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PCR amplification of the 3′ external transcribed and intergenic spacer of the ribosomal DNA repeat unit in three species of Saccharomyces
Abstract
Two spacer regions outside the ribosomal DNA (rDNA) transcriptional unit in three species of Saccharomyces, S. cerevisiae, S. carlsbergensis and S. pastorianus, were amplified using the polymerase chain reaction. These regions were composed of the 3' external transcribed spacer (ETS) and the intergenic spacer (IGS). Primers were developed from sequence alignments and by taking the reverse complement of a previously described sequence. The region amplified spanned base position 3110 on the 26S rRNA to base position 27 on the 5S rRNA of S. cerevisiae. Nine of the twelve strains used in this study exhibited different restriction profiles, showing that the spacers are highly variable between species. The results suggest that PCR fingerprinting of the non-coding spacer regions can be used to distinguish between closely related Saccharomyces species and may have potential in DNA profiling of other yeasts.
... 1475 and 1882, isolated from California and Huelva win-However, these differences and the relationships among es, respectively. The white wine cluster is formed by white restriction patterns are more easily observed in a cluster wine strains 1476, 1477, 1483, 1881, 1883, 1887, 1888, Table 2. Above the diagonal, Euclidean distances between all pairs of wine Saccharomyces strains obtained from mtDNA restriction fragments according to the metric of Excoffier et al. (1992). This measure corresponds to the total number of band differences between strains. ...
... Frezier and Dubourdieu (1992) studied natural strains from different fermentations on the basis of chromosomal profiles and mtDNA restriction patterns, and they found that a small number of S. cerevisiae strains were capable of dominating fermentation in all vats of the same winery independently of the grapevine cultivar and over two vintages. ...
... Vezinhet et al. (1992) analyzed the evolution of S. cerevisiae strains isolated during 6 consecutive years in two vineyards also based on chromosomal profiles and mtDNA restriction analysis. They concluded that the wide distribution of some strains in the studied areas and their presence over years, constitute an evidence for the occurrence of specific native strains representative of a wine re-glOn. ...
We identify and characterize 31 Saccharomyces strains from different wine regions, deposited at the Spanish Type Culture Collection, according to mtDNA restriction patterns and chromosomal profiles. By using this kind of information we analyze the correlation between genetic distances and ecological or geographical factors by means of a cluster analysis, assessed by an analysis of the molecular variance (AMOVA). From these analyses, red wine strains are significantly grouped according to their geographic origin, independently of the wine type and the grapevine cultivar, and white wine strians according to ecological factors (wine type of grapevine cultivars). This study also confirms the usefulness of the analysis of interpopulation genetic polymorphism, such as that resulting from mtDNA restriction fragment analysis of wine yeast strains, to determine the distribution of variation in natural populations of Saccharomyces cerevisiae.
... Spacer regions between the ss rDNA and the large-subunit rDNA (ls rDNA), i.e., the internal transcribed spacer (ITS) (25), and the spacer between the ribosomal gene clusters, i.e., the nontranscribed spacer (NTS) (15,24), are highly variable between and within a species. This could be explained by the fact that both regions were conserved to a lesser degree than were the rRNA-encoding regions, since they are under fewer evolutionary constraints (14). The nucleotide sequences of rRNA genes and spacer regions have been used to analyze phylogenetic relationships among prokaryotes and eukaryotes (10,25). ...
... The definition of the primer JV52ET for the amplification of the NTS region was based on the comparison of the known ss rDNA sequences of several yeast species as described by Baleiras Couto et al. (2). Primer JV51ET was defined on the basis of the work of Molina et al. (14) and by comparison of the conserved regions in the ls rRNA of the yeast species C. albicans, S. cerevisiae, and Schizosaccharomyces pombe by alignment with the PC/GENE program (version 6.8; Amos Bairoch, University of Geneva, Geneva, Switzerland). The sequences of the primers used for the amplification of the NTS were, for JV51ET, 5Ј-TGA ACG CCT CTA AGY CAG AAT C and, for JV52ET, 5Ј-TTA TAC TTA GAC ATG CAT GGC. ...
... In earlier work, Molina et al. (14) have shown that MspI digests of only the left part of the NTS region (designated as the 3Ј external transcribed spacer and the intergenic spacer) generated different banding patterns in each of the four strains of S. cerevisiae tested. These authors suggested that restriction enzyme analysis of the 3Ј external transcribed spacer and intergenic spacer should be useful in the identification of strains in which the 5S rDNA is part of the rDNA repeated gene cluster. ...
Discrimination of strains within the species Saccharomyces cerevisiae was demonstrated by the use of four different techniques to type 15 strains isolated from spoiled wine and beer. Random amplified polymorphic DNA with specific oligonucleotides and PCR fingerprinting with the microsatellite oligonucleotide primers (GAC)5 and (GTG)5 enabled discrimination between the strains tested. Additionally, restriction enzyme analysis, with TaqI and MseI, of PCR-amplified fragments from the complete internal transcribed spacer and nontranscribed spacer, both present in the rRNA-encoding gene cluster, proved to be suitable for generating intraspecies-specific patterns. Random amplified polymorphic DNA with primers 24 and OPA-11 and PCR fingerprinting with primer (GTG)5 appeared to generate the highest degree of diversity. However, the results indicated that there was no single PCR-mediated typing technique enabling discrimination on the strain level. Discrimination of each individual strain was nevertheless possible by combining the results obtained with all typing techniques.
... Pieniä 5 S (~120 bp) ja 5,8 S (~160 bp) rRNA-geenejä voidaan käyttää kaukaisempien sukulaisuussuhteiden selvittämiseen (Kurzman & Blanz, 1998). rDNA:n välialueet eivät ole yhtä hyvin konservoituneet kuin rRNA-geenit ja niiden sekvensseissä on osoitettu esiintyvän lajinsisäistä ja lajien välistä vaihtelua (Molina et al., 1993;Baleiras Couto et al., 1995;Esteve-Zarzoso et al., 1999). ...
... ITS-ja IGS-alueiden RFLP:tä on käytetty Saccharomyces-kantojen tyypitykseen vaihtelevalla menestyksellä (Molina et al., 1993;Baleiras Couto et al., 1996b;Yamagishi et al., 1999). ITS-alueen restriktioanalyysin on kuitenkin osoitettu soveltuvan erinomaisesti useimpien elintarvikkeissa ja juomissa esiintyvien hiivalajien tunnistamiseen. ...
... However, owing to the much larger regions involved, IGS sequences have not been exploited in a routine manner. In fact, complete IGS regions are known for only a few organisms that include Neurospora crassa (Dutta & Verma, 1990), Saccharomyces cerevisiae (Molina et al., 1993), Filobasidiella (Cryptococcus) neoformans (Fan et al., 1995), Neotyphodium lolii (Ganley & Scott, 1998), Collybia fusipes and Trichosporon species (Sugita et al., 2002). ...
... However, owing to the much larger regions involved, IGS sequences have not been exploited in a routine manner. In fact, complete IGS regions are known for only a few organisms that include Neurospora crassa (Dutta & Verma, 1990), Saccharomyces cerevisiae (Molina et al., 1993), Filobasidiella (Cryptococcus) neoformans (Fan et al., 1995), Neotyphodium lolii (Ganley & Scott, 1998), Collybia fusipes and Trichosporon species (Sugita et al., 2002). In the present study, we describe the complete IGS1 sequences of several P. anomala strains for the first time and evaluate and compare the ITS and IGS1 regions of clinical and non-clinical strains of P. anomala for the development of a simple typing method. ...
... Molecular genetic methods have started to have a major impact on yeast identification and characterisation compared to traditional methods based on phenotypic characteristics. Among these molecular methods, those based on the analysis of restriction fragment length polymorphism of the DNA that encodes the ribosomal RNA genes (5S, 5.8S, 18S and 26S) and the non-coding ITS (internal transcribed spacers) and IGS (intergenic spacer) regions appear to be useful for the detection of many yeast and fungal species (e.g., Hopfer et al. 1993;Molina et al. 1993;Redecker et al. 1997;Wyder & Puhan 1997). The interest in these molecules for species identification comes from the concerted fashion in which they evolve showing a low intraspecific polymorphism and a high interspecific variability (Li 1997). ...
... These studies also failed to differentiate between S. bayanus-S. pastorianus (Molina et al. , 1993Messner & Prillinger 1995;Smole Mozina et al. 1997), at least with the wide range of restriction endonucleases tested. However, it seems obvious that other molecular markers should be used to decipher relationships between these two closely related species, such as RAPDs (Molnár et al. 1995a, b;Fernández-Espinar et al. in preparation) and PCR-fingerprinting based in the intron splice sites (de Barros Lopes et al. 1998). ...
