CA2262519A1 - Identification of and cloning a mobile transposon from aspergillus - Google Patents

Abstract

There are provided transposable elements isolated from Aspergillus. Also provided are fragments comprising the inverted repeat(s) of the transposable elements, such fragments being useful as probes to isolate transposable elements from other filamentous fungi.

Description

CA 02262~19 1999-02-03 WO 98/08g60 PCrlUS97/14g78 IDENTIFICATION OF AND CLONING
A MOBILE TRANSPOSON FROM ASPERGILLUS

Field of the Invention The present invention is directed at the identification, cloning and sequencing of mobile transposons or transposable elements from Aspergil/us nigervar. awamon. The transposable elements, referred to as Vader and Tan1, are approximately 437 base pair (bp) and 2.3 kb elements, respectively. The Vader and Tan1 elements are bounded by inverted repeat sequences of 44 and 45 base pairs, respectively. The transposable elements target a "TA" sequence in target DNA during insertion. In addition, the present invention is directed at the identification, cloning and sequencing of one or more transposable element(s) from other filamentous fungi using as a probe DNA comprising the Vader element 44 bp or the Tan1 element 45 bp inverted repeat isolated from Aspergillus nigervar. awamon. Also provided are methods for utilizing either the Vader or Tan1 elements to inactivate genes (for example, by inserting the transposon into the gene to be inactivated), to overexpress a gene (by, for example, inserting a known promoter or other regulatory gene within the inverted repeats of Vader or Tan 1 and allowing the DNA of the IR-promoter-lR to jump in front of (and overexpress) a gene of interest) or to act as an activation marker to, for example, identify new promoters.

Back~round of the Invention It is well know that transposons are a class of DNA sequences that can move froman episome to a chromosomal site or from one chromosomal site to another. Transposons are known in both prokaryotes, such as bacteria, as well as in eukaryotes, although there have been few transposons isolated from filamentous fungi.
Several groups have looked for transposons in filamentous fungi. The element pogo, which exists in multiple copies and at different sites in different strains of Neurospora crassa, was described by Schectman (1) and is believed to be a transposon. To date the most characterized transposon in filamentous fungi is Tad. Tad was isolated as aspontaneous mutant in the am (glutamate dehydrogenase) gene in an Adiopodoume strain of N. crassa isolated from the Ivory Coast. To detect mutations caused by insertion of a transposable element, Kinsey and Helber (2) isolated genomic DNA from 33 am mutant CA 02262~l9 l999-02-03 Wo 98/08960 PCT/US97tl4978 transposable element, Kinsey and Helber (2) isolated genomic DNA from 33 am mutant strains which were then screened by Southern analysis for restriction fragment size alterations. In two of the mutant strains, the mutation was shown to be caused by the - insertion of a 7 kb element (Tad) into the am gene. Subsequently Kinsey (3) demonstrated that Tad was able to transpose between nuclei of heterokaryons, conri""ing that Tad was a retrotransposon and that there was a cytoplasmic phase involved in the retro~,ansposition events. More recently, Cambareri et al. (4) demonstrated that Tad was a LlNE-like DNA
element with two major open reading frames (ORFs) on the plus strand. Typical of LINE-like elements, Tad had no terminal repeats. Attempts to isolate mobile transposons in 10 laboratory strains of N. crassa were unsuccessful.
A second retrotransposon was cloned by McHale et al. (5), who reported the isolation of CfT-1, an LTR-re~ nsposon from Cladosporium fulvum. This transposonwas 6968 bp in length and bounded by identical long terminal repeats of 427 bp, a 5 bp target site duplication. Virus-like particles were detected which co-sediment with reverse transcriptase activity in homogenates of this fungus.
Daboussi et al. (6) were the first to successfully use the niaD (nitrate reductase) gene as a transposon trap. The niaD mutants can be isolated by a direct selection for chlorate resistance (7) . The strategy employed was to isolate niaD mutants amongst six isolates belonging to different races of the fungus Fusarium oxysporum. More than 100 niaD mutants were isolated from each isolate and examined for instability. One strain, F24, yielded up to 10% unstable niaD mutants. Assuming that the genetic instability of the niaD
mutants was caused by transposable elements, it seemed plausible that this isolate contained mobile transposons. A stable niaD mutant in the F24 was transformed with the cloned niaD gene from A. nidu/ans because the F. oxysporum niaD gene had not been cloned. Unstable niaD mutants were isolated in transformants containing the A. nidulans niaD gene. Two unstable niaD mutants were shown by Southern blot analysis to contain a insertion of 1.9 kb in size. Analysis of this element, Fot1, revealed it was 1928 bp long, had a 44 bp inverted terminal repeats, contained a large open reading frame, and was flanked by a 2 bp (TA) target site duplication. Very recently, Daboussi et al. (8) have reported the 30 cloning of a new transposable element from an unstable niaD mutant. This element, FML
(Fusarium mariner-like), is 128~ bp long and has inverted repeats of 27 bp. The FML
element inserts into a TA site and excises imprecisely.
Using the characterization of unstable niaD mutants strategy, Lebrun et al. (9) were able to isolate a transposon from Magnaporti7e grisea. However, in this case the A.

CA 02262~l9 l999-02-03 WO 98/08960 PCTtUS97114978 nidu/ans niaD gene which was transformed into M. grisea by transro"naLion was used as a transposon trap. The element inserted into the niaD gene was shown to belong to a family of M. gnsea LTR~ t~ol~ansposons, Fos1 (Schull and Hamer, unpublished) and Mag1 (Farman and Leong, unpublished). The cloned retro-element was 5.6 kb and the target site (ATATT) was shown to be duplicated. All revertants from this mutant examined had one copy of the LTR left at the point of insertion. A second transposon, Pot2, from M. grisea was recently cloned by Kachroo et al. (10). The strategy used to clone Pot2 was to analyze the fingerl rint patterns of repetitive DNA's which were cloned from the M. gnsea genome.
A repetitive family present in both rice and non-rice pathogens of M. grisea in high copy number was cloned. The element, 1857 bp in size, has a 43 bp perfect terminal inverted repeats (TIR) and 16 bp direct repeats within the TlRs. An open reading frame was shown to display extensive identity to that of Fot1 of F. oxysporum. As with Fot1, the Pot2 element duplicates the dinucleotide TA at the target insertion site. Pot2 was shown to be present at a copy number of approximately 100 per haploid genome.
Several groups have reported looking without success for transposons in laboratory strains of A. nidulans (Kinghorn personnel communication, 5). One explanation for the lack of transposons in laboratory strains is that the desirable features of strain stability required for genetic analysis may preclude strains with mobile transposon. By using the niaD gene as a transposon trap we have identified and isolated a transposable element from the industrially important fungus A. nigervar. awamori. This element, Vader, is present in approximately 15 copies in A. niger and A. nigervar. awamori. Southern analysis of A. nidulans with this element indicates that this transposable element was absent from one laboratory strain and only present as a single copy in a second laboratory strain. These results support the notion that laboratory strains of A. nidulans contain very few transposons.

