System. Appl. Microbiol.

15, 439-446 (1992)

© Gustav Fischer Verlag, StuttgartlNew York

A Comparative Study of Different Methods of Yeast Strain

Characterization

AMPARO QUEROL\ ELADIO BARRI02 , and DANIEL RAM6N 3 "

1 Departament de Microbiologia, Universitat de Valencia

2 Departament de Genetica, Universitat de Valencia
3 Unidad de Bioingenierfa de Alimentos, Instituto de Agroquimica y Tecnologfa de Alimentos (C.S.I.C.), Valencia, Spain

Received January 21, 1992

Summary
An extensive survey of different methods of yeast strain identification (classical microbiological tests,
whole-cell protein electrophoresis, chromosomal patterns, DNA hybridization and mitochondrial DNA
restriction analysis) has been carried out in order to differentiate, with industrial purposes, strains present
in the Alicante wine ecosystem. Only chromosomal patterns and mitochondrial DNA (mtDNA) restriction
analysis show differences between strains. Both techniques are very complex to be used in biotechnological
industries. For this reason, we have developed a new, simple, unexpensive and rapid method based on
mtDNA restriction analysis.

Key words: Wine yeast - Saccharomyces cerevisiae - Identification - Protein electrophoregrams - mtDNA
restriction analysis - Electrophoretic karyotype - DNA hybridization

Introduction

Numerous yeast strains are being used for commercial
wine fermentation, beer brewing and bakery products.
The importance of using a suitable strain for producing an
exclusive alcoholic beverage or bakery product with
sought-after characteristics has prompted the selection and
development of new yeast strains. Two reasons have made
necessary the characterization of these new strains: quality
control in dry yeast production to ensure that final pro-
duct is identical to the original strain, and fermentation
process control to ensure that it is really conducted by the
inoculated yeast. As most of the industrial yeast strains
belong to the S. cerevisiae group and can not be readily
distinguished and identified by classical biochemical
methods, molecular methods (widely used in micro-
biological taxonomy) have been recently employed for
yeast strain characterization. These comprise analyses of
protein patterns (Van Vuuren and Van der Meer, 1987),
DNA sequence polymorphisms (Degre et al., 1989),
chromosomal DNA patterns (Vezinhet et al., 1990), and
mitochondrial DNA (mtDNA) restriction patterns (Du-
bourdieu et al., 1984). The complexity of some of these
* Corresponding author
techniques renders their industrial application difficult. In
this sense, a method applied to biotechnology should be
fast, reliable and economic and should have no require-
ments for sophisticate equipment and special personal
skills.
During the last few years we have studied the wines
produced in the Mediterranean region of Alicante (Spain).
In the final stages of grape maturation, the variable cli-
mate of this geographic area strongly affects the composi-
tion of the microbial flora. For this reason, the quality and
organoleptical characteristics of the wine produced varied
considerably from one year to another (Querol et al.,
1990). In order to solve this problem, we have isolated and
characterized a S. cerevisiae strain (namely T73) from
Alicante musts, that used as a dry yeast produces an excel-
lent wine (Querol et al., 1991). Our results indicate the
predominance of S. cerevisiae strains in the Alicante
musts. The use of our selected strain in the winemaking
process requires the development of techniques that can
clearly differentiate between the inoculated strain and the
wild strains present in the must. This is a convenient con-
trol in order to ensure that the fermentation process is
really conducted by the inoculated yeast: This is the reason
440 A. Querol, E. Barrio, and D. Ramon

