WO2005056587A1 - Inteins within fungal prp8 genes, uses thereof and assays - Google Patents

Abstract

The invention relates to inteins derived from the PRP8 gene obtainable from an organism from the genus Aspergillus, the genus Neosartorya or the genus Histoplasma or the species Cryptococcus laurentii and to nucleic acids encoding these and to assays for organisms.

Description

INTEINS WITHIN FUNGAL PRP8 GENES, USES THEREOF AND ASSAYS

TECHNICAL FIELD

The invention relates to novel inteins identified in the PRP8 protein of Aspergillus nidulans, Aspergillus fumigatus and its close relative Neosartorya fischeri, and in Histoplasma capsulatum, together with an intein in Cryptococcus laurentii. The inteins are useful for a number of purposes. These include use as a molecular target for testing drugs to inhibit microbial function. Polynucleotides encoding the intein or portions thereof are useful in detecting or measuring presence of the organisms in samples, especially clinical samples. Such polynucleotides also are useful as biocontrol agents, exploiting the homing endonucleases that they encode.

BACKGROUND ART

Aspergillus is a filamentous, cosmopolitan and ubiquitous fungus found in nature. It is commonly isolated from soil, plant debris and indoor air environment. The genus Aspergillus includes over 185 species. Around 20 species have so far been reported as causative agents of opportunistic infections in humans. Among these infections, Aspergillus fumigatus is by far the most commonly isolated species, followed by Aspergillus flavus and Aspergillus niger, Aspergillus clavatus, Aspergillus nidulans and Aspergillus oryzae. Immunosuppression is the major factor predisposing to development of opportunistic infections. These infections may present in a wide spectrum, varying from local involvement to dissemination and as a whole called aspergillosis. Among all filamentous fungi, Aspergillus is in general the most commonly isolated one in invasive infections. It is the second most commonly recovered fungus in opportunistic mycoses following Candida.

Some Aspergillus species produce various mycotoxins. These mycotoxins, by chronic ingestion, have proven to possess carcinogenic potential particularly in animals. Among these mycotoxins, aflatoxin is well-known and may induce hepatocellular carcinoma. It is mostly produced by Aspergillus flavus and contaminates foodstuff, such as peanuts.

Histoplasma is a thermally dimorphic fungus found in nature. Although it is claimed to exist worldwide, it is more frequently encountered in tropical areas. It is endemic in the Tennessee- Ohio-Mississippi river basins. The genus Histoplasma contains one species, Histoplasma capsulatum. Histoplasma capsulatum is the causative agent of a systemic mycosis called histoplasmosis. The spectrum of the disease is wide, varying from an acute benign pulmonary infection to a chronic pulmonary or fatal disseminated disease.

Aspergillus and Histoplasma are related even though classified in different orders. They last shared a common ancestor some 125 million years ago. Aspergillus species have been diverging for about 20 million years. Neosartorya fischeri is very closely related to A. fumigatus, despite being presently classified in different genera.

Cryptococcus laurentii is a basidiomycete and thus very distantly related to the ascomycete fungi mentioned above. The species is a very occasional pathogen in immuno-compromised people.

Rapid and early diagnosis and quantification of Aspergillus infections is a high priority. Invasive infections with Aspergillus species are increasing, resulting in high mortality rates or causing severe illness. Delayed diagnosis and therapy of invasive pulmonary aspergillosis worsen the outlook. The increasing incidence of life-threatening systemic fungal infections emphasizes the need to improve the presently limited diagnostic tools for the detection and monitoring of these infections. The highest risk occurs during induction of treatment for acute leukemia or after bone marrow transplantation. Due to this poor prognosis, all diagnostic approaches primarily aim at an early confirmation of the infection. There have been limited diagnostic protocols for early detection of invasive aspergillosis, with the systemic infection frequently being diagnosed late or confirmed only at autopsy (Denning, 1998. Invasive aspergillosis. Clin. Infect. Dis. 26:781-803). Early diagnosis of invasive fungal infections is hampered by a lack of sensitive and specific assays, especially for invasive aspergillosis, and standard sensitive methods based on commercially available tests are still missing. Similar problems arise with histoplasmosis. Besides fungal detection, quantification of the fungal burden is also of great clinical relevance, since the individual fungal burden may allow therapeutic monitoring.

Diagnosis of infectious disease is moving towards molecular methods (for example, detecting specific stretches of DNA present in the particular infecting organism). This is partly because these molecular methods are faster. They are also less subjective, compared to culture methods and/or morphological methods they require less experienced personnel to interpret the results. Molecular methods can also be more specific (ie only the relevant species is detected).

The polymerase chain reaction (PCR) is the present method of choice for many molecularly- based diagnostic systems. In this technique, a small region of DNA is preferentially amplified in vitro from a preparation of DNA extracted from the whole infecting organism or clinical sample.

For diagnosis especially of fungi, there is often difficulty in finding an appropriate, species- specific DNA target to amplify; most PCR applications so far rely on conserved sequences that are similar in many species and this can lead to false positive results.

Inteins are genetic elements present within protein-coding sequences (intervening proteins). Inteins perform protein splicing. This is a self-catalyzed process by which the intein removes itself from the protein creating the functioning enzyme. Most inteins have an endonuclease domain. Inteins lacking an endonuclease domain have also been identified. These "mini- inteins" account for less than 20% of all inteins and while they can still be spliced, they are not capable of spreading to new sites. Inteins from bacteria and higher organisms share a similar structure and mechanism of operation. Of the bacteria which are associated with humans, the only genus that carries inteins is Mycobacterium. This includes the causative agent of tuberculosis.

These elements are rare in living organisms but examples are found in all three kingdoms. Inteins that are present at the same location within homologous proteins from different organisms are termed 'allelic' inteins. To date, five non-allelic inteins have been described in eukaryotic organisms. Porphyra purpurea (a red alga) chloroplasts and Guillardia theta (a cryptomonad alga) plastids have allelic mini-intein genes in their DNA B helicase genes. The green alga Chlamydomonas eugametos has a DODendonuclease-containing intein gene in its chloroplast clp-A gene. There is an intein in the Chilo iridescent virus ribonucleotide reductase class 1 gene. An allelic intein is in the homologous gene from a Bacillus subtilis prophage (Spbeta). Only two inteins have been described in the nuclear genetic material of higher organisms. One is present in the vacuolar ATPase (VMA) genes of Saccharomyces cerevisae (See VMA; 3) and related species including Candida tropicalis (Ctr VMA; 4). The See VMA and Ctr VMA allelic inteins are of similar length (454 and 471 amino acids, respectively) and are 37% identical. Both have an endonuclease domain in addition to the splicing domains. The endonuclease of See VMA has been shown to cleave unoccupied target sites in the intein-less VMA genes during meiosis, resulting in 'homing' of the intein gene to the previously unoccupied allele.

The applicant has discovered the only other described nuclear intein allele. It is within the PRP8 gene of the basidiomycete Cryptococcus neoformans (see published PCT application WO02/095036), the contents of which are fully incorporated herein by reference. The applicant has also discovered homologous inteins in the PRP8 genes of the ascomycetes Aspergillus nidulans, Aspergillus fumigatus Neosartorya fischeri, and Histoplasma capsulatum as well as in the basidiomycete Cryptococcus laurentii.

The PRP8 gene product is one of the most highly conserved proteins known (Luo et al RNA, 5, 893-908 (1991), comprising the core of the spliceosome. The PRP8 gene product is an indispensable component of the spliceosome and therefore essential for cell viability. Loss of PRP8 function would result in an inability to process introns from all mRNA transcripts. PRP8 is needed in very large amounts during rapid growth. Every intron in every message has to be removed before the mRNA can be active.

Whereas the intein in the Cryptococcus neoformans PRP8 gene consists only of the splicing structure (a mini-intein), the allelic Aspergillus, Neosartorya, Histoplasma and C. laurentii inteins also contain a DNA cutting function ('homing' endonuclease). The homing endonuclease, HEG, enables the intein to move from its initial position to a comparable 'empty' allelic site. This movement is dependent on the highly specific sequence recognition of the homing endonuclease which recognises a unique site in the genome.

