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Applied and Environmental Microbiology, February 1999, p. 450-456, Vol. 65, No. 2
0099-2240/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.
Multiple 
-Glucoside Transporter Genes in
Brewer's Yeast
Lene
Jespersen,1
Lene B.
Cesar,1
Philip G.
Meaden,2,* and
Mogens
Jakobsen1
Department of Dairy and Food Science, Royal
Veterinary and Agricultural University, 1958 Frederiksberg C,
Denmark,1 and
The International Centre
for Brewing and Distilling, Heriot-Watt University, Edinburgh EH14
4AS, Scotland2
Received 20 July 1998/Accepted 1 November 1998
 |
ABSTRACT |
Maltose and maltotriose are the two most abundant fermentable
sugars in brewer's wort, and the rate of uptake of these sugars by
brewer's yeast can have a major impact on fermentation performance. In
spite of this, no information is currently available on the genetics of
maltose and maltotriose uptake in brewing strains of yeast. In this
work, we studied 30 brewing strains of yeast (5 ale strains and 25 lager strains) with the aim of examining the alleles of maltose and
maltotriose transporter genes contained by them. To do this, we
hybridized gene probes to chromosome blots. Studies performed with
laboratory strains have shown that maltose utilization is conferred by
any one of five unlinked but highly homologous MAL loci
(MAL1 to MAL4 and MAL6). Gene 1 at
each locus encodes a maltose transporter. All of the strains of
brewer's yeast examined except two were found to contain
MAL11 and MAL31 sequences, and only one of
these strains lacked MAL41. MAL21 was not present in the
five ale strains and 12 of the lager strains. MAL61 was not
found in any of the yeast strains. In three of the lager strains, there
was evidence that MAL transporter gene sequences occurred
on chromosomes other than those known to carry MAL loci. Sequences corresponding to the AGT1 gene, which encodes a
transporter of several 
-glucosides, including maltose and
maltotriose, were detected in all but one of the yeast strains.
Homologues of AGT1 were identified in three of the lager
strains, and two of these homologues were mapped, one to
chromosome II and the other to chromosome XI. AGT1 appears
to be a member of a family of closely related genes, which may have
arisen in brewer's yeast in response to selective pressure.
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INTRODUCTION |
The three major fermentable sugars
found in brewer's wort are glucose and the -glucosides maltose and
maltotriose. Maltose is by far the most abundant of these sugars,
typically accounting for 50 to 60% of the total fermentable sugar in
an all-malt wort (8); glucose and maltotriose account
for 10 to 15 and 15 to 20%, respectively. Sucrose and fructose
are also found in wort but at much lower levels (1 to 2%)
(8). Of the three major wort sugars, glucose is
preferentially utilized by brewing strains of yeast
(Saccharomyces cerevisiae or Saccharomyces
pastorianus) (23), but efficient fermentation requires
rapid and complete utilization of both maltose and maltotriose. Gene
dosage studies performed with laboratory strains of yeast have shown
that the transport of maltose into the cell may be the rate-limiting
step in the utilization of this sugar (12). In addition,
constitutive expression of a maltose transporter gene in a lager strain
of yeast has been found to accelerate the fermentation of maltose during high-gravity brewing (17). Information on the maltose and maltotriose transporter genes present in brewer's yeast may therefore be of some value in selecting suitable strains and in predicting fermentation performance.
Brewing strains of yeast are polyploid, aneuploid, or, in the case of
lager strains, alloploid (reviewed in references 13 and 16). Such strains sporulate poorly, and even
when spores can be obtained, they are frequently not viable. In
rare cases, spores may germinate, but the vegetative cells lack
the ability to mate. Consequently, genetic analysis of brewing strains
of yeast by classical methods has been severely hampered. Advances in
the molecular genetics of yeast, including the complete sequencing of
the genome (10, 11), have provided an opportunity to examine in more detail the genetic constitution of brewing strains of yeast.