we are interested in the development of fast and easy SDS-PAGE was performed according to the technique of
methods that permits us clear strain differentiations. Laemmli (1970). A sample of 100 Ilg of whole-cell proteins was
In this study, we have carried out different methods loaded in each electrophoresis well.
currently available for yeast strain characterization in or- DNA hybridizations. Total yeast DNA was isolated by a stan-
dard method based on spheroplast lysis (Sherman et aI., 1986).
der to determined the most sensitive method to differenti-
Yeast DNA was digested with the following restriction endonuc-
ate among yeast isolated from the same must. On the other leases (Boehringer, Mannheim): BeoRI, EeoRV, HaeIII, HindIII,
hand, as an alternative, we have developed a new techni- HinfI, MspI, PstI and XbaI . Restriction fragments were separated
que that clearly simplifies the characterization of yeast on slab horizontal 0.8-1.2% agarose gels, and transferred to
strains. nylon membranes (Hybond-N, Amersham International) by
Southern blotting (Sambrook et aI., 1989).
The following plasmids were used for DNA hybridization:
Materials and Methods YIPS, carrying URA3-probe (PstI-SmaI fragment of 0.901 kb),
and pRS304, carrying TRP1-probe (PvuI fragment of 2.871 kb) .
Plasmid DNA was prepared by the method of Birnboim and Doly
Yeast strains. A total of fourteen strains of S. cerevisiae was
(1979). Hybridization probes were cut from the vectors using t!i.e
analysed. The origin and source of these strains are listed in
Table 1. restriction enzymes described above. The fragments were elec-
trophoresed, and bands carrying the desired fragments were cut
Yeast identification. The methods used for the identification of
out of the gels. DNA was eluted using the Unidirectional Elec-
yeast were according to the general methods described by Barnett
troeluter-Analytical Electrolution System (Industrial Biotech-
et al. (1983) and Kreger-van Rij (1984).
nologies Inc.). The isolated fragments were labelled with Digoxi-
Protein patterns. Yeast cells (5 ml of an exponential phase cul-
genin-11-dUTP using the Nonradioactive DNA Labeling and De-
ture) were harvested and washed twice with 1 ml of buffer I (3.2
tection Kit from Boehringer Mannheim.
mM Tris-HCI pH 7). Cells were precipitated at 4,000 X g for 5
Hybridizations were performed according to the Boehringer
min, and the pellet resuspended in 0.3 ml of buffer II (1 mM
Mannheim protocol at 65 °C for 18-20 h. Membranes were
phenylmethyl-sulphonyl-fluoride in isopropanol). The same vol-
washed twice 5 min in 2 x SSC, 0.1 % SDS at room temperature,
ume of 0.5 mm diameter glass beads was added. Cells were dis-
and twice 15 min in 0.5 x SSC, 0.1 % SDS at 6ZOC. Probe DNA-
rupted by vortexing 1 min and cooling the tube on ice for another
target DNA hybrids were detected by following the manufactur-
minute. This operation was repeated until cells were completely
er's instructions (Boehringer Mannheim).
disrupted. Intact cells and debris were removed by centrifugation
A synthetic poly (GGGT)4 oligonucleotide was also used as a
at 4,000 x g and 4°C for 15 min. The supernatant was transfer-
probe. The oligonucleotide was labelled with digoxigenin-11-
red to a fresh tube, and after addition of 3 % SDS (final concen-
dUTP (Boehringer Mannheim). Hybridizations were performed
tration) and boiling for 10 min, the supernatant was stored at
according to the Boehringer Mannheim instructions at 42 °C for
-80°C. Protein concentrations were determined by the Lowry
method (Plummer, 197 1). 18-20 h. Filters were washed five times for 1 h in 2 x SSC, 0.1 %
SDS at two different temperatures: either 42 °C or room tempera-
ture.
Chromosomal polymorphisms. Chromosomal DNA was pre-
Table 1. List of the strains used in this work pared in agarose plugs (Carle et aI., 1985). Chromosome separa-
tions were carried out as described previously (Querol et aI.,
Strain Species Reference Source 1991 ).
Mitochondrial DNA restriction analysis. In the first experi-
R08 S. cerevisiae 'CECT-1895 Alicante wine ments, mitochondrial DNA (mtDNA) was purified as described
var. bayanus previously (Querol and Barrio, 1990), and digested with 7 re-
R32 S. cerevisiae CECT-1896 Alicante wine striction endonucleases, which recognize 6-bp (EcoRI, EcoRV,
var. cerevisiae PstI), 5-bp (DdeI, HinfI), and 4-bp (A luI, RsaI) sequences.
T38 S. cerevisiae CECT-1891 Alicante wine Later, a new method for the detection of mtDNA polymorph-
var. cerevisiae isms was developed. Yeast cells were grown in an overnight cul-
T56 S. cerevisiae CECT-1892 Alicante wine ture of 5 ml YEPD (1 % yeast extract, 2% peptone, 2% glucose).
var. eerevisiae Cells were spun down in a centrifuge and resuspended in 0.5 ml
T71 S. eerevisiae CECT-1893 Alicante wine of 1 M Sorbitol, 0.1 M EDTA pH 7.5. Then they were transferred
var. cerevisiae to a 1.5 ml microfuge tube, where 0.02 ml of a solution of zy-
T73 S. cerevisiae CECT-1894 Alicante wine molyase 60 (2.5 mg/ml) was added. Tubes were incubated at
var. bayanus 37°C for 30-60 min (the obtaining of spheroplasts was optional-
P22 S. eerevisiae bP.Sanz bread ly monitored). Spheroplasts were centrifuged for 1 min in a mi-
P39 S. eerevisiae bP.Sanz bread crofuge and resuspended in 0.5 ml of 50 mM Tris-HCl, 20 mM
1170 S. cerevisiae CECT-1170 distillery EDTA pH 7.4. After resuspension, 0.05 ml of 10% SDS were
1189 S. eerevisiae CECT-1l89 beer added, and the mixture was incubated at 65 °C for 30 min. Im-
var. carlsbergensis mediately, 0.2 ml of 5 M potassium acetate was added and the
1331 S. cerevisiae CECT-1331 beer tubes were placed on ice for 30 min. Then, they were centrifuged
var. earlsbergensis at maximum speed in a microfuge for 5 min. Supernatant was
1383 S. eerevisiae CECT-1383 distillery transferred to a fresh microfuge tube, and the DNA was precipi-
1462 S. cerevisiae CECT-1462 beer tated by adding 1 vol of isopropanol, and after incubation at
2180 S. eerevisiae X-2180 laboratory room temperature for 5 min was centrifugated for 10 min. The
strain DNA was washed with 70% ethanol and vacuum dried and dis-
solved in 50 III TE pH 7.4. Yeast DNA (2 fll) was digested with
• Deposited in the Spanish Type Culture Collection (CECT). restriction enzymes that recognize 5-bp or 4-bp sequences, and
b Kindly supplied by Dr. P. Sanz, Instituto de Agroqufmica y the mtDNA restriction patterns were observed by agarose elec-
Tecnologfa de alimentos (CSIC), Valencia, Spain. trophoresis.
 Yeast Strain Characterization Methods 441

