Beruflich Dokumente
Kultur Dokumente
www.fems-microbiology.org
Abstract
The transcriptome of a lager brewing yeast (Saccharomyces carlsbergensis, syn. of S. pastorianus), was analysed at 12 different time
points spanning a production-scale lager beer fermentation. Generally, the average expression rapidly increased and had a maximum value
on day 2, then decreased as the sugar got consumed. Especially genes involved in protein and lipid biosynthesis or glycolysis were highly
expressed during the beginning of the fermentation. Similarities as well as significant differences in expression profiles could be observed
when comparing to a previous transcriptome analysis of a laboratory yeast grown in YPD. The regional distribution of various expression
levels on the chromosomes appeared to be random or near-random and no reduction in expression near telomeres was observed.
5 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
Keywords : Expression ; Gene regulation; Chromatin structure; Telomeres; Hierarchical clustering ; Transcriptome analysis ; Saccharomyces cerevisiae ;
Saccharomyces pastorianus
1. Introduction
During the process of beer fermentation, carbohydratecontaining wort is converted into alcohol-containing beer.
From the point of view of the yeast the growth conditions
change radically during this process. Prior to the fermentation the yeast is kept in storage tanks at a very high cell
density and basically without any nutrients. The yeast is
then transferred to fresh wort, which, due to a content of
approximately 100 g l31 of carbohydrates, subjects the
cells to high osmotic stress. The progress of fermentation
gradually causes new forms of stress for the cells in the
form of nutrient depletion and an increasing concentration
of alcohol. When all fermentable carbohydrates have been
utilised, the temperature is lowered from typically 11^14C
to 7^8C to mature the beer and, in some cases, to assist
harvesting of the yeast. Ultimately the yeast is collected
and transferred back to storage tanks. Presumably, the
yeast responds to all these changes in growth conditions
* Corresponding author. Tel. : +45 3327 5309 ; Fax: +45 3327 4764.
E-mail address : kol@crc.dk (K. Olesen).
1567-1356 / 02 / $22.00 5 2002 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.
PII : S 1 5 6 7 - 1 3 5 6 ( 0 2 ) 0 0 1 5 5 - 1
564
versus SGDID numbers (gf100_nal_data_071201, Research Genetics, Invitrogen Corp.) and SGDID numbers
versus ORF name, gene name, ORF length, ORF homologies, genetic function and cellular process [19]. It should
be noted that prior to December 2001 the data le which
Research Genetics was supplying to describe the identity
of spots on the GeneFilters was based on partially incorrect annotations made by MIPS/SGD. Prompted by our
discovery of these errors, Research Genetics has in collaboration with SGD produced an updated version (gf100_nal_data_071201) which now identies spots based on the
unique SGDID number. The updated data le was used
for this investigation. The resulting data have been included in the Gene Expression Omnibus repository at
NCBI [20] with the accession number GSE79.
2.5. Hierarchical clustering
Clustering was performed using the programmes Cluster and Treeview [21] (both available at http://rana.lbl.gov/EisenSoftware.htm). Cluster was used for hierarchical
clustering while Treeview was used for visualisation of the
resulting clustered tree. Parameters used for Cluster are
described in the Fig. 11 legend.
2.6. Northern blotting
Electrophoretic separation of RNA was done on a 1.2%
agarose gel (1U MOPS buer, 6.5% formaldehyde, and
0.05 Wg ml31 ethidium bromide). Running buer was 1U
MOPS. RNA samples were prepared of 40 Wl denaturation
buer (50% formamide, 2.2 M formaldehyde, 1U MOPS),
9 Wl loading buer (5% Ficoll, 0.125% bromphenol blue,
0.125% xylene cyanol, 50% formamide) and 5^10 Wg RNA
in a volume of 10 Wl. Samples were denatured for 10 min
at 65C and chilled on ice until loading. After separation
of the RNA, an image was stored for use in normalisation
of the amounts of RNA loaded. Transfer of the RNA was
performed with conventional capillary blotting (over
night) using nylon membrane (Hybond-XL, Amersham
Biosciences, Sunnyvale, CA, USA) and 10USSC. UVirradiation (UV Stratalinker 1800, Stratagene, La Jolla,
CA, USA) was applied to cross-link the RNA to the membrane.