Maltose utilization in yeast is conferred by any one of five
MAL loci, MAL1 to MAL4 and
MAL6 (reviewed in reference 26). Each locus consists of three genes; gene 1 encodes a maltose
transporter, gene 2 encodes a maltase (-glucosidase), and gene 3 encodes a transcriptional activator of the other two genes. Thus, for
example, the maltose transporter gene at the MAL6 locus is
designated MAL61. The five MAL loci each map to a
different yeast chromosome, as follows: MAL1,
chromosome VII; MAL2, chromosome III; MAL3,
chromosome II; MAL4, chromosome XI; and MAL6,
chromosome VIII. The MAL loci exhibit a very high degree of
homology and are telomere linked, suggesting that they evolved by
translocation from telomeric regions of different chromosomes
(18). Since a fully functional or partial allele of the
MAL1 locus is found in all strains of S. cerevisiae, this locus has been proposed as the progenitor of the
other MAL loci (5).
Han et al. (14) have described a yeast gene,
AGT1, which encodes a general -glucoside transporter that
is capable of taking up maltotriose, isomaltose, -methylglucoside,
palatinose, trehalose, and melezitose in addition to maltose
(and turanose). AGT1 is an allele of MAL11
on chromosome VII. The AGT1 protein is 57% identical to the
MAL61-encoded maltose transporter MAL61 and thus far is the
only S. cerevisiae protein that has been demonstrated to be
a transporter of maltotriose.
Sequencing of the yeast genome has revealed two other open
reading frames (ORFs) that encode products with strong homology to MAL61. These ORFs are YDL247w and YJR160c, which are located on chromosomes IV and X, respectively (20). Although
no results of a functional analysis of the genes or their products have
been published, it is quite possible that YDL247w and YJR160c could play some role in -glucoside transport.
In this work, we surveyed brewing strains of yeast for the
presence of maltose and maltotriose transporter gene sequences. In addition to identifying alleles of known -glucoside transporter genes, we also obtained evidence that related sequences map to other
chromosomes in some of the genomes examined.
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MATERIALS AND METHODS |
Yeast strains.
Five ale strains of yeast (S. cerevisiae KVL011 to KVL015) and 25 lager strains (S. pastorianus KVL001 to KVL010 and KVL016 to KVL030) were included
in this work. All of these strains are currently used in beer
production at a number of different brewery sites. Three of the lager
strains (KVL028 to KVL030) originated from the same brewery, but their
relationship to one another is not known. KVL025 is a single-colony
isolate obtained from KVL007; these are the only two strains used in
this study that are known to be very closely related or possibly identical.
PFGE.
Yeast strains were propagated in yeast
extract-peptone-glucose broth containing (per liter of distilled water)
10 g of yeast extract (Difco), 20 g of Bacto Peptone (Difco),
and 40 g of glucose. For preparation of chromosomes, strains were
initially grown with shaking at 25°C for 48 h in 100 ml of broth
and then propagated twice (24 h each) in the same broth. Cells of each
strain were harvested by centrifugation at 3,000 × g
for 5 min and washed once with 8 ml of spheroplasting buffer (1.2 M
sorbitol, 10 mM Tris-HCl, 10 mM CaCl2; pH 7.5) before they
were resuspended in 6 ml of the same buffer. Following addition of
Zymolyase 100-T (0.2 ml of a 5-mg ml
1 suspension;
Seikagaku America, Ijamsville, Md.), the yeast cells were incubated for
1 h at 37°C. An aliquot (1 ml) of the spheroplast suspension was
mixed with an equal volume of 1.5% (wt/vol) low-melting-point agarose
(Sigma) dissolved in TES buffer (10 mM Tris-HCl [pH 7.5], 10 mM NaCl,
1 mM EDTA) containing 10.3% (wt/vol) sucrose. This mixture was
dispensed into a mold (Pharmacia Biotech) in order to produce small
blocks that were used in pulsed-field gel electrophoresis (PFGE) and
allowed to solidify. The blocks were then immersed in a protease
solution (5 mg of pronase E [Sigma] per ml, 1% [wt/vol] N-laurylsarcosine, 500 mM EDTA; pH 8) and incubated at
45°C overnight. After the blocks were washed twice (1 h each) at
50°C with TE buffer (10 mM Tris-HCl [pH 7.5], 1 mM EDTA), between
one-quarter and one-half of each block was transferred to a 1.2%
(wt/vol) NA-agarose (Pharmacia Biotech) gel. PFGE was performed at
10°C in TBE buffer (45 mM Tris base, 44 mM boric acid, 1 mM EDTA; pH 7.5) by using a Gene Navigator pulsed-field system (Pharmacia Biotech)
with the following settings: 100 to 120 mA; 165 V; and a 90-s pulse for
14 h, a 105-s pulse for 12 h, and a 120-s pulse for 14 h. Yeast DNA PFGE markers (Pharmacia Biotech) were used for molecular
weight calibration. Following electrophoresis, the gel was stained with
ethidium bromide in TBE buffer and photographed.