Homing endonuclease genes (HEGs) can spread through populations or 'gene pools' owing to their biased 'super-Mendelian' inheritance. HEGs are found at unique sites in the genome. They encode an enzyme that recognizes and cuts a 20-30 bp DNA sequence representing an 'empty' allelic site (an allelic site not containing a copy of the HEG). The HEG produces a double strand break in the DNA molecule at the target site. The broken HEG^" chromosome will be repaired by the cell's recombinational repair system, which uses the intact HEG⁺ containing allele as a template. As a result of the action of the HEG the HEG-encoding allele is copied into the empty allelic site. This process is an example of what is known as gene conversion. After repair, both chromosomes will contain a copy of the HEG, and a heterozygote will have been converted into a homozygote. The HEG found in the VMA gene of Saccharomyces acts during meiosis. In this system a diploid cell heterozygous for the HEG produces haploid meiotic products all of which carry the HEG. Thus, the biased meiotic inheritance arises from a combination of a sequence-specific endonuclease and the cell's own recombinational repair pathway. A recent paper by Burt Proc. Roy. Soc Lord. B 270,921-928 (2003) and the review by Morton New Scientist 2387, 30-33 (2003) explain a theoretical system for using homing endonucleases as bio-control elements. The critical element missing from the Burt proposal is a candidate homing endonuclease gene (HEG) having the desired properties. Burt says 'if such genes can be engineered to target new host sequences then they can be used to manipulate natural populations... '.

The intein in the PRP8 gene of Aspergillus, Neosartorya, Histoplasma and C. laurentii includes a homing endonuclease which has all the features needed for a bio-control element.

Inteins have been identified as promising antimicrobial targets (US 5,795,731 incorporated herein by reference). To be useful as a target an intein needs to be present in most or all strains of the microbe being targeted, and in a microbe which is of significant pathogenic concern. Eukaryotic inteins are also particularly useful because this group is as yet poorly characterised.

Polynucleotides encoding eukaryotic microbial inteins are also useful in assays for detecting the microbes in samples, especially clinical samples. It is an object of one aspect of the present invention to provide an assay for the detection and/or quantitation of pathogenic Aspergillus, Neosartorya, Histoplasma and C laurentii species.

DNA encoding inteins is potentially useful in biocontrol agents, exploiting the homing endonucleases that they encode.

It is therefore an object of this invention to provide an intein and/or a polynucleotide which goes at least some way towards meeting some or all of these requirements, or at least provides the public with a useful choice. DISCLOSURE OF THE INVENTION

The inventors have now unexpectedly identified eukaryotic inteins present in the ascomycetes Aspergillus nidulans, Aspergillus fumigatus Neosartorya fischeri, and Histoplasma capsulatum as well as in the basidiomycete Cryptococcus laurentii.. It is towards these and the polynucleotides encoding these and their uses that the present invention is broadly directed.

Accordingly, in a first aspect the present invention provides an intein derived from the PRP8 gene obtainable from the organism from the genus Aspergillus or the genus Histoplasma, or the genus Neosartorya, or the species Cryptococcus laurentii or a functionally equivalent, or functionally altered, fragment or variant thereof.

In a preferred embodiment, the intein is derived from the PRP8 gene of a member of the genus Aspergillus such as Aspergillus nidulans or Aspergillus fumigatus.

In a further preferred embodiment, the intein is derived from the PRP8 gene of Histoplasma capsulatum.

In a further preferred embodiment, the intein is derived from the PRP8 gene of. Aspergillus fumigatus

In a further preferred embodiment, the intein is derived from the PRP8 gene of Neosartorya fischeri.

In a further preferred embodiment, the intein is derived from the PRP8 gene of Cryptococcus laurentii. The intein comprises in a preferred aspect an amino acid sequence selected from the group consisting of the intein sequences shown in Figure 1, Figure 2, Figure 3, Figure 4 and Figure

5..

Preferably, the intein has the amino acid sequence set forth in Figure 1, Figure 2, Figure 3, Figure 4 and Figure 5 or is a functionally equivalent variant or fragment thereof.

The invention also provides an isolated intein which has an amino acid sequence which has greater than about 35%, preferably greater than about 50% identity with the sequence of SEQ ID NO:l, preferably greater than about 60%, more preferably greater than about 70%, more preferably greater than about 80%, more preferably greater than about 90%, and even more preferably greater than about 95% identity with the sequence an intein shown in Figure 1,

Figure 2, Figure 3, Figure 4 and Figure 5.

In a further aspect, the present invention provides an isolated nucleic acid molecule encoding an intein of the invention.

In a still further aspect, the present invention provides an isolated nucleic acid molecule comprising the intein nucleotide sequence set forth in Figure 6 Figure 7, Figure 8, Figure 9 or Figure 10 or fragments or variants thereof, which encode an intein of the invention, or a fragment or variant with a biological activity of the intein.

The invention also provides an isolated nucleic acid molecule which comprises a sequence encoding an intein selected from the group consisting of the intein sequences of Figure 6 Figure 7, Figure 8, Figure 9 and Figure 10, or a fragment or variant with a biological activity of an intein.

The nucleic acid molecules can be RNA or cDNA, but are preferably DNA molecules. Preferred molecules include those also including PRP8 flanking sequences.

Still further, the invention provides a vector or construct, which includes a nucleic acid molecule of the invention or a fragment or variant thereof as defined above.

Hosts transformed with a vector of the invention and capable of expressing an intein of the invention are also provided.

The invention further comprises a transgenic organism which includes a nucleic acid molecule of the invention and which is capable of expressing an intein of the invention.

The invention further comprises a transgenic organism which includes a nucleic acid molecule of the invention encoding a sequence selected from the group comprising the intein sequences of Figure 1, Figure 2, Figure 3, Figure 4 and Figure 5, or a fragment or variant with a biological activity of the intein. In still another aspect, the present invention provides an intein or intein construct for use in medicine.

Preferably, the use is as a target for testing agents for antimicrobial activity.

The invention also provides a composition comprising an intein of the invention.

The invention in a further aspect provides a protein including an intein of the invention.

In one embodiment, the protein comprises an intein of the invention flanked by N- and C- terminal exteins.

Preferably, the N- and C-terminal exteins comprise proximal and distal extein reporter portions which together form a reporter protein.

In an alternate embodiment, the protein comprises a binding protein portion, an intein of the invention, and a reporter protein portion.

Preferably, the intein separates the binding protein portion and the reporter protein portion.

The reporter protein may be selected from an enzymatic assay protein, a protein conferring antibiotic resistance, a protein providing a direct colorimetric assay, or a protein assayable by in vivo activity.

Preferably, the reporter protein is selected from the group consisting of: thymidylate synthase, β-galactosidase, orotic acid decarboxylase, galactokinase, alkaline phosphatase, β-lactamase, luciferase, and green fluorescent protein.

In a further aspect, the invention provides a method for producing a protein, the method comprising subjecting a protein containing an intein of the invention to cleavage conditions.

In one embodiment, the protein is a fusion protein.

The invention also provides an isolated nucleic acid molecule which encodes a protein of the invention. The invention also provides a method for screening an agent for antimicrobial activity against a microorganism, the microorganism having an intein of the invention in a gene encoding a protein which facilitates growth of the microorganism, the method comprising detecting inhibition of said intein, which comprises:

(a) preparing recombinant clones of an inducible expression vector containing: (i) an altered reporter gene comprising a silent restriction site within a reporter gene, and (ii) said intein;

(b) detecting production of extein product of said intein by said recombinant clones in the presence of said agent;

wherein reduced production of said extein product indicates inhibition of said intein, and antimicrobial activity of said agent against said microorganism.