The PCR amplification and subsequent restriction analysis of the region spanning the internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene was applied to the identification of yeasts belonging to the genus Saccharomyces. This methodology has previously been used for the identification of some species of this genus, but in the present work, this application was extended to the identification of new accepted Saccharomyces species (S. kunashirensis, S. martiniae, S. rosinii, S. spencerorum, and S. transvaalensis), as well as to the differentiation of an interesting group of Saccharomyces cerevisiae strains, known as flor yeasts, which are responsible for ageing sherry wine. Among the species of the Saccharomyces sensu lato complex, the high diversity observed, either in the length of the amplified region (ranged between 700 and 875 bp) or in their restriction patterns allows the unequivocal identification of these species. With respect to the four sibling species of the Saccharomyces sensu stricto complex, only two of them, S. bayanus and S. pastorianus, cannot be differentiated according to their restriction patterns, which is in accordance with the hybrid origin (S. bayanus S. cerevisiae) of S. pastorianus. The flor S. cerevisiae strains exhibited restriction patterns different from those typical of the species S. cerevisiae. These differences can easily be used to differentiate this interesting group of strains. We demonstrate that the specific patterns exhibited by flor yeasts are due to the presence of a 24-bp deletion located in the ITS1 region and that this could have originated as a consequence of a slipped-strand mispairing during replication or be due to an unequal crossing-over. A subsequent restriction analysis of this region from more than 150 flor strains indicated that this deletion is fixed in flor yeast populations.
... Other studies have demonstrated variability in S. cerevisiae and between species of the genus Saccharomyces (21). Those studies used PCR to amplify small-subunit rRNA genes and RFLP analysis of the amplified genes (21). ...
... Other studies have demonstrated variability in S. cerevisiae and between species of the genus Saccharomyces (21). Those studies used PCR to amplify small-subunit rRNA genes and RFLP analysis of the amplified genes (21). In addition, wine strains of S. cerevisiae show a high degree of polymorphism in the mitochondrial and chromosomal DNAs (3,41). ...
We have previously described differences in phenotype and virulence among clinical and nonclinical isolates of Saccharomyces. To further characterize these isolates, a comparison of restriction fragment length polymorphism (RFLP) patterns and genetic analysis were done. The cellular DNA of each of 49 clinical and 11 nonclinical isolates of Saccharomyces was digested with the endonuclease EcoRI, and the resultant fragments were separated by electrophoresis. Sixty isolates were grouped on the basis of the presence (group B) or absence (group A) of a 3-kb band. Group A contained 43 isolates (35 clinical and 8 nonclinical isolates) in 31 discernible subgroups, and group B had 17 isolates (14 clinical and 3 nonclinical isolates) in 10 subgroups. Interestingly, six of eight known vaginal isolates were group B, with four of those six being identical. Virulence of isolates was associated with membership in group A (P = 0.03). Comparison of known members of sibling species within the genus Saccharomyces, which cannot be distinguished by standard biochemical tests, showed that S. paradoxus, S. bayanus, and S. cerevisiae could be differentiated by RFLP analysis. Genetic analysis of the isolates forming viable spores showed that most group A isolates were diploid and members of the species S. cerevisiae. Those group A and B isolates unable to form viable spores may be diploid hybrids between Saccharomyces species. The group B isolates that formed viable spores were tetraploid and may also be interspecific hybrids. Overall, clinical isolates of Saccharomyces were very heterogeneous and exhibited little clonality. RFLP pattern analysis could be a useful method of demonstrating transmission in patients with infection or between environmental sources and patients.
... The polymerase chain reaction (PCR) provides a means for rapidly identifying yeast species; several studies have used restriction fragment length polymorphism (RFLP) in rRNA genes (rDNA) (Vilgay and Hester 1990). Molina et al. (1993) reported RFLP in the rDNA of closely related Saccharomyces species. Random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) and specific PCR using d sequence primers have been used to characterize lager yeasts, ale yeasts and non-brewing yeasts (Coakley et al. 1996;Laidlaw et al. 1996;Tompkins et al. 1996). ...
... For the amplification of rDNA, primers 5S2 (5? CACCGTTTCCCGTCCGATC 3?) and EST2 (5? CTGAACGCCTCTAAGTC 3?) were used. These primers, designed for amplification of rDNA, were previously described by Molina et al. (1993). The region amplified by these PCR primers spanned base position 3110 on the 26S rRNA to base position 27 on the 5S rRNA. ...
In order to differentiate brewing from non-brewing yeasts, a specific polymerase chain reaction (PCR) which targeted the open reading frame of FLO1 was employed. Non-brewing yeasts include 'non-brewing Saccharomyces yeasts' and 'non-Saccharomyces yeasts'. The molecular sizes of the PCR products differed between brewing and non-brewing Saccharomyces yeasts. No FLO1 PCR products were obtained from non-Saccharomyces yeasts. Specific PCR, using oligonucleotide primers that targeted the region between the 5S and 26S rRNA genes, could be used to differentiate brewing yeasts from some non-brewing yeasts. These PCR products were digested with restriction enzymes, Scr FI and Msp I. Different restriction profiles were obtained from brewing and non-brewing yeasts which could not be differentiated using specific PCR of rDNA. These results suggest that it is possible to identify brewing from non-brewing yeasts using specific PCR of FLO1 and rDNA, and detection of restriction fragment polymorphism of rDNA.
... T h e entire sequence of this region is k n o w n only for a few fungal rDNAs, e.g. Neurospora crassa (Dutta & Verma, 1990), Saccharomyces cerevisiae (Molina et al., 1993), Filobasidiella (Cryptococcus) neofoymans (Fan et al., 1995) and Neotyphodium lolii (Ganley & Scott, 1998). For the basidiomycetes, the complete IGS sequence has only been characterized for Collybia fusipes (B. ...
... T h e entire sequence of this region is k n o w n only for a few fungal rDNAs, e.g. Neurospora crassa (Dutta & Verma, 1990), Saccharomyces cerevisiae (Molina et al., 1993), Filobasidiella (Cryptococcus) neofoymans (Fan et al., 1995 ...
The nuclear rDNA intergenic spacer (IGS) of the ectomycorrhizal basidiomycete Laccaria bicolor was amplified and sequenced to identify the source of its intraspecific polymorphism. The IGS was amplified by PCR in several L. bicolor strains and shown to exhibit multiple bands and length polymorphism. The IGS loci were shown to segregate in a 1:1 ratio within haploid progenies in three dikaryotic strains, suggesting that divergent IGS haplotypes were present in the two nuclei of these strains. The two haplotypes of L. bicolor S238N were sequenced: the beta-haplotype was 4160 bp in length, whereas the size of the alpha-haplotype was estimated to be about 4700 bp. These represent the largest published fungal IGS sequences to date. These sequences can be subdivided into three main regions, IGS1, 5S rDNA and IGS2. The IGS sequences are AT-rich and contain numerous occurrences of three types of subrepeats (e.g. T2-AC3). The length polymorphism, observed between the IGS sequence of the alpha- and beta-haplotypes, results from the insertion of various numbers of a 71 bp subrepeat, called B, occurring in IGS2. This variation in subrepeat number suggests that the two haplotypes resulted from unequal cross-overs. The L. bicolor IGS was aligned with IGS sequences of two other Tricholomataceae (i.e. Tricholoma matsutake and Collybia fusipes). No sequence similarity was observed between these IGSs, but homologous subrepeats were found in L. bicolor and T. matsutake. Analysis of IGS length polymorphism is therefore an efficient tool for investigating genetic relationships between genets and within progenies in natural fungal populations.
... Molecular genetic methods have started to have a major impact on yeast identification and characterisation compared to traditional methods based on phenotypic characteristics. Among these molecular methods, those based on the analysis of restriction fragment length polymorphism of the DNA that encodes the ribosomal RNA genes (5S, 5.8S, 18S and 26S) and the non-coding ITS (internal transcribed spacers) and IGS (intergenic spacer) regions appear to be useful for the detection of many yeast and fungal species (e.g., Hopfer et al. 1993;Molina et al. 1993;Redecker et al. 1997;Wyder & Puhan 1997). The interest in these molecules for species identification comes from the concerted fashion in which they evolve showing a low intraspecific polymorphism and a high interspecific variability (Li 1997). ...
... These studies also failed to differentiate between S. bayanus-S. pastorianus (Molina et al. , 1993Messner & Prillinger 1995;Smole Mozina et al. 1997), at least with the wide range of restriction endonucleases tested. However, it seems obvious that other molecular markers should be used to decipher relationships between these two closely related species, such as RAPDs (Molnár et al. 1995a, b;Fernández-Espinar et al. in preparation) and PCR-fingerprinting based in the intron splice sites (de Barros Lopes et al. 1998). ...
Yeasts involved in velum formation during biological ageing of sherry wine have to date been classified into four races of Saccharomyces cerevisiae (beticus, cheresiensis, montuliensis, rouxii) according to their abilities to ferment different sugars. It has been proposed that race succession during biological ageing is essential for the development of the organoleptical properties of sherry wines. In this work we studied the physiological characteristics, the molecular differentiation and the phylogenetic relationships of the four races employing type and reference strains from culture collections and natural environments. Using restriction analysis of the ribosomal region that includes the 5.8S rRNA gene and internal transcribed regions (5.8S-ITS) we were able to differentiate 'flor' and non-'flor' S. cerevisiae yeast strains. However, no correlation between fermentation profile, mitochondrial DNA restriction analysis or chromosomal profiles and these races was found. Moreover, sequences of the D1/D2 domain of the 26S rRNA gene and the 5.8S-ITS region from these strains were analysed and no genetic differences were noted suggesting that 'flor' yeast cannot be grouped into four different races and the four races are identified as S. cerevisiae. Since the yeasts isolated from velum in sherry wine present a unique 5.8S rRNA pattern different from the rest of the Saccharomyces cerevisiae strains we propose that they should be included as a single race or variety inside the S. cerevisiae taxon.