Probes for Northern blots were prepared using a Random Primed DNA labelling kit (Roche Applied Science,
Rotkreuz, Switzerland). Template for making probe was a
PCR fragment of the desired ORF and its promoter.
Amount of template DNA was approximately 10 Wg.
The membrane was soaked in 2U SSC and placed in a
hybridisation tube (Hybaid, Ashford, UK) containing 10
ml hybridisation buer (ULTRAhyb, Ambion, Huntingdon, UK). Prehybridisation was for at least 30 min at
42C. After hybridisation overnight the membrane was
washed twice (3 and 30 min at 65C) with wash buer-1
(2U SSC, 0.1% SDS), followed by one wash in buer-2
565
3. Results
3.1. Data set construction
mRNA purication followed by GeneFilter analysis was
performed for lager brewing yeast harvested from the
yeast storage tank either just prior to or just after the
actual fermentation, as well as from 10 dierent time
points during the fermentation. A set of Research Genetics
GF100 GeneFilters, which were used for the analysis, contained 6144 spots. In some cases the GeneFilters contained
two spots for each ORF (e.g. FLO8) while in a few cases
the production of a particular spot failed (annotated with
an error ag). Also, some ORFs have been dened after
the design of the lters, and hence were not represented.
Thus, merging of the analysed data with data tables (SGD
and Research Genetics) of SGDID, ORF names, gene
names, ORF length, and function resulted in a data set
containing data for 6084 ORFs. Thus, a total number of
73 008 data points of arbitrary magnitude were generated.
It should be noted that an updated and corrected Research Genetics data le (gf100_nal_data_071201) was
used for describing the identities of spots on the GeneFilters (see Section 2.4).
566
Fig. 2. X^Y plot of ORF length (SGD) versus GeneFilter spot intensities.
Fig. 4. Expression prole of TPI1 (YDR050C) as determined by GeneFilters (a) and Northern blot (b).
567
Fig. 5. Average expression value (a) of all ORFs and sugar content of
maltose (E), maltotriose (F), and glucose (b) plotted against time.
blots were normalised to each other by measuring the relative expression values on day 2 on dot blots containing
equal amounts of DNA of the genes being analysed (data
not shown). Based on the correlation of all corresponding
expression values we estimated that the obtained expression values were accurate within a factor of Y 3 (Fig. 3). It
was found that data for highly expressed ORFs correlated
well between the GeneFilter analysis and the Northern
analysis, especially with respect to the expression proles
(Fig. 4), while relative expression strengths correlated less
well. However, based on the Northern analysis it was also
evident that the expression data derived from GeneFilter
Table 1A
The 25 ORFs with the lowest average expression values
ORF
Gene
Average
YLR235C
YLR041W
YLR315W
YGL024W
YKL131W
YDR457W
YLR233C
YAL001C
TOM1
EST1
TFC3
656
721
756
848
902
917
935
943
YLR124W
YML092C
PRE8
953
968
YPL221W
YKR027W
YLR042C
YKL139W
YKL123W
YIR020C
YKL055C
YFR019W
YPR130C
YLR318W
YDR431W
YAL058C-A
YLR423C
BOP1
CTK1
OAR1
FAB1
EST2
KRE20
APG17
974
976
988
990
1000
1015
1082
1082
1091
1103
1112
1118
1166
Fig. 6. Plot of distribution of minimum, average and maximum expression values throughout the fermentation.