Brief Description of the Invention In accordance with the present invention, novel eukaryotic transposable elementsfrom Aspergillus nigervar. awamon are provided. The larger transposable element,referred to herein as Tan1, is 2.3 kb in size. The smaller transposable element, referred to herein as Vader, is a 437 bp element ~SEQ ID NO:3). Vader is found within the larger element Tan1. The Vader transposable element is a 437 bp element which comprises a 44 bp inverted repeat sequence at either end of the transposable element. Tan1 is approximately a 2325 bp element which comprises 45 bp inverted repeats at eitherterminus and internal IRs. Tan1 comprises a 555 aa open reading frame (ORF) which CA 02262~19 1999-02-03 codes for a transposase which allows the elements (Tan1 or Vader) to "hop" or insert themselves in the genome of a host. The target for insertion of these novel transposable elements is a "TA" sequence in the target DNA for insertion. The "TA" sequence is repeated at either end of the transposon upon insertion of the transposable element into the target DNA. Therefore, the present invention provides the larger Tan1 transposable element as well as the smaller element (Vader) internal thereto, as well as the DNA
encoding each.
Another embodiment of the present invention comprises a fragment of the Vader orTan1 transposable elements which comprise the 44 or 45 bp (respectively) inverted repeat 10 sequences found at either terminus of the transposable element from A. nigervar.
awamori, as well as the use of said fragments as probes to hybridize under low stringency conditions to DNA of other filamentous fungi for the isolating and/or cloning of transposable elements from such other filamentous fungi. While the exact 44 bp IR of Vader or the 45 bp IR of Tan1 can be utilized, it is well understood by those skilled in the art that variation of such DNA would also work as a suitable probe. For example, at a minimum, the imperfect direct repeats within the IRs of Tan1 would be suitable to use as probes for isolating transposable elements from other filamentous fungi. Initially the inverted repeat of Vader was used to clone Tan1 using PCR techniques. This work was followed by obtaining a genomic copy of Tan1 from a partial library.
Another embodiment of the present invention is the transposase activity coded for by the ORF of Tan1. This transposase is 555 aa (SEQ ID NOS:7 or 14, PCR and genomic, respectively).
In a process embodiment of the present invention there are provided methods for gene tagging comprising using the transposable elements of the present invention (Vader 25 or Tan1 or any transposable element isolated using the IRs of either) to inactivate genes via insertion of the element into a given gene, thus disrupting or inactivating gene expression. Alternatively, the transposable element can be used in activation tagging (to activate or turn on genes) rather than for gene disruption. For example, by inserting DNA
coding a promoter into the transposable element and then allowing such transposable 30 element to become inserted 5' to a desired gene, the promoter may be activated to drive the expression of the desired gene product or to turn on cryptic pathways. Additionally, gene tagging can be utilized to activate marker genes by inserting a marker gene within the IRs of a transposon of the present invention. This marker gene can then "hop" into targeted DNA and, if expression of the marker is selected for, it will be possible to identify CA 02262~l9 l999-02-03 the pro",oter driving such expression. This may lead to identification of isolation of new strong promoters.