... Thus, it is possible to establish ITS references (RFLPs or sequences) without the need to include a large number of isolates/vouchers from each reference species. Similar conclusions have been drawn from surveys of the ectomycorrhizal community in natural stands of bishop pine (Gardes and Bruns, 1996a), black alder (Pritsch et al., 1997) In higher fungi, the noncoding regions IGS1 (between the 25S and 5S coding units) and IGS2 (between the 5S and 17S coding units) (figure lA), are known to present length and sequence variations within a species (Molina et al., 1993;Iraçabal and Labarr6re, 1994). We examined the polymorphism of nuclear PCR-amplified rDNA IGS to evaluate its intraspecific genetic variability in an attempt to identify specific isolates of Laccaria bicolor, L. laccata, L. ...
Les techniques de diagnostic moléculaire permettant d’identifier les différentes espèces de champignons ectomycorhiziens, ou les génotypes au sein de ces espèces, se sont considérablement développées au cours des dernières années. Plusieurs types de marqueurs moléculaires ont été mis au point pour identifier des souches de champignons ectomycorhiziens, aussi bien dans les pépinières forestières que dans les sites de reforestations et les écosystèmes naturels. Diverses méthodes, basées sur la PCR, sont utilisées pour caractériser le polymorphisme des ADN ribosomaux nucléaires et mitochondriaux de ces champignons. Ce polymorphisme a permis d’aborder la structuration et la dynamique des communautés et des populations naturelles de symbiotes ectomycorhiziens. L’analyse RAPD et les empreintes génétiques obtenues par amplification des régions intermicrosatellitaires permettent d’identifier les différents génotypes et de décrire la genèse des populations mycorhiziennes. Cet article fait la synthèse des méthodes de diagnostic moléculaire développées afin d’identifier les mycélia et fructifications de champignons ectomycorhiziens, ainsi que leurs ectomycorhizes. Ces outils nous ont permis d’analyser la persistance et la dissémination d’une souche fongique exotique (Laccaria bicolor S238) introduite en pépinières, puis en plantations.
... ; Molina et al.(1993);Baleiras et al. (1996);Masneuf et al., (1996) and Fermadez-Espinar et al., (2000) المعاملة أدت وقد للساللة D5 إلى للخمٌرة الجسمً الوراثً العبور طفرات حدوث ( Latouche et al.,1997 andLavallee et al., 1994 ) للسرطان ...
... In fungi, the noncoding spacer regions of rDNA, which evolve rapidly, have been utilized in inferring phylogeny among more closely related taxa. The IGS region have been examined in the course of evolutionary and taxanomic studies of fungi (Erland et al., 1994;Molin et al., 1993;Aminnejad et al., 2009). Nowadays, the noncoding spacer regions (ITS/IGS) have been found valuable to study and establish relationships among closely related taxa particularly in fungi and other organisms. ...
... These regions are highly variable among and within species, because they were under a lower degree of conservation. Length and sequence polymorphism of these regions may be visualized upon resFiction enzyme digestion of PCR amplified fragments, enabling strain differentiation at the subspecies level (10,136). We have assessed genomic variability among S. cerevisiae strains using RAPD analysis, PCR fingerprinting, and restriction enzyme analysis of the ITS and NTS regions (10). ...
... Partial DNA fragments of the IGS regions can exhibit intraspecific variation, e.g. variation in the DNA sequences of individual strains within a species [53,67,111,112]. The two most frequently referenced DNA regions for identifying yeast at the species level are the 18S genes and the D1/D2 domains, which are located at the 5' end of the 26S genes [72,89]. ...
During the production of beverages or in terms of quality control, the question often arises as to which yeast species occur in which beverage as starter cultures or as spoilers? The species name should direct beverage microbiologists and technologists to information that is available about species' specific spoilage and fermentation characteristics. This review provides an overview regarding the yeast flora present in beverage processes in general and provides guidance on how this yeast flora can be identified at the level of genus, species or strain. Additionally, beverage-specific technological and microbiological information about specific yeast species is included. This review also provides an overview of the literature and information on the methods currently available for species identification from the point of view of beverage production, designed to aid microbiologists or technologists in coping with the challenges they face. Descriptions of the yeast flora found in beer, wine, sparkling wine and the mash used for the production of distilled beverages, as well as indigenous fermented beverages are provided below. Yeasts are grouped as either inoculated or spontaneous starter cultures and as direct or indirect spoilage organisms. Spoilage yeasts present in carbonated soft drinks are classified according to their spoilage and fermentative potential in a specific matrix. Detailed information about beverage-relevant characteristics is summarized in alphabetical order according to yeast genus and species name. Fermentation characteristics, fermentation by-products, sources of spoilage, resistance to preservatives and ethanol, osmotolerance, growth conditions and temperatures, characteristics in culture media and other beverage-specific background information are described.
... Intergenic spacer (IGS) region, which separates rDNA repeat units located between the 28S and 18S subunit genes, evolves more rapidly than ITS region and is more suitable for analyzing intraspecific and interspecific variation among the closely related species (Anderson and Stasovski, 1992;Erland et al., 1994;Hills and Dixon, 1991;Appel and Gordon, 1995). Polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) of the IGS region has been widely applied to the identification of the closely related species or strains (Banik et al., 1996;Guidot et al., 1999;Morina et al., 1993;Selosse et al., 1996) and could be a potential method to assess the intraspecific and interspecific variation of fungal endophytes from locoweeds. ...
Locoweeds including the toxic species of Astragalus spp and Oxytropis spp. are widely distributed in the western region of China and result in a chronic neurological disease known as locoism in animals. To determine the presence of swainsonine-producing fungal endophyte of major locoweed species in China, endophytes were isolated from 8 locoweed species that including A. variabilis, A. strictus, O. glacialis, O. kansuensis, O. ochrocepala, O. sericopetala, O. glabra and O. latibracteata. Seven species of locoweed were confirmed contain substantial amounts of swainsonine and infect swainsonine-producing fungal endophyte. These endophytes were classified as Undifilim oxytropis according to the fungal morphology and phylogenetic analysis based on sufficient ITS sequences. PCR-RFLP analysis of IGS region showed that the interspecific or intraspecific variations were present among the endophytes from different locoweed species.
... Thus, it is possible to establish ITS references (RFLPs or sequences) without the need to include a large number of isolates/vouchers from each reference species. Similar conclusions have been drawn from surveys of the ectomycorrhizal community in natural stands of bishop pine (Gardes and Bruns, 1996a), black alder (Pritsch et al., 1997) In higher fungi, the noncoding regions IGS1 (between the 25S and 5S coding units) and IGS2 (between the 5S and 17S coding units) (figure lA), are known to present length and sequence variations within a species (Molina et al., 1993;Iraçabal and Labarr6re, 1994). We examined the polymorphism of nuclear PCR-amplified rDNA IGS to evaluate its intraspecific genetic variability in an attempt to identify specific isolates of Laccaria bicolor, L. laccata, L. ...
Deoxyribonucleic acid (DNA) markers have been used in many studies to distinguish related species of ectomycorrhizal fungi or genotypes of a single species. These markers have been designed to identify defined strains of ectomycorrhizal fungi in forest nurseries and within the complex microbial communities of reforestation sites and natural ecosystems. Various polymerase chain reaction (PCR)-based methods revealed sequence polymorphisms in nuclear and mitochondrial ribosomal DNA of ectomycorrhizal fungi that can be used as highly informative markers for the structure and dynamics of genomes at the level of communities and populations. Markers based on random amplified polymorphic DNA (RAPD) or on microsatellite-primed PCR are suitable for identifying clones (genotypes) and describing the differentiation of populations of ectomycorrhizal fungi. Here we describe the procedures that allow the rapid typing of an ectomycorrhizal isolate from vegetative mycelium, fruiting bodies, as well as a single ectomycorrhizal root tip. Specific applications illustrated are: identification of fungal species and isolates; analysis of the persistence and dissemination of introduced exotic strains; and detection of gene flow between introduced strains and local forest populations.
... IGS has been used as a tool for identification of yeasts at species level, using PCR amplification of the complete IGS region or of only part of it, followed by endonuclease restriction analysis or DNA sequencing. PCR/restriction fragment length polymorphism (RFLP) of the NTS1 (Molina et al., 1993) was used to identify the species of the Saccharomyces sensu stricto complex. It was shown that the NTS2 could be even more informative and convenient, because PCR/RFLP of the NTS2 digested with AluI and BanI endonucleases generated simple patterns that clearly differentiated the sibling species S. cerevisiae, Saccharomyces paradoxus, Saccharomyces pastorianus as well as Saccharomyces uvarum from Saccharomyces bayanus (Nguyen et al., 2000a). ...
The intergenic spacer rDNA amplification and AluI fingerprinting (IGSAF) method detected four distinct groups among 170 Debaryomyces hansenii strains: D. hansenii var. hansenii; Candida famata var. famata; D. hansenii var. fabryi and C. famata var. flareri. IGS sequence comparison of representative strains showed that D. hansenii var. hansenii and C. famata var. famata belonged to one species, whereas D. hansenii var. fabryi and C. famata var. flareri belonged to two different ones. This confirmed the following three species recently reinstated: D. hansenii (=C. famata), Debaryomyces fabryi and Debaryomyces subglobosus (=Candida flareri). Accordingly, growth at 37 degrees C may no longer be used to differentiate D. hansenii from D. fabryi. Riboflavin production is more specific for D. fabryi and D. subglobosus strains. IGSAF identified all the other 17 species of the genus Debaryomyces, six of them sharing with D. hansenii an rRNA gene unit harbouring two 5S rRNA genes. The phylogenetic tree established with IGS sequences was congruent with the one based on ACT1, GPD1 and COX2 sequences depicting a distinct D. hansenii clade close to the D. subglobosus, Debaryomyces prosopidis and D. fabryi clade. Description of Debaryomyces vietnamensis sp. nov. (type strain CBS 10535(T), MUCL 51648(T)), closely related to Debaryomyces nepalensis is given.