Results and Discussion Differentiation of yeast strains based on electrophoretic

whole-cell protein patterns
Microbiological analysis
Electrophoresis of whole-cell proteins has been used
In order to differentiate between the six strains isolated
successfully to distinguish between and within yeast
from the Alicaote wine ecosystem, classical microbiologi-
species (Van Vuuren and Van der Meer, 1987; Degre et
cal tests based on morphological and physiological char-
ai., 1989; Vancanneyt et ai., 1991). Fig. 1 shows the pro-
acteristics of yeasts (Barnett et ai., 1983; Kreger-van Rij,
tein pattern of the strains analysed. As it can be seen, the
1984) were carried out. The results of fermentation and
same patterns is present in all strains. No difference can be
assimilation tests are shown in Table 2. Other analyses, as
observed even between the varieties bayanus and cere-
morphological traits of the vegetative cells, production
visiae. Such results are in accordance with those reported
and number of ascospores, etc., did not show any differ-
by Van Vuuren and Van der Meer (1987) and Degre et ai.
ence between strains (data not shown). The possibility of
(1989), who could not differentiate both varieties. Prob-
assimilation and fermentation of different carbon sources
ably, this technique is of value when it is used to discern
only allows us to differentiate between two varieties very
between species or even between ecologically and geog-
usual in wines: S. cerevisiae var. bayanus (T73 and R08
raphically separated strains (Van Vuuren and V~n der
strains), and S. cerevisiae var. cerevisiae (the other four
Meer, 1987; Vancanneyt et ai., 1991), but as It was
strains), but not within varieties. Furthermore, just a single
pointed out by the last authors, the infraspe~ific variatio~s
character distinguishes one variety from the other (galac-
are mainly due to quantitative differences m the protem
tose fermentation), and methodological differences in
patterns. This fact was not observed in our study.
these assessments can lead to differing results. For these
reasons, the application of these tests could not differenti-
ate between our selected yeast strain inoculated in the
must and the rest of wild strains present there. ~ CD CO CO N
,... It) 0 to') to)
... t- It t- a::
94-
66-

