Physical localization of the flocculation gene FLO1 on chromosome I of Saccharomyces cerevisiae

The genetics of flocculation in the yeast Saccharomyces cerevisiae are poorly understood despite the importance of this property for strains used in industry. To be able to study the regulation of flocculation in yeast, one of the genes involved, FLO1, has been partially cloned. The identity of the gene was confirmed by the non-flocculent phenotype of cells in which the C-terminal part of the gene had been replaced by the URA3 gene. Southern blots and genetic crosses showed that the URA3 gene had integrated at the expected position on chromosome I. A region of approximately 2 kb in the middle of the FLO1 gene was consistently deleted during propagation in Escherichia coli and could not be isolated. Plasmids containing the incomplete gene, however, were still able to cause weak flocculation in a nonflocculent strain. The 3′ end of the FLO1 gene was localized at approximately 24 kb from the right end of chromosome I, 20 kb centromere-proximal to PHO11. Most of the newly isolated chromosome I sequences also hybridized to chromosome VIII DNA, thus extending the homology between the right end of chromosome I and chromosome VIII to approximately 28 kb.     

Characterization of Kluyveromyces lactis subtelomeric sequences including a distal element with strong purine/pyrimidine strand bias

Telomeres are the specialized structures at the ends of eukaryotic chromosomes and are composed of short T/G-rich DNA repeats and the proteins that interact with them. Internal to telomeres are subtelomeric regions that are species-specific and often repetitive. The yeast Kluyveromyces lactis has telomeric tracts of 10-20 copies of a 25 bp repeat, but the subtelomeric regions have not previously been characterized in detail. Here we have cloned and characterized subtelomeric regions from 10 of the 12 chromosome ends. The amount of sequence examined was 0.7-10 kb for each subtelomeric region. We have identified a K. lactis subtelomeric element, the R element, which has a strong purine/pyrimidine strand bias and extends for about 2 kb. Internal to the R element, we found extensive similarity that is shared among half of the chromosome ends reported here. This similarity appears to include three putative gene families, two of which are also subtelomeric in Saccharomyces cerevisiae.     

Genetic analysis of the karyotype instability in natural wine yeast strains.

Yeast strains isolated from the wild may show high rates of changes in their karyotypes during vegetative growth. We analysed over 500 karyotypes from mitotic and meiotic derivatives of strain DC5, which has a chromosome rearrangement rate of 8.2 x 10(-3) changes/generation. About 70% of the meiotic derivatives of DC5 had low rearrangement rates, with an average of 5.8 x 10(-4) changes/generation, suggesting that karyotype instability behaved as a dominant phenotype. Diploid derivatives with low karyotype variability in mitosis also had low rates of chromosomal rearrangement during meiosis, suggesting that the two phenotypes may be linked. DC5 and some of its meiotic derivatives (both with high and low karyotype variability) had chromosome XII hypervariable bands. Their distribution among the meiotic products indicates that they are not indicators for genetic instability. To our knowledge, data in this paper are the first to indicate that karyotypically unstable yeast strains may give stable progeny at high rates. Understanding of the relevant mechanism(s) may allow the design of genetic strategies to stabilize karyotypes from natural and/or industrial wine yeasts with unacceptable karyotype rearrangement rates.     

CONSTRUCTION OF FLOCCULENT BREWER'S YEAST BY CHROMOSOMAL INTEGRATION OF THE YEAST FLOCCULATION GENE FLO1

Yeast flocculation gene FLO1, located on chromosome I of Saccharomyces cerevisiae, has been cloned previously¹⁶. However, it has recently been found that the gene was an in-frame deletion derivative of the chromosomal intact FLO1 gene¹⁹. When introduced into non-flocculent industrial strains, including brewer's yeast, the latter gene, FLO1L, containing an open reading frame of 4,611 bp, conferred stronger flocculation than the former gene, FLO1S, containing an open reading frame of 2,586. By chromosomal integration of the ADH1-controlled FLO1L gene, “gene therapy” of the flocculation behaviour of the parent non-flocculent brewer's yeast was successfully achieved.     