... Likewise, in human rDNA arrays, individual IGS units can vary considerably in length, ranging from 9–72 kb, with an average length of 34.2 6 5.4 kb (Caburet et al. 2005 ). Indeed, for this very reason, the rDNA IGS has been specifically used in a number of yeast phylogenetic studies to examine both inter-and intraspecific relationships (Molina et al. 1993; Fell and Blatt 1999; Diaz et al. 2000 Diaz et al. , 2005). However, even within the IGS, it is apparent that the distribution of polymorphisms is uneven . ...
Ribosomal DNA (rDNA) plays a key role in ribosome biogenesis, encoding genes for the structural RNA components of this important cellular organelle. These genes are vital for efficient functioning of the cellular protein synthesis machinery and as such are highly conserved and normally present in high copy numbers. In the baker's yeast Saccharomyces cerevisiae, there are more than 100 rDNA repeats located at a single locus on chromosome XII. Stability and sequence homogeneity of the rDNA array is essential for function, and this is achieved primarily by the mechanism of gene conversion. Detecting variation within these arrays is extremely problematic due to their large size and repetitive structure. In an attempt to address this, we have analyzed over 35 Mbp of rDNA sequence obtained from whole-genome shotgun sequencing (WGSS) of 34 strains of S. cerevisiae. Contrary to expectation, we find significant rDNA sequence variation exists within individual genomes. Many of the detected polymorphisms are not fully resolved. For this type of sequence variation, we introduce the term partial single nucleotide polymorphism, or pSNP. Comparative analysis of the complete data set reveals that different S. cerevisiae genomes possess different patterns of rDNA polymorphism, with much of the variation located within the rapidly evolving nontranscribed intergenic spacer (IGS) region. Furthermore, we find that strains known to have either structured or mosaic/hybrid genomes can be distinguished from one another based on rDNA pSNP number, indicating that pSNP dynamics may provide a reliable new measure of genome origin and stability.
... They are located at one or more loci and have identical sequences in a given organism due to concerted evolution (Srivasta and Schlessinger 1991 ). In higher fungi, the 25s/17s intergenic spacer (IGS; seeFig. 1 A) is known to present length and sequence variations within a species (Henrion et al. 1992; Molina et al. 1993; Iraiabal andLabarè re 1994). PCR/RFLP analysis of the IGS in¸.in¸. ...
The aim of this study was to clarify the inheritance of the nuclear ribosomal DNA (rDNA) in the ectomycorrhizal basidiomycete Laccaria bicolor S238N in order to resolve inter- and within-strain relationships in forest ecosystems. PCR amplification of the intergenic spacer (IGS) was carried out in the dikaryotic mycelium and its haploid progeny. In the dikaryotic mycelium, multiple amplification products were produced for the 25s/5s (IGS1) and 5s/17s (IGS2) intergenic spacers. The 4.5- and 4.0-kb fragments of IGS2 (haplotypes alpha and beta, respectively) were observed to occur in a 1:1 ratio within the haploid progeny as a result of divergent IGS haplotypes in the two separate nuclei. Recombinant monokaryons having both types of IGS2 occurred at a low frequency (6.5%; 60 kb per centimorgan) during meiosis. Haplotypes alpha and beta of IGS1 cross-hybridized forming heteroduplexes during the PCR temperature cycle. The two IGS1 haplotypes differed only by the repeat number of a TA2C3 motif and co-segregated with the IGS2 haplotypes. Heteroduplex formation and IGS polymorphism provide information that is helpful in distinguishing between introduced exotic L. bicolor S238N and indigenous populations of Laccaria spp. in forest ecosystems.
... Length variations was also reported by Carlotti et al. (1997) in Candida krusei, and was explained by the existence of variable number of copies of a tandemly repeated sub-element in the IGS. The external transcribed spacers (ETS) have rarely been used in yeast taxonomy, although Molina et al. (1993) have amplified and digested the 3′ETS plus part of the IGS for identification of Saccharomyces species. The internal transcribed spacers (ITS), on the other hand, are being used not only for yeasts, but also for other fungi and for plants (Baldwin et al. 1995, Hamelin and Rail 1997, Holst-Jensen et al. 1997). ...
The literature on sequencing as a tool for yeast molecular taxonomy is reviewed. Ribosomal DNA has been preferred for sequencing over other molecules such as mitochondrial DNA, and a large database is now available. rDNA consists of regions that evolve at different rates, allowing comparison of different levels of relationship among yeasts. Sequences of the 18S rDNA and the 25S rDNA have been largely used for yeast systematics and phylogeny, but the search for regions with increased resolving power has led to the study of the spacer regions of the rDNA. Few studies are concerned with signature sequences.
... However, owing to the much larger regions involved, IGS sequences have not been exploited in a routine manner. In fact, complete IGS regions are known for only a few organisms that include Neurospora crassa (Dutta & Verma, 1990), Saccharomyces cerevisiae (Molina et al., 1993), Filobasidiella (Cryptococcus) neoformans (Fan et al., 1995), Neotyphodium lolii (Ganley & Scott, 1998), Collybia fusipes and Trichosporon species (Sugita et al., 2002). ...
Pichia anomala is an emerging nosocomial pathogen and there is a need for methods that distinguish between different P. anomala strains. In the typing of several clinical as well as non-clinical P. anomala strains, the sequence variation of the internal transcribed spacer (ITS) was found to be inadequate for typing purposes. The intergenic spacer 1 (IGS1) region of the rDNA of several P. anomala strains was therefore investigated in detail. The IGS1 region (which varied from 1213 to 1231 bp in length) was interspersed with repeats and had more variation than the ITS regions. Comparative analysis in cases where analysis by the ITS was ambiguous clearly revealed the IGS1 region to be a more discriminatory tool in the typing of P. anomala strains.
Abstract
This study aimed to isolate and identify saccharomyces boulardii from Mangosteen fruits (Garcinia mangostana L.) by traditional and molecular identification methods, as well as some of its physiological and probiotic properties were studied. Then, the identified isolates were mutagenized to obtain uracil auxotrophic mutants. These mutants were transformed to generate transformants, which are suitable for genetic engineering and have the potential to produce of recombinant proteins. This study was performed on steps:
The first step included two stages namely, isolation and identification. Ninety isolate were obtained from the fruit. These isolates with two commercial ones were subjected to cultural, morphological and biochemical tests and then followed by investigating some of their physiological characteristics such as growth in different temperatures, resistance to low pH and high concentration of bile salt. Eleven isolates shown best growth at 37 o C, resistance to low pH and tolerance to high concentration (3%) of bile salts. Adhesion ability (Auto aggregation ability), antagonistic activity for cells and cell free extract and reduction of cholesterol which are considered as probiotic properties were also investigated for these isolates. These isolates shown an average auto aggregative ability of 37.11 – 69.14 %. The isolates SbR1, SbR2, SbR3, SbR4, SbR6, SbR7, SbR9, SbC1 and SbC2 shown antagonistic activity toward all tested pathogenic microorganisms. However, isolates SbR7 and SbC2 shown best antagonistic activity toward E. coli O 157:H7, in which inhibition zone was 18 millimeter. In addition, although cell free extract of SbR1, SbR7 and SbC2 isolates shown inhibition ability toward all tested microorganisms, cell free extract of SbR7 isolate shown the best inhibition activity toward E cali O157:H7, in which inhibition zone was 10 millimeter.
The results shown variations in the ability of isolates to utilize and reduce cholesterol in modified culture media. The best ability in cholesterol reduction during 24 and 48 hrs. of incubation is shown by SbR7 and SbR3 isolates and the reduction percentages were 21.69, 21.04% and 56.54, 53.90% respectively.
S. boulardii isolates cannot be identified accurately by cultural, morphological and biochemical characteristics. Therefore, the eleven isolates in the second step were subjected to molecular identification. This was performed by amplification of internal transcribed spacer (ITS) for conservative 5.8S rRNA gene with specific primer for this region using Polymerase Chain Reaction. The amplified fragments were sequenced and the data were compared with the sequence data of the same region for the available strains in gene bank using NCBI – BLAST program. The SbR7 isolate was selected for its best physiological and probiotic properties. According to molecular identification, this isolate was almost genetically identical (99%) with S. boulardii standard strains which are available in gene bank data base.
The third step included mutagenesis of SbR7 isolate using UV light and isolation of uracil auxotrophic mutant on 5-FOA medium. The results demonstrated that the percentages of survival of SbR7 cells were decreased by increasing the exposure time of UV radiation. The killing percentage was 100% at exposure time 50sec., while the UV exposure time required to kill 90% of SbR7 cells was 25sec. Auxotroph mutants were isolated from this treatment (25sec), and four mutants shown stable mutation during 10 days of incubation. Tow mutants SbR7M7 and SbR7M10 were chosen for transformation according to their similar behavior with SbR7 isolate related to optimum temperature for growth, resistance to low pH and high concentration of bile salt.
