GB2230605A - Genetic characterisation of fungi - Google Patents

Abstract

A first method of characterising a yeast or other fungus comprises probing a restriction enzyme digest of the chromosomal DNA of the fungus with a probe which has a polyGT or polyCA sequence. A second method of characterising a yeast or other fungus to establish its relationship to a control fungus comprises probing a restriction enzyme digest of the chromosomal DNA of the fungus under investigation with a probe which will hybridise to a sequence which flanks, i.e. is within 5kb of, a poly GT sequence of the chromosomal DNA of the control fungus, and comparing the results with those obtained for the control fungus. The fungus to be characterised may be A. Nidulans or a Candida sub-species or may be a yeast such as Zygosaccharomyces bailii, Hansenula anomola or a Saccharomyces sub-species.

Description

CHARACTERISATION OF FUNGI The present invention relates to the identification or characterisation of fungi, particularly but not exclusively yeasts.

The identification of yeasts is important for a number of purposes. For example, in the brewing, baking or biotechnology industries identification may be important for quality control to ensure that the correct organism or mix of organisms is being used, or for security reasons to ensure that competitors have not dishonestly acquired strains. Unfortunately, existing methods for the identification of yeasts can be complicated and time consuming to carry out.

The general definition that includes all yeasts is that they are fungi that do not produce asexual spores and spend at least part of their vegetative cycle as single cells. Asexual reproduction occurs either by budding, fission or bud fission. Within this rather broad physical description there are examples such as Saccharomyces cerevisiae and Schizosaccharomyces pombe which appear to be only distantly related by biochemical and molecular biological criteria.

Classification of the yeasts using keys depends upon the definition of various different physical or physiological parameters.

These include texture, colour and shape of yeast colonies grown on malt extract media, vegetative cell shape, size and budding characteristics, ascospore numbers and shapes, and the ability to utilise different carbon and nitrogen sources. Such analysis is time consuming, and methodological differences can yield differing results.

At the species and strain (sub species) level, classification becomes more difficult. Firstly, the definition of a new species by virtue of its inability to interbreed with previously defined species (species as "closed gene pools") is of limited use for organisms which have no sexual life cycle. This is frequently the case for industrial brewing strains as well as for some laboratory strains. Secondly a strain may be initially defined only by a single character that distinguishes it from related organisms. This single character may be a specific biochemical effect (overproduction of a certain protein) or a more complex character such as the taste of a fermented drink product, or dough-rising quality of a bakery product.These characteristics, though definable, may be more difficult or time-consuming to check, though in the interests of quality control or perhaps even security they may need to be ascertained rapidly. It is at this level that the need arises for fast and unambiguous identification.

For many laboratory strains identity "tags" are provided by means of mutation. Those most commonly used are mutations in amino acid or nucleic acid biosynthetic pathways that give rise to auxotrophy - the cells cannot grow without the appropriate supplements. Unfortunately this is not a system that is easily used in the common brewing strains as they are often polyploid and so less amenable to mutation. Alternative strategies include the addition of genetic tags by recombinant methods. A dominant gene, such as those which confer resistance to an antimicrobial agent may be added or alternatively a unique DNA sequence may be added to provide a diagnostic hybridisation target. The principal objection to these approaches is concern about consumer acceptance of genetic engineered foods.The final complication for some of the brewers is that they sometimes use a mixture of different yeasts such that a full check of the system also requires a primary separation into individual yeast clones (streaking out etc.) before identification can be attempted.

Genetic fingerprinting is unlike key-driven taxonomy and has demonstrated the power of molecular hybridisation probes. Keys rely on phenotype or, in a different context, the end products of biological information flow ("DNA gives RNA gives protein"). The genetic fingerprint is in essence a rather specialised genotype - it reflects differences at the DNA level. There is a very substantial amount of DNA which does not get transcribed or translated, and cannot therefore contribute to the phenotype. Whilst in some cases this DNA still carries information necessary to gene expression, much of it, perhaps as much as eighty percent is either junk, or information which biologists cannot yet decode (proteins with no known function, or untranslatable regions).All of the DNA in the cell is subject to mutation, though mutations in essential genes are generally more likely to be lost from populations because of adverse affects.