Localization of the dominant flocculation genes FLO5 and FLO8 of Saccharomyces cerevisiae

In the yeast Saccharomyces cerevisiae three dominant flocculation genes, FLO1, FLO5 and FLO8 have been described. Until now only the FLO1 gene, which is located at chromosome I, has been cloned and sequenced. FLO5 and FLO8 were previously localized at chromosomes I and VIII respectively (Vezinhet, F., Blondin, B. and Barre, P. (1991). Mapping of the FLO5 gene of Saccharomyces cerevisiae by transfer of a chromosome during cytoduction. Biotechnol. Lett. 13 , 47–52; Yamashita, I. and Fukui, S. (1983). Mating signals control expression of both starch fermentation genes and a novel flocculation gene FLO8 in the yeast Saccharomyces. Agric. Biol. Chem. 47 , 2889–2896). This was not in agreement with our results. Here, we report the location of FLO5 and FLO8 on chromosomes VIII and I respectively. By induced chromosome loss and genetic mapping, the FLO5 gene was localized at the right end of chromosome VIII approximately 34 cM centromere distal of PET3. This is part of the region that is present both at chromosome I and chromosome VIII. The location of FLO5 in this area of chromosome VIII made it necessary to re-evaluate the localization of FLO8, which was previously thought to occur in this region. Both genetic and physical mapping showed that FLO8 is allelic to FLO1. Hence, there are only two known dominant flocculation genes, FLO1 and FLO5. Analysis of the nucleotide sequence of chromosome VIII of a non-flocculent strain revealed an open reading frame encoding a putative protein that is approximately 96% identical to the Flo1 protein. This suggests that both dominant flocculation genes encode similar, cell wall-associated, proteins with the same function in the flocculation mechanism.     

The telomere-associated MAL3 locus of Saccharomyces is a tandem array of repeated genes.

Saccharomyces strains capable of fermenting maltose contain any one of five telomere-associated MAL loci. Each MAL locus is a complex of three genes encoding the three functions required to ferment maltose: maltose permease (GENE 1), maltase (GENE 2) and the MAL trans-activator (GENE 3). All five loci have been cloned and all are highly sequence homologous over at least a 9.0 kbp region containing these GENEs (Charron et al., Genetics 122, 307-331, 1989). Our initial studies of strains carrying the MAL3 locus indicated the presence of linked, repeated MAL-homologous sequences (Michels and Needleman, Mol. Gen. Genet. 191, 225-230, 1983). Here we report our analysis of the centromere-proximal MAL3-linked sequences and show that the complete MAL3 locus spans approximately 40 kbp and consists of tandemly arrayed, partial repeats of the three GENE sequences described above. In addition, the structure of the MAL3 locus is compared to that of three partially functional alleles of MAL3. These alleles were shown to contain only MAL31 and MAL32 and their structure suggests that they resulted from MAL3 deletions removing the sequences centromere-proximal to MAL31. The amplification and rearrangement of the telomere-linked MAL3 sequences are discussed in the context of studies on other telemere-associated sequences from yeast and other species.     

Yeast flocculation: reconciliation of physiological and genetic viewpoints.

Yeast flocculation results from surface expression of specific proteins (lectins). Two flocculation phenotypes were suggested by physiological and biochemical tests, whereas genetic data suggested a larger number of mechanisms of flocculation. After reviewing the biochemistry, physiology and genetics of flocculation, a new hypothesis combining the data available from these different sources, is proposed. Flocculation results when lectins present on flocculent cell walls bind to sugar residues of neighbouring cell walls. These sugar receptors are intrinsic to the mannan comprising cell walls of Saccharomyces cerevisiae. Two lectin phenotypes were revealed by sugar inhibition studies. The gluco- and mannospecific NewFlo phenotype is not, as yet, found in genetically defined strains. Mannospecific flocculation (Flo1 phenotype) is found in strains containing the genes FLO1, FLO5 and FLO8. This phenotype is also found following mutation of the TUP1 or CYC8 loci, in previously non-flocculent strains. It is therefore proposed that the structural gene for mannospecific flocculation is common or possibly ubiquitous in non-flocculent strains and in consequence, FLO1, FLO5 and FLO8 are probably regulatory genes, exerting positive control over the structural gene. Flocculation expression requires lectin secretion to the cell surface. Many of the observed 'suppressions' of flocculation may be due to mutations of the secretory process, involved in transporting structural proteins to the cell wall. The possible involvement of killer L double-stranded RNA with flocculation is suggested, given the lectin properties of viral coat proteins and an association between L double-stranded RNA and the Flo1 phenotype.     