Table 2. Carbon assimilation and fermentation characteristics of 45-

the six yeast strains

Characteristics R08 R32 T38 T56 T71 T73 29-

Fermentation 18-
Glucose + + + + + +
Galactose + + + + 14-
Sucrose + + + + + +
Maltose + + + + + + Fig. 1. Protein patterns of the strains analysed.
Lactose
Melibiose
Raffinose + + + + + +
DNA sequence polymorphisms as markers in yeast
Asimilation strain differentiation
Sucrose + + + + + +
Maltose + + + + + + The use of specific gene probes as tools in yeast tax-
Galactose + + + + + + onomy and strain differentiation has been proposed by
Lactose several authors (Braus et ai., 1985; Seehaus et ai., 1985;
Melibiose Pedersen, 1983, 1985, 1986a, 1986b; Degre et ai., 1989).
Melezitose Different probes that contain genes codifying enzymes of
Trehalose + + + primary metabolism (HIS4, LEU2, TRP1, TRP2, TRP3,
Raffinose + + + + + + TRP4) have been used in these studies. We have carried
Inositol
Cellobiose out this kind of analysis with two different gene probes
Xylose that contain the URA3 and TRPl genes (Fig. 2 shows the
Rhamnose results with some of the enzymes). The fragment lengths
Erythritol determined by restriction analysis of the URA3 and TRPl
Ribitol regions of the strains under study are given in Table 3.
Starch TRPl region was monomorphic in the strains analysed
Arabinose with all the restriction enzymes. Similar results were found
Mannitol by Braus et ai. (1985), who analysed the TRPl region of
2-keto-D-Gluconate
Citrate 11 S. cerevisiae strains, eight of them (73 %) showed the
Glucose + + + + + + same restriction patterns with the three restriction enzy-
mes used.
442 A. Querol, E. Barrio, and D. Ramon

Table 3. Fragment sizes (kb) for each restriction pattern obtained plotypes present in each strain. However, this kind of
with the URA 3 and TRP 1 probes in the seven yeast strains anal- analysis is not good enough to differentiate each one of the
ysed strains.
In opinion of some authors, an ideal probe for genetic
Enzymes URA3 TRPl
fingerprinting hybridizes to a conserved, dispersed repeti-
Eco RI A: >30 A: 1.4 tive DNA sequence, that is not associated with essential
Eco RV A: 4.6 A: 0.7 genomic sequences as this allows the sampling of all the
B: 4.2 different genomic locations that flank the repeat. Several
Hae III A: 0.35, 1.8 A: 0.7 types of repetitive sequences have been used as probes:
Hind III A: 1.17 A: 3.2 ribosomal RNA genes (Pedersen, 1983 and 1985), trans-
Hinf I A: 1.2 A: 0.55, 1.0 posable elements (Ty elements, Pedersen, 1985; Y1 ele-
Msp I A: 0.85 A: 0.87 ments, Degre et aI., 1989), polyGT repetitive oligonuc-
Pst I A: 3.7 A: 3.2 leotide (Walmsley et aI., 1989), etc. For this reason, a
B: 4.0
polyGT repetitive oligonucleotide (see Materials and
Xba I A: 3.0 A: 1.5
B: 3.75 Methods) was used as a probe to differentiate our strains.
The results obtained are given in Fig. 3. Two different con-
ditions of stringency were used to determine the restriction
patterns, but only minor differences between strain pat-
For the URA3 region, only 3 out of the 8 restriction terns could be observed. Moreover, the analysis of these
enzymes used (EeaRV, PstI and XbaI) yielded variable results become very difficult: the more stringent the condi-
patterns (two per enzyme). Nevertheless, this variability tions, the higher the difficulty to observe differences be-
can not be explained by gain or lose of restriction sites tween strains; the less stringent the conditions, the higher
caused by nucleotide substitutions, it can only be ex- the difficulty to determine the patterns.
plained if most of the strains (T56, T71, T73, R32, T38,
2180) are heterozygotic for this region (two different nuc-
Chromosomal polymorphisms as tools for differentia-
leomorphs or haplotypes for the same nucleon, according
tion and identification of yeast strains
to Nei, 1987). The nucleomorphs or haplotypes found in
each strain are given in Table 4. These results allows us to Since the first orthogonal-field-alternation gel elec-
cluster the strains in three groups according to the ha- trophoresis karyotyping performed by Carle and Olson