In a further aspect, the invention provides a method for screening an agent for antimicrobial activity against a microorganism, the microorganism having an intein of the invention in a gene encoding a protein which facilitates growth of said microorganism, the method comprising detecting inhibition of said intein by monitoring intein function, which comprises:

(a) creating a silent restriction site within a reporter gene which results in an altered reporter gene;

(b) cloning said altered reporter gene into an inducible expression vector;

(c) cloning said intein into said inducible expression vector containing said altered reporter gene to generate recombinant clones; and (d) detecting the production of extein product of said intein by said recombinant clones in the presence of said agent;

wherein reduced production of said extein product indicates inhibition of said intein, and antimicrobial activity of said agent against said microorganism.

In these methods the inteins may further comprise an additional conserved distal amino acid residue selected from cysteine, serine and threonine.

The microorganism may be selected from a broad range of microbial pathogens, yeasts and bacteria. Preferably, the microorganism is selected from the group consisting of Aspergillus, Histoplasma, E. coli and Saccharomyces species.

Preferably, the protein is a PRP8

A preferred reporter gene is β-galactosidase. A preferred inducible expression vector is pUC19. Another preferred inducible expression vector is pTYB2.

Detection of extein production is conveniently achieved by phenotype characterisation.

In a further aspect, the invention provides a method or the detection and/or assay of an Aspergillus, or Histoplasma organism in a sample, especially a clinical sample, comprising detecting or measurement of a polynucleotide in the sample encoding the intein of the PRP8 gene of the organism.

In a preferred embodiment of the invention, DNA of the sample is contacted with a polynucleotide that binds to the DNA encoding the intein and the binding is detected and/or measured. The polynucleotide may alternatively or additionally bind to DNA complementary to the DNA encoding the intein.

In a further preferred embodiment the DNA encoding the intein in the sample is amplified.

Preferably the DNA to be detected and/or amplified comprises at least a part of the DNA encoding the intein of the PRP8 intein of the organism and a portion of the adjacent PRP8 sequence.

Preferably the DNA detected is the whole intein sequence plus a portion of the immediately adjacent flanking sequences of PRP8.

Alternatively, the DNA detected is a portion of the intein sequence plus a portion of the immediately adjacent flanking sequence of PRP8.

Alternatively, the DNA detected is a portion of the intein sequence. Currently the most preferred method is PCR.

PCR methods are well known by those skilled in the art (Mullis et al., Eds 1994 The Polymerase Chain Reaction, Birkhauser). The template for amplification may be selected from genomic DNA, mRNA or first strand cDNA derived from a sample obtained from the sample under test (Sambrook et al., 1989 Molecular Cloning - A Laboratory Manual, Cold Spring Harbour Laboratory, Cold Spring Harbour NY). The LightCycler (Roche Molecular Biochemicals, Mannheim, Germany) technology uses PCR and is capable of detecting and quantifying specific fungal DNA simultaneously.

The amplified DNA may be detected by any one of a number of techniques for example by gel electrophoresis or using fluorescently labelled probes. In real-time PCR protocols, such as the LightCycler, the PCR process is monitored either by fluorescence quantification of the DNA-binding dye SYBR Green I for the general detection of double-stranded DNA or by hybridization probes. LightCycler hybridization probes consist of a pair of oligonucleotides annealing next to each other on a given nucleic acid sequence and labeled with two different fluorescent dyes on their 3' and 5' ends, respectively. In the presence of the specific target sequence, the probes hybridize head-to-tail, bringing the two fluorescent dyes into close physical proximity. When brought into proximity the fluorescent dyes can interact. The fluorescence intensity is directly proportional to the amount of specific target sequence present in the amplification mixture and measured during each PCR cycle.

Real time PCR is capable of providing sequence confirmation for the amplified product by a simple 'melting curve analysis'. A melting curve analysis relies on the fact that a double- stranded DNA (such as a PCR product and the associated, labelled fluorescent probes) will denature or disassociate at a specific sharply defined temperature. This temperature is determined by the length of the duplex, the base composition of the duplex and the presence of any mismatched bases. In a melting curve analysis, after the PCR process is completed, the temperature in the thermal chamber is slowly increased. When one of the probes melts off and the two fluorescent dyes are no longer in close proximity, fluorescence decreases and a characteristic melting point is observed for the given target sequence. Similar, single hybridisation probes are used in other 'real-time' PCR protocols, such as the TaqMan system of Applied Biosystems.

Real time PCR is capable of providing quantification of the infection by measuring the number of cycles needed to produce the amplified product. A low level infection will result in a low concentration of template in the clinical sample and this will require more cycles of amplification to achieve a given level of duplex product.

Real time PCR is capable of discriminating species of Aspergillus and related fungi by analysing the amplified product by a simple 'melting curve analysis'. A melting curve analysis relies on the fact that a double-stranded DNA (such as a PCR product and the associated, labelled fluorescent probes) will denature or disassociate at a specific sharply defined temperature. This temperature is determined by the length of the duplex, the base composition of the duplex and the presence of any mismatched bases. Every mismatch present in the duplex formed by the PCR product and the hybridization probe will lower the observed melting point. It is possible to design hybridisation probes which will give distinct melting curves for various species. These probes can be designed on the basis of the DNA sequences which we have described for Aspergillus, and Histoplasma.

In another aspect the invention comprises a method of diagnosing a microbial infection using a method as described above.

The PRP8 intein is thus ideal in that it has conserved flanking sequences for primer design, these flanking regions are not too far apart for PCR amplification and the presence of the intein is rare in fungi.

The conserved sequence of the PRP8 and intein means that it is possible to design a PCR assay using specific primers (the flanking sequences from which the amplification process is initiated). Preferred primer pairs have one member that binds to the junction of the PRP8 gene and the start of the intein and one member which binds to the end of the intein and the adjacent PRP8 sequence. That is to say, prefeoed primer pairs have one member that binds to the PRP8 gene upstream from the intein and to the start of the intein and a second member which binds to the end of the intein and the PRP8 gene downstream of the intein. Prefeoed primers have a length of 15-25 nucleotides, preferably 20 nucleotides.

Prefeoed primers have a similar composition, preferably approximately 50% guanine and cytosine.

Prefeoed primers may be equimolar mixtures of several very similar molecules (redundancy) so as to provide perfect complementarity to slightly divergent fungal sequences.

In another aspect, the prefeoed primers may consist of one which is complementary to a conserved internal region of the intein and another which binds to the junction of the PRP8 gene and the intein. This alternative would allow the amplification of a smaller PCR product

(~500bp) which would be suitable for real-time PCR analyses.

In another aspect, the prefeoed primers may consist of primers complementary to a conserved internal region of the intein. This alternative would allow the amplification of a smaller PCR product (~500bp) which would be suitable for real-time PCR analyses.

A prefeoed primer pair comprises:

FW_Aspdiag 5' _gA[CT]Ag[CT]gA[CT]gg[ACgT]TggTA 3' and Aspdiag_RV 5' TC[Ag]AA[ACgT]cc[AgJCT[Ag]TT[Ag]Tg 3' where [ct] indicates redundancy, equimolar amounts of two oligonucleotides one with c and the other with t at that position, [ ag] redundancy indicates either an a or a g at that position.

[AgCT] indicates equimolar amounts of all 4 alternatives. In this prefeoed pair FW Aspdiag is complementary to a highly conserved endonuclease-encoding region. Aspdiag_RV is complementary to the junction between the end of the intein and the adjacent PRP8 gene. The

PCR product of these primers is ~500bp, a size suitable for real-time PCR. The 500bp sequence contains many species-specific nucleotides.

In one embodiment sequences specific to a particular organism are used. For example a 202- residue insertion is specific to A. fumigatus intein Afu PRP8 may be used as a target for probes and primers in detection and assay of that organism.

In a further aspect the invention provides a diagnostic kit which can be used for diagnosis of an Aspergillus, or Histoplasma infection comprising the step of detecting the PRP8 intein. One kit includes a set of primers of the invention and reagents useful for amplifying DNA. Such a kit is useful for detecting the presence of the organism in a sample from an infected patient.

In a further aspect the invention provides a kit which can be used for quantification of an Aspergillus, or Histoplasma infection in a real time PCR system. One kit includes a set of primers of the invention and reagents useful for amplifying DNA. Such a kit is useful for assessing the effectiveness of an antibiotic treatment against an infection with these fungi in a patient.