Crief Descri~ti~-- of the Dla~ q;~
Fig. 1 shows the Southern blot analysis of unstable niaD mutants. PCR-amplified geno",ic niaD gene from four niaD mutants and UVK143f were digested with Bglll (sites are 3 of all inserts). Blot probed with 500 bp fragment of Sall digested PCR product of niaD1 and niaD2. Wild-type band hybridizes at 2.5 kb while gene with insertion hybridizes at 2.9 kb. Lanes: 1=MW marker lll (Boehringer Mannheim); 2=UVK143f; 3=niaD410; 4=niaD436;
10 5=niaD 587; 6=niaD392.
Fig. 2 depicts the mapping of Vader insertions within the niaD gene. The positions of Vader insertions 1-4 (niaD410, niaD436, niaD587 and niaD392, respectively) are shown relative to the six introns of the structural gene coding region. Because the exact site of insertion for Vader-1 and Vader-4 is still unknown they have been presented using the 15 approximate area of insertion. Relevant resl,iclion sites are shown using the following letters: E=EcoRI S=Sall, Sp=Sphl, K=S<pnl, and B=Bglll.
Fig. 3 shows Southern blot analysis to determine Vader genomic copy number.
Four A. nigervar. awamori niaD mutants and UVK143f were digested with EcoRV to completion. EcoRV cuts the Vader sequence once. Hybridization indicates that Vader is 20 present in the genome in more than 14 copies. The hybridizing bands of niaD 392, which are different from the other mutants and UVK143f suggest that the Vader sequence is mobile. Lanes: 1=MW marker lll 2=UVK143f 3=niaD410, 4=niaD 436, 5=niaD 587, 6=niaD 392.
Fig. 4. Southern blot to determine presence of Vader sequence in other fungi.
25 Other filamentous fungi an industrial production strain and niaD mutant 392 were digested with EcoRV to completion. Low stringency hybridization (32) indicates that sequences homologous to Vader are present in A. nidulans (FGSC A237), A. cinnamomeus, A.
phoenicis, A. foeWus, an industrial A. nigerstrain. Lanes: 1=MW marker, 2=A. foetidus, 3=an industrial glucoamylase production strain of A. niger(ETC #2663), 4=A. nigervar.
30 awamon niaD mutant 392, 5=A. phoenicis (ATCC #11362), 6=A. nidulans (FGSC A691), 7=A. wentii (ATCC #10593~, 8=A. versicolor, 9= A. cinnamomeus (ATCC #1027), 10=A.
nidulans (FGSC A237) Fig. 5. Southern blot to determine Tan1 (transposon from A. niger') genomic copynumber. Four niaD mutants A. nigervar. awamori mutants and UVK143f were digested CA 02262~19 1999-02-03 with EcoRI to cG",plet:on. EcoRI cuts the Tan1 sequence once. A probe corresponding to the ORF region (see Fig. 9) was used in the hybridization. Hybridi~alion ind;cales that Tan1 is present as a single copy in the genome. Lanes: 1=MW marker lll, 2=UVK143f, 3=niaD410, 4=niaD 436, 5=niaD 587, 6=niaD 392.
Figs. 6A-6C. Southern blots to determine if the inverted repeats of transposableelements Fot1 and Pot2 will hybridize to elements in A. nigervar. awamon. Four niaD
mutants A. nigervar. awamori mutants were digested with EcoRI to completion. EcoRI cuts the Tan1 sequence once. Inverted repeat oligonucleotide probes of Vader (SEQ ID NO:5), Fot1 and Pot2 were labeled with digoxigenin (Boehringer Mannheim). Lanes: 1=MW
10 marker lll, 2=niaD436, 3=niaD587. Blot A (lanes 1-3) and B and C were probed with the labeled inverted repeat probes of Vader, Fot1 and Pot2, respectively.
Fig. 7 shows the sequence of the Vader insertion (SEQ ID NO:3) as generated by PCR. Vader was found to be 437 bp in length. The 44 bp inverted repeat of the Vader insert corresponding to SEQ ID NO:4 (the 5' IR) and SEQ ID NO:5 (the 3' IR), respectively, 15 from the 5' end to the 3' end of Vader are underlined, the single mismatch which occurs in the inverted repeats is identified in bold, and the TA 2 bp duplication is shown in bold print.
niaD sequences flanking the element are shown in lower case letters.
Figs. 8A and 8D show the entire DNA sequence of the Tan1 element (SEQ ID
NO:6) as generated by PCF'c, as well as the putative amino acid sequence of the 20 transposase coded for by Tan1 tSEQ ID NO:7). Tan1 as generated by PCR is 2320 bp in length (excluding the unknown nucleotides shown as "N" in the figure) and has a large open reading frame of 1668 bp which encodes for 555 amino acids (SEQ ID NO:7). Tan1 comprises the sequences of four inverted repeats (underlined) similar to those found in Vader.
2s Fig. 9 shows a schematic presentation of Vader and Tan1 elements. Dark boxes represent the 45 bp (Tan1) and 44 bp (Vader) inverted repeats. The unique EcoRI site in the Tan1 element was used for digestion of genomic DNA in Southern analysis (Figs. 5 and 10). Bold, horizontal lines above the Tan1 element indicate the probes corresponding to the end of the ORF and Vader used in Southern analysis shown in Fig. 10 and Fig. 5.
Fig. 10 shows Southern analysis of A. nigervar. awamori niaD mutants (niaD410, niaD436, niaD587, niaD392) and the wild-type UVK143f: lane 1, molecular weight marker lll (Boehringer Mannheim); lane 2, UVK143f; lane 3, niaD410; lane 4, niaD436; lane 5, niaD587; lane 6, niaD392. This blot was probed for the Vader element (see Fig. 9). When this blot (Fig. 10) was superimposed with the blot shown in Fig. 5, one of the illuminated 3s bands from the Vader-probe hybridization overlaid the single band in the ORF-probe CA 02262~19 1999-02-03 W 098/08960 PCT~US97/14978 hyb~idi~ation indicating that the Tan1 element is composed of contiguous ORF and Vader elements.
Figs. 11A and 11 D show the nucleotide sequence (genomic copy) of Tan1 (SEQ ID
NO:13). The amino acid sequence encoding the putative transposase (555 aa) (SEQ ID
NO:14) is shown below the DNA sequence in the one-letter amino acid code. The inverted repeats are underlined (SEQ ID NOS:1, 2, 15 and 16, respectively, 5' to 3') and the imperfect direct repeats within the inverted repeats are shown with arrows above or below the sequence. The gaps within the arrows indicate the imperfect nucleotides within the direct repeats. Undetermined sequence is denoted in the figure by question marks and in 10 the sequence listing as "N." The figure shows the DNA sequence as 2324 base pairs, excluding the unknown nucleotides indicated by "?" in the figure.
Detailed DescriPtion of the Invention While the specification concludes with claims particularly pointing out and distinctly claiming the subject matter regarded as forming the present invention, it is believed that the invention will be better understood from the following detailed description of preferred embodiments.
Standard biochemical nomenclature is used herein in which the nucleotide bases are designated as adenine (A); thymine (T); guanine (G); and cytosine (C). N connotes any of these nucleotides. As is conventional for convenience in the structural representation of a DNA nucleotide sequence, only one strand is usually shown in which A
on one strand connotes T on its complement and G connotes C.
Applicanls have isolated two transposable elements from A nigervar. awamori. Thecloned element Vader was identified by screening unstable nitrate reductase (niaD) mutants for insertion. This element is present in approximately fifteen copies in the genome of A. niger strains examined. In contrast, the Vader element is present in one copy in only one of the two A. nidulans strains studied. These results explain why several groups have been unsuccessful in isolating active transposons in laboratory A. nidulans - strains. A plausible assumption is that "domesticated" strains of A. nidulans have lost their transposons due to repeated manipulation of such strains and the possible discarding of aberrant A. nidulans strains displaying genetic instability.
The Vader element shows similarities to transposable elements cloned from the plant pathogens Pot1 from M. gnsea (12) and Fot1 from F. oxysporum (8). The target site for duplication in all three fungi is a 2 bp TA sequence. In the case of Fot1, this transposon does not excise precisely. In two niaD revertants examined, the excision products retained a 4 bp insertion relative to the wild-type gene (TAATTA versus TA). The insertion studied CA 02262~l9 l999-02-03 was integrated into an intron, therefore, imprecise excision of Fot1 did not effect the functionality of the niaD gene product. There is no published evidence that Pot2 is a functional element.
A homology search made at the nucleotide level gave a strong 60.7% homology s between Tan1 and a 1230 bp overlap to the A. oryzae agdA gene coding for an a-glucosiçlase (33). This homology search revealed that the last 1.2 kb of a total of 5.2 kb of the a-glucosid~se sequence submitted to GenBank is, in fact, part of a novel transposon, hereinafter called Tao1 (transposon Aspergillus oryzae), which also belongs to the Fof 1 family. Only the 5' half of the Tao1 element is included in the GenBank sequence, thus, for 10 the lack of comparison, the exact size of the inverted repeat cannot be determined.
However, it can be concluded that there are 13 bp perfect direct repeats within the inverted repeat. The inverted repeat is flanked by a TA-dinucleotide, suggesting a commonly occurring TA-insertion site. Direct analyses gave only short ORFs, but when the often-occurring stop codons were ignored, a long ORF was obtained which shared over 50%
identity to the Tan1 transposase. Multiple stop codons indicate that the A. oryzae Tao1 is a defective element. This transposable element from A. oryzae, thus, is within the scope of the present invention as, based on the high degree of sequence homology between the Tan1 and Tao1, it is believed that Tao1 would hybridize to a probe comprising Tan1 or Vader IRs or variations thereof. The sequence of the IR of Tao1 is provided as SEQ ID
NO:17. This IR (Tao1) or the IRs from Tan1 or Vader may be used to isolate othertransposable elements from filamentous fungi.
In an attempt to determine if there were transposons similar to those reported for F.
oxysporum and M. grisea, synthetic oligomers were made corresponding to the inverted repeats of both Fot1 (7) and Pot2 (10). When Southern analysis of A. niger var. awamori was conducted using the Vader 44 bp inverted repeat (SEQ ID NO:5) as a control, no conclusive hybridizations could be detected with either the Fot1 or Pot2 oligomeric probe.
These results indicate that elements with high identity to F. oxysporum Fot1 and M. grisea Pot2 are not found from A. nigervar. awamori genome.
With regard to the structure of the Vader element, elements which transpose directly through DNA copies are typified by having inverted terminal repeats. Elements which transpose through reinsertion of the product of reverse transcription of an RNA copy of the element (retroelements) can be without long terminal repeats such as the Drosphilia I
element (for a review see (16)). Alternatively, retrotransposons can have long terminal repeats such as the Drosphilia copia element. The Vader inverted repeats shown in Fig. 7, CA 02262~19 1999-02-03 W 098/08960 PCTrUS97/14978 __ 9 __ SEQ ID NOS:4 and 5, respectively, have a single mismatch. Elements which transpose through DNA copies typically have open reading frame(s) which encode a transposase activity. The Fot1 element is 1.9 kb in length and the Pot1 element 1.8 kb in length. Both the Fot1 and Pot1 elements have ORF encoding for a putative transposase-like protein.
The Vader element, although mobile, does not have an ORF and hence it was deduced that the mobility of Vader was dependent upon a transposase activity present elsewhere in the genome. A synthetic 44 bp oligomer of the inverted repeat of Vader (SEQ ID NO:5) was used to clone, via PCR, a 2.3 kb element. This element, called Tan1 (SEQ ID NO:6), cG",prises fourinverted repeats (SEQ ID NOS:1, 2, 15 and 16 from 5' to 3', respectively) 10 similar to those in Vader and has a unique organization IR-ORF-lR-lR-Vader-lR. Tan1 is 2324 bp in length and has a large open reading frame (1668 bp) which encodes a putative transposase comprising 555 amino acids (shown in SEQ ID NOS:7 and 14), which is homologous to Fot1 and Pot2 transposases. Immediately 3' to the second IR (SEQ ID
NO:2), which bounds the transposase, is a copy of the Vader element. We hypothesize ~5 that at some stage the independent Vader element, although inactive by itself, has arisen from Tan1, resulting in current strains with only one copy of Tan1 providing transposase activity and numerous mobile copies of Vader dispersed in the genome.
Thus, applicants have been the first to identify a transposable element(s) with certain Aspergillvs species. These transposable elements are believed to be quite useful in the deveiopment of gene tagging systems for Aspergillus or other microorganisms. Basic requirements for developing a gene tagging system are that the tagging element can be distinguished from the endogenous elements, it displays little sequence specificity for transposition and that excision is followed by integration at a new site. More refined tagging systems include ability to monitor excision and reinsertion by, e.g., activation of antibiotic resistance genes and ability to stabiiize the mutations by, e.g., a two transposons system (23, 24 and 25).
For development of a tagging system for Aspergillus, it is proposed that the system is tested first in A. nidulans, which we have already shown does not have endogenous Tan1 or Vader sequences. However, at this stage the Vader element is altered from the original in such a way that the same construction can be later used in A. nigervar. awamon and be distinguished from the endogenous Vader elements.
In a model tagging system using Vader as the "mutator," a first vector can be constructed for expression of the Vader element, similar to the non-autonomous maize Dc.
The internal sequence of the Vader element is altered to contain translation initiation and stop codons in three different frames. This sequence can later be used as a recognition CA 02262~l9 l999-02-03 site for a probe in PCR analysis of the mutants. This altered Vader element, Vader-S, is inserted within an expression cassette conferring antibiotic resistance such as hygromycin resistance. Since excision of Vader may not always be precise, Vader-S is inserted in the promoter area (e.g., oliC) between the transcription and translation ini~ialion sites. This disrupted hygromycin phosphol,dnsferdse cassette is flanked by marker genes - oralternatively the marker gene upstream of the hygromycin promoter can be placed within Vader. These marker genes can be used for monitoring whether the hygromycin gene, and Vader within it, have integrated in full length. A vector, for example, Vector 1, containing these elements will be transferred to A. nidulans and transrunl)allls expressing the two 10 marker genes, but sensitive to hygromycin, are selected. Screening of mutants at later stages is easier, if the transformant selected for mutagenesis has only one to two copies of Vector I sequences integrated in its genome.
A transformant with only a few (preferentially one) intact Vader-S/hygromycin phosphotransferase cassettes integrated in its genome is retransformed with Vector ll, 15 which is an autonomously replicating vector carrying the transposase encoding gene. The autonomously replicating vector, pHELP, used as a basis for DNA construction work, can be segregated away by methods known to those skilled in the art. This enables stabilization of the Vader-S element after the mutagenesis step. Vader-S is activated by a transposase (from Tan1) in pHELP, which can be monitored by activation of the 20 hygromycin resistance gene. Tan1 is not cloned into the vector in full length to disrupt its mobility. Again, Vector ll contains a marker gene used for screening of transformants and also for monitoring its segregation after the sporulation phase.
Marker genes can either complement host mutations or be dominant markers such as benomylR, acetamidase or ,~-glucuronidase (GUS).
In a model system for gene tagging the target gene for mutagenesis should be onewith a simple plate screen, e.g., disruption of the niaD gene (by insertion of Vader), which can be screened by selection of chlorate resistant mutants and the gene disruption can be further mapped by a plate test using different nitrogen sources ~no growth on nitrate, growth on nitrite, xanthine and uric acid). Another target gene for mutagenesis could be an 30 acid protease gene. It has been shown previously for A. nigerthat disruption of this one protease is sufficient to abolish halo formation almost completely on skim milk plates.
The advantage of using transposon tagging is that the mutants produced can be identified by subsequent isolation of the mutated gene. There are several methods available for PCR amplification of genomic sequences when only one end of the sequence 35 iS known - which, in this case, is the transposable element. PCR methods developed for CA 02262~19 1999-02-03 W O 98/08960 PCT~US97/14978 genomic walking are, e.g., "Inverse PCR" (27 and 28), "Vectorette PCR" (29) and "Panhandle PCR" (30).
Setting up the transposon tagging system can be followed by studies of excision frequency, environmental influences on transposition frequency (24, 31), activation of the transposase by a heterologous promoter and effect of altered inverted repeats on transposition.
Transposon tagging does need to be applied for inactivation of genes.
Alternatively, tagging can be used to insert promoter sequences in Vader and therein activate genes. A third option is to insert a promoterless marker gene in Vader, in which case the transposon can be used in search for novel, strong fungal promoters.