A family of laboratory strains of Saccharomyces cerevisiae carry rearrangements involving chromosomes I and III

In order to study meiotic segregation of chromosome length polymorphism in yeast, we analysed the progeny of a cross involving two laboratory strains FL100trp and YNN295. Analysis of the parental strains led us to detect an important length polymorphism of chromosomes I and III in FL100trp. A reciprocal translocation involving 80 kb of the left arm of chromosome III and 45 kb of the right arm of chromosome I was shown to be the cause for the observed polymorphism in this strain. The characterization of the translocation breakpoints revealed the existence of a transposition hot-spot on chromosome I: the sequence of the translocation joints on chromosomes I and III suggests that the mechanism very likely involved homologous recombination between Ty2 transposable elements on each chromosome. Analysis of FL100, FL200 and FL100trp ura, which are related to FL100trp, shows that this reciprocal translocation is present in some of the strains of the FL series, whereas the parental strain FL100 does not carry the same rearrangement. We evidenced instead the duplication of 80 kb of chromosome III on chromosome I and a deletion of 45 kb of the right arm of chromosome I in this strain, indicating that secondary events might have taken place and that the strain currently named FL100 is not the common ancestor of the FL series. © 1998 John Wiley & Sons, Ltd.     

Analysis of chromosomal DNA patterns of the genus Kluyveromyces

Using an improved procedure of pulsed field gel electrophoresis, yeast chromosomes were separated over a wide range of molecular size (250-4000 kbp) on single gels. The chromosomal DNA patterns of all the species belonging to the genus Kluyveromyces were examined. Within the species K. marxianus, the varieties lactis, drosophilarum and vanudenii showed closely related patterns: very different from them, the varieties bulgaricus and marxianus were related to each other, forming a distinct group; the strains commonly called 'K. lactis' and 'K. fragilis' were unambiguously different from each other in chromosome patterns. These differences were correlated with the presence of characteristic repetitive sequence elements in the mitochondrial DNA of the former group and not in the latter. Analysis of Candida macedoniensis, which had been considered to be an anamorph of K. marxianus var. marxianus, showed that these two yeast species were indeed similar in chromosome patterns and in mitochondrial DNA restriction patterns.     

FLO11 is essential for pellicle formation by wild pellicle-forming yeasts isolated from contaminated wines

The Δflo11/Δflo11 disruptant strains constructed from three wild yeast strains, which were independently isolated from pellicles formed on the surface of contaminated stored wines, completely lost their ability to form pellicles. The FLO11 expression level of the pellicle-forming strains was much higher than that of non-pellicle-forming wine strains. Cell surface hydrophobicity of the Δflo11/Δflo11 disruptant strains was as high as that of the parental pellicle-forming strains. These findings indicate that FLO11 is essential for unfavorable pellicle formation of wild pellicle-forming yeasts by mechanisms other than increasing cell surface hydrophobicity.     

Common industrial sake yeast strains have three copies of the AQY1-ARR3 region of chromosome XVI in their genomes