The intein in the PRP8 gene of Aspergillus & Histoplasma includes a homing endonuclease which has all the features needed for a bio-control element. Specifically, a HEG-containing PRP8 intein has been detected in Aspergillus nidulans, A. fumigatus and Histoplamsa capsulatum. Additionally HEG-containing PRP8 inteins have been detected in Neosartorya fischeri strain FRR0181, a close relative of A. fumigatus. Additionally a HEG-containing intein has been detected in Cryptococcus laurentii. These inteins are allelic with the Cryptococcus PRP8 mini-inteins (published PCT application WO 02/095036). The applicant has cloned and sequenced the Aspergillus, Neosartorya and C laurentii inteins. The applicant has partially characterised this intein (for example it has been demonstrated that the intein splices in E. coli).

The insertion site of the intein is a very conserved site in the PRP8 and the HEGs are typically a bit imprecise when it comes to recognising cognate target DNA so we expect the HEG will cut the PRP8 sequences of many organisms.

The HEG (not the whole intein) can be put behind a suitable promoter, the composite flanked on both sides by pest PRP8 target sequences. The construct will be injected into a pest, for example, into the eggs of an insect.

The HEG should cut the pest chromosomal PRP8 and trigger a double strand break DNA repair pathway. This repair should replace the genomic PRP8 with the HEG construct. That is the empty site will be converted into an HEG-containing site. If a late meiotic promoter is used then a heterozygous cell will produce only HEG gametes. This HEG-containing allele will initially spread through the population represented by heterozygotes showing biased meiotic products. Once the heterozygotes reach a certain proportion, lethal homozygotes will occur at increasing frequency followed by the collapse of the population. PRP8 is used because there are only two described nuclear HEGs in eukaryotes which recognise coding sequences. These are the PRP8 and the VMA intein. Only the PRP8 is a suitable conserved essential target. An alignment of the PRP8 target sequences of diverse eukaryotes is given in Figure 11.

The mammalian sequences (mouse, human) are very similar and suggest that all mammals, including marsupials such as the pest species the possum will have similar sequence.

The insect sequences are also very similar and suggest that other insect pest species will be similar in sequence.

The fish and toad sequences are also very similar and serve as an indication of the sequence of pest species such as the cane toad and carp.

The HEG-containing allele will spread only meiotically within a species. The PRP8 HEG allele cannot get out of the target species, it is not infectious. However within a target species it could double in each generation (without any assistance) reaching xl million in 20 generations .The system may be used to control any eukaryote, including for example, the malarial mosquito Anopheles and the possum.

Therefore the invention also provides a method for controlling a pest species comprising (a) inserting a HEG into the DNA of a pest; and (b) allowing the organism to mate with the wild pest population. Preferably the HEG is put behind a suitable promoter, the composite being flanked on both sides by pest PRP8 target sequences.

Preferably the HEG is from the intein in the PRP8 gene of Aspergillus, Neosartorya, Histoplasma or Cryptococcus laurentii. The intein of the invention can include its entire amino acid sequence or can include only parts of that sequence where such parts constitute active fragments. Such activity will be as an intein. One important group of fragments have homing endonuclease activity.

The invention also provides a transgenic organism comprising a recombinant HEG. Preferably the prefeoed HEG described above is included.

Extended inteins which include a conserved residue distal to the intein are also provided. The conserved residue immediately distal to the intein may be selected from the group consisting of cysteine, serine and threonine.

One extended intein for use in the invention has the amino acid sequence of the shaded portion of Figure 1 - the sequence of the PRP8 intein of Aspergillus nidulans with an additional alanine at the N-terminus and an additional serine at the C terminal.

As noted above, the invention also includes within its scope functionally equivalent variants of the intein of Figure 1, Figure 2, Figure 3, Figure 4, and Figure 5.

The phrase "functionally equivalent variants" recognises that it is possible to vary the amino acid of a protein while retaining substantially equivalent functionality. For example, a protein can be considered a functional equivalent of another protein for a specific function if the equivalent peptide is immunologically cross-reactive with and has at least substantially the same function as the original intein.

Functionally altered variants are also contemplated. It will be appreciated by the skilled reader that highly conserved sequences appear at the junction of inteins and exteins (see InBase http ://www. ineb.com/inteins) . Serine(S), Threonine(T) or Cysteine (C) occur at the N-terminal end, while Histidine (H) and Asparagine (N) occur at the C-terminal end of the intein. It is recognised in the art that mutation or deletion of these amino acid residues alters intein function for example to yield cleavage at one or both of the intein-extein junctions, or to result in an intein incapable of splicing or cleavage. (See Chong et al. (1998b) J. Biol. Chem. 273:10567-10577, and WO 01/12820). The term "functionally altered" as used herein includes all such inteins.

It will be appreciated by the skilled reader that highly conserved sequences appear in the homing endonuclease domain of the intein. Although these are not extensive they include the 'LAGLIDAG' motif which cooesponds to the active site of the HEG. Functionally altered variants of the HEG are also contemplated.

The functionally equivalent or altered protein need not be the same size as the original. The equivalent or altered protein can be, for example, a fragment of the protein, a fusion of the protein with another protein or carrier, or a fusion of a fragment with additional amino acids.

Active fragments may be obtained by deletion of one or more amino acid residues of full length intein. It is also possible to substitute amino acids in a sequence with equivalent amino acids using conventional techniques. Groups of amino acids normally held to be equivalent are: (a) Ala, Ser, Thr, Pro, Gly; (b) Asn, Asp, Glu, Gin; (c) His, Arg, Lys; (d) Met, Leu, He, Val; and (e) Phe, Tyr, Trp.

That equivalent may, for example, be a fragment of the intein containing, for example, from 550 to 604 amino acids, a substitution, addition, or deletion mutant of the intein, or a fusion of the protein or a fragment or a mutant with other amino acids.

Polypeptide sequences may be aligned, and percentage of identical amino acids in a specified region may be determined against another sequence, using computer algorithms that are publicly available. The similarity of polypeptide sequences may be examined using the BLASTP algorithm. BLASTP software is available on the NCBI anonymous FTP server (ftp://ncbi.nlm.nih.gov) under /blast/executables/. The use of the BLAST family of algorithms, including BLASTP, is described at NCBI's website at URL http://www.ncbi.nlm.nih.gov/BLAST/newblast.html and in the publication of Altschul, Stephen F., et al. (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-34023.

Accordingly, in a further aspect, the invention also provides an intein which includes one or more active peptides from within the amino acid sequence set forth in Figure 1, Figure 2, Figure 3, Figure 4, and Figure 5. .

A specific intein of the invention identified has the amino acid sequence of the shaded portion of Figure 1, Figure 2, Figure 3, Figure 4, and Figure 5.

or a functionally equivalent variant or fragment thereof.

Polypeptides of the invention also include homologous polypeptides having an amino acid sequence with about 35%, preferably about 50%, preferably at least 60%, more preferably at least 70% identity to the intein of the invention, preferably at least about 80% identity, more preferably at least about 90% identity, as well as those polypeptides having an amino acid sequence at least about 95% identical to the intein.

An intein of the invention together with its active fragments and other variants may be generated by synthetic or recombinant means. Synthetic polypeptides having fewer than about 100 amino acids, and generally fewer than about 50 amino acids, may be generated by techniques well known to those of ordinary skill in the art. For example, such peptides may be synthesised using any of the commercially available solid-phase techniques such as the Meoyfield solid phase synthesis method, where amino acids are sequentially added to a growing amino acid chain (see Meoyfield, J. Am. Chem. Soc 85: 2146-2149 (1963)). Equipment for automative synthesis of peptides is commercially available from suppliers such as Perkin Elmer/Applied Biosystems, Inc. and may be operated according to the manufacturers instructions.

Fragments may be obtained by deletion of one or more amino acid residues of the full length intein. This may be by stepwise deletion of amino acid residues, from the N- or C-terminal end of the intein, or from within the intein.