Chromosome blotting and hybridization.
The chromosomes
separated by PFGE were transferred to a nylon membrane (Boehringer
Mannheim) by using a VacuGene XL blotting system (Pharmacia Biotech) as
recommended by the manufacturer. Following this transfer, the DNA was
UV cross-linked to the nylon membrane. For hybridization, the membrane
was initially incubated for 1 h at 67°C in a solution containing
5× SSC, 0.1% (wt/vol) N-laurylsarcosine, 0.02% (wt/vol)
sodium dodecyl sulfate (SDS), and 2% (wt/vol) blocking reagent
(Boehringer Mannheim) (1× SSC is 0.15 M NaCl plus 15 mM sodium
citrate). The denatured, digoxigenin (DIG)-labeled probe (10 ng) was
then added to the solution, and the preparation was incubated at 67°C
overnight. The membrane was washed twice (5 min each) at room
temperature with 2× SSC-0.1% (wt/vol) SDS and then twice (15 min
each) at 67°C with 0.1× SSC-0.1% (wt/vol) SDS. Chemiluminescent
detection of DIG hybrids on the membrane was performed by using CSPD
(Boehringer Mannheim) as directed by the manufacturer. X-ray film
(Kodak BioMax MR) was exposed to the membrane for 1 to 3 h before
it was developed.
Preparation of DIG-labeled probes.
DIG-labeled probes were
prepared by PCR as described previously (27). Genomic DNA
from yeast strain 1403-7A (MATa MAL4 MGL3 gal3 gal4
ura3; Yeast Genetic Stock Center, Berkeley, Calif.) was generally
used as the template for PCR. This strain has been shown to contain the
AGT1 gene (14). For production of a probe hybridizing to the MAL-encoded maltose transporter gene,
plasmid pSC138 containing MAL61 (4) was used as
the template. The primers used for amplification of DNA by PCR (Table
1) were designed by using the program
Primer3
(http://www.genome.wi.mit.edu/cgi-bin/primer/primer3.cgi) and specifying a product length of 750 to 1,000 bp. Probes were stored
at 
20°C and were denatured immediately prior to use by diluting
them to a volume of 0.1 ml with distilled water and heating them at
100°C for 10 min.
Other methods.
Nucleotide sequences of yeast genes or ORFs
were obtained from the Saccharomyces Genome Database
(http://genome-www.stanford.edu/Saccharomyces/). Updates on the sizes
of yeast chromosomes were obtained from the Munich Information Center
for Protein Sequences
(http://speedy.mips.biochem.mpg.de/mips/yeast/index.htmlx).
| |
RESULTS |
Maltose transporter genes in brewing strains of yeast.
Following separation by PFGE, chromosomes from each of the yeast
strains were blotted onto nylon membranes and hybridized with the
MAL61 probe. Representative results are shown in Fig. 1, and the complete results obtained for
all 30 strains are summarized in Table 2.
It is quite possible that some of the MAL alleles detected
in the yeast strains are mutated and therefore do not encode functional
products. Indeed, naturally occurring mutations at the MAL
loci, in particular at MAL1, have been reported previously (19). For this reason, the genotypes shown in Table 2 should be considered tentative. Nevertheless, brewing strains of yeast must be
able to take up maltose efficiently, and so it is reasonable to suppose
that the majority of the alleles detected with the MAL61
probe do in fact encode a functional transporter.