The fourth step included extraction of pYES2 from E. coli Top10 and digested with either Dra l or EcoR V restriction enzymes. Results of gel electrophoresis revealed that two clear bands were observed after digestion with Dra l, in addition to the third band which was appeared in the early stage of electrophoresis, while two closely bands were appeared after digestion with EcoR V .
The two uracil auxotrophic mutants (SbR7M7 and SbR7M10) were transformed with pYES2 and two transformants (SbR7T7, SbR7T10) obtained. To confirm the transformation of these isolates with pYES2, this plasmid was isolated from these transformants and was also used to transform E. coli Top10 cells. The plasmid was isolated again from this bacterium to confirm the transformation. Some probiotic properties were studied for these transformants (SbR7T7, SbR7T10) which included auto aggregation ability, antagonistic ability for cells and their free extract and reduction of cholesterol. The two transformants shown that there was a noticed improvement in auto aggregation ability, which was 69.72 and 68.95% compared with 65.37% for wild type. The results also shown an improvement of antagonistic activity of the two transformants toward E. coli: O157:H7 and C. albicans. The diameter of inhibition zone caused by SbR7T7 was 20 and 21 millimeters respectively, while that caused by SbR7T10 was 19 and 21 millimeters respectively, compared with inhibition zone for wild type, which was 18 and 19 millimeters respectively. In addition, cell free extract for both transforments shown best inhibition of C. albicans when compared with W. T.
The transformants (SbR7T7 and SbR7T10) were reduced cholesterol ratio to 27.78 and 26.42% respectively after incubation period for 24hrs and these percentages increased to 48.72 and 52.86% after 48hrs incubation period when compared with wild type, which reduced 25.59% and 53.90 of cholesterol in the same incubation periods.
With an advent of modern molecular markers technology, it has become easier for the rapid identification, characterization and detection of important plant fungus that were previously a major bottleneck to detect by traditional microbiological methods. Present molecular tools in fungal biodiversity can have potential to characterize in very rapid manner by small quantity of DNA/RNA of fungal pathogens in short period. Different molecular markers viz.; RFLP, RAPD, SSR, AFLP, ITS, IGS and other markers have accelerated the identification and characterization of important plant fungus since previous decades. Internal transcribed spacer (ITS) region of ribosomal RNA genes is widely accepted technique for explanation of the diversity among fungal communities due to its utility for the identification of fungi to genus/ species taxonomic levels, due to its several important including its high representation in public sequence databases. Investigation of fungal diversity has also promoted since introduction of next-generation DNA sequencing technologies. Presently, high-throughput sequencing techniques are widely adopted by the several researchers for the detection of biodiversity of fungal populations. At present, construction of sequence databases of different fungal species that have broaden the representation across fungal population by bioinformatics and taxonomic experts is a critical next step towards the assessments of fungal diversity.
Variation within the intergenic spacer (IGS) of the ribosomal DNA gene for twenty-two strains of E oxysporum and its formae speciales was examined by PCR, coupled with RFLP analysis. The length of the amplified IGS region was about 2.6 kb in all strains except F oxysporum f. sp. cucumerinnin from Korea and E oxysporum f. sp. niveum. Those two strains were 2.5 kb long. Restriction digestion of IGS-RFLP regions by EcoRI, NruI, HincII, SalI, SmaI, BglII, HindIII, XhoI, and KpnI gave rise to nine IGS haplotypes among all strains. Cluster analysis based on the presence or absence of comigrating restriction fragments show the two groups based on 44% genetic similarity. These results demonstrated that analysis of IGS showed some difference within and between F oxysporum formae speciales.
The internal transcribed spacer (ITS) of ribosomal DNA (rDNA) was used as a marker to identify genetic differences among 24 accessions of species and hybrids of the Oncidiinae based on PCR-amplified RFLP analysis. The ITS region of rDNA in 24 respective accessions of the Oncidiinae was amplified by a pair of complementary primers to conserved regions at the 3’ end of 17S and 5’ end of 25S of rRNA genes. The ITS was found to be about 730 bp in length, and there was no obvious variation in length among the 24 accessions studied. The PCR-amplified samples were digested with ten restriction enzymes and subjected to electrophoresis. Among the 166 bands that appeared, 159 were polymorphic. Cluster analysis was then done based on the pattern of band distribution in all samples studied. Four major groups and two individual clusters not belonging to any of the four groups were identified based on molecular data. The results of the study provide a DNA marker that can serve as a good tool to identify new hybrids of Oncidiinae flowers at the molecular level. This technique could be applied to protect the rights of plant breeders in the future.
Yeasts are of benefit to mankind because they are widely used for production of foods, wine, beer, and a variety of biochemicals. Yeasts also cause spoilage of foods and beverages, and are of medical importance. At present, approximately 700 yeast species are recognized, but only a few are commonly known. Relatively few natural habitats have been thoroughly investigated for yeast species; consequently, we can assume that many more species await discovery. Because yeasts are widely used in traditional and modern biotechnology, the exploration for new species should lead to additional novel technologies.
Molecular analyses of nucleic acid polymorphisms have proved to be very helpful in avoiding taxonomic ambiguities and simplifying yeast identification schemes. PCR mediated typing assays have the advantage of a rapid amplification of yeast target DNA in the laboratory thus enabling rapid analysis of DNA polymorphisms on different levels. We used two approaches - restriction analysis of amplified rDNA fragments (RFLPs of 2550 bp long 18S-ITS1-5.8S-ITS2 and 3350 bp long 25S rDNA amplicons) and AP-PCR fingerprinting of yeast DNA with six non-specific oligonucleotide primers for the characterization of Hanseniaspora (anamorph Kloeckera) yeasts. Species-specific restriction patterns were derived from type strains analysis. Two restriction analyses of 18S-ITS1-5.8S-ITS2 rDNA and/or 25S rDNA fragment were sufficient for species delineation among Hanseniaspora/Kloeckera yeasts. They were used for rapid species identification of thirthy-three Hanseniaspora/Kloeckera strains isolated from grapes and musts at the start of spontaneous fermentation in two geographically distinct wine-producing subregions in Slovenia. All identified strains were H. uvarum/K. apiculata. AP-PCR fingerprinting revealed great heterogeneity of the isolates at the intraspecies level. No correlation between genetic similarity, calculated from AP-PCR fingerprints, and the geographical origin of H. uvarum/K. apiculata strains was evidenced.
Background and Objectives: Nowadays RFLP test from IGS region is a used for determination of interspecies diversities. The aim of this study was to determine pathogenic species of Alternaria from leaf spots of citrus in Mamasani region based on morphological and ITS1 sequencing and to determine genetic variation of isolates based on RFLP-IGS.
Materials and Methods: This descriptive study was performed on 70 leaf spotted samples collected from different citruses species and cultivars of Mamasany orchards including orange (Valencia, Navel and leaf spoon Cv.), lime and sweet lemon (Wikova and local Cv.) during In autumn 2011. The isolates were cultured on PDA medium and purified by using single spore method and their species were identified based on morphological characters. The pathogenicity of the isolates was evaluated by based on Koch’s rules through exposure of isolates to leaf. DNA was extracted from mycelia by CTAB and the ITS1 and IGS regions were amplified by using specific primers. The ITS1 region was sequenced and the IGS region was digested by HinfI, BsnI, RsaI, BssmI and AluI restricted enzymes.
Results: All isolates were identified as Alternaria alternata based on morphological characters and sequencing of ITS1 region. Constructed dendrogram based on RFLP patterns showed existence of three divergent groups in 51% similarity. Orange, lime and sweet lemon isolates grouped in divergent groups. Also Wikova isolates grouped in different clade.
Conclusion: The obtained results clearly showed that the enzyme patterns of IGS can reveal the classification defects of Alternaria alternata form citrus hosts.
A PCR-RFLP method has been improved for the rapid identification of the four species of Saccharomyces sensu stricto. We used the NTS2-ETS sequence of the rDNA as target and the amplification reaction was realized by chosing a pair of primers that could anneal to the end and the beginning of the 5S and 18S conserved sequences, respectively. PCR products obtained from S. cerevisiae, S. bayanus, S. paradoxus and S. pastorianus (synonym S. carlsbergensis) type strains displayed a clear polymorphism when digested with several restriction enzymes. Using only three enzymes: Ban I, Alu I and Taq I we were able to differentiate S. cerevisiae, S. paradoxus and the S. bayanus/S. pastorianus complex which shared the same pattern. The S. bayanus taxon now regroups the former species S. abuliensis, S. globosus, S. heterogenicus, S. intermedius, S. inusitatus, S. uvarum. PCR-RFLP of these species showed that, except for S. abuliensis and S. uvarum, they shared the same patterns as S. bayanus type strain. That lead us to conclude that there are two subgroups in S. bayanus and that S. abuliensis and S. uvarum belong to a distinct subgroup. Remarkably, wine strains that were previously identified as S. bayanus by genetic hybridization techniques, as well as strains isolated from cider fermentation, belong to the S. uvarum subgroup.