An intein of the invention, or a fragment or variant thereof, may also be produced recombinantly by inserting a polynucleotide (usually DNA) sequence that encodes the protein into an expression vector and expressing the protein in an appropriate host. Any of a variety of expression vectors known to those of ordinary skill in the art may be employed and include plasmids, pUC and pET series plasmids, particularly pUC19 and pTYB2. Expression may be achieved in any appropriate host cell that has been transformed or transfected with an expression vector containing a DNA molecule which encodes the recombinant protein. Suitable host cells include procaryotes, yeasts and higher eukaryotic cells. Preferably, the host cells employed are E. coli, yeasts or a mammalian cell line such as COS or CHO, or an insect cell line, such as SF9, using a baculovirus expression vector. E. coli and Saccharomyces species are particularly prefeoed. Aspergillus and Histoplasma species may of course also be used as host cells. The DNA sequence expressed in this manner may encode the naturally occuoing intein, fragments of the naturally occuoing protein or variants thereof.

Variants of the intein may also be prepared using standard mutagenesis techniques such as oligonucleotide-directed site specific mutagenesis. In a yet further aspect, the invention provides an isolated nucleic acid molecule encoding an intein of the invention.

A specific nucleic acid molecule of the invention includes the nucleotide sequence of Figure 6, Figure 7, Figure 8, Figure 9 and Figure lOor a fragment or variant thereof.

The invention also includes within its scope, homologues or variants of an isolated nucleic acid molecule encoding an intein of the invention. Specifically contemplated are allelic variants, which occur even though they have low (under 50%) percentage identity. Polynucleotide sequences may be aligned, and percentage of identical nucleotides in a specified region may be determined against another sequence, using computer algorithms that are publicly available.

Two exemplary algorithms for aligning and identifying the similarity of polynucleotide sequences are the BLASTN and FASTA algorithms. The BLASTN software is available on the NCBI anonymous FTP server (ftp://ncbi.nlm.nih.gov) under /blast/executables/. The BLASTN algorithm version 2.0.4 [Feb-24-1998], set to the default parameters described in the documentation and distributed with the algorithm, is prefeoed for use in the determination of variants according to the present invention. The use of the BLAST family of algorithms, including BLASTN, is described at NCBI's website at URL http://www.ncbi.nlm.nih.gov/BLAST/newblast.html and in the publication of Altschul, Stephen F, et al (1997). "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402. The computer algorithm FASTA is available on the Internet at the ftp site ftp://ftp.virginia.edu.pub/fasta/. Version 2.0u4, February 1996, set to the default parameters described in the documentation and distributed with the algorithm, is prefeoed for use in the determination of variants according to the present invention. The use of the FASTA algorithm is described in the W R Pearson and D.J. Lipman, "Improved Tools for Biological Sequence Analysis," Proc. Natl. Acad. Sci. USA 85:2444-2448 (1988) and W.R. Pearson, "Rapid and Sensitive Sequence Comparison with FASTP and FASTA," Methods in Enzymology 183:63-98 (1990).

All sequences identified as above qualify as "variants" as that term is used herein.

The invention also includes nucleic acid molecules or polynucleotides that comprise a polynucleotide sequence encoding an intein having at least about 35% identity, preferably at least about 50%, preferably at least about 60%, preferably at least about 70%identity, preferably at least about 85% identity, more preferably at least about 90% identity, as well as those polynucleotides having a nucleic acid sequence at least about 95%, 97%, 98%, or 99% identical to the intein nucleotide sequence of Figure 6, Figure 7, Figure 8, Figure 9 and Figure 10.

An intein, intein fragment or nucleic acid molecule of the invention may be generated by synthetic or recombinant means by techniques well known to those of ordinary skill in the art. Variants may be prepared using standard mutagenesis techniques or may be isolated from organisms.

Once obtained, the intein is readily purified if desired. This may involve affinity chromatography. Other approaches to purification (e.g. gel-filtration or anion exchange chromatography) can also be used. Where the intein or fragment is produced in the form of a fusion protein, the caoier portion of the fusion protein can prove useful in this regard.

Furthermore, if viewed as desirable, additional purification steps can be employed using approaches which are standard in the art. The approaches are fully able to deliver a highly pure preparation of the intein. Preferably, the intein preparation comprises at least about 50% by weight of the protein, preferably at least about 80%, preferably at least about 90%, and more preferably at least about 95% by weight of the protein.

The purification procedure will of course depend on the degree of purity required for the use to which the intein, fusion protein or fragment is to be put.

Once obtained, the intein and/or its fragments and/or its functionally equivalent variants can be formulated into a composition. The composition can be, for example, a therapeutic composition for application as a veterinary, pharmaceutical, or diagnostic composition. For these purposes it is generally prefeoed that the intein be present in a pure or substantially pure form. Again, standard approaches can be taken in formulating such compositions (see, for example, Remington's Pharmaceutical Sciences, 18^th Edition, Mack Publishing (1990)). The inteins and proteins and/or primers therefore of the present invention can also be included in assay kits. Polymerase chain reactions using appropriate primers flanking the intein or from within the intein may be used to diagnose infection. The kit may further include PCR primers, thermostable polymerase, deoxyribose triphosphates, and buffer. The assay carried out may be by RealTime PCR or by agarose gel electrophoresis detecting an amplified DNA band of the appropriate size. The PCR product could be subjected to DNA sequencing to identify specific types or strains.

In a further aspect, the invention also provides a process for producing a protein of interest, particularly fusion proteins comprising a protein of interest and an intein of the invention. The intein may, for example, be used in conjunction with an affinity group to purify a desired protein. Affinity fusion-based protein purification is taught, for example in Chong et al. (1997b) Gene 192: 271-281; Chong et al. (1998b) Nucl Acids Res. 26: 5109-5115, and WO 01/12820 all incorporated herein by reference. Where self-cleavage of the intein occurs, rather than splicing the desired protein is released without the need for protease addition, simplifying purification. Cleavage may be achieved using standard art protocols by blocking the later stages of intein splicing or using an intein mutant in one of the amino acids critical for completion of splicing. For example mutants lacking the conserved N-terminal and C- teoninal residues as discussed above. It is likely that conditions favouring cleavage of the PRP8 intein will differ from others such as the NMA intein, these points of difference may be usefully employed.

The strategies are usefully represented as follows: Splicing Ν_x - 1 - C_x where N_x and C_x are the proximal and distal exteins respectively and I is an intein of the invention, splice to form N_xC_x + I

Cleavage N_x - 1 - C_x cleave to form N_XI + C_x The N- and C-terminal flanking exteins may be from the same protein (cis splicing or cleavage), or from different proteins (trans splicing or cleavage).

In one embodiment, the N- and C-terminal proximal and distal exteins comprise the protein A. nidulans PRP8. The protein or fusion protein may as discussed above be produced by chemical synthesis, or recombinantly or according to other known art techniques. Preferably, the protein is produced recombinantly by preparing a vector containing nucleic acid sequences and/or DNA encoding the protein or fusion protein, transforming a host cell with the vector, and expressing the nucleic acid/DNA in the host cell. Vectors and host cells as discussed may be employed.

The protein produced may be purified as described above, using standard art techniques.

The invention also provides a method for purifying a protein of interest. The method comprises producing a fusion polypeptide comprising a binding protein portion, an intein of the invention and a protein of interest portion, binding the fusion polypeptide to a binding moiety, subjecting the intein to cleavage conditions, and separating the desired protein. Binding may comprise binding of the fusion polypeptide to an affinity matrix (e.g. beads, membranes, columns, or material in a column). Separation can include subjecting the matrix (e.g. column contents) to a chemical or physical change such as pH or temperature shift, and eluting the desired protein. Useful cleavage conditions are known in the art for example in WO 01/12820 incorporated herein by reference.

In the situation where the protein is synthesised as a protein of interest/intein/binding moiety fusion, and the binding moiety is recognised by and retained on a column, a one-step purification method is feasible. For example, if the protein of interest is fused to an intein with a distal chitin binding domain the tripartite protein may be bound to a chitin column in the presence of zinc. Elution of the column with EDTA will chelate the zinc and allow the intein to cleave from the protein of interest. The intein can be modified so as to prevent the splicing reaction. The result is that the protein of interests elutes from the column while the intein and chitin binding domain remain. Tripartite fusions and purification methods are discussed in WO 01/12820 above. Cleavage is also achieved as described above.