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FIG. 1.
Detection of MAL transporter genes in brewing
strains of yeast. (A) Separation of chromosomes of yeast strains by
PFGE. (B) Detection of MAL transporter genes after
chromosomes were blotted onto a nylon membrane and hybridized with a
probe for MAL61. Images were obtained by scanning with a
Color OneScanner 600/27 scanner operated from an Apple Macintosh and
were annotated in Adobe Photoshop 3.0 (Macintosh version). The Roman
numerals on the left in panel A are the chromosome numbers, in order of
decreasing size where more than one numeral is given on a line. The
values on the right in panel A are the sizes (in kilobase pairs) of
selected chromosomes. The positions of chromosomes carrying
MAL11, MAL21, MAL31, and
MAL41 are indicated on the right in panel B. Bands a and b
are bands for specific chromosomes, as described in the text. Lanes 1 and 12, markers; lanes 2, KVL011; lanes 3, KVL012; lanes 4, KVL001;
lanes 5, KVL004; lanes 6, KVL005; lanes 7, KVL006; lanes 8, KVL018;
lanes 9, KVL021; lanes 10, KVL024; lanes 11, KVL026.
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MAL11,
MAL31, and
MAL41 sequences were
detected in all of the brewing strains examined except the lager yeast
strains KVL021
(which lacked
MAL11) and KVL026 (which lacked
all three sequences)
(see below). Although all of the ale strains
lacked
MAL21 (Fig.
1B, lanes 2 and 3), this gene was found
in 13 of the 25 lager
strains (Table
2). For many of these strains, the
signal corresponding
to
MAL21 was weak (Fig.
1B, lanes 8 and
10). One explanation for
this could be mutation which resulted in the
loss of homology
to the
MAL61 probe. Alternatively, there
may be fewer copies of
MAL21 in the genome compared to the
number of
MAL11,
MAL31, and
MAL41
copies. We favor the second of these possibilities, as there
is no
obvious reason why
MAL21 should have a higher level of
mutation
than the other
MAL transporter genes have. Brewing
strains of
yeast are polyploid or, in the case of lager strains,
alloploid
(
16), and different copies of the same chromosome
are not necessarily
expected to be identical in structure and to
contain the full
complement of genes.
MAL61 (on chromosome
VIII) was not detected
in any of the strains
examined.
In general, the sizes of the different chromosomes carrying the
MAL genes were highly conserved in different yeast strains,
although chromosome length polymorphisms were evident in some
of the
strains. Size doublets were clearly observed for chromosome
VII
(carrying
MAL11) of KVL001 and KVL005 (Fig.
1B, lanes 4 and
6). In KVL001, the same doublet was detected with a probe for
BGL2 (another gene mapping to chromosome VII) (results not
shown),
which confirmed that both chromosomes were chromosome VII. A
similar
test was not carried out with KVL005, but it is very likely
that
in this strain the doublet also resulted from two copies of
chromosome
VII that were different sizes (as observed for KVL001).
Similarly,
a doublet that may have corresponded to two copies of
chromosome
II (which contains
MAL31) was obtained for KVL018
(Fig.
1B, lane
8). However, only the smaller chromosome in the doublet
hybridized
to a probe for
LYS2 (which maps to chromosome II)
(results not
shown). The identity of the larger chromosome (length,
approximately
850 kbp) is therefore
unclear.
KVL021 and KVL026 were significantly different from the other lager
strains with respect to the patterns of hybridization
of their
chromosomes to the
MAL61 probe. In the case of KVL021,
a
chromosome with an estimated size of 1,350 kbp exhibited relatively
weak hybridization to the
MAL61 probe (Fig.
1B, lane 9, band
a).