TRPI URA3

M_COIOIO('I° (")_CDIOIO('I
........ .,.,OMM IO ...... .,., 0 (") (")
1-~~a:~a:N ~~~a:I-a:

lcoNI /linfl /loe III Pstl

Fig. 2. Restriction patterns of the TRPl and URA3 regions obtained by DNA hybridization. Fragment lengths are given in Table 3.
 Yeast Strain Characterization Methods 443

Table 4. Restriction patterns and composite patterns for the seven yeast strains with the URA3 probe. Fragment sizes for each
restriction pattern are given in Table 3

Restriction enzyme patterns

Composite
Strain ECD RI ECD RV Hae III Hind III Hinf I Msp I Pst I Xba I pattern

R08 A A A A A A A A 1
R32 A A A A A A A A and B 2
T38 A A A A A A A A andB 2
T56 A A and B A A A A A and B A andB 3
T71 A A and B A A A A A and B A and B 3
T73 A A A A A A A A andB 2
2180 A A and B A A A A A and B A and B B3

(1985), pulsed field electrophoresis techniques have made
possible the separation of the most part of chromosomes
of S. cerevisiae and other yeasts. Chromosomal poly-
morphisms are due to deletions, insertions and transloca-
tions of DNA fragments long enough to be detectable by
electrophoresis (at least 10 kb). Although these phenome-
na could be considered as much less frequent than nuc-
leotide substitutions and insertions/deletions of few nuc-
leotides, karyotype analysis of a number of yeast strains
has shown a high level of polymorphism (De Jonge et aI.,
1986; Johnston and Mortimer, 1986; Casey et aI., 1988;
Johnston et aI., 1988; Vezinhet et aI., 1990).
The application of these techniques to our strains per-
mits to differentiate each strain by its specific chromosom-
al pattern (Fig. 4). The number of bands detected ranges
between 12 and 15 depending on the strain. As it can be
seen in the Figure, the most important part of the variabili-
ty is observed from the sixth band, where each strain ex-
hibits an specific pattern. Similar results were previously
obtained in the karyotyping analysis of other wine strains
(Degre et aI., 1989; Vezinhet et aI., 1990).
Evidently, this technique allows us to differentiate each
one of the strains present in the same ecosystem. However,
it is a complex and time-consuming technique (35 h for the

A B

lc() 1(1 Nihf/ Msp/ Pstl

Fig. 3. Restriction patterns obtained by DNA hybridization with the poly (GGGT)4 repetitive oligonucleotide used as a probe. Filters
were washed under two different stringency conditions: A, five times for 1 h in 2 x SSC, 0.1 % SDS at 42°C; B, five times for 1 h in 2 x
SSC, 0.1 % SDS at room temperature.
444 A. Querol, E. Barrio, and D. Ramon

1
2
4
5
6

12
13
14
15

Fig. 4. Chromosome profiles of the yeast strains as detected after chromosome electrophoresis on agarose gels by the CHEF method.
Numbers at left indicate the different chromosome bands observed.

sample preparation and 24 h for the electrophoresis) that Yeast strain characterization based on mitochondrial
requires sophisticate equipment and does not permit the DNA restriction analysis '
analysis of a large number of samples.
In the search of a method that allows us to differentiate
our selected strain (T73 ) from the rest of yeast strains, we
carried out mitochondrial DNA restriction analysis, a
marker widely employed for industrial strain differentia-
tion (brewery yeasts, AigIe et al., 1984; Lee and Knudsen,
1985; and enological strains, Dubourdieu et al., 1984;
(,,) ... (OO)o)N
,....,.... It) 0 (") (") Hallet et al., 1988; Vezinhet et al., 1990).
e ............ a:: .... a:: Several methods have been developed for the isolation
of yeast mitochondrial DNA (AigIe et al., 1984; Gargouri,
1989). Recently, we developed a method that does not
require the use of caesium chloride gradients for the purifi-
cation of yeast mtDNA (Querol and Barrio, 1990). The
results of the restriction analysis of the mtDNA purified by
this method are depicted in Fig. 5, and the patterns that
emerge from this analysis are given in Table 5. Several
restriction enzymes do not determine a specific pattern for
each strain, thus, AluI and DdeI yield the same pattern for
three strains (T71, T56, T38), and EcoRV the same pat-
tern for two strains (T71 and T38). However, each strain
shows a specific pattern with EcoRI, HinfI and RsaI.
We have combined the patterns obtained with the seven
endonucleases in order to determine the different haploty-
f1lul [co I(V pes present in the strains (Table 5). Hence, all seven strains
Fig. 5. Mitochondrial DNA restriction analysis with several re-
analysed have distinctive haplotypes. According to these
striction endonucleases. mtDNA was purified by the method of results, mtDNA restriction analysis could be a good
Querol and Barrio (1990). Size markers in kb correspond to a technique to differentiate yeast strains from the same
mixture of lambda phage DNA fragments obtained with HindIII ecosystem, but it depends on the restriction enzymes used
and with EcoRI-HindIII. in the analysis.
 Yeast Strain Characterization Methods 445