In one embodiment, the protein of interest portion is a reporter protein portion. The intein may separate the binding protein portion and the reporter protein portion, or protein of interest portion. The binding portion may for example be maltose binding protein of E. coli or a His-tag.

Accordingly, in a further aspect, the invention also provides a protein including an intein of the invention. Prefeoed proteins include those with N- and C-terminal proximal and distal exteins, as well as binding protein/intein/reporter protein fusion proteins as discussed above. It will also be appreciated that in some cases the protein is a precursor protein produced prior to intein splicing or cleavage.

The invention further provides isolated nucleic acid molecules which encode the proteins of the invention. These nucleic acid molecules may be produced according to the methods discussed above.

As noted above, the invention also has application in screening agents for antimicrobial activity against a microorganism. In this method, an intein of the invention is present in a gene encoding a protein which facilitates growth of a microorganism. The microorganism may be selected from a broad range of microbial pathogens such as Candida and Aspergillus; yeasts such as Saccharomyces; and bacteria such as E. coli. Preferably, the microorganism is selected from the group consisting of A. fumigatus, E. coli, and Saccharomyces species. E. coli is particularly useful to facilitate initial screening. Screening requires the preparation of an inducible expression vector containing an altered reporter gene which contains a silent restriction site therein, and the intein of the invention therein. The vector is expressed in a host cell. Production of extein product of the intein is detected and/or measured in the presence of an agent of interest.

A reduction in the amount of extein produced indicates that the intein has been inhibited, and that the agent has inhibitory activity against the intein. From this it may be reasonably infeoed that the agent may inhibit the growth of a microorganism incorporating the intein, particularly natural pathogens. The agent tested may be employed in the screening methods of the invention at varying concentrations. In this way the most effective concentrations of the agent can also be determined.

In one embodiment, the invention relates to a genetic system to monitor intein function based on the cloning of the Aspergillus fumigatus PRP8 intein into the α peptide of the β- galactosidase of E.coli. This may conveniently be in a plasmid such as pUC19. The uninterrupted α peptide, which encodes the amino fragment of the β-galactosidase, has been developed as a cloning reporter gene. The uninterrupted α-peptide gene can complement E. coli cells deficient in β-galactosidase for growth on medium with lactose as a sole carbon source (minimal Medium + lactose), confeoing a Lac+phenotype. In contrast, if sequences are inserted within the α peptide coding sequence they may interfere with translation or the activity of the peptide. In-frame fusions of the PRP8 intein will be made to the α peptide exteins. If bacteria carrying this construct are Lac+ this indicates the α peptide sequence is active, the activity of the enzyme indicating that the intervening protein sequence (the intein) has been excised from the precursor protein.

Similarly, a wide range of reporter genes/proteins may be employed in the proteins, protein preparation, protein purification and screening methods of the present invention. Prefeoed reporter genes/proteins are easily assayable either in vivo or in vitro, or both, and include but are not limited to β-galactosidase, galactokinase, luciferase, alkaline phosphatase (for enzymatic assays), β-lactamase (a reporter conferring antibiotic resistance), orotic acid decarboxylase and green fluorescent protein, a reporter useful in direct colorimetric assays.

β-galactosidase is particularly prefeoed for use. The presence of extein is readily measured by spectrophotometric assays using this enzyme. Alternatively it can be assayed in vivo by liquid growth assay of bacteria in minimal medium + lactase (turbidity) or on petri plates (using various synthetic galactosides).

Detection of extein product may be achieved by standard analytical methods such as phenotype characterisation, protein characterisation, for example by amino terminal sequence mapping of tryptic peptides and mass spectroscopy enzyme assays, and colorimetric methods all of which are well known to those versed in the art.

As will be appreciated using this method a precursor protein is synthesized comprising exteins interrupted by an intein. Protein splicing then results in intein excision, and extein ligation, which restores the uninterrupted reading frame to the intein-containing protein. Highly conserved sequences appear at the junction of the inteins and the exteins. Ser (S), Thr (T) or Cys (C) occur at the N-terminal end while His (H) and Asn (N) occur at the C-terminal end of the intein. In addition there is a highly conserved extein residue immediately adjacent to the C-terminal Asn of the intein, either a Cys, Thr or Ser. The presence of these conserved amino-acids is employed in the detection of inteins in genomic sequence. An intein presents as an in phase insertion in the coding sequence of a gene, there should be homologues of the gene which lack the intein, the intein will display the characteristic N-terminal and C-terminal amino acids and there will be some degree of protein sequence homology to other inteins.

The invention will now be described more fully in the following experimental section, which is provided for illustrative purposes only.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the PRP8 amino acid sequence of Aspergillus nidulans. The shaded area cooesponds to the intein.

Figure 2 shows the PRP8 amino acid sequence of Aspergillus fumigatus. The shaded area cooesponds to the intein.

Figure 3 shows the PRP8 amino acid sequence of Histoplasma capsulatum. The shaded area cooesponds to the intein.

Figure 4 shows the PRP8 intein amino acid sequence of Neosartorya fischeri.

Figure 5 shows the PRP8 intein amino acid sequence of Cryptococcus laurentii.

Figure 6 shows the nucleotide sequence encoding PRP8 of Aspergillus nidulans. The shaded area cooesponds to the intein.

Figure 7 shows the nucleotide sequence encoding PRP8 of Aspergillus fumigatus. The shaded area cooesponds to the intein. The uppercase area cooesponds to an intron.

Figure 8 shows the nucleotide sequence encoding PRP8 of Histoplasma capsulatum. The shaded area cooesponds to the intein. The uppercase area cooesponds to an intron. Figure 9 shows the nucleotide sequence encoding the PRP8 intein of Neosartorya fischeri.

Figure 10 shows the nucleotide sequence encoding the PRP8 intein of Cryptococcus laurentii.

Figure 11 shows an alignment of PRP8 sequences in diverse eukaryotes.

Figure 12 shows the sequence for the 648 bp insertion of A fumigatus.

EXAMPLE 1

Identification of Inteins in Aspergillus and Histoplasma.

The ascomycetous fungus Aspergillus nidulans is one of the simplest multicellular eukaryotes. Its estimated 31-megabase genome with low repetitive DNA content, distributed among eight chromosomes, is sufficiently complex to direct multicellular development. With its sophisticated forward and reverse genetics, A. nidulans has been used to address fundamental questions in cell and molecular biology and has become a model system for the study of pathogenic and biotechnologically useful relatives.

Methods

Aspergillus. Preliminary sequence data for Aspergillus fumigatus were obtained from The

Institute for Genomic Research website at http://www.tigr.org. Aspergillus nidulans data were available at the Aspergillus nidulans Sequencing Project; Whitehead Institute/MIT Center for Genome Research (http://www-genome.wi.mit.edu).

Histoplasma capsulatum. Genomic sequence data were from the Genome Sequencing Center at Washington University in St. Louis (http://www.genome.wustl.edu/projects/hcapsulatum/). Two distinct strains of H. capsulatum, G217B and G186AR, are being sequenced.

Isolates of Neosartorya were obtained from Food Science Australia (http://www.foodscience.afisc.csiro.au/fcc/). Isolate CBS 139 of Cryptococcus laurentii was obtained from the CBS (Utrecht). The strain of Aspergillus nidulans used in this analysis (R20) was obtained from the University of Leicester, United Kingdom. Strains were grown at 27°C or 37 °C in ANA medium (0.1% Difco Yeast extract, 0.1% peptone, 0.2% glucose, 6g/L NaNO₃, 0.52g/L MgSO₄, 0.52g/LKCl, 1 crystal each of FeSO₄ and ZnSO₄, lmg/L biotin; solidified with 1.5% agar when necessary).