At first, we thought that this chromosome probably corresponded
to
chromosome VII, which is the largest chromosome (length, 1,091
kbp)
(
25) known to carry a
MAL locus. Even so, a
substantial
addition of DNA (250 kbp) would have been necessary to
account
for the size of the 1,350-kbp chromosome in KVL021. However,
this
chromosome could not be detected with probes for the
BGL2 or
SER2 genes, both of which map to
chromosome VII (Table
1). Recently,
Tamai et al. (
24)
identified a 1,350-kbp chromosome in
S. pastorianus that
appeared to originate from the non-
S. cerevisiae parent
(namely,
Saccharomyces bayanus) (
22).
Translocation of
MAL to the 1,350-kbp
chromosome would
account for the hybridization pattern which we
observed. With KVL021
there was no strong hybridization at the
expected position for
chromosome VII (1,091 kbp) (
25).
The pattern obtained for KVL026 was very different from the patterns
obtained for all of the other yeast strains included
in this study,
both ale and lager, when chromosomes were probed
with
MAL61.
Although the
MAL61 probe detected a chromosome at
1,150 kbp
(Fig.
1B, lane 11) that was thought most likely to be
chromosome VII,
the
BGL2 probe specific for chromosome VII failed
to detect
this chromosome (results not shown). Another KVL026
chromosome that
hybridized to the
MAL61 probe migrated at approximately
900 kbp (Fig.
1B, lane 11, band b). The size of this chromosome
is about 85 kbp greater than the size of chromosome II (
9),
the closest
size match for a chromosome that carries a known
MAL locus
(in this case,
MAL3). However, a probe for
LYS2
(which maps
to chromosome II) (Table
1) failed to detect the 900-kbp
chromosome.
Detection of the general -glucoside transporter gene
AGT1.
DNA that hybridized to the AGT1 probe was
found in all of the yeast strains except KVL026. The results obtained
for some of the strains are shown in Fig.
2. For the majority of the strains that exhibited hybridization to the AGT1 probe, the
signal mapped, as expected, to chromosome VII. In the case of the five
ale strains (KVL011 to KVL015) (Fig. 2B, lanes 2 to 6), two bands that
migrated close together were observed. These bands were probably
chromosome length polymorphisms of chromosome VII, as demonstrated for
KVL001 with the BGL2 probe (see above). A similar pattern
was also observed for some of the lager strains, including KVL001 (Fig.
2B, lane 8). For KVL021, a chromosome that hybridized to the
AGT1 probe was detected at 1,350 kbp (Fig. 2B, lane 10, band
a), as was the case for the MAL61 probe. In contrast to the
findings obtained with the MAL61 probe, a copy of
chromosome VII that hybridized to the AGT1 probe was also
detected in KVL021 at the expected position, about 1,100 kbp (Fig. 2B,
lane 10). Two of the lager strains in addition to KVL021 showed
evidence of AGT1-related sequences located on other
chromosomes. For KVL006, a signal was observed at approximately 860 kbp
(Fig. 2B, lane 9, band b); this signal may have corresponded to either
chromosome II (813 kbp) or chromosome XIII (924 kbp) (2, 9).
Similarly, strong hybridization to a chromosome migrating at 670 kbp
(tentatively identified as chromosome XI at 666 kbp) (6) was
observed for KVL021 (Fig. 2B, lane 10, band c).
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FIG. 2.
Detection of the AGT1 gene and homologues in
brewing strains of yeast. (A) Separation of chromosomes of yeast
strains by PFGE. (B) Detection of AGT1 after chromosomes
were blotted onto a nylon membrane and hybridized with a probe for
AGT1. Images were obtained by scanning with a Color
OneScanner 600/27 scanner operated from an Apple Macintosh and were
annotated in Adobe Photoshop 3.0 (Macintosh version). The Roman
numerals on the left in panel A are the chromosome numbers, in order of
decreasing size where more than one numeral is given on a line. The
values on the right in panel A are the sizes (in kilobase pairs) of
selected chromosomes. The position of chromosome VII carrying
AGT1 is indicated on the right in panel B; bands a, b, and c
are bands for specific chromosomes, as described in the text. Lanes 1 and 12, markers; lanes 2, KVL011; lanes 3, KVL012; lanes 4, KVL013;
lanes 5, KVL014; lanes 6, KVL015; lanes 7, KVL024; lanes 8, KVL001;
lanes 9, KVL006; lanes 10, KVL021; lanes 11, KVL026.