Genomic analysis of industrial yeast strains is important for exploitation of their potential. We analysed the genomic structure of the most widely used sake yeast strain, Kyokai no. 7 (K7), by DNA microarray. Since the analysis suggested that the copy number of the AQY1-ARR3 region in the right arm of chromosome XVI was amplified, we performed Southern blot analysis using the AQY1 gene as a probe. The probe hybridized to three bands in the widely used sake strains derived from K7, but only to one band of 1.4 kb in the laboratory strains. Since the extra two bands were not observed in old sake strains, or in other industrial strains, the amplification of this region appeared to be specific for the widely used sake strains. The copy number of the AQY1-ARR3 region appeared to have increased by chromosomal translocation, since chromosomal Southern blot analysis revealed that the AQY1 probe hybridized to chromosomes IV and XIII, in addition to chromosome XVI, in which AQY1 of the laboratory strain is encoded. The chromosomal translocation was also confirmed by PCR analysis using primers that amplify the region containing the breakpoint. Cloning and sequencing of cosmids that encode the AQY1-ARR3 region revealed that this region is flanked by TG(1-3) repeats on the centromere-proximal side in chromosomes IV and XIII, suggesting that amplification of this region occurred by homologous recombination through TG(1-3) repeats. These results demonstrate the genomic characteristics of the modern widely used sake strains that discriminate them from other strains.     

Multi-locus genotyping of bottom fermenting yeasts by single nucleotide polymorphisms indicative of brewing characteristics

Yeast plays a capital role in brewing fermentation and has a direct impact on flavor and aroma. For the evaluation of competent brewing strains during quality control or development of novel strains it is standard practice to perform fermentation tests, which are costly and time-consuming. Here, we have categorized DNA markers which enable to distinguish and to screen brewing strains more efficiently than ever before. Sequence analysis at 289 loci in the genomes of six bottom fermenting Saccharomyces pastorianus strains revealed that 30 loci contained single nucleotide polymorphisms (SNPs). By determining the nucleotide sequences at the SNP-loci in 26 other S. pastorianus strains and 20 strains of the top fermenting yeast Saccharomyces cerevisiae, almost all these strains could be discriminated solely on the basis of the SNPs. By comparing the fermentative phenotypes of these strains we found that some DNA markers showed a strong association with brewing characteristics, such as the production of ethyl acetate and hydrogen sulphide (H2S). Therefore, the DNA markers we identified will facilitate quality control and the efficient development of brewing yeast strains.     

Mapping the putative RNA helicase genes by sequence overlapping.

An 'electronic' gene mapping procedure based on computer-aided search for overlapping gene sequences was used to identify adjacent genes and localize several putative RNA helicase genes to different chromosomes. PRP28 and AMD1 genes map to the right arm of chromosome IV next to sup2, which encodes a tyrosine tRNA. PRP16, previously mapped to chromosome XI, is tightly linked to MRP-L20. PRP22 is adjacent to PRE1, whose chromosomal location is currently unknown. The utility of this approach in yeast gene mapping is evaluated.     

The 2μm plasmid of laboratory yeast strains is a type-1/type-2 hybrid

Industrial yeast strains carry one of two homeologous 2μm plasmids designated as type-1 or type-2. The 2μm plasmid, Scp1, found in common laboratory strains of Saccharomyces cerevisiae is considered a type-2 plasmid, since the ori, STB, RAF and REP1 loci and intergenic sequences of the right-unique region of Scp1 are homologous to the corresponding loci in industrial strain type-2 plasmids. However, within both its 599 bp inverted repeats Scp1 has 142-bp sequences homologous to the bakers' yeast type-1 plasmid. DNA sequence analyses and oligonucleotide hybridizations indicate that the 142-bp insertion in Scp1 was probably due to homeologous recombination between type-1 and type-2 plasmids. These results suggest that some of the plasmid and chromosomal sequence polymorphisms seen in laboratory yeast strains result from homeologous recombination in their ancestral breeding stock.     

III. Yeast sequencing reports. Corrected sequence for the right telomere of Saccharomyces cerevisiae chromosome III

A comparison of the sequences of telomere regions from several yeast chromosomes revealed an apparent cloning artifact for the right end of chromosome III. An integrating vector containing G_1–3T telomere sequences was used to clone the right end of chromosome III from a strain related to S288C. The sequence of this clone confirmed that the published sequence was incorrect and demonstrated that the right telomere region of chromosome III is similar to other telomeres.     