Genomic DNA was isolated from 50ml overnight cultures essentially via the method of Muller et al., (1998, J Clin Microbiol. 36(6): 1625-9). Amplification of the intein sequence and flanking regions was accomplished with the Expand High Fidelity PCR system (Roche, Mannheim, Germany) as outlined in Butler et al., Yeast 18, 1365-70(2001). Primers were synthesised by Proligo, Singapore. The resulting PCR products were purified with Qiagen columns (Hilden, Germany) prior to automatic sequencing at the Centre for Gene Research at Otago University (http://microbes.otago.ac.nz/cgr/home.htm) using an ABI 377 DNA Sequencer.

General sequence analyses were done using the Wisconsin GCG package (Genetics Computer Group, 575 Science Drive, Madison, Wis.) and the Australian National Genomic Information Service node located at the University of Otago (http://angis.otago.ac.nz ). Sequence similarity searches were performed using the National Center for Biotechnology Information BLAST server (http://www.ncbi.nlm.nih.gov/BLAST and at the various fungal genome sequencing project web sites (see below). Multiple sequence alignments were constructed using CLUSTAL_X at the European Bioinformatics Institute server (http://www.ebi.ac.uk/clustalw/) edited with Seaview (Galtier et al, 1996) and shaded with MacBoxshade (http://www.isrec.isb-sib.ch/sib-isrec/boxshade/macBoxshade). Phylogenetic trees were constructed using PAUP4M0 (Swofford 1998).

Results

We have previously analysed an immobile mini-intein in two pathogenic species of

Cryptococcus (C neoformans and C. bacillisporus). We then used PCR and degenerate primers designed to anneal to various regions of the highly conserved PRP8 gene to screen other, closely related, basidiomycete species for the presence of an intein in PRP8. Some of these species were drawn from the Tremellales group of basidiomycetes which includes Cryptococcus neoformans and C. bacillisporus (Fell et al., Int JSyst Evol 50,1351-71(2000)). None of these species, including C. amylolentus (also known as Filobasidiella amylolentus), the species most closely related to C neoformans (Fell et al.,- 2000), contained an intein in PRP8. Unexpectedly, we detected a PCR product from C laurentii (CBS 139) which was considerably larger than that found for C. neoformans or C. bacillisporus. We sequenced this PCR product directly with several specifically designed primers. The conceptual translation of these aligned sequences yielded an open reading frame of 1566bp encoding not only sequences similar to the splicing domains of CnePRPδ and CbaPRP8 but also internal sequences cooesponding to a homing endonuclease of the LAGLIDADG type

We then looked for the presence of the intein in other fungi using the available genome sequence data. There are no inteins in the PRP8 of the basidiomycetes Phanerochaete chrysosporium, Ustilago maydis or Coprinopsis cinerea; these are three of the few basidiomycetes other than Cryptococcus neoformans for which significant amounts of sequence data are available.

Our search of the PRP8 gene in other eukaryotes revealed a HEG-containing intein to be present in the PRP8 of five ascomycete fungi: Aspergillus nidulans, Aspergillus fumigatus, Neosartorya fischeri and Histoplasma capsulatum. All three inteins occur at the same site within the PRP8 gene as the Cryptococcus PRP8 inteins. All three inteins, however, have internal sequences suggestive of an endonuclease of the DOD type found in most other full- length inteins (and in some mobile introns). The distant relationship between Aspergillus and Histoplasma (the species are from different orders) suggests a widespread occuoence and an ancient origin of the intein in the euascomycetes, although Giberella zeae (anamorph: Fusarium graminearum), Neurospora crassa and Coccidiodes posadasii do not have an intein in PRP8. An intein is not present in the PRP8 gene of any species from either of the other ascomycete classes, hemiascomycetes (for example, Candida albicans, Saccharomyces cerevisiae) or archiascomycetes (Schizosaccharomyces pombe), for which PRP8 sequence data are available. There is also no intein present in the highly conserved PRP8 of any vertebrate or insect for which significant sequence information is available in the public databases (October, 2003).

The phylogeny of the PRP8 proteins of these species follows the expected host phylogeny. The absence of an intein gene in the PRP8 gene of species closely related to Cryptococcus neoformans and C. bacillisporus is, however, puzzling. The presence of an allelic EN- containing intein in the PRP8 gene of Aspergillus nidulans, A. fumigatus and Histoplasma capsulatum, all distantly related to Cryptococcus, suggests that horizontal gene transfer from this ascomycete group may be the source of the Cryptococcus intein. Finding very closely related elements in relatively distantly related hosts has previously been taken as evidence of horizontal transfer.

EXAMPLE 2 In-frame fusions of the CnePRPδ intein were made to 'α peptide exteins' caoied by expression plasmids such as pTYB2 or pUC19 (AmpR) and derivatives by restriction and ligation. The intein DNA was generated by polymerase chain reaction and plasmid DNA was obtained commercially from New England Biolabs, USA. These constructs were transformed into E.coli DH5αLac- on Ampicillin selective medium. The transformed cells were able to grow on this medium as they had acquired ampicillin resistance. Plasmids were isolated from these cultures and sequenced. It was confirmed that they caoied a plasmid in which intein sequences from Aspergillus nidulans had been inserted into the plasmid at the intended site in the expected in-frame orientation. The colonies were blue in the presence of X-gal.

The blue colour indicates α peptide complementation of β-galactosidase activity. The activity of this enzyme in bacteria containing the plasmid with the cloned Aspergillus intein indicates that the intervening protein sequence (the intein) has been excised from the precursor protein leaving a functional α peptide. This indicates the intein is active in this bacterial host and at this site in the α peptide coding sequence. If the intein function was blocked the cultures would grow white on X-gal.

EXAMPLE 3

The intein peptide reporter construct may be grown in the presence and absence of an agent (usually a drug to be tested) at one, or varying concentrations of the drug and their phenotypes are scored at different drug concentrations. A drug might be for example zinc salts. Mills and Paulus (2001, Reversible inhibition of protein splicing by zinc ion. J Biol Chem. 276(14): 10832-8) taking advantage of recently developed in vitro systems in which protein splicing occurs in trans (N- and C-exteins from different proteins) to assay for protein-splicing inhibitors, discovered that low concentrations of Zn(2+) inhibited splicing mediated both by the RecA intein from Mycobacterium tuberculosis and by the naturally split DnaE intein from Synechocystis sp. PCC6803. 90% inhibition was achieved by 0.2mM zinc (6parts/million), a relatively low concentration. The inhibition of intein processing by zinc salts in vivo could be tested by inoculating each of two strains of E. coli into a series of broths. One strain of E. coli would carry the plasmid (into which the Aspergillus intein had been cooectly configured) and as a control the other strain of E. coli would carry the original plasmid without the intein. Zinc salts would be added to the broths at various concentrations (0/0.2/2/20 inM). Intein inhibition would be infeoed if the growth of the intein-caoying cultures was inhibited at a zinc concentration that failed to inhibit the control culture.

EXAMPLE 4

The polymerase chain reaction (PCR) is the present method of choice for many molecularly- based diagnostic systems. In this technique, a small region of DNA is preferentially amplified in vitro from a preparation of DNA extracted from the whole infecting organism or clinical sample. For diagnosis especially of fungi, there is often difficulty in finding an appropriate, species-specific DNA target to amplify; most PCR applications so far rely on conserved sequences that are similar in many species and this can lead to false positive results. The conserved sequence of the PRP8 and intein means that it is possible to design a PCR assay using specific primers (the flanking sequences from which the amplification process is initiated).