Different mutations in the inessential regions may however accumulate in a population. It is these differences which are at the core of a genetic fingerprint. A single base change may cause the creation or loss of a restriction endonuclease (RE) cleavage site; a spontaneous deletion of a segment of DNA will change spacing between RE sites, insertions or other rearrangements caused by transposable elements will similarly alter spacing of RE sites. A good fingerprinting probe will hybridise to a dispersed repetitive sequence. Differences in sequences that flank the probe will result in restriction fragment length polymorphisms (RFLPs) between strains.

It is an object of the present invention to provide a method of identifying yeasts based on the use of molecular hybridisation probes.

According to a first aspect of the present invention there is provided a method of genetically characterising a yeast comprising obtaining a restriction enzyme digest of the chromosomal DNA of the yeast being investigated, probing the fragments of the digest with a polynucleotide (a poly dG.dT sequence or a poly dC.dA sequence) detecting fragments of the digest which are hybridised to the polynucleotide, and comparing the results with those obtained for a control yeast.

The comparison may, for example, be with a library of results obtained by the technique from other yeasts so as to characterise the yeast under investigation.

More particularly, this probing method may be a genetic fingerprinting technique in which the restriction endonuclease digest fragments are separated by electrophoresis on an agarose gel, the separated fragments are transferred to a suitable carrier (e.g.

nitro-cellulose) by standard blotting techniques, denatured, and then hybridised (again using standard techniques) to the polynucleotide probes. The probes may be radio-labelled (e.g. 32p) to facilitate detection of the hybridised fragments by auto-radiography. It is however possible to use any other suitable method of detection.

The chromosomal DNA may be obtained from the yeast culture by well known methods, and the digests prepared using any suitable restriction endonuclease, e.g. Sau3al Hind III or EcoRI, used in accordance with manufacturers instructions.

Synthetic 'poly GT', i.e. synthetic DNA comprised of strands containing repeating GT sequences base paired to strands containing repeating CA sequences may be used as the precursor for the polynucleotide probes, which may be generated simply bynick translation and denaturation (by heat) of the DNA. Conveniently, the poly GT which provides the precursor for the probes will have a minimum length of about 30 base pairs and conveniently also it is possible to use a heterogeneous mix of synthetic poly GT fragments of different length. Typical conditions for the hybridisation are at 55-58OC in 3+SSC (1*SSC is 0.15M sodium chloride, 0.015M sodium citrate).

The result of this probing technique is a genetic fingerprint which is characteristic of the yeast strain being investigated. With more specific reference to S. cerevisiae, this has about one hundred poly GT tracts which is a sufficiently small number for them to be separately distinguished by blotting techniques.

Previous work has shown that at least half of the hybridising sequences in the S. cerevisiae genome are found at the chromosomal termini, the telomeres. The rest appear to be fairly randomly distributed around the rest of the genome.

The telomeric sequences are of two types; those which occur at the very tip of the chromosome and those which are embedded between the telomere associated sequences denoted X and Y'. The actual sequence detected by the probe at these locations is polydCl-3dA.polydGl-3dT.

The terminal tract is heterogeneous in length within cells of a given strain, but has a characteristic mean. In comparing strains this mean value is often found to be different, and the variation has been used in a genetic analysis of telomere length. It appears that length is determined by several genes. The difference in mean size is often only subtle but is frequently a useful diagnostic.

The sequences between X and Y' occur on different sized restriction fragments for two reasons. X elements are heterogeneous making XY boundary fragments variable. Y' copy number varies from zero to four per end such that each chromosome has different numbers of XY and YY boundary fragments. Since there are (probably) 32 telomeres in S. cerevisiae strains this variation contributes significantly to a genetic fingerprint.

The non-telomeric poly GT hybridising sequences are largely uncharacterised, though sequence data suggests that those from the yeasts are similar in length to those found in the other eukaryotes.

They are valuable in the genetic fingerprint for two reasons.

Firstly, they are conserved dispersed and repetitive. Secondly, they have been shown to be recombination hotspots so they are more likely than other sequences to be associated with restriction fragment length polymorphisms.

Turning now to a second aspect of the invention, the abovementioned conservation of the poly GT sequences in the chromosomal DNA of the yeast suggests that at least some of them may be associated with conserved functions. We have investigated this possibility by looking for conservation of poly GT-flanking sequences.