Inferences of evolutionary relationships from a population survey of LTR-retrotransposons and telomeric-associated sequences in the Saccharomyces sensu stricto complex

The Saccharomyces sensu stricto complex consists of six closely related species and one natural hybrid. Intra- and inter- species variability in repetitive elements can help elucidate the population structure and evolution of these close relatives. The chromosome positions of several telomeric associated sequences (TASs) and LTR-retrotransposons have been determined, using PFGE, in 112 isolates. Most of the repetitive elements studied are found in multiple copies in each strain, although in some subpopulations these elements are present in low copy number or are absent. Hybridization patterns and copy numbers of the repetitive elements correlate with geographic distribution. These patterns may yield interesting clues as to the origins and evolution of some TASs and retrotransposons, e.g. we can infer that Y' originated on the left end of chromosome XIV. There is strong evidence for horizontal transfer of Ty2 between S. cerevisiae and S. mikatae. Ty1 and Ty5 are either lost easily or frequently horizontally transferred. We have also found some gross chromosomal rearrangements in isolates within species and a few new natural hybrids between species, indicating that these processes occur in the wild and are not limited to conditions of human influence. DNA sequences have been deposited with the EMBL/GenBank database under Accession Nos AJ632279-AJ632293.     

Molecular identification of yeast species associated with 'Hamei'--a traditional starter used for rice wine production in Manipur, India

In Manipur state of North-Eastern India, wine from glutinous rice using traditional solid state starter called 'Hamei' is particularly interesting because of its unique flavour. A total of 163 yeast isolates were obtained from fifty four 'Hamei' samples collected from household rice wine preparations in tribal villages of Manipur. Molecular identification of yeast species was carried out by analysis of the restriction digestion pattern generated from PCR amplified internal transcribed spacer region along with 5.8S rRNA gene (ITS1-5.8S-ITS2). Seventeen different restriction profiles were obtained from the size of PCR products and the restriction analysis with three endonucleases (Hae III, Cfo I and Hinf I). Nine groups were identified as S. cerevisiae, Pichia anomala, Trichosporon sp., Candida tropicalis, Pichia guilliermondi, Candida parapsilosis, Torulaspora delbrueckii, Pichia fabianii and Candida montana by comparing this ITS-RFLP profile with type strains of common wine yeasts, published data and insilico analysis of ITS sequence data available in CBS yeast database. ITS-RFLP profile of eight groups was not matching with available database of 288 common wine yeast species. The most frequent yeast species associated with 'Hamei' were S. cerevisiae (32.5%), P. anomala (41.7%) and Trichosporon sp. (8%). The identity of major groups was confirmed by additional restriction digestion of ITS region with Hind III, EcoRI, Dde I and Msp I. The genetic diversity of industrially important S. cerevisiae group was investigated using Pulsed Field Gel Electrophoresis (PFGE). Although most of the 53 strains of S. cerevisiae examined were exhibited a common species specific pattern, a distinct degree of chromosomal length polymorphism and variable number of chromosomal DNA fragments were observed with in the species. Cluster analysis showed seven major karyotypes (K1-K7) with more than 83% similarity. The karyotype pattern K1 was the most frequent (67.9%) among the strains from different samples. Other karyotypes K2-K7 were very unique with less than 80% similarity. Finally using mitochondrial DNA restriction analysis (mt-DNA RFLP), S. cerevisiae strains belonging to the major karyotype K1 were distinctly differentiated with highly polymorphic bands by Hinf I and Hae III endonucleases.     

Possible gene interchange between plasmid and chromosome in yeast

Genomic DNAs isolated from 420 yeast strains stocked in the Department of Fermentation Technology, Hiroshima University (HUT) were screened for the presence of a plasmid sequence both as plasmid or in the chromosome. Five DNA samples gave rise to a positive hybridization signal when 32P-labelled Zygosaccharomyces plasmid pSR1 was used as a probe. Two among these contain hybridizing sequences as plasmids while the other three apparently were chromosomal. Two chromosomal DNA segments of HUT 7195 (Zygosaccharomyces spp.) which hybridized with pSR1 probe were cloned and sequenced. Both DNAs hybridized with a plasmid sequence covering the P gene of pSR1. One of the two segments contains a large open reading frame which can encode 410 amino acid residues. The deduced amino acid sequence is closely related with that of the P gene of pSR1. The present finding suggests that there was an interchange(s) of a gene between yeast plasmid(s) and chromosomes.