We have isolated DNA from Aspergillus nidulans and subjected it to PCR using primers designed to the regions flanking the PRP8 intein and also to internal regions of the PRP8 intein. An example of such a primer pair is: aspnid Nterm (5' ctgggagaaagcttgtcttgc 3') and aspnid_spl (5' cgatgaaggaccaggatatgg 3')

The sequence of these primers perfectly match the A. nidulans sequence and provide a PCR product of ~223bp. As described elsewhere in this application, degenerate primers may be used to amplify targets from divergent strains of the same species, the same genus or related genera carrying the intein. The example given above of such redundant primers is: FW_Aspdiag 5' _gA[CT]Ag[CT]gA[CT]gg[ACgT]TggTA 3' and Aspdiag_RV 5' TC[Ag]AA[ACgT]cc[Ag]CT[Ag]TT[Ag]Tg 3' where [ct] indicates redundancy, equimolar amounts of two oligonucleotides one with c and the other with t at that position, [ ag] redundancy indicates either an a or a g at that position. [AgCT] indicates equimolar amounts of all 4 alternatives. In this prefeoed pair FW_Aspdiag is complementary to a highly conserved endonuclease-encoding region. Aspdiag_RV is complementary to the junction between the end of the intein and the adjacent PRP8 gene. The PCR product of these primers is ~500bp, a size suitable for real-time PCR. The 500bp sequence contains many species-specific nucleotides.

Alternative redundant primers may be designed, depending on the anticipated clinical application. For example, primers which would recognise all Aspergillus but not other related fungi. Such alternative redundant primers can be designed by consideration of the nucleotide sequence of the appropriate fungi.

EXAMPLE 5

Aspergillus fumigatus is a serious fungal pathogen, responsible for a large number of deaths in immunocompromised patients. A. fumigatus contains an intein in the PRP8 gene. Comparisons of this intein sequence with those in species of Aspergillus such as A. nidulans, indicated that the A. fumigatus intein contained an additional, 648bp, unique sequence. There is also an intein in the PRP8 of Neosartorya fischeri. Neosartorya fischeri is the species most closely related to the 'fumigatus' group of Aspergillus (Varga et al., 2000, Antonie van Leeuwenhoek 77:235-239). The intein in N fischeri does not contain the extra 648bp insertion found in A. fumigatus. The insertion is found in all of the 7 strains of A. fumigatus which we analysed. We have therefore designed PCR primers from within this A. fumigatus- specific insertion and are testing them in a LightCycler (real-time PCR) assay. The primers are highly specific, since they are designed to a region not present in other species, even if they did have a PRP8 intein.

The first part (only) of the intein sequences of N fischeri and A. fumigatus are aligned in Figure 12. The protein sequence is also included. The sequence of the A. fumigatus insertion is bolded as being the region which is specific to A. fumigatus

Primers were prepared by the methods described in Example 1. The primers used were:

Setl : β 5' CTGTTACCAATCCTCAGC 3' rv3 5 ' GCTGGCTAC ACTTCGTC A 3 '

Set2: f5 5' TTTCTGGTCTGCTTTGAAGAGTG 3' rv8 5' CCCTCAACGAACGAGCATCAAG 3' These match regions in the 202-residue insertion that is specific to the A. fumigatus intein Afu PRP8.

These primers will not provide a PCR product with any of the species listed below (determined empirically):

Aspergillus nidulans Neosartorya fischeri Neosartorya glabra Neosartorya spinosa Aspergillus brevipes Aspergillus viridinutans

Other species shown to have an intein (but not the extra 202-residue insertion)

Histoplasma capsulatum

Other, variously related, Aspergillus species were shown by Liu & Yang FEBS Letters 572, 46-50 (2004) not to have an intein in PRP8

Aspergillus flavus Aspergillus niger Aspergillus oryzae Aspergillus parasiticus Aspergillus teoeus Aspergillus ustus

Those persons skilled in the art will understand that the above description is provided by way of illustration only and that the invention is not limited thereto.

Claims

1. An isolated intein derived from the PRP8 gene obtainable from an organism from the genus Aspergillus, Neosartorya or the genus Histoplasma or from Cryptococcus laurentii or a functionally equivalent, or functionally altered, fragment or variant thereof.

2. An intein as claimed in claim 1 wherein the intein is derived from the PRP8 gene of a member of the genus Aspergillus.

3. An intein as claimed in claim 1 wherein the Aspergillus species is Aspergillus nidulans or Aspergillus fumigatus.

4. An intein as claimed in claim 1 wherein the intein is derived from the PRP8 gene of Histoplasma capsulatum.

5. An intein as claimed in claim 1 wherein the intein is derived from the PRP8 gene of. Aspergillus fumigatus 6. An intein as claimed in claim 1 wherein the intein is derived from the PRP8 gene of Neosartorya fischeri

I. An intein as claimed in claim 1 wherein the intein is derived from the PRP8 gene of Cryptococcus laurentii.

8. An intein with an amino acid sequence selected from the group consisting of the intein sequences shown in Figure 1, Figure 2, Figure 3, Figure 4, and Figure 5.

9. An isolated nucleic acid molecule encoding an intein as claimed in any one of claims 1-8.

10. An isolated nucleic acid molecule as claimed in claim 9 comprising the intein nucleotide sequence set forth in any one of Figure 6, Figure 7, Figure 8, Figure 9, or Figure 10 or fragments or variants thereof, which encode an intein of the invention, or a fragment or variant with a biological activity of the intein.

I I. An isolated nucleic acid molecule as claimed in claim 9 which comprises a sequence encoding an intein selected from the group consisting of the intein sequences of Figure 6, Figure 7, Figure 8, Figure 9, and Figure 10 or a fragment or variant with a biological activity of an intein.

12. A vector or construct including a nucleic acid molecule of the invention or a fragment or variant thereof as defined in any one of claims 9- 11. .

13. A host transformed with a vector of or construct of claim 12, capable of expressing an intein.

14. A transgenic organism which includes a nucleic acid molecule of any one of claims 9 - 11 and which is capable of expressing an intein of the invention.

15. A transgenic organism which includes a nucleic acid molecule encoding a sequence selected from the group comprising the intein sequences of Figure 1, Figure 2, Figure 3, Figure 4, and Figure 5, or a fragment or variant with a biological activity of the intein.

16. A protein including an intein as claimed in any one of claims 1-8.

17. A protein as claimed in claim 12 comprising proximal and distal extein reporter portions which together form a reporter protein.

18. A protein as claimed in claim 12 comprising a binding protein portion, an intein of the invention, and a reporter protein portion.

19. A method for screening an agent for antimicrobial activity against a microorganism, the microorganism having an intein of any one of claims 1-8 encoded within a gene encoding a protein which facilitates growth of the microorganism, the method comprising detecting inhibition of said intein, which comprises:

(a) preparing recombinant clones of an inducible expression vector containing: (i) an altered reporter gene comprising a silent restriction site within a reporter gene, and (ii) said intein;

(b) detecting production of extein product of said intein by said recombinant clones in the presence of said agent;

wherein reduced production of said extein product indicates inhibition of said intein, and antimicrobial activity of said agent against said microorganism.

20. A method for screening an agent for antimicrobial activity against a microorganism, the microorganism having an intein of any one of claims 1-8 encoded within a gene encoding a protein which facilitates growth of said microorganism, the method comprising detecting inhibition of said intein by monitoring intein function, which comprises:

(a) creating a silent restriction site within a reporter gene which results in an altered reporter gene; (b) cloning said altered reporter gene into an inducible expression vector;

(c) cloning said intein into said inducible expression vector containing said altered reporter gene to generate recombinant clones; and

(d) detecting the production of extein product of said intein by said recombinant clones in the presence of said agent;

wherein reduced production of said extein product indicates inhibition of said intein, and antimicrobial activity of said agent against said microorganism.

21. A method or the detection and/or assay of an Aspergillus, or Histoplasma organism in a sample, comprising detecting or measurement of a polynucleotide in the sample encoding the intein of the PRP8 gene of the organism.

22. A method as claimed in claim 21 wherein DNA of the sample is contacted with a polynucleotide that binds to the DNA encoding the intein or a portion thereof and the binding is detected and/or measured.

23. A method of claim 21 or 22 wherein the polynucleotide or a further polynucleotide binds to DNA complementary to the DNA encoding the intein.

24. A method as claimed in any one of claims 21-23 wherein the DNA encoding the intein or a portion thereof in the sample is amplified.

25. A method as claimed in any one of claims 21-24 wherein the method is a PCR method. 26. A method as claimed in any one of claims 21-25 wheoein the organism is an Aspergillus fumigatis organism.