E~peri..,ental Materials and Methods Strains. Vader and Tan1 elements were isolated from Aspergillus nigervar.
awamon UVK143f, derived from Northern Regional Research Laboratories (NRRL) #3112.
E. coliJM101 [F' traD361ac1q~(1acZ)M15proA'B~lsupEthi~(laGproAB)] and Epicurian co/i SURE 2 (Stratagene Cloning Systems, La Jolla, CA) were used for propagation of Vader and Tan1 subclones, respectively.
Spontaneous chlorate resistant mutants were derived from Aspergillus nigervar.
awamori UVK143f (NRRL #3112). The following Aspergillus strains were obtained from the ATCC: A. cinnamomeus (ATCC #1027), A. wentii (ATCC #10593), and A. phoenicis (ATCC #11362). A. nidulans (FGSC #A237), a nitrate reductase structural gene mutant (niaD15), and A. nidulans (FGSC #A691), a tryptophan requiring mutant (trpC801), were obtained from Fungal Genetics Stock Center (FGSC), Dept. of Microbiology, University of Kansas Medical Center. A. versicolor, A. foetidus, and a proprietary A. niger glucoamylase strain are from the Genencor International Inc. culture collection.
Mutant Selection. Spore suspensions (1 x 108) of UVK143f were plated on CM
agar (11) containing 600 mM KC103 and 10 mM glutamic acid. Chlorate (KCI03), a toxic analog of nitrate, allows selection of mutants in the nitrate assimilation pathway by chlorate resistance. Plates were incubated at 37~C until individual colonies of spontaneous mutants could be identified. Single mutants resistant to KC103 were allowed to sporulate on CM
plates and spores from these plates were then streaked onto minimal media (11) with various soie nitrogen sources (10 mM): NaN03 (nitrate), NaN02 (nitrite), hypoxanthine, uric acid or NH4CI (ammonium chloride). Each of these compounds are intermediate products CA 02262~l9 l999-02-03 of the nitrate assimilation pathway. niaD mutants were identified as those resistant to KClO3and able to grow in the presence of all pathway intermediates, except for NaNO3.
Isolation of Vader via PCR Amplification. Genomic DNA oF A. niger var. awamon niaD mutants and UVK143f was used as template (see Southem Analysis). Primers (50 pmol) used for amplification of the niaD gene were NiaD1 (position 142-165 relative to the initiation site of niaD): 5'-CCAACCGAGTCCTCAGTATAGAC-3' (SEQ ID N0:8) and NiaD2 (position 2738-2715): 5'-CAACGCTTCATAGGCGTCCAGATC-3' (SEQ ID NO:9). Deep Vent (exo~) DNA polymerase (New England Biolabs) was used with the buffer and dNTPs provided by the manufacturer. For optimal amplification of the niaD gene the reaction 10 mixture contained 4 mM MgSO4. Denaturation of template DNA,2 min. at 94~C, was followed by 30 cycles of denaturation (30 sec. at 94~C), annealing of primers (45 sec. at 55~C) and extension (4 min. at 72~C). PCR fragments were purified from gel using the Qiaex DNA gel extraction kit (Qiagen), digested and used for restriction enzyme analysis by standard procedures (12).
Confirmation of Excision Foot Print by PCR Amplification and Sequencing.
Template DNA from niaD436 was used in a PCR reaction in an attempt to amplify both the larger niaD sequence with an insert and the shorter niaD fragment resulting from excision of the Vader element. The PCR reaction was conducted as previously described, except for using primers MA003 (positions 359-378): 5'-2D ATATGAATTCCTTCTTGACTTCCCCGGAAC-3' (SEQ ID NO:11) and NiaD5 (position 1125-1144): 5'-ATATAAGCTTGTCACTGGACGACATTTCAG-3' (SEQ ID NO:12). The gel purified fragment (ca. 800 bp) resulting from the excision event was submitted for sequencing.
Isolation of Tan1 via PCR Amplification. Fungal genomic DNA for PCR and 2~ Southern analyses was isolated from mycelia grown in CSL supplemented with 5% fructose (21). Genomic DNA of A. nigervar. awamori ntaD 436 mutant (22) was used as a template. A single primer (100 pmol), IR1, was used for amplification of Tan1. The 54-mer IR1 was derived from the 44 bp inverted repeat sequence of Vader preceded by a restriction enzyme recognition site for EcoRI: 5'-ATATGAATTC ACGTAATCAA
30 CGGTCGGACG GGCCACACGG TCAGGCGGGC CATC-3' (SEQ ID NO:10). Deep Vent (exo-) DNA polymerase (New England Biolabs) was used with the buffer and dNTPs provided by the manufacturer. Denaturation of template DNA,10 min. at 94~C, was followed by 30 cycles of denaturation (1 min. at 94~C), annealing of primers (1 min. at 55~C) and extension (6 min. at 72~C). PCR fragments were purified from agarose gels CA 02262~19 1999-02-03 W O 98t08960 PCT~US97/14978 using the Qiaex DNA gel extraction kit (Qiagen) and subcloned as blunt-ended inserts into EcoRV cut pSL1180 (Pharmacia Biotech).
Estimation of niaD Mutant Reversion Frequency. Spores from niaD mutants niaD392, niaD410, niaD436 and niaD587 were streaked onto minimal media containing NaN03 as a sole nitrogen source. Nitrate non-utilizing colonies of niaD mutants, which had a spidery appearance and did not sporulate, were streaked onto CM containing 600 mM
potassium chlordle (KCI03) and incubated to confluency at 37~C. Ten-fold dilution series of spore suspensions (in 0.8% NaCI-0.25% Tween 80) of niaD392, niaD410, niaD436, niaD587 and UVK143f wild-type spores were plated on minimal media with nitrate (10 mM) 10 to determine reversion frequency, and on CM to determine viability.
Southern Analysis. Genomic DNA for PCR and Southern analysis was isolated (13) from mycelia grown in CSL (13), which contained 600 mM KCI03 in order to reduce reversion of niaD back to the wild-type during cultivation. DNA (10 ,ug) was digested with either Bglll, which leaves the insertion intact in the niaD gene, or with EcoRV, which cuts the insertion element (Vader) once, and thus enables determination of its copy number in the genome. Genomic DNA (approximately 10 ~ug) of A. nidulans, A. cinnamomeus, A.
versicolor, A. wentii, A. phoenicis, A. foetidus and of an industrial A. niger strain were digested with EcoRV to obtain an estimate of Vader copy number in these fungal genomes.
The digested and gel-separated DNA was transferred to a positively-charged nylon membrdne (Boehringer Mannheim) by capillary action.
The DNA probe for the niaD gene was derived from the PCR product (UVK143f DNA template amplified with primers NiaD1 (SEQ ID N0:8) and NiaD2 (SEQ ID N0:9)), which was digested with Sall, resulting in a 528 bp probe fragment. The probe for the insertion element, Vader, was derived from a PCR reaction in which niaD436 DNA was used as a template. This PCR product was purified and digested with Sall and Sphl and subcloned into the vector pUC19. This subclone was digested with Scal and Xbal to yield a 236 bp fragment which was used for esli",alion of the copy number of Vader sequences in the genomes of various fungi.
A DNA labeling and detection kit (Genius1, Boehringer Mannheim) was used for - 30 random primed labeling of probe DNA with digoxigenin, and for detection with alkaline-phosphatase labeled antibody to digoxigenin.
Hybridization and washing conditions for homologous probes were conducted as recommended by the manufacturer using hybridization buffer without formamide at 68~C
(Boehringer Mannheim). Hybridizations for heterologous Southern analysis (i.e., analysis CA 02262~l9 l999-02-03 of DNA from other Aspergit/us sp.) was conducted using hybridization buffer with 25%
formamide at 37~C. Washes were performed as in stringent wash protocol.
Nitrate R~ ct~se Assays. Nitrate reduct~se assays were performed as described in Dunn-Coleman, et al. (18).
DNA Analysis and Sequence Del~, ."ination. Sequences were determined using fluorescent-labeled dideoxynucleotide terminators and Taq cycle sequencing on the 373A
sequencer (ABI). Commercially available universal and reverse (New England Biolabs) primers were used. Alignment of sequences and prediction of amino acid sequences were performed using DNASTAR (DNASTAR, Inc.). The nucleotide and deduced amino acid 10 sequences were analyzed and compared to those in GenBank, EMBL and Prot-Swiss using Fast A and BLAST programs (Genetics Computer Group, Inc. software package, Madison, Wl).
Other Probes Used for Southern Analysis. The Tan1 probe was prepared by digesting Tan1 with Hindlll and Stul resulting in a 650 bp fragment corresponding to the 3' 15 end of the transposase coding region (ORF-probe in Fig. 9). The Vader element was digested with Xbal and Scal to yield a 236 bp fragment to be used for recognition of internal Vader sequence in Southern analysis (Vader-probe in Fig. 9).
Southern Analysis to Determine Tan1 Copy Number. Aspergillus genomic DNA
(10 ~9) was digested with EcoRI, which cuts the Tan1 element once in the transposase 20 coding region and upstream of sequences corresponding to the Vader and Tan1 probes used in hybridizations (Figs. 5, 9 and 10). DNA labeling and detection kit (Genius 1, Boehringer Mannheim) was used for random primed labeling of probe DNA with digoxigenin and for detection with alkaline-phosphatase labeled antibody to digoxigenin.
Hybridization and washing conditions were conducted as recommended by the 25 manufacturer (Boehringer Mannheim).
Isolation of Tan1 from a Partial Genomic Library. It was known from the sequence of the PCR-amplified Tan1 element that Tan1 did not have restriction enzyme recognition sites for Bglll and Xhol. A Bglll-Xhol digested Southern blot of Aspergillus niger var. awamon genomic DNA, hybridized with the 650 bp Hindlll-Stul Tan1 probe, resulted in 30 identification of a 4.5 kb genomic fragment containing Tan1. A. nigervar. awamori niaD436 DNA was digested with Bglll and Xhol and fragments in a size range of 4-5 kb were cloned into pSP73 (Promega). This partial genomic library was screened by colony hybridization using the nonradioactive nucleic acid labeling and detection system from Boehringer Mannheim.