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Detection of the maltose transporter gene homologues YDL247w
and YJR160c.
YDL247w (chromosome IV) and YJR160c (chromosome
X) are two ORFs that encode products that exhibit 96% identity with
each other, 74% identity with MAL61, and 53% identity with
AGT1. The high levels of homology exhibited by the products of
YDL247w and YJR160c to MAL61 and AGT1 suggest that these proteins
play a role in -glucoside transport. The 5' noncoding regions of
YDL247w and YJR160c are more than 99% identical (for at least
2,000 nucleotides upstream of the translational start), but
they lack any significant similarity to the 5' noncoding regions
of MAL61 and AGT1. This suggests that YDL247w and
YJR160c may be regulated rather differently than MAL61 and
AGT1 are.
Hybridization of the YDL247w-YJR160c probe to chromosome blots revealed
that whereas most of the yeast strains contained nucleotide
sequences
corresponding to YDL247w, none contained YJR160c. Figure
3 shows the results obtained for 10 of
the strains examined. As
only YDL247w was detected, it is likely that
this ORF represents
the ancestral sequence. Only 4 of the 30 strains
that were investigated
(ale strains KVL011 [Fig.
3B, lane 2], KVL014,
and KVL015 and
lager strain KVL026 [Fig.
3B, lane 11]) lacked
YDL247w.
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FIG. 3.
Detection of YDL247w in brewing strains of yeast. (A)
Separation of chromosomes of yeast strains by PFGE. (B) Detection of
MAL transporter genes after chromosomes were blotted onto a
nylon membrane and hybridized with a probe for YDL247w and YJR160c.
Images were obtained by scanning with a Color OneScanner 600/27 scanner
operated from an Apple Macintosh and were annotated in Adobe Photoshop
3.0 (Macintosh version). The Roman numerals on the left in panel A are
the chromosome numbers, in order of decreasing size where more than one
numeral is given on a line. The values on the right in panel A are the
sizes (in kilobase pairs) of selected chromosomes. The positions of
chromosomes carrying YDL247w (and, for the markers, YJR160c) are
indicated on the right in panel B; bands a and b are bands for specific
chromosomes, as described in the text. Lanes 1 and 12, markers; lanes
2, KVL011; lanes 3, KVL012; lanes 4, KVL013; lanes 5, KVL024; lanes 6, KVL001; lanes 7, KVL004; lanes 8, KVL006; lanes 9, KVL018; lanes 10, KVL021; lanes 11, KVL026.
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Evidence that other chromosomes exhibited strong hybridization to the
YDL247w-YJR160c probe was obtained for four of the lager
strains
examined (KVL004, KVL006, KVL018, and KVL021) (Fig.
3B,
lanes 7 to 10).
In two of these strains, KVL004 and KVL006, the
chromosome containing
the homologous sequence is about 1,050 kbp
long (Fig.
3B, band a). This
chromosome most likely corresponds
to either chromosome VII or
chromosome XV (both of which are 1,091
kbp long) (
7,
25). In
KVL018 and KVL021, the chromosomes
containing sequences homologous to
YDL247w and YJR160c sequences
are 960 and 990 kbp long, respectively
(Fig.
3B, band b). The
closest match to these chromosomes is
chromosome XVI (length,
948 kbp) (
3).
Mapping of AGT1 homologues to chromosomes.
Additional blotting and hybridization experiments were carried out to
map two of the homologues identified with the AGT1 probe. Chromosomes from yeast strains KVL006 and KVL021 were subjected to PFGE
in duplicate and blotted. One blot of each duplicate was hybridized
with the AGT1 probe, and the other was hybridized with a
gene probe that hybridized to a known chromosome (Fig.
4).
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FIG. 4.