A total of 79 isolates of Fusarium semitectum were characterized by morphological and IGS-RFLP analysis to assess its intraspecific variation. Based on morphological characteristics, the isolates of F. semitectum were classified into 2 distinct groups, morphotypes I and II. Morphotype I was characterized by longer macroconidia (3 - septate: 31.03 ± 2.57 µm; 5 - septate: 40.17 ± 1.85 µm), 0 - 7 septate with 5 - septate was the most common, absence of chlamydospores, presence of sporodochia, abundant- floccose mycelium, peach colony appearance, peach to orange pigmentations and fast growing. While isolates of morphotype II produced shorter macroconidia (3 - septate: 24.98 ± 1.87 µm; 5 - septate: 35.24 ± 2.07 µm), 0 - 5 septate with 3 - septate was the most common, with (56%) or without chlamydospores (44%), without sporodochia, abundant-floccose and abundant-powdery mycelium, beige to brown colonies, brown to dark brown pigmentations and slow growing. Corresponding to the morphological characterization, IGS-RFLP analysis indicated that the 79 isolates could be divided into 2 different clusters assigned as RFLP groups I and II. 49 IGS haplotypes were produced by 8 restriction enzymes (AluI, Bsu15I, BsuRI, Eco881, Hin6I, MspI, PstI and TaqI) which indicated a high level of intraspecific variation and polymorphism among the 79 isolates. This is the first report of F. semitectum associated with H. polyrhizus.
The non-wine Saccharomyces cerevisiae strain of 96581 was found to be a promising candidate for the production of white wine. It produced wines with fusel alcohols that were 57% higher than those produced by the wine yeasts studied and was also more efficient in the production of 2-phenethyl acetate and 3-methyl-1-butanol acetate. This study also shows that there is a difference in the ester-formation efficiency for acetates relative to C6, C8 and C10 fatty acid esters for all the studied yeast strains. Therefore, it supports the view that other unidentified enzymes besides those regulated by ATF1 and ATF2 genes are involved in the production of ethyl esters of C6–C10 fatty acids. DNA analysis of the 25S, 18S, 5.8S and 5S ribosomal DNA genes in these strains showed high conservation. Despite the closely related nature of these yeast strains, the chemical profiles of the wines produced were significantly different.
Macrophomina phaseolina is a global pathogen that inflicts losses on many agriculturally important crops worldwide, particularly in warm and tropical
environments. Efforts to divide M. phaseolina into subspecies have been unsuccessful largely due to the extreme intraspecific variations in morphology and pathogenecity.
The failure to adequately identify and detect M. phaseolina using conventional culture-based morphological techniques has led to the development of nucleic acid-based molecular approaches.
PCR-based methods are highly sensitive and specific and have the potential to replace traditional technologies. Recently,
species-specific oligonucleotide primers and digoxigenin (DIG)-labeled probe were designed at internal transcribed spacer
(ITS) region for identification and detection of M. phaseolina. Accurate diagnosis and early detection of pathogens is an essential step in agriculture and environmental monitoring including
plant disease management. The main objective of this review is to outline various molecular tools used for detection, identification,
and characterization of M. phaseolina isolates. We also emphasize the significance of advanced technique such as real-time polymerase chain reaction (PCR) for
qualitative and quantitative assays.
Probiotics are viable microorganisms which upon ingestion confer health benefits to the host. Any microorganism irrespective
of its origin, capable of surviving in the digestive tract of host and exerting such effects can be a candidate. Most of the
currently used probiotics belong to prokaryotic origin. Unlike prokaryotes, several eukaryotic microorganisms can also be
very useful to animal’s health. Since a long time, eukaryotes are used as single cell protein and/or as components of food
starters for human and animal consumption throughout the world. Apart from these uses, certain eukaryotic microorganisms are
also used as probiotics since they can withstand the harsh milieu of gut and execute beneficial effects in host. While bacterial
probiotics are common, only limited eukaryotic probiotics belonging to fungi/moulds/yeasts are used in human and animal practices.
Nowadays interest in eukaryotic probiotics is on the rise and in most of the cases, their efficacy and usefulness has been
confirmed by firm scientific evidences. Among the eukaryotic probiotics, yeasts especially Saccharomyces species are dominant and routinely used in a broad range of hosts. This chapter deals with the occurrence, distribution,
taxonomic characterization, and detail modes of action of eukaryotic probiotics with special reference to yeasts in human
and other animals.
A total of 166 food-related yeast species were selected and used as a basis for a taxonomic study. An extensive survey has been conducted on the cellular long-chain fatty acid composition (LCFA), G+C values, coenzyme Q analyses and orthogonal field alternation gel electrophoresis (OFAGE) of yeasts associated with food in order to obtain rapid and reliable identification of the yeasts. In the above survey, the yeast strains were cultured under standardized conditions, after which the different profiles of each species were examined using the above-mentioned techniques. The chemotaxonomic data were compared with data obtained by means of standard conventional methods using a comparative model. None of the chemotaxonomic techniques proved to be solely adequate in characterizing isolated species. Our data suggest, however, that the coordinate use of all the above techniques provide a reliable approach for differentiating between food yeast species.
Recent changes in the epidemiology of candidiasis highlighted an increase in non- Candida albicans species emphasizing the need for reliable identification methods. Molecular diagnostics in fungal infections may improve species characterization, particularly in cases of the closely related species in the Candida complexes. We developed two PCR/restriction fragment length polymorphism assays, targeting either a part of the intergenic spacer 2 or the entire intergenic spacer (IGS) of ribosomal DNA using a panel of 270 isolates. A part of the intergenic spacer was used for discrimination between C. albicans and C. dubliniensis and between species of the C. glabrata complex (C. glabrata/C. bracarensis/C. nivariensis). The whole IGS was applied to C. parapsilosis, C. metapsilosis, and C. orthopsilosis, and to separate C. famata (Debaryomyces hansenii) from C. guilliermondii (Pichia guilliermondii) and from the other species within this complex (ie, C. carpophila, C. fermentati and C. xestobii). Sharing similar biochemical patterns, Pichia norvegensis and C. inconspicua exhibited specific IGS profiles. Our study confirmed that isolates of C. guilliermondii were frequently mis-identified as C. famata. As much as 67% of the clinical isolates phenotypically determined as C. famata were recognized mostly as true P. guilliermondii. Conversely, 44% of the isolates initially identified as C. guilliermondii were corrected by the IGS fingerprints as C. parapsilosis, C. fermentati, or C. zeylanoides. These two PCR/restriction fragment length polymorphism methods may be used as reference tools [either alternatively or adjunctively to the existing ribosomal DNA (26S or ITS) sequence comparisons] for unambiguous determination of the Candida species for which phenotypic characterization remains problematic.
In beverage industries yeasts are considered as spoilage yeasts on the one hand and on the other hand they are of importance as starter cultures for fermented alcoholic beverages. In this study real-time PCR assays, PCR-DHPLC methods, sequencing methods and FT-IR spectroscopy methods were being developed, optimised and transferred to the microbiological analysis of beverages with the aim to identify and differentiate beverage-relevant yeasts. A combination of sequencing methods, real-time PCR assays and FT-IR spectroscopy methods enabled the identification (on species level) of 363 isolated strains from 53 species. The isolates originated from breweries, beverage companies and starter cultures. For the first time Saccharomyces kudriavzevii was isolated and identified from a brewing environment. Specific real-time PCR assays enable the identification of Saccharomyces sensu stricto species.
Fifty-six isolates of Fusarium oxysporum, including F. oxysporum f. sp. melonis and nonpathogenic strains, were chosen from a larger collection to represent diversity in vegetative compatibility groups (VCGs), mitochondrial DNA (mtDNA) haplotype, geographic distribution, and virulence. Using PCR, a 2.6-kb fragment including the intergenic spacer (IGS) region of the ribosomal DNA was amplified from each isolate. The enzymes EcoRI, Sau3A, CfoI, and AvaII, cut this fragment differentially, revealing 5, 6, 6, and 7 patterns, respectively. Among the 56 isolates, a total of 13 unique IGS haplotypes was identified. Among most F. o. melonis isolates. IGS haplotype correlated with VCG and mtDNA haplotype, but did not differentiate among races. However, a race 1 isolate found in VCG 0131 shared virulence, mtDNA, and IGS haplotypes characteristic of VCG 0134; this isolate may represent a conversion in VCG from 0134 to 0131. Four nonpathogens shared the pathogen vegetative compatibility phenotypes. One race 1, 2 isolate associated with VCG 0134 shared both IGS haplotype and VCG with a nonpathogen, but these isolates did not share the same mtDNA haplotype. Another nonpathogenic isolate shared mtDNA and IGS haplotypes with pathogen group 0131 and may simply be an avirulent mutant of a pathogenic strain. For the other two nonpathogenic isolates, vegetative compatibility indicated a close relationship to the pathogen, but differences in both mtDNA and IGS haplotype suggest otherwise. Overall, the IGS haplotype was more variable among the nonpathogenic F. oxysporum VCGs among which 12 of the 13 IGS haplotypes were found. Nonpathogenic isolates that shared a common mtDNA haplotype, but were associated with different VCGs, often had different IGS haplotypes.
Several strains of the four sibling species of the genus Saccharomyces (S. bayanus, S. cerevisiae, S. paradoxus, and S. pastorianus) were characterized by using a rapid and simple method of restriction analysis of mitochondrial DNA. Patterns obtained with four-cutter endonucleases (such as AluI, DdeI, HinfI, and RsaI) made it possible to differentiate each species. S. cerevisiae and S. paradoxus presented a greater number of large fragments than S. pastorianus and S. bayanus with all the assay enzymes. With AluI and DdeI, species-specific bands clearly permitted differentiation between S. pastorianus and S. bayanus. To test the resolution of this method, wild Saccharomyces strains were analyzed. The correct assignment of these strains to a known taxon by this rapid method was confirmed by means of electrophoretic karyotyping.