GT flanking sequences are, for the purpose of the description, sequences from a yeast genome which are within 5kb of a poly GT sequence. Such flanking sequences may be most conveniently prepared as restriction fragments from poly GT-hybridising clones.

We have established that such flanking sequences can be conserved and provide a further way to distinguish yeasts.

According to a second aspect of the invention there is provided a method of characterising a yeast to establish its relationship to a control yeast comprising probing a restriction enzyme digest of the chromosomal DNA of the yeast under investigation with a probe which will hybridise to a sequence which flanks a poly GT sequence of the chromosomal DNA of the control yeast, and comparing the results with those obtained for the control yeast.

This method may be a genetic fingerprinting technique in which the restriction digest fragments are separated (e.g. on agarose), and subsequently denatured and probed (e.g. after transfer to a suitable membrane by blotting techniques) with a probe (e.g. radioactively labelled) based on the aforementioned flanking sequence.

The poly GT and the flanking sequence (which is most likely to be a unique sequence of DNA) will be located on the chromosomal DNA between restriction sites (the 'first sites') at which a particular endonuclease "cuts" the DNA strand. Generally there will be at least one further (different) restriction site (the 'second' site) between the poly GT sequence and the flanking sequence.

The probe is preferably a single strand of the same nucleotide sequence as occurs in the flanking sequence between the first and second restriction sites. In the simplest form of the second aspect of the invention, this probe is used to probe digests of the control yeast and the yeast under investigation which have been prepared with a restriction endonuclease which cuts at the first site. Generally however there will be a further restriction site (the 'third' site) on the flanking sequence between the first and second sites. It is more preferred to effect the method of the second aspect of the invention on a digest of the chromosomal DNA prepared with a restriction enzyme which 'cuts' at the first site and one which cuts at the 'third' site.

In this case the probe will hybridise to each of the (different length) fragments cut from between the two 'first' sites in the control yeast.

The use of the two restriction enzymes provides a greater amount of information for comparing yeast B with yeast A than is the case where any one enzyme is used.

Probes for use in the second aspect of the invention may be obtained from a genomic library comprising a collection of plasmids each containing a different insert of yeast genomic DNA. The library may be probed to identify poly GT hybridising clones, ie., clones which contain copies of a restriction fragment containing a poly GT sequence.

Any poly GT hybridising clone may then be selected for further investigation. The selected clone is restriction mapped, from which the position of the poly GT sequence itself may be identified. A sequence which flanks the poly GT sequence (ie., which is separated therefrom by at least one restriction site) may then be 'cut' fr ;.i the poly GT hybridising clone (using the appropriate restriction enzyme).

Radioactive probes may be prepared by conventional methods from these flanking sequences.

The radioactive probes may then be used for probing a restriction enzyme digest of the chromosomal DNA of a yeast under investigation.

Such a probing operation may be a genetic fingerprinting technique in which the restriction fragment digests (of the yeast under investigation) are electrophonetically separated (eg., on agarose), and subsequently denatured and probed (eg., after transfer to a suitable membrane by blotting techniques with the radioactively labelled probe, the genetic fingerprint being recorded by autoradiography.

In a modification which avoids the need to prepare radioactive probes and also the need to use autoradiography, the probes (ie., the DNA flanking sequence) may be sequenced using conventional techniques so as to determine their absolute nucleotide sequence.

Abundant quantities of the targe genomic sequence may then be sythesized using the Polymerase Chain Reaction (PCR). RFLPs within the targe sequence may be detected by subsequent restriction endonucleolytic cleavage of the amplified DNA.

The 'synthetic' probes may then be used to obtain a genetic fingerprint of the yeast under investigation. The 'abundant' quantities of the probe which may be used in this technique is such that the bands in the spectrum on the gel (comprising nucleotide sequences from the yeast under investigation hybridised to a nucleotide sequence of the probe) are visible to the naked eye and thus the need to use autoradiography is avoided.

Applications of the methods of the first and second aspects of the invention are as in the following, non-exhaustive list.

(i) Quality control for the brewing and baking industries.

(ii) Diagnosis and epidemiology of human pathogenic fungi, including Candida species.

(iii) Taxonomy.