Mapping of the AGT1 homologues found in yeast
strains KVL006 and KVL021. Lane 1, chromosome blot of KVL006 hybridized
with a probe for AGT1; lane 2, same as lane 1 except that
the blot was hybridized with a LYS2 probe; lane 3, chromosome blot of KVL021 hybridized with a probe for AGT1;
lane 4, same as lane 3 except that the blot was hybridized with a
TRP3 probe. Images were obtained by scanning with a Color
OneScanner 600/27 scanner operated from an Apple Macintosh and were
annotated in Adobe Photoshop 3.0 (Macintosh version). The position of
chromosome VII containing AGT1 is indicated on the right in
lanes 1 and 3. Band a, KVL006 chromosome containing a homologue of
AGT1; bands b and c, KVL006 chromosome II (containing
LYS2); band d, KVL021 chromosome containing a homologue of
AGT1; band e, KVL021 chromosome XI (containing
TRP3).
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For KVL006, chromosome blots were separately hybridized with the
AGT1 probe and a probe for
LYS2 (a gene that is
located on
chromosome II). The
LYS2 probe detected two
copies of chromosome
II whose sizes differed by about 60 kbp (Fig.
4,
lane 2, bands
b and c). The larger copy comigrated with the chromosome
carrying
the homologue of
AGT1 (Fig.
4, lane 1, band a),
confirming that
this homologue does indeed lie on chromosome II. A
similar experiment
was carried out in order to map the
AGT1
homologue found in KVL021
to chromosome XI. A probe for
TRP3, which hybridized to chromosome
XI (length, 666 kbp)
(
6), produced a single band on the developed
blot (Fig.
4,
lane 4, band e). This band comigrated with the chromosome
carrying the
AGT1 homologue in KVL021 (Fig.
4, lane 3, band
d).
| |
DISCUSSION |
It is likely that because of the demands of brewery fermentation,
brewing strains of yeast have been subjected to selection for more
efficient fermentation of maltose and maltotriose. The finding that
most strains examined in this study contained sequences corresponding
to the maltose transporter gene at three or four different
MAL loci is therefore not surprising. The complete absence of MAL61 was more unexpected. However, in a study of 28 strains of S. cerevisiae isolated from natural sources,
Naumov et al. (19) identified only 1 strain that carried the
MAL6 locus. Similarly, Oda and Tonomura (21)
found that only one of seven baking strains of S. cerevisiae
which they examined contained MAL6. These findings and the
results of this study suggest that the evolution of MAL6 may
have been a comparatively recent event.
A majority of the yeast strains used in this study, both ale and lager,
contained MAL transporter gene sequences that mapped to
chromosomes previously shown to carry MAL loci. For three of the lager strains, KVL018, KVL021, and KVL026, there is evidence that
MAL transporter genes occur on other chromosomes. In the case of the 1,350-kbp chromosome of KVL021 that hybridizes to the
MAL61 probe, the best size match is with a chromosome from S. bayanus rather than S. cerevisiae.
Telomeric translocation of a MAL locus from a chromosome of
S. cerevisiae would account for this. The most likely source
of the translocated DNA is chromosome VII, since the MAL1
locus on this chromosome is thought to be the progenitor of the other
MAL loci (5). The 1,350-kbp chromosome could not
be detected with a probe for BGL2, which is located about 18 kbp from the MAL1 locus (23). This suggests that
either the translocated DNA sequence is relatively short or it was
derived from a chromosome other than chromosome VII.
With the exception of KVL026, all of the yeast strains examined in this
study contained chromosomes that hybridized to the AGT1
probe. Most of these strains contained both AGT1 and
MAL11 sequences on chromosome VII, and there are three
possible explanations for this. One is that different copies of
chromosome VII in the same strain carry either MAL11 or
AGT1. Since brewing strains of yeast are polyploid or
aneuploid, this is quite plausible. Alternatively, AGT1 and
MAL11 may be closely linked on the same chromosome. This
seems less likely since AGT1 has been shown (at least in
laboratory strains of yeast) to be an allele of MAL11 (14). A third possibility, the least likely possibility, is that hybrid genes with homology to both the AGT1 and
MAL61 probes are present. Cloning and sequencing of the
MAL11 and AGT1 alleles and adjacent DNA would
allow these possibilities to be explored.