CA 02262~l9 l999-02-03 Wo 98/08960Pcrluss7ll4978 Example 1 Isolation of Spontaneous High Frequency Reverting niaD Mutants of A. ni.qervar. awamori Assuming that niaD mutants which arise from the insertion of a transposable s element would be unstable, a total of 152 niaD mutants, isolated on the basis of spontaneous resistance to chlorate were characterized. To determine if the niaD mutation was unstable, spores from 43 niaD mutants were plated onto medium with nitrate as the sole nitrogen source. Fourteen of the mutants reverted to the wild-type phenotype at a frequency of greater than 1 X 10 5. Table 1 summarizes the niaD mutant reversion studies.
Table 1 Reversion Conidia Plated No. Wild-Type Frequency Mutant No. x 103 Colonies x 10-4 niaD392 2.9 27 93 niaD410 7.7 5 6.5 niaD436 3.7 164 443 niaD587 18.9 12 6.3 There appeared to be two classes of niaD mutants which reverted at high frequency. The niaD mutants niaD436 and niaD392 reverted at high frequency, while mutants niaD410 and niaD587 yielded smaller numbers of revertant colonies.
The level of nitrate reductase activity was determined using the assay described in (18) from revertant colonies isolated from the niaD 436 mutant. Nitrate reductase activity was detected in 14 of 15 revertants analyzed (see Table 2). A spectrum of activities was detected, suggesting that excision of Vader may not always be precise.
Table 2 % Nitrate Reductase Activity Strain Compared to Wild-Type UVK143f (wild-type) 100 niaD436 (niaD mutant) ND' Revertants of niaD436:
34.7 2 42.8 3 27.7 4 3.5 ND' ' Activity non-detectable CA 02262~l9 l999-02-03 WO98/08960 Pcr~S97/14978 % Nitrate Reductase Activity StrainCompared to Wild-Type 6 47.4 7 90.4 8 9.8 9 25.4 28.9 11 38.2 12 6.9 13 71.7 14 71.7 49.7 Example 2 Cloninq of a Vader Element To determine if an insertion sequence was located within the niaD gene, two primers were synthesized. The first primer, niaD1 (SEQ ID NO:8), corresponded to position 142-165 of the niaD gene, and niaD2 (SEQ ID NO:9) corresponded to position 2738-2715 of the niaD gene. Genomic DNA was isolated from 14 unstable niaD mutants. This genomic DNA served as a template for the PCR primers. PCR reaction products with 4 niaD mutants (410, 436, 587 and 392) revealed an approximately 440 bp insertion (Vader) 10 in the niaD gene.
For Southern blot analysis, genomic DNA isolated from the wild-type and four niaD
mutants (410, 436, 587 and 392) was digested with Bglll. The probe used was a Sall digestion fragment of the 500 bp PCR product generated using the niaD1 (SEQ ID NO:8) and niaD2 (SEQ ID NO:9) oligomeric probes. The probe hybridized to a 2.5 kb fragment 15 with wild-type DNA (lane 5, Fig.1). In the case of the niaD mutants 410 (lane 1, Fig. 1), 436(1ane 3, Fig. 1) and 392 (lane 4, Fig. 1), the probe hybridized to a 2.9 kb fragment.
These results indicate that these three niaD mutants contain an approximately 440 bp insertion. Interestingly, with the mutant niaD587, the probe hybridized to both a 2.5 kb and 2.9 kb fragment, although mycelium had been grown in the experiment in the presence of KCI03 to favor growth of the niaD mutant and not revertant cells, the detection of two hybridizable sequences indicated that in some cells Vader had been excised from the niaD
gene.
The approximate location of the insertion was determined in each of the four unstable niaD mutants by restriction mapping analysis. The location of the insertion in each of the four mutants examined is shown in Fig.2. All four mutants had an approximately 440 bp insertion located at different sites within the niaD gene.