Table 1B
The 25 ORFs with the highest maximum expression values
ORF
Gene
Max
YBR118W
YJL052W
YJR009C
YPR080W
YGR192C
YHR007C
YGR254W
YGL055W
YHR174W
YKL060C
YLR167W
YEL034W
YER011W
TEF2
TDH1
TDH2
TEF1
TDH3
ERG11
ENO1
OLE1
ENO2
FBA1
RPS31
HYP2
TIR1
988 290
809 362
765 585
759 467
737 481
622 612
563 517
541 299
521 654
498 716
475 732
462 765
446 434
YDR502C
YIL018W
YGL135W
YJR047C
YMR015C
YGR175C
YBR299W
YER102W
YGL123W
YFR031C-A
YBR181C
YLR044C
SAM2
RPL2B
RPL1B
ANB1
ERG5
ERG1
MAL32
RPS8B
RPS2
RPL2A
RPS6B
PDC1
433 826
395 883
393 986
393 541
389 381
381 017
375 972
369 718
355 866
348 313
347 943
341 951
protein
protein
protein
protein
biosynthesis
biosynthesis
biosynthesis
biosynthesis
568
Table 2
Day-to-day correlation coecients of expression values
Day
Day
Day
Day
Day
Day
Day
Day
Day
Day
Day
Day
0
1
2
3
4
5
6
7
8
9
11
12
Day 0
Day 1
Day 2
Day 3
Day 4
Day 5
Day 6
Day 7
Day 8
Day 9
Day 11
Day 12
1.00
0.22
1.00
0.29
0.84
1.00
0.44
0.69
0.86
1.00
0.49
0.59
0.82
0.94
1.00
0.51
0.48
0.67
0.87
0.87
1.00
0.64
0.38
0.58
0.81
0.86
0.89
1.00
0.67
0.32
0.43
0.71
0.70
0.80
0.92
1.00
0.79
0.33
0.47
0.69
0.71
0.78
0.91
0.91
1.00
0.77
0.30
0.39
0.63
0.65
0.70
0.81
0.87
0.86
1.00
0.65
0.29
0.32
0.49
0.54
0.65
0.60
0.65
0.63
0.79
1.00
0.89
0.24
0.31
0.45
0.54
0.53
0.65
0.65
0.71
0.78
0.73
1.00
Table 3A
Correlation coecients between DeRisi et al., [1] expression values and GeneFilter expression values
09
11
13
15
17
19
21
h
h
h
h
h
h
h
Day 0
Day 1
Day 2
Day 3
Day 4
Day 5
Day 6
Day 7
Day 8
Day 9
Day 11
Day 12
0.16
0.17
0.16
0.18
0.19
0.25
0.27
0.57
0.55
0.52
0.54
0.48
0.33
0.22
0.47
0.46
0.45
0.46
0.42
0.33
0.24
0.38
0.38
0.38
0.39
0.37
0.33
0.26
0.33
0.34
0.33
0.34
0.33
0.31
0.25
0.23
0.23
0.25
0.24
0.22
0.22
0.19
0.20
0.21
0.22
0.21
0.21
0.23
0.22
0.21
0.22
0.22
0.24
0.23
0.30
0.29
0.18
0.19
0.20
0.21
0.20
0.28
0.29
0.19
0.20
0.19
0.21
0.23
0.32
0.32
0.08
0.08
0.10
0.10
0.08
0.12
0.13
0.13
0.14
0.12
0.15
0.16
0.20
0.19
Table 3B
Correlation coecients between DeRisi et al., [1] expression values and GeneFilter expression values
09
11
13
15
17
19
21
h
h
h
h
h
h
h
Day 0
Day 1
Day 2
Day 3
Day 4
Day 5
Day 6
Day 7
Day 8
Day 9
Day 11
Day 12
0.16
0.18
0.15
0.19
0.20
0.27
0.29
0.65
0.63
0.59
0.61
0.54
0.39
0.25
0.54
0.53
0.50
0.51
0.47
0.38
0.26
0.43
0.44
0.42
0.44
0.41
0.38
0.31
0.37
0.38
0.37
0.38
0.36
0.36
0.29
0.26
0.26
0.27
0.27
0.25
0.26
0.22
0.23
0.24
0.24
0.24
0.24
0.29
0.27
0.24
0.26
0.25
0.27
0.26
0.35
0.34
0.23
0.24
0.24
0.27
0.25
0.34
0.34
0.22
0.24
0.21
0.26
0.27
0.40
0.40
0.10
0.10
0.10
0.12
0.11
0.16
0.17
0.13
0.15
0.12
0.15
0.16
0.21
0.20
The GeneFilter data set was ltered to only include ORFs not having neighbouring spots of three times or higher intensity.