Homologues of AGT1 were identified in two of the lager
strains of yeast (KVL006 and KVL021), and these homologues were
separately mapped to chromosomes II and XI. Interestingly, each of
these chromosomes carries a copy of the MAL locus
(MAL3 on chromosome II and MAL4 on chromosome
XI). Since AGT1 is an allele of MAL11 (on
chromosome VII), it is quite possible that the homologues of
AGT1 identified on chromosomes II and XI are themselves
alleles of MAL31 and MAL41, respectively. Cloning
of the AGT1 homologues followed by DNA sequencing would have
to be carried out to investigate this further.
Han et al. (14) proposed that AGT1 arose from two
recombination events. The first of these events was the translocation of AGT1 sequences to the telomere of chromosome VII, and the
second event brought AGT1 into position within the
MAL locus (MAL1) on that chromosome. A similar
set of events may have been responsible for the origin of the
AGT1 homologues found on chromosomes II and XI.
Alternatively, the translocations that gave rise to the spread of
MAL loci from the ancestral locus on chromosome VII may also
have been responsible for the dispersal of AGT1 sequences to
chromosomes II and XI. Han et al. (14) speculated that
AGT1 might be just one member of a gene family in
Saccharomyces spp., and the findings reported here provide
the first evidence that such a family of genes does indeed exist.
Naumov et al. (19) were not able to detect AGT1
sequences in the yeast strains which they examined, but this is
probably because they used a MAL61 probe in their
hybridization analysis. As shown here, under stringent conditions there
is no detectable hybridization between AGT1 and MAL-encoded transporter genes. Nevertheless, it remains to
be seen how widespread AGT1 is among nonbrewing
Saccharomyces strains. The presence of maltotriose in
brewer's wort may have imposed selection for AGT1 and other
closely related genes in brewing strains of yeast, and such selection
should not apply to many nonbrewing strains, such as wine yeast strains.
An analysis of YDL247w and YJR160c sequences in brewing strains of
yeast was included in this study because it has been predicted that
both of these ORFs encode products that are highly homologous to the
MAL-encoded transporter. The fact that YDL247w and YJR160c were discovered not through genetic analysis of maltose utilization but
by genome sequencing suggests that their products do not play a major
role in maltose transport. One possible function of the proteins
encoded by YDL247w and YJR160c might be in low-affinity transport of
maltose, although this phenomenon has been ruled out in one study as an
artifact (1). The finding that YJR160c was absent from all
of the yeast strains surveyed in this study leads to the conclusion
that YDL247w, which was found in all but four strains, is the
progenitor sequence. Both YDL247w and YJR160c are subtelomeric,
suggesting that a mechanism involving translocation of telomeric
sequences (as proposed for the MAL loci) may have given rise
to this gene family. The YDL247w and YJR160c homologues identified in
some of the brewing strains which we examined (Fig. 3) are therefore
also likely to be telomere associated.
This work was undertaken to obtain information on maltose and
maltotriose transporter genes in brewing strains of yeast, because the
products of these genes are expected to have a major influence on yeast
fermentation performance in brewing. As expected, MAL transporter gene sequences were widespread. We also found evidence that
there is a family of genes related to AGT1, which may
contribute to efficient fermentation of maltotriose. Additional work
involving a functional analysis of the AGT1-related
sequences will be necessary to investigate this possibility.
| |
ACKNOWLEDGMENTS |
This work was supported by the FØTEK program, which was
sponsored by the Danish Ministry of Education through LMC (the Centre for Advanced Food Studies) and Alfred Jørgensen Laboratory Ltd. (Copenhagen, Denmark).
| |
FOOTNOTES |
*
Corresponding author. Mailing address: The
International Centre for Brewing and Distilling, Heriot-Watt
University, Riccarton, Edinburgh EH14 4AS, Scotland. Phone: 44 131 451 3641. Fax: 44 131 451 3009. E-mail:
P.G.Meaden{at}hw.ac.uk.
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Top
Abstract
Introduction