The invention will be further described by way of example only with reference to the accompanying drawings, in which: Fig. 1 schematically illustrates a portion of the chromosomal DNA of two yeasts A and B; Fig. 2 schematically illustrates the genetic fingerprints obtained by the method of the first aspect of the invention from the DNA portion represented in Fig. 1; Fig. 3 is similar to Fig. 1 but annotated to illustrate the second aspect of the invention.

Fig. 4 schematically illustrates the genetic fingerprints obtained by the method of the second aspect of the invention from the DNA represented in Fig. 3.

Figs. 5 are autoradiographs obtained from yeasts using the first aspect of the invention; Fig. 6 is a restriction map; and Figs. 7 are autoradiographs obtained from yeasts using the second aspect of the invention.

In Figure 1, there are schematically depicted portions of the chromosomal DNA of two different yeasts which are members of the same sub group, one of which is designated A, and the other B. Along each strand are shown different restriction sites as represented by the "o", "*", and "+" markers. Within each strand are poly GT sequences as shown. Strand A is shown to differ from strand B in having an extra "o" marker as indicated.

In accordance with the first aspect of the invention, the yeast may be distinguished from each other by preparing digests of the chromosomal DNA with a restriction enzyme which cuts at the "o" sites.

By probing with radio-labelled poly GT as described previously, two spectra are obtained somewhat as shown in Fig. 2 in which the hybridised fragments labelled as Al, A2, B1, B2 correspond to the correspondingly labelled sequence lengths in Fig. 1. The dotted bands shown in Fig. 2 represent additional bands which would be obtained in the spectrum from poly GT sequences not shown in Fig. 1, ie the rest of the genome.

Thus Fig. 2 illustrates that different spectra are stained for the yeasts A and B. It will be appreciated that the spectra obtained with the use of actual yeasts will be considerably more complicated than that illustrated in Fig. 2 due to the large number of poly GT tracts in the chromosomal DNA. In actual practice, the hybridisation spectrum generated by the use of the poly GT probes is unique and complex. Some of the complexity in S. cerevisiae species can be accounted for by known differences that occur between the telomeres.

Qualitative and quantitative differences between non-telomeric poly GT tracts occur with sufficiently high frequency to generate a unique spectrum for any of these organisms.

Thus, this method of the first aspect of the invention may be used to test whether a particular yeast, say yeast B, is exactly the same as a control standard, i.e. yeast A. The method is comparatively rapid and will therefore be useful for quality control or security purposes in those industries which rely on the use of yeast, e.g. the brewing industry. The method is equally applicable to mixes of yeasts.

The second aspect of the invention relies on probes produced from nucleotide sequences which flank the poly GT sequences in the chromosomal DNA in a yeast. Consider yeast A. The sequence illustrated as A "+"-"o" (Fig. 3) will be a unique sequence along the length of the chromosomal DNA of yeast A. It is assumed for the purpose of this description that yeast B is a mutation of yeast A (or a closely related strain) and that the sequence A "+"-"o" has been substantially conserved and is virtually identical to the sequence represented by the length B "+"-"m" in yeast B, the only difference between the sequences A "+"-"o" and B "+"-"m" being the mutation in the latter which has caused the loss of the "o" restriction site.

If the sequence A "+"-"o" can be cloned, it will be a useful fingerprinting probe, for a comparison of yeasts A and B. In this technique, the chromosomal DNA of each of yeast A and B are digested separately firstly with a restriction enzyme which cuts at the "o" site and then one which cuts at the "*" site. Amongst the fragment obtained from A will be the two sequences A "o"-"*" and A "*"-'o", whereas the digest of B will include the fragments B "o"-"*" (identical to A "o"-"*") and B "*"-"o" (see Fig. 3).

The length of DNA A "+"-"o" has sequences common to each of the four fragments mentioned in the preceding paragraph. Thus by electrophonetically separating the fragments and probing the separated fragments with a radioactively labelled probe based on the sequence A "+"-"o" an autoradiogram similar to that shown in Fig. 4 is obtained, in which the sequences indicated for the individual bands correspond to the similarly labelled sequences in Fig. 3.

It should be noted that each of yeasts A and B will yield only two bands in this spectrum (in contrast to the multitude of bands they each produce in the method of the first aspect of the invention). This is because the DNA sequence A "+"-"o" will be a unique sequence on the chromosomal DNA of yeast A and will be substantially identical to the sequence B "+"-"m" in yeast B. Thus probes based on sequence A "+"-"o" will be specific for only those sequences represented in Fig.4.