CA 02262~19 1999-02-03 W O 98/08960 PCT~US97/14978 Example 3 Determination of Vader CoPy Number To determine the Vader copy number a 236 bp Scal-Xbal internal fragment of Vader-2 (cloned from the mutant niaD436) was hybridized to EcoRV cleaved genomicDNA. There is only one EcoRV site within the Vader transposon. Southern blot analysis indicatPd that there are approximately fifteen copies of Vader sequences in the genome of A. nigervar. awamon. (Fig. 4). The Vader sequences were integrated at identical genomic locations in the three niaD mutants, 410, 436 and 587. However, in the niaD392 mutant, Vader sequences were located in five different locations compared to the three niaD
10 mutants examined. This result was somewhat surprising considering that all four niaD
mutants were isolated from the same strain, but provides good evidence for the high mobility of the Vader element in this strain. When a propriety A. niger glucoamylase production strain (ETC #2663) was also examined, approximately 15 hybridization signals cou~d be detected. Although some of the hybridization patterns appeared to be identical, clear differences could be seen between A. nigervar. awamori and A. niger.
Example 4 Isolation of Vader in Other Fun~al Species In an atterrlpt to determine if this transposable element was found in other filamentous fungi, genomic Southern blot analysis was performed using the 236 bpfragment (Xbal-Scal) of Vader sequence as per Example 3, as a probe (Fig. 5). Two strains of A. nidulans were obtained from Fungal Genetics Stock Center (FGSC), FGSC
#A691, a nitrate reductase structural gene mutant (niaD15), and FGSC #A237, a tryptophan-requiring mutant (trpC801). No hybridization signals could be visualized with strain A691, and a single strong hybridization signal could be detected with strain A237.
These results support the notion that the lack of success in cloning transposable elements from laboratory strains of A. nidulans is due to low copy number or absence. Similarly, only one hybridi~alion signal could be detected in A. foetidus and A. phoenicis, while two hybridi~a~ion signals were detected in A. cinnamomeus. No hybridizations could be detected in A. wentii and A. versicolor. In addition, no hybridi~ation signals could be detected with Humicola grisea var. thermoidea, Neurospora crassa and Trichodermareesei (results not shown). These results indicate that the Vader element is most commonly found in A. nigervar. awamori and A. niger.

CA 02262~l9 l999-02-03 Example 5 Excision of the Vader Element Part of the niaD gene from niaD436 containing the Vader element was amplified using PCR. The PCR amplification resulted in the expected 1200 bp fragment of the Vader s element flanked by n/aD sequences and a shorter 800 bp fragment resulting from the excision event. Sequencing of the shorter fragment indicated that the Vader element had excised precisely. However, when several revertants of niaD436 and niaD410 were assayed for their nitrate reduct~se activity (18), a spectrum of activities was detected, suggesting that excision of the Vader element may not always be precise (results not shown).
Example 6 Isolation of Tan1 The previously isolated Vader element, although mobile, did not have an ORF
encoding transposase activity presumed to be required for excision (22). This observation led to a search for a transposase-encoding larger element, thus an oligomer corresponding to the Vader inverted repeat was synthesized and used for PCR amplification of the genomic A. niger var. awamor7 DNA. The PCR amplification resulted in the generation of three DNA fragments: the 0.4 kb Vader element, as expected, and fragments of 1.9 kb and 2.3 kb in length.
Both of the larger PCR-generated fragments were sequenced and the sequences were identical with an exception that the 2.3 kb fragment had an additional 400 bp at the 3' end. Surprisingly this additional sequence at the 3' end was a Vader element, which differed only by a few nucleotides from the previously isolated Vader. The 5' end sequence, shared by both of the 1.9 kb and 2.3 kb fragments, had a single ORF (1668 bp) coding for a protein of 555 amino acids flanked by inverted repeats (IRs). Thus, the 1.9 kb fragment, devoid of the Vader element, had an organization of IR-ORF-IR. The larger 2.3 kb fragment had a unique organkdLion, IR-ORF-lR-lR-Vader-lR, with a total of four inverted repeats (Figs. 9 and 11). In this larger element the two central inverted repeats, side by side, potentially form a tight hairpin structure, and despite many sequencing attempts with varying conditions, we were unable to determine the sequence between the two inverted repeats. However, the overall length of the PCR product, as determined by electrophoresis, corresponded to the size of the sequence shown in Fig. 11, suggesting that the two central contiguous IRs are not separated by a large segment of DNA.Due to the organization of the 1.9 kb and 2.3 kb fragments, it was believed that the 1.9 kb fragment could have arisen in PCR from a partial amplification of the 2.3 kb CA 02262~l9 l999-02-03 W O 98/08960 PCT~US97/14978 fragment if the 3' IR-primer had annealed to the first central IR instead of the IR in the end of the Vader element. Southern analysis was conducted in order to determine if the 1.9 kb element existed in the genome without the associated Vader element, or whether it was a PCR-artifact derived from a partial amplification of the 2.3 kb element. The two probes used in Southern analysis corresponded to the internal sequence of Vader and to the carboxyterminal part of the ORF (Fig. 9). The genomic DNA from A. niger var. awamol7 niaD mutants and UVK143f were digested with EcoRI, which cuts once in the coding region of the ORF upstream from the ORF-probe and does not cut Vader. The Southern analysis showed numerous bands for the Vader element (Fig. 10), similar to previous Southern 10 analyses (22). However, only one fragment lit up with the probe corresponding to the ORF
and a fragment of the same size (1.6 kb) was recognized by the Vader probe (Fig. 10). It was concluded that the actual element in the genome was the 2.3 kb fragment and that the shorter 1.9 kb had only been a PCR-artifact. The isolated 2.3 kb fragment was designated as Tan1.
A genomic clone of the Tan1 element (2.3 kb) was isolated from a partial genomiclibrary. Restriction enzymes, which were shown not to have any recognition sites in the PCR-amplified Tan1, were used separately and in combinations in Southern analysis of the genomic DNA. A double digestion with Bglll and Xhol resulted in a relatively short, 4.5 kb, fragment which hybridized with the ORF-specific probe (data not shown). Genomic DNA
20 fragments cleaved by Bg/ll and Xhol and between 4 kb and 5 kb in size were cloned into pSP73 (Promega). The correct clone containing the Tan1 element was isolated by colony hyL,ridi~alion using the ORF-specific probe. Differences between the sequences of the genomic clone and the PCR-generated Tan1 were minor, even for the flanking IRs which were almost identical even though in the PCR-generated Tan1 the IRs were derived from 25 the Vader IRs (PCR primers). It was seen from the genomic clone of Tan1 that immediately outside of the terminal IRs there were TA-dinucleotides, suggesting a TA target site and its duplication upon insertion. Sequence of the Tan1 genomic clone is shown in Figs. 11A and 11B [SEQ ID NO:13 (DNA) and SEQ ID NO:14 (amino acid)].
Example 7 Insertional Inactivation/Gene Ta~qin~
Vader was cloned by insertional inactivation of the target gene niaD, which encodes nitrate reductase. The target sequence for integration of Vader is TA, a sequence which must be very common in the genome of fungi. Nitrate reductase mutants cannot grow on nitrate and inconsequence are resistant to the toxic analog of nitrate, KCI03.