The random amplified polymorphic DNA (RAPD) assay and the restriction enzyme analysis of PCR amplified rDNA are compared for the identification of the common spoilage yeasts Zygosaccharomyces bailii, Z. rouxii, Saccharomyces cerevisiae, Candida valida and C. lipolytica. Both techniques proved to be adequate tools for yeast identification. Since the RAPD does provide less stable patterns than restriction enzyme analysis of PCR amplified rDNA, and a large amount of data had to be compared without data reduction, Principal Component Analysis (PCA) was applied successfully for clustering the RAPD patterns. The success of PCA is highly influenced by the primer used in RAPD and the amount of reference samples. A large amount of reference samples improves the performance of clustering in PCA. The primer of choice was shown to be important with respect to the discriminatory power of the RAPD method. Some primers used enabled discrimination on the subspecies level. The results collected with both typing methods justify the conclusion that the present typing system can be applied for taxonomical purposes.
Molecular and chemical taxonomic characteristics, including DNA base composition, electrophoretic karyotype, restriction fragment length polymorphism of genes coding for rRNA, cellular fatty acid composition, and ubiquinone systems, were studied for 19 strains of Candida boidinii Ramirez. Electrophoretic karyotype and restriction fragment length polymorphism demonstrated marked differences among these strains. A combination of molecular and chemical analyses can serve as a reliable tool for culture authentication and quality control of industrial strains.
The rapid identification of spoilage microorganisms is of eminent importance to the food industry. It provides the food industry with the opportunity to reduce economical losses by designing adequate intervention measures. The use of identification systems based on biochemical and physiological characteristics resulted often in disappointing identification results and misidentifications. This will inevitably lead to inappropriate strategies to prevent spoilage. This review discusses the potential of the DNA based identification technology including the polymerase chain reaction (PCR) for the identification and specific detection of microorganisms. Fingerprinting methods based on the DNA-probe technology enable a clear insight in the identity of microorganisms on different levels, varying from genus to strain level depending on the systems used. Discrimination between subspecies and strain level is shown to be helpful for investigating routes and sources of contamination. Differentiation at the species level is demonstrated to be essential in order to design a highly specific detection system enabling to signalize a microorganism that belongs to a particular species. Also indicated in this review is the necessity and the technical approach to detect microorganisms that display a particular undesirable trait.
Several wild and collection strains of the genus Zygosaccharomyces were characterized using a rapid and simple method of restriction analysis of mitochondrial DNA. Patterns obtained with three endonucleases (HaeIII, HinfI and RsaI) made it possible to differentiate each species and to identify the wild strains, isolated from the same spoiled concentrated must, as belonging to the species Z. rouxii. The HinfI restriction enzyme produced a strain-specific pattern which allowed us to recognize that the seven wild isolates belonged to only three strains.
In this study, we identified a total of 33 wine yeast species and strains using the restriction patterns generated from the region spanning the internal transcribed spacers (ITS 1 and 2) and the 5.8S rRNA gene. Polymerase chain reaction (PCR) products of this rDNA region showed a high length variation for the different species. The size of the PCR products and the restriction analyses with three restriction endonucleases (HinfI, CfoI, and HaeIII) yielded a specific restriction pattern for each species with the exception of the corresponding anamorph and teleomorph states, which presented identical patterns. This method was applied to analyze the diversity of wine yeast species during spontaneous wine fermentation.
The polymerase chain reaction was used to amplify a targeted region: an internal transcribed spacer region of the ribosomal DNA from 114 Candida isolates and 65 reference strains. Unique product sizes were obtained for Candida glabrata, C. guillermondii and C. inconspicua. Isolates of C. albicans, C. tropicalis, C. dubliniensis and C. krusei could be identified following restriction digestion of the PCR products. The methods proved to be both simple and reproducible and may offer potential advantages over phenotyping methods.
DNA sequences of the single-copy gene coding for the 42 kDa endochitinase enzyme (EC 3.2.1.14) were used for phylogenetic analysis in Trichoderma. A set of 12 primers was developed and the entire gene was sequenced for 16 strains, and nucleotide and deduced amino acid sequences were compared to data from GenBank for additional Trichoderma strains. Analysis of the sequences revealed parsimony informative variation from 2.4 to 43.6% depending on the part of the gene (exons/introns) and the taxonomic level considered. Results are discussed in comparison to previous data from ITS-1 and ITS-2 rDNA sequencing and suggest the 42 kDa endochitinase gene as a potential molecular marker for reconstructing phylogenetic relationships in the genus Trichoderma at species level.
We used microsatellite fingerprinting and RAPD analysis to characterize 28 wild European strains of Saccharomyces paradoxus. In contrast to our results from a previous allozyme survey [Naumov et al. Int. J. Syst. Bacteriol. 47: 341-344 (1997a)], these methods revealed extensive genetic variation. The RAPD primers 5'AATCGGGCTG and 5'GGGTAACGCC and the microsatellite primer (GTG)5 yielded reproducible amplification patterns of sufficient clarity and variability to distinguish individual strains from the wild. UPGMA analysis tended to group the strains according to climatic and geographic origin. A comparative study of Ty1 sequence having multiple chromosomal location was also done. Each wild S. paradoxus isolate shows a unique hybridization pattern allowing discrimination to the strain level.
PCR/RFLP of the NTS2 sequence of rDNA was shown to be suitable for differentiating Saccharomyces sensu stricto species. We previously showed that, within the presently accepted S. bayanus taxon, strains formerly classified as S. uvarum represented a distinct subgroup (Nguyen and Gaillardin, 1997). In this study, we reidentified 43 more strains isolated recently from wine, cider and various fermentation habitats, and confirmed by karyotyping, hybridization and mtDNA analysis the homogeneity of strains from the S. uvarum subspecies. Molecular typing of nuclear and mitochondrial genomes of strains preserved in collections, and often originating from beer like S. pastorianusNT, revealed the existence of hybrids between S. uvarum and S. cerevisiae. Surprisingly, S. bayanusT CBS380 appeared itself to be a hybrid between S. uvarum and S. cerevisiae. This strain has a mitochondrial genome identical to that of S. uvarum, and a very similar karyotype with 13 isomorphic chromosomes, six of which at least hybridize strongly with S. uvarum chromosomes or with a S. uvarum specific sequence. However, four of the chromosome bands of S. bayanusT bear Y' sequences indistinguishable from those of S. cerevisiae, a feature that is not observed among presently isolated S. uvarum strains. Because of the hybrid nature of S. bayanus(T) and of the scarcity of similar hybrids among present days isolates, we propose to reinstate S. uvarum as a proper species among the Saccharomyces sensu stricto complex.
During the elaboration process of prepared and frozen foods, Clostridium sp. have been reported. From those microorganisms, C. perfringens and C. botulinum may pose a high risk for the consumers. To avoid these pathogenic organisms an HACCP program should be implemented, but in addition sensitive and moderately time consuming microbiological methods for monitoring C. perfringens and C. boulinum should be established. In this work, an RFLP analysis of the 16S rDNA will be developed to differentiate C. perfringens from other Clostridium sp. In addition, a PCR protocol, will be assayed to detect C. botulinum. Both two methods will be compared with biochemical characterization by API system. The restriction analysis of the 16S rDNA with Taq I and Rsa I showed at least the same sensitivity to differentiate C. perfringens from clostridial isolates as biochemical identification. However, the former method takes only 8-10 h of analysis as compared with 24-48 h required for biochemical characterization. With the specific PCR protocol to detect C. botulinum a band of 1.1 kbp was obtained derived from the specific amplification of BoNT genes, taking 6-8 h for analysis. Both two molecular DNA based methods should be considered as verification techniques of pathogenic clostridia in the HACCP program.
The yeasts represent a diverse assemblage of fungi having in common a vegetative stage that is predominantly unicellular. Although from the end of the last century investigators have attempted to classify yeasts based on morphological and physiological differences, none of the various systems currently in use can be considered satisfactory, and no consensus exists among mycologists regarding the natural relationships among these organisms. We have therefore determined the deoxyribonucleic acid (DNA) base sequence relatedness among a phenotypically similar group of yeasts to establish whether the existing taxonomic criteria in fact reflect the evolutionary affinities within the group. Our results provide evidence that the methodology generally used for delimiting yeast species is inadequate to define natural yeast taxa.
Polymerase chain reaction conditions were established for the in vitro amplification of eukaryotic small subunit ribosomal (16S-like) rRNA genes. Coding regions from algae, fungi, and protozoa were amplified from nanogram quantities of genomic DNA or recombinant plasmids containing rDNA genes. Oligodeoxynucleotides that are complementary to conserved regions at the 5' and 3' termini of eukaryotic 16S-like rRNAs were used to prime DNA synthesis in repetitive cycles of denaturation, reannealing, and DNA synthesis. The fidelity of synthesis for the amplification products was evaluated by comparisons with sequences of previously reported rRNA genes or with primer extension analyses of rRNAs. Fewer than one error per 2000 positions were observed in the amplified rRNA coding region sequences. The primary structure of the 16S-like rRNA from the marine diatom, Skeletonema costatum, was inferred from the sequence of its in vitro amplified coding region.