The autoradiogram of Fig. 4 provides a good indication that yeast B is simply a mutation of yeast A but not necessarily conclusive proof. However, additional poly GT flanking probes may be prepared from other regions of the chromosomal DNA of yeast A in a manner entirely analogous to that described above. These flanking probes may be used to investigate digests of yeasts A and B with the appropriate restriction enzymes. If each such flanking probe produces identical bands in the autoradiogram then this will be conclusive that the yeast B is simply a mutation of yeast A.

The spectra obtained using the method of the second aspect of the invention are simpler and more straightforward to analyse than those for the first aspect. The spectra of the second aspect are sufficient in cases where one wishes routinely to distinguish between one or two different organisms. The principal strength of the full spectrum (first aspect) is that it represents information from all of the chromosomes instead of only one part of one chromosome. This is particularly important if one is concerned about the appearance of contaminants that do not hybridise to one of the single copy probes.

The first and second aspects of the invention are further illustrated by the following description.

Figure 5 shows genetic fingerprints from E collection of yeasts. Each panel is a photograph of an autoradiograph. A key to the strains is given in Table 1. DNA was cut with the restriction enzymes Hind III track (i), MspI (tracks (ii)) (II) and EcoRI (tracks (iii)). The resulting fragments were separated by gel electrophoresis, Southern blotted onto nitrocellulose then probed with polyGT. It is clear that all the yeasts shown have different fingerprints. It should be noted especially however that although a,b,c and d are all strains of one species Saccharomyces cerevisiae they are still readily distinguishable.

Table 1 a. Saccharomyces cerevisiae 625. R.B. Gilliland (Saccharomyces diastacticus, flocculent strain, used in protoplast fusion studies) 1960.

b. Saccharomyces cerevisiae 505. C.B.S. (CBS 1171, ATCC 18824,T, from brewing yeast, 5:1:5:5:1) 1957.

c. Saccharomyces cerevisiae 509. C.B.S. (Saccharomyces uvarum, CBS 395, IFO 0615,T from Saccharomyces uvarum, from currant juice) 1957.

d. Saccharomyces cerevisiae 74 ATCC (Saccharomyces carlsbergensis, ATCC 9080, ATCC 24904, CBS 2354, Hillman Hospital strain 4228).

e. Saccharomyces exigus 1476. T.F. Brocklehurst (E18, from spoiled coleslaw) 1983.

f. Saccharomyces dairensis 1477. T.F. Brocklehurst (Y2-80, from shredded cabbage) 1983.

g. Saccharomyces kluyveri 543. H.C. Phaff (UCD 55-91, CBS 3082, IFO 1685,T, from Drosophila).

h. Saccharomyces unisporus 971 C.B.S. (CBS 398, ATCC 10612, NRRL,Y-1556, IFO 0316,T) 1980.

i. Zygosaccharomyces bailii 1427 K Painting 1982.

j. Hansenula anomala 682 F.R. Elliot, B.R.F. (from pitching yeast, produces high amounts of ethyl acetate).

The poly GT sequence hybridises to a large number of restriction fragments on the Southern blott, each corresponding to a different genomic location. It is also possible to probe for these fragments individually. This is useful because it allows simpler fingerprints to be generated - patterns of only a few bands. Clearly, if two organisms differ at only one position in the poly GT fingerprint, a probe corresponding to this position will be sufficient to distinguish between them.

To isolate probes corresponding to individual poly GT locations, a genomic library of Saccharomyces cerevisiae restriction fragments was probed with poly GT. This yielded a collection of three hundred poly GT-hybridising clones. If these clones are used as probes themselves a fingerprint essentially the same as the poly GT fingerprint is generated due to the presence of the poly GT sequence.

For this reason the following procedure is followed for each individual clone. The plasmid is restriction mapped and the position of the poly GT located. Using this information restriction fragments containing genomic DNA but not poly GT are isolated. These are called "poly GT-flanking sequences". The following example illustrates the use of poly GT flanking sequences.

Figure 6 shows the restriction maps of two poly GT hybridising genomic clones denoted pGT34 and pGT66. These clones were selected from a library of fragments generated by partial Sau3A digestion inserted in YEP24. For simplicity the vector sequences are not shown.