CA 02262~19 1999-02-03 W O 98/08960 PCT~US97114978 lt is possible that one of the reasons heterologous protein production in fungi is lower than that of homologously produced protein using the same proi"oter is that the heterologous protein is being degraded by the cell. If there are genes whose products are responsible for degrading/se~ues~ring foreign protein, it would be advantageous to inactivate those genes. In order to achieve this, a strain is constructed using gene disruption, which lacks the Tan1 gene. Such strain is then used to transform and express a heterologous protein such as the mammalian chymosin protein. It would be advantageous if the activity of such genes could be visualized or selected for on petri dishes. For example chymosin produced in A. niger results in a halo of clearing around a colony grown 10 on skim milk. (See US Patent 5,364,770, the disclosure of which is incorporated herein by reference.) Having transformed the strain with a construct comprising the desired heterologous protein or polypeptide, one would transform the strain a second time with Vader and Tan1 appropriately modified for gene tagging purposes.
The transformants are then plated on medium which can be used to visualize heterologous protein production, such as skim milk plates in the case of chymosin.
The plates are then screened for increased halo size, which is the result of inactivation of a gene whose product limits foreign protein production.
The inactivated gene can be cloned using the transposon sequences as a marker 20 for cloning strategies. (See generally (19).) Example 8 Elevation of Gene ExPression Usin~ TransPosons A reason that heterologous protein production is lower than expected in fungi ispresumed to be that genes essential for foreign (heterologous) gene production are NOT
2s expressed at sufficiently high levels in the fungi.
In order to overcome this problem, utilizing the transposable element(s) of the present invention, a strain is constructed in which the native Tan1 gene is inactivated by gene disruption.
This strain is used to express a heterologous protein whose expression can be 30 easily visualized, such as chymosin (US Patent 5,364,770). A second transformation is made with Vader and Tan1, appropriately modified for gene tagging purposes. The internal sequence of Vader is replaced by a promoter sequence. One of the many integration events possible will be the integration of this promoter carrying Vader element into 5' to a gene beneficial to heterologous protein (e.g., chymosin) expression or secretion. Upon 35 insertion, this beneficial gene is activated and such integrant colonies can be screened for, CA 022625l9 l999-02-03 e.g., increased halo size (chymosin). The activated gene can be cloned using the transposon sequences as a marker for cloning strategies.

CA 02262~19 1999-02-03 References 1. Schectman, M.G. (1987) Mol. Cell. Biol. 7:3168-3177 2. Kinsey, J.A. & Hebler, J. (1989) Proc. Natl. Acad. Sci. USA 86:1929-1933 3. Kinsey, J.A. (1993) Proc. Natl. Acad. Sci. USA 90:9384-9387 4. Cambareri, E.B., Helber, J. & Kinsey, J.A. (1994) Mol. Gen. Genet. 242:658-665 5. McHale, M.T., Roberts, l.N., Noble, S.M., Beaumont, C., Whitehead, M.P., Seth, D. & Oliver, R.P. (1992) Mol. Gen. Genet. 233:337-347 6. Daboussi, M.J., Langin, T. & Brygoo, Y. (1992) Mol. Gen. Genet. 232:12-16 7. Cove, D.J. (1976) Heredity 36: 191-203 8. Daboussi, M.J. & Langin, T. (1994) Genetica 93:49-59 9. Lebrum, M.-H. Chumley, F. & Valent, B. (1994) Fungal Genetics News Leffer 41 A: 52 10. Kachroo, P., Leong, S.A. & Chattoo, B.B. (1994) Mol. Gen. Genet. 245:339-348 11. Rowlands, R.T. &Turner, G. (1973) Mol. Gen. Genet. 126:201-216 12. Sambrook, J., Fritsch, E.F. & Maniatis, T. (1989) Molecular Clonin~: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Plainview, NY, pp. 1.11-1.85 13. Timberlake, W.E. & Barnard, E.C. (1981) Cell26:29-37 14. Yanisch-Peron, C., Vieira, J. & Messing, J. (1985) Gene 33:103-119 15. Meseselson, M . & Yuan, R. (1968) Nature 217: 1110-1114 16. Charlesworth, B., Snlegowski, P. & Stephan, W. (1994) Nature 371:215-220.