The tempo of evolutionary change determines in what manner any class of characters is informative in evolutionary studies and at which taxonomic levels. Here we describe and summarize some fundamental features of the evolution of the DNA sequences that encode ribosomal RNA genes in the nuclear genome of higher plants. By analyzing a sample of angiosperm species having known phylogenetic relationships at five different taxonomic levels ranging from the intraspecific to the interfamilial, we show that plant ribosomal DNA determines at least eleven classes of characters that can be distinguished by comparisons at the DNA level. These classes are temporal and physical subsets of three basic modes of variation: length variation, single base pair substitution, and DNA modification. We also discuss the impact of length variants on population genetic studies and the implications of these studies for understanding the molecular mechanisms of rDNA evolution.
Restriction fragment length polymorphisms in the rDNA internal transcribed spacer region (ITS) of 18 yeast strains currently assigned toSaccharomyces cerevisiae, S. pastorianus, andS. bayanus were examined. Primers complementary to the ITS region were used to amplify the ITS rDNA by the polymerase chain reaction (PCR). The products were digested with 10 endonucleases and cluster analysis was used to generate a phenogram from the restriction fragment data. Three strains ofS. cerevisiae (ATCC 10609, 26250, and 66162) exhibited restriction patterns that were different from the type strain but identical to those of theS. bayanus-S. pastorianus cluster. In contrast,S. pastorianus (ATCC 76671) showed restriction profiles that were different from its type strain but were identical to the type strain ofS. cerevisiae (ATCC 18824). These results suggest that the three strains ofS. cerevisiae should be reassigned to eitherS. pastorianus orS. bayanus, and the strain ofS. pastorianus (ATCC 76671) should be reclassified asS. cerevisiae.
Deoxyribonucleic acid reassociation studies of 24 different wine and beer-associated strains of Saccharomyces confirmed the presence of three separate species. S. cerevisiae and S. bayanus strains had only 22% of their genomes in common. S. pastorianus, with intermediate hybridization values between S. cerevisiae and S. bayanus (52 and 72%, respectively) could possibly be a natural hybrid of the two species. S. pastorianus replaces S. carlsbergensis with which it is homologous for 93% of its genome, since the former species was described first by Hansen in 1904. These data do not agree with the results of traditional physiological tests.
Genes and Genomes provides a well-directed excursion into the realms of modern-day molecular genetics with all the excitement of new discoveries clearly presented to the reader. The first 215 pages of the book present a review of basic genetic concepts that laid the groundwork for the approaches and techniques that have led to an explosion of knowledge in the field of molecular genetics. The second part is an extensive guide to tools and experimental systems that are being used to explore the mysteries of the eukaryotic genome. With these tools in hand, the authors now launch the reader into a 400-page account of current understanding of the anatomy, the expression, and the regulation of eukaryotic genes. The final section of the book is an introduction to more complex biological systems.
The so-called wine yeasts Saccharomyces cerevisiae, S. chevalieri, S. bayanus, S. italicus and S. uvarum are characterized by high ethanol tolerance and fermentation velocity. They are ecologically related, being predominantly associated with grape must and wine, and are taxonomically indistinguishable. The only significant physiological differences are between the ability to ferment certain sugars. A taxonomic revision of more than 1,000 strains isolated during the past 50 years and belonging to the above species showed extreme instability in the ability to ferment different sugars. The relationships between these yeasts were examined for DNA base composition and DNA-DNA reassociation. The G+C ranged from 37.6% to 39.0% while optical reassociation experiments defined a first group of species (Saccharomyces cerevisiae, S. chevalieri and S. italicus) exhibiting high base sequence complementarity (>90%). S. bayanus and S. uvarum also showed a high degree of relatedness. Low homology values (30%) indicate that the two groups of species are not closely related. While it is proposed to combine S. cerevisiae, S. chevalieri and S. italicus into one single species under the oldest epithet Saccharomyces cerevisiae, a study of a larger number of strains is recommended before considering the taxonomic position of S. bayanus and S. uvarum.
The intergenic spacers between some adjacent tRNA genes were shown to be polymorphic in length when closely related Staphylococcus species were compared. A simple procedure was developed to detect and sequence these tRNA intergenic length polymorphisms (tRNA-ILPs). A comparison of homologous tRNA gene sequences flanking these ILPs in three Staphylococcus species was used to derive primers for high-stringency amplification of the ILPs by the polymerase chain reaction (PCR). The detection of tRNA-ILPs by PCR allowed the classification of virtually all strains from the five species of Staphylococcus that were examined. The procedure used to identify, sequence and derive primers for PCR detection of tRNA-ILPs in Staphylococcus should be applicable to many other genera of eubacteria. These primers could be used on uncultured material such as clinical samples.
This chapter describes the DNA of Saccharomyces cerevisiae strains used in genetic studies. Discussion of DNA isolation procedures is followed by a description of useful information derived from analysis of restriction spectra. Interpretation of the band patterns can be quite useful. First, the extent of cleavage (partial or complete) can be deduced for many restriction endonucleases. Second, the average copy number of newly introduced plasmids can be estimated. Third, restriction site polymorphisms in repeated DNA can be detected. The sequence organization and the location of restriction sites for the four classes of repeated DNA (rDNA, 2-μm plasmid, Ty elements, telomeric Y' sequences) which are the origin of most bands seen in restriction spectra are discussed in the chapter. The ribosomal DNA consists of a cluster of usually 100 to 120 tandem copies of a 9.08-kb repeat unit. Cell clones with higher and lower copy number are observed.
Detailed restriction analyses of many samples often require substantial amounts of time and effort for DNA extraction, restriction digests, Southern blotting, and hybridization. We describe a novel approach that uses the polymerase chain reaction (PCR) for rapid simplified restriction typing and mapping of DNA from many different isolates. DNA fragments up to 2 kilobase pairs in length were efficiently amplified from crude DNA samples of several pathogenic Cryptococcus species, including C. neoformans, C. albidus, C. laurentii, and C. uniguttulatus. Digestion and electrophoresis of the PCR products by using frequent-cutting restriction enzymes produced complex restriction phenotypes (fingerprints) that were often unique for each strain or species. We used the PCR to amplify and analyze restriction pattern variation within three major portions of the ribosomal DNA (rDNA) repeats from these fungi. Detailed mapping of many restriction sites within the rDNA locus was determined by fingerprint analysis of progressively larger PCR fragments sharing a common primer site at one end. As judged by PCR fingerprints, the rDNA of 19 C. neoformans isolates showed no variation for four restriction enzymes that we surveyed. Other Cryptococcus spp. showed varying levels of restriction pattern variation within their rDNAs and were shown to be genetically distinct from C. neoformans. The PCR primers used in this study have also been successfully applied for amplification of rDNAs from other pathogenic and nonpathogenic fungi, including Candida spp., and ought to have wide applicability for clinical detection and other studies.
Quantitative methods for estimating the extent of raffinose fermentation were applied to cultures ofSaccharomyces pastorianus andSaccharomyces bayanus received from culture collections. Four cultures ofSaccharomyces pastorianus fermented two-thirds only of the raffinose molecule. This was in contrast to recently published reports and confirmed the standard description ofSaccharomyces pastorianus. Two cultures ofSaccharomyces bayanus each consisted of two components, the major component was able to ferment two-thirds raffinose and the minor to ferment raffinose completely. Isolates of the major component could readily mutate to the minor. Three cultures ofSaccharomyces bayanus corresponded to the standard description in their ability to ferment only one-third raffinose. The taxonomic implications of these findings were considered.
Eukaryotic 5S rRNA sequences from 34 diverse species were compared by the following method: (1) The sequences were aligned; (2) the positions of substitutions were located by comparison of all possible pairs of sequences; (3) the substitution sites were mapped to an assumed general base pairing model; and (4) the R-Y model of base stacking was used to study stacking pattern relationships in the structure. An analysis of the sequence and structure variability in each region of the molecule is presented. It was found that the degree of base substitution varies over a wide range, from absolute conservation to occurrence of over 90% of the possible observable substitutions. The substitutions are located primarily in stem regions of the 5S rRNA secondary structure. More than 88% of the substitutions in helical regions maintain base pairing. The disruptive substitutions are primarily located at the edges of helical regions, resulting in shortening of the helical regions and lengthening of the adjacent nonpaired regions. Base stacking patterns determined by the R-Y model are mapped onto the general secondary structure. Intrastrand and interstrand stacking could stabilize alternative coaxial structures and limit the conformational flexibility of nonpaired regions. Two short contiguous regions are 100% conserved in all species. This may reflect evolutionary constraints imposed at the DNA level by the requirement for binding of a 5S gene transcription initiation factor during gene expression.
NTSYS-pc: Numerical taxonomy and multivariate analysis system
- F J Rohif
Rohif, F.J. (1990) NTSYS-pc: Numerical taxonomy and
multivariate analysis system. Exeter Publishing, NY.
Genes and genomes. University Science Books
- M Singer
- P Berg
Singer, M. and Berg, P. (1991) Genes and genomes. University Science Books, Mill Valley, CA.
PCR-amplified length polymorphisms in tRNA intergenic spacers for categorizing staphylococci
- J Welsh
- M Mccleuand
Welsh, J. and McCleUand, M. (1992) PCR-amplified length
polymorphisms in tRNA intergenic spacers for categorizing staphylococci. Mol. Microbiol. 6, 1673-1680.
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