Table 5. Restriction and haplotype for the seven yeast strains it is possible to determine the RFLP differences between
with mtDNA yeast mtDNAs without needing to purify mitochondria
(sucrose gradient) and/or mtDNA (CsCI gradient).
Restriction enzymes patterns So, as it can be observed in Fig. 6, the same resolution of
Haplo-
Strains Alu I DdeI Eco RI Eco RVHinfI Rsa I type the mtDNA restriction patterns is obtained by this method
and by digestion of purified mtDNA with the 4-bp or 5-bp
R08 A A A A A A I restriction enzymes. The method is rapid (3 h of extrac-
R32 B B B B B B II tion, 2 h of digestion, and 2 of electrophoresis) and simple
T38 C C C C C C III (only a microfuge and a DNA electrophoresis system is
T56 C C D D D D IV required), being the only limitation the number of tubes
T71 C C E C E E V that the user can manipulate.
T73 D D F E F F VI
This method combine the advantage of the mtDNA re-
2180 E E G F G G VII
striction analysis as a good marker in yeast strain differen-
tiation and the simplicity of the technique. Therefore, this
method will be of particular interest to industrial users of
Even though this method is easier than chromosome yeast (wine-makers, brewers, bakers and other biotech-
profiles, the use of ultracentrifuge do not permit the nologists), and it also has a potential interest in characteri-
routine analysis of a high number of samples. zation of yeast cultures from Type Culture Collections (as
In order to avoid this problem, we have developed a it can be observed in Fig. 7).
new method which consists on the standard miniprep iso-
lation of total yeast DNA and the use of specific restriction
endonucleases that recognize 4 bp (AluI, HaeIII, HpaII,
MaeI, MaeII, MboI, Sau3AI, RsaI, TaqI) or 5 bp (DdeI,
Hinfl, MaeIII) . These enzymes recognize a high number of
~ites in the yeast nuclear DNA, but few sites in the
mtDNA. Hence, mitochondrial restriction fragments are
easily observed by agarose gel electrophoresis. In this way,

111111 Hil1f I
Fig. 7. Potential applications of the new method of mtDNA re-
striction analysis. Patterns of different industrial strains obtained
according to the new method. The origin of these strains and the
source whence they were obtained are listed in Table 1. Size
markers as in Fig. 6.

Fig. 6. Mitochondria DNA restriction patterns of S. cerevisiae
obtained with the new method. Lanes marked with the strain
reference correspond to 14 I1L of purified mtDNA of several
strains (obtained according to Querol and Barrio, 1990) digested
with RsaI. Lanes marked with the strain reference and an "a"
correspond to 2 111 of DNA of the same strains digested with RsaI
according to the new method. Lane m: a mixture of lambda DNA
digested with HindIII, and with HindIII-EcoRI was used as size
marker.
Conclusions
From the several methods of yeast strain characteriza-
tion analysed, only chromosomal and mtDNA restriction
patterns could differentiate between our inoculated S.
cerevisiae strain (T73) and the rest of wild S. cerevisiae
strains present in the must. Nevertheless, both techniques
are expensive, time-consuming and very complicate to be
used in biotechnological industries.
The development of a new method of detection of yeast
mtDNA restriction patterns, that does not require mtDNA
purification, simplifies the characterization of yeast
strains.
 446 A. Querol, E. Barrio, and D. Ramon
Acknowledgements. This work was supported by a grant
ALI90-0949 from e.I.e.Y.T. A.Q. and E.B. were recipients of
fellowships from Conselleria de Cultura, Educaci6 i Ciencia de la
Generalitat Valenciana.
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