroform: isoamyl alcohol
(24:24:1) followed by chloroform: isoamyl alcohol
(24:1). It was then precipitated with cold absolute ethanol,
electrophoresis in 1-2% agarose containing TAE buffer.7
Results
Twenty
different
restriction
endonucleases
which
used to digest mtDNA from two closely related production
strains of S. uvarum and a laboratory strain of S. cerevisiae.
The mtDNA fragment phenotypes produced by digestion
with these enzymes is shown in Figs. 2-5. Mitochondria]
DNA from both strains of S. uvarum is digested by all
TABLE 1. Restriction endonucleascs used for analysis of mtDNA
Digestion Conditions
Enzyme
Ava I
Bam HI
bbiii
EcoRI
Haell
Haelll
Hhal
Hind 111
Hinfl
Hpall
Kpnl
Mboll
Salt*
medium
medium
low
high
low
medium
medium
medium
medium
low
low
low
Incubation
Temperature
Recognition
Sequence
37°C
GIPyCGPuG
GJGATCC
AJGATCT
GIAATTC
PuGCGQPy
GGICC
GCG1C
AJAGCTT
GjANTC
C|CGG
GGTACJ.C
GAAGANNNNNNNN1
CTTCTNNNNNNN
CiCGG
CTCGAJG
GAGiCTG
GiTCGAC
1GATC
G1GNCC
GAGCTiC
CiTCGAG
37'C
37°C
37°C
37°C
37°C
37CC
37-55°C
37°C
37°C
37°C
37°C
as above.
Mspl
Pstl
Pvull
Sail
Sst3A
Sau 961
Sstl
Xhol
Restriction Endonuclease Analysis.—Purified mtDNA
was subjected to restriction endonuclease digestion using
*Thc salt concentrations designated as low, medium or high are as
described in reference 5.
Aval
Bam HI
Bglll
EcoRI
low
medium
high
high
medium
medium
low
high
37°C
2l-37°C
37°C
37°C
37°C
37°C
37°C
37°C
Haell
uncut
Figs. 2-5. Electrophoretic patterns obtained after restriction endonucleasc digestion of S. uvarum LA (lane
A), S. uvarum LC (lane B), and S. cerevisiae D273-1OB (lane C). The restriction endonucleases used
arc indicated.
Vol. 91,1985]
171
LEE ETALl DIFFERENTIATION OF YEAST STRAINS
Hoe III
Hhal
Hindi
Hind III
Hint I
uncut
^W ^W r^
Fig. 3.
Hpall
Kpnl
Mspl
Mboll
Pstl
uncut
Fig. 4.
enzymes tested except Pstl, Sal I, and Xhol. The fragment
phenotypes produced by BamHI, BglH, EcoRI, Haell,
Hhal, Hindi, Hindlll, Hinfl, Hpal, Kpnl, Mbo II, Mspl,
PvuII, Sau3A, and Sau96I are essentially identical for these
two strains. Two enzymes, Ava I and Hae III, produce frag
ment patterns which are slightly different in these two
strains. In each case the two strains differ in a single restric
tion fragment, with a longer form being present in LA and
a shorter form in LC. Considered together, these data indi
cate that strains LA and LC differ in the positioning of
one recognition site for Ava I and Hae III. They also indi
cate that aside from these differences these strains have
essentially identical mitochondrial genomes.
By comparison to 5. uvarum, mtDNA from S. cerevisiae
is not cut by Ava I, Bglll, Kpnl, Mspl, Pstl, Sail, Sau96I,
and Xhol. The fragment phenotypes produced by BamHI,
EcoRI, Haell, Haelll, Hhal, Hindi, Hindlll, Hinfl,
Hpall, Mboll, PvuII, and Sau3A all differ from those seen
in both strains of S. uvarum. These results indicate that the
organization of restriction endonuclease recognition sites in
mtDNA from these two species of Saccharomyces is very
different.
172
[J. Inst. Brew.
LEE ETAL\ DIFFERENTIATION OF YEAST STRAINS
Pvull
Sail
Sau3A
Sau961
Sst I
Xho I
5.
Discussion
The restriction endonuclease fingerprints shown in Figs.
2-5 confirm several earlier studies1'6'8 which have demon
strated polymorphism in mtDNA of different strains and
species of Saccharomyces. More importantly, however, they
demonstrate that restriction endonuclease fingerprints pro
duced by Aval and Haelll (Fig. 6) can be used to differen
tiate two closely related production strains of S. uvarum
which previously could not be distinguished by other
diagnostic procedures. Since both Aval and Haelll produce
Aval
I
C
Haelll
I
A
kb
fingerprints which differ in a single fragment, it is likely that
mtDNA from these two strains differ in the positioning of a
single recognition site for each of these endonucleases.
Our success in differentiating two closely related produc
tion strains of brewery yeast by restriction endonucleases
fingerprinting attests to the usefulness of this method as a
diagnostic tool in the classification of brewery yeast strains.
In our experience this method is no more time-consuming
than other diagnostic procedures used to identify yeast
strains. However, it is far more specific and sensitive.
Although the procedure outlined in Fig. 1 is straight
forward, two steps need special comment. First, the dye
DAP1 is more readily extractable from mtDNA than the
dye Hoechst #33258. This observation is important because
either dye, if incompletely extracted, will inhibit the diges
tion of mtDNA by restriction endonucleases. And second,
we find that the final extraction of mtDNA with phenolchloroform-isoamylalcohol and chloroform-isoamylalcohol
is essential in order to obtain reproducible restriction
endonuclease fingerprints, especially from mtDNA of the 5.
uvarum strains used here. These final extractions serve to
remove any residual carbohydrates, proteins, and lipids
which may be bound to, or co-purify with, mtDNA during
CsCl gradient centrifugation.
In summary, we have found that restriction endonuclease
fingerprints of mtDNA are useful both for differentiating
one Saccharomyces species from another and for differenti
ating strains within the same Saccharomyces species. This
approach to yeast strain identification should have a
number of applications in fermentation microbiology.
Acknowledgements—We would like to thank the Adolph
Coors Company for their support of this research. We also
thank Drs J. McEwen, R. M. Wright, V. Cameron and G.
Bellus for their advice and assistance.
References
Fig.
6. Restriction endonuclease fingerprints of digests which
demonstrate clear differences in mtDNA from all three yeast
stragis analyzed. The size (kilobase pairs) of restriction fragments
was determined by comparison to a gel lane on which a Hind III
digest of phage X DNA was electroporesed.
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