569
Fig. 7. X^Y plot of expression values for time point day 1 versus time
point 9 h from DeRisi et al., [1]. Only data points for spots not having neighbours with more than three times higher intensity are included.
The correlation coecient between the two data sets is 0.65.
Fig. 8. X^Y plot of expression ratios for day 1/day 9 versus 9 h/21
h from DeRisi et al. [1]. Only data points for spots not having neighbours with more than ve times higher intensity are included. The correlation coecient between the two data sets is 0.36.
570
571
Table 4
Distribution of common genetic functions within clusters
Cluster
Total
% of cluster
all
Biosynthesis
1
Cell cycle
0
Chaperone
1
Glycolysis
0
Kinase
0
Metabolism
2
mRNA
0
Protein biosyn0
thesis
Protein degrada1
tion
Protein folding
0
RNA
0
rRNA
0
Stress response
4
Structural protein
0
of ribosome
Transcription
2
Transcription fac- 0
tor
Transport
6
tRNA
0
Ubiquitin
2
Unknown
46
Sum
65
TOTAL entries
73
in cluster
98
2
4
3
9
1
9
94
36
1
7
2
6
5
3
33
14
3
2
1
8
13
1
4
23
8
2
1
20
15
8
13
61
12
6
15
30
34
25
24
12
4
5
1
10
7
8
7
12
7
2
0
9
8
15
6
sum
257
37
29
23
92
85
69
181
1
0
1
0
0
3
0
0
30
1
1
1
3
0
3
29
17
0
3
1
3
2
1
16
4
1
1
0
2
3
0
1
3
1
0
0
3
2
1
2
4
1
0
1
2
2
2
2
3
1
1
0
2
2
2
2
2
1
0
0
1
1
2
1
6
1
1
1
2
2
2
4
26
10
48
4
24
6
5
93
5
16
2
3
31
2
12
1
7
4
0
34
6
4
7
6
90
9
9
17
4
32
1
3
6
1
37
1
8
4
22
245
26
43
162
0
0
0
5
0
1
7
2
2
29
2
8
1
1
15
1
3
0
2
1
0
5
1
1
1
0
7
1
1
1
1
7
0
1
1
0
5
0
1
1
1
6
1
1
4
8
3
8
6
16
9
34
16
63
24
20
11
25
12
176
81
3
0
2
1
4
3
4
2
5
2
5
2
4
2
4
2
4
2
12
2
3
94
476
326
12
7
0
74
257
208
14
1
5
223
341
394
21
3
10
410
640
718
51
15
34
690
1241
1362
29
8
12
233
423
457
22
7
5
439
623
685
167
43
71
2209
4066
4215
8
0
3
63
89
4
1
1
29
146
6
3
0
36
124
4
0
1
57
87
3
0
1
57
89
4
1
2
51
91
6
2
3
51
93
3
1
1
64
91
4
1
2
52
96
Numbers represent the number of ORFs in each cluster that contains the query word in either the SGD-annotated Process or Function. Thus, e.g. the
query unknown represents all ORFs with either unknown process or unknown function. Some query words, e.g. metabolism, will return any process or
functions in which metabolism is a substring. Several ORFs will be returned by more than one query.
572
4. Discussion
To our knowledge, this work represents the rst example of a genome-wide transcription analysis of a lager
brewing yeast during actual production fermentation conditions. Although the transcriptome data obtained as a
result of this investigation was associated with some sources of inaccuracy, such as the ability to distinguish between expression from the two dierent subgenomes,
they nevertheless provide signicant new insight into the
genetics and physiology of an important industrial microorganism.
573
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identical to the ACB1 gene from S. monacensis. Yeast 13, 1409^1421.
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