The shaded areas locate the poly GT sequence. E,H and S correspond to cleavage sites for the enzymes EcoRl, HindIII and Sau3Al respectively. Underlined areas are poly GT flanking sequences and were excised and used as probes.

The probe derived from pGT66 was used to screen a small collection of yeasts. DNA from the yeast strains was cut with HindIII and Southern Blotted then probed with the nick translated fragment.

The autoradiogram is shown in Figure 7a. The probe hybridised to some strains but not others.

A probe comprising the poly GT-flanking sequences from pGT34 was used to screen the same collection of yeasts. The same subgroup of strains hybridised but with a different pattern in each case (Fig.

7b).

The results obtained with poly GT flanking sequences show that some of the poly GT locations from S. cerevisiae are sufficiently conserved to allow cross hybridisation between organisms. In one case (pGT66) it appeared that this conservation was very high since the same sized restriction fragments were detected in three different strains and species. The pGT66 probe would be useful in distinguishing between subgroups of yeasts. In the case of pGT34 there are clearly differences in the local environment apparent as RFLPs. The pGT34 probe is useful in distinguishing between members of a subgroup of yeasts.

The genetic fingerprints shown in Figs. 7a and 7b are autoradiograms obtained by using radioactively labelled probes. In an alternative method of carrying out the invention, the flanking sequences to be used as probes may be sequenced using standard techniques and at least a portion of the sequence may be amplified by the Polymerase Chain Reaction (particularly by using TAQ polymerase).

The result of this procedure is that significant quantities of the desired sequence are generated and thus a comparatively large quantity of the sequence may be used as the probe. As a result, there will be a sufficient quantity of the hybridised fragments to be visible to the naked eye without the need for the probes to be radioactively labelled probes.

Although the invention has been described with specific reference to yeasts, it is also applicable to other fungi. For example we have used a poly GT probe with the filamentous fungus (Aspergillas nidulans) which has sufficiently few bands to be examined with the probe.

In the foregoing nucleic acid analyses, restriction fragments were separated by electrophoresis through 0.8 per cent agarose gels at 2v/M for 16 hours. Southern blotting (to Hybond-N, Amersham International) and nick translations were carrried out by standard procedures. Hybridisations were performed at 55-58C in 3 x SSC.

Claims (13)

1. A method of genetically characterising a yeast or other fungus comprising obtaining a restriction enzyme digest of the chromosomal DNA of the fungus being investigated, probing the fragments of the digest with a polynucleotide having a poly dG.dT sequence or a poly dC.dA sequence, and detecting fragments of the digest which are hybridised to the polynucleotide.

2. A method as claimed in claim 1, wherein the fragments of the digest are separated by electrophoresis and then probed with said polynucleotide.

3. A method as claimed in claim 1 or 2 in which the polynucleotide is radiolabelled.

4. A method as claimed in any one of claims 1 to 3 wherein said polynucleotide consists of a poly dG.dT sequence or a poly dC.dA sequence.

5. A method as claimed in any one of claims 1 to 3 wherein the probes are derived from synthetic DNA comprised of strands containing repeating GT sequences.

6. A method as claimed in any one of claims 1 to 5 wherein the polynucleotide probe comprises a minimum length of 30 bases in the poly dG.dT or poly dC.dA sequence.

7. A method of characterising a yeast or other fungus to establish its relationship to a control fungus comprising probing a restriction enzyme digest of the chromosomal DNA of the fungus under investigation with a probe which will hybridise to a sequence which flanks a poly GT sequence of the chromosomal DNA of the control fungus, and comparing the results with those obtained for the control fungus.

8. A method as claimed in claim 7 wherein the fragments of the digest are separated by electrophoresis and then probed.

9. A method as claimed in claim 7 or 8 wherein the probes are radioactively labelled.

10. A method as claimed in claim 7 or 8 wherein the fingerprints have been generated from the flanking sequence by a polymerase chain reaction.

11. A probe which comprises a nucleotide sequence which is the same as or will hybridise to a poly GT-flanking sequence of the chromosomal DNA of a fungus.

12. A probe as claimed in claim 11 wherein the fungus is a yeast.

13. A probe as claimed in claim 12 wherein the yeast is S.

cerevisiae.