17. Fedoroff, N,V., Furtek, D.B. & Nelson, O.E. (1984) Proc. NatlAcad. Sci. USA 81 :3829-3835 18. Dunn-Coleman, N.S., Tomsch, A.D. & Garrett, R.H. (1981) Molec. Gen. Getet.
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awamori," Curr. Genet. 29:468-473 CA 02262sl9 l999-02-03 W 098/08960 PCTrUS97/14978 23. Bancroft, 1., Bhatt, A., Sjodin, C., Scofield, S., Jones, J. & Dean, C. (1992) Mol.
Gen. Genet. 233:449-461 24. Bancroft, 1. & Dean, C. (1993) Mol. Gen. Genet. 240:65-67 25. Long, D., Martin, M., Sundberg, E., Swinburne, J., Puangsomlee, P. & Coapland, G.
(1993) Proc. Natl. Acad. Sci. 90:10370-10374 26. Berka et al. (1990) Gene 86:153-162 27. Ochman, H., Gerber, A.S. & Hart, D.L. (1988) Genetics 120:621-623 28. Williams, J.F. (1989) Biofechniques 7:762-769 29. Arnold, C. & Hodgson, I.J. (1991) PCR Methods Appl. 1:39-42 30. Jones, D.H. & Winistorfer, S.C. (1993) PCR Methods Appl. 2:197-203 31. Brujin, F.J. & Lupski, J.R. (1984) Gene 27:131-149 32. Brown, T. Current Protocols in Molecular Biology~ Supplements 21,24,26 and 29 33. Minetoki, T., Gomi, K., Kitamoto, K., Kumagai, C., Tamura, G. (1995) "Nucleotide sequence and expression of alpha-glucosidase-encoding gene (agdA) from aspergillus oryzae," Biosci. Biotechnol. Biochem. 59:1516-1521 CA 02262~l9 l999-02-03 Sequence Listinq (1) GENERAL INFORMATION:
(i) APPLICANT: Amutan, Maria Dunn-Coleman, Nigel Nyyssonen, Eini M.
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ATGGGTCACT CCAATTGAGT GTATTCGC'rC TAATGGAGAG GTTCTACCTT CGACCCTGAT 780 AAGTGGCTCA GTGCTTCAAA CACCACATAA TATAAAACAC CTTTTGGAGC ATCCA~AATC 1380 CA 02262~l9 l999-02-03 W O 98/08960 PCT~US97/14978 (2) INFORMATION FOR SEQ ID NO: 7:
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(A) LENGTH: 555 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7:
Met Pro Pro Lys Ala Ser Ile Pro Ser Lys Ser Gln Val Glu Gln Glu Gly Arg Ile Leu Leu Ala Ile Glu Ala Ile Gln Lys Gly Gln Ile Thr Ser Ile Arg Glu Ala Ala Arg Val Tyr Asp Val Ala Arg Thr Thr Leu Gln Ala Arg Leu Ser Gly Arg Val Phe Ala Lys Asn Met Thr Asn Ala Arg Gln Lys Leu Ser Asn Asn Glu Glu Glu Ser Leu Val Lys Trp Ile Leu Ser Leu Asp Lys Arg Gly Ala Ser Pro Arg Pro Leu Asp Ile Arg Asp Met Ala Asn Leu Ile Ile Ser Lys Arg Gly Tyr Ser Thr Val Glu . .

CA 02262~19 1999-02-03 Gln Val Gly Ile Asn Trp Ala Tyr Ser Phe Val Lys Arg His Glu Ser Leu Arg Thr Arg Phe Ala Arg Arg Leu Asn Tyr Pro Arg Ala Lys Met Glu Asp Pro Glu Val Ile Lys Asp Trp Phe Gln Arg Val Gln Glu Val ~le Gln Glu Tyr Gly Ile Ser Ser Asp Asp Ile Tyr Asn Phe Asp Glu ~hr Gly Phe Ala Met Gly Met Ile Ala Thr Tyr Lys Val Val Thr Ser Ser Gln Arg Ala Gly Arg Pro Ser Leu Val Gln Pro Gly Asn Arg G~ u Trp Val Thr Pro Ile Glu Cys Ile Arg Ser Asn Gly Glu Val Leu Pro Ser Thr Leu Ile Phe Lys Gly Lys Thr His Leu Lys Ala Trp Tyr Glu ~ly Gln Ser Ile Pro Pro Thr Trp Arg Phe Glu Val Ser Asp Asn Gly ~rp Thr Thr Asp Lys Ile Gly Leu Arg Trp Leu Pro Lys His Phe Ile Pro Leu Ile Arg Gly Lys Ser Val Gly Lys Tyr Ser Leu Leu Val Leu Asp Gly His Gly Ser His Leu Thr Pro Glu Phe Asp Gln Ser Cys Ala Glu Asn Glu Val Ile Pro Ile Cys Met Pro Ala His Ser Ser His Leu ~eu Gln Pro Leu Asp Val Gly Cys Phe Ser Val Leu Lys Arg Thr Tyr ~ly Gly Met Val Pro Lys Gln Met Gln Tyr Gly Arg Asn His Ile Asp Lys Leu Asp Phe Leu Glu Val Tyr Pro Lys Ala His Gln Cys Ala Leu Ser Lys Ser Asn Ile Ile Ser Gly Phe Arg Ala Thr Gly Leu Val Pro Leu Asp Pro Asp Gln Val Leu Ser Arg Leu His Ile Arg Leu Lys Thr ~ro Pro Thr Pro Asp Ser Gln Ser Ser Gly Ser Val Leu Gln Thr Pro ~is Asn Ile Lys His Leu Leu Glu His Pro Lys Ser Val Glu Arg Leu ~eu Arg Lys Arg Gln Ala Ser Pro Thr Ser Pro Thr Asn Ser Thr Leu CA 02262~l9 l999-02-03 W O 98/08960 PCT~US97/14978 Arg Gln Leu Leu Lys Gly Cys Glu Leu Ala Ile Thr Asn Ser Ile Ile Leu Ala Lys Glu Asn Ala Glu Leu Arg Ala Ser His Glu Lys Gln Leu Pro Lys Arg Lys Arg Ser Arg Lys Gln Val Ile Tyr Thr Glu Gly Thr Thr Val Glu Glu Ala Gln Arg Ala Ile Gln Glu Val Glu Glu Val Gln Asn Asp Glu Asp Ile Glu Val Glu Pro Gln Ser Gln Tyr Thr Glu Thr Pro Ser Arg Ala Pro Pro Arg Cys Ser Asn Cys Phe Asn Ile Gly His Arg Arg Thr Gln Cys Ser Lys Pro Pro Thr Asn (2) INFORMATION FOR SEQ ID NO: 8:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 23 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8:

(2) INFORMATION FOR SEQ ID NO: 9:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 24 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
~xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9:

(2) INFORMATION FOR SEQ ID NO: 10:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 54 base pairs (B) TYPE: nucleic acid ~C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10:

CA 02262~l9 l999-02-03 (2) INFORMATION FOR SEQ ID NO: 11:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11:

~2) INFORMATION FOR SEQ ID NO: 12:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12:

(2) INFORMATION FOR SEQ ID NO: 13:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 2329 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 13:

CA 02262~l9 l999-02-03 W 098/08960 PCT~US97/14978 ACCATCCAGG TCTAGGAGAA TTAGATTTCT TCTATATAGA TTTTAAATAA TATA~ATAAT 2040 (2) INFORMATION FOR SEQ ID NO: 14:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 555 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: single CA 02262~l9 l999-02-03 W 098/08960 PCT~US97114978 (D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein ~xi) SEQUENCE DESCRIPTION: SEQ ID NO: 14:
Met Pro Pro Lys Ala Ser Ile Pro Ser Lys Ser Gln Val Glu Arg Glu ~ly Arg Ile Leu Leu Ala Ile Glu Ala Ile Arg Lys Gly Gln Ile Thr Ser Ile Arg Glu Ala Ala Arg Val Tyr Asp Val Ala Arg Thr Thr Leu Gln Ala Arg Leu Ser Gly Arg Val Phe Ala Lys Asn Met Thr Asn Ala Arg Gln Lys Leu Ser Asn Asn Glu Glu Glu Ser Leu Val Lys Trp Ile ~eu Ser Leu Asp Lys Arg Gly Ala Ser Pro Arg Pro Leu Asp Ile Arg ~sp Met Ala Asn Leu Ile Ile Ser Lys Arg Gly Tyr Ser Thr Val Glu Gln Val Gly Ile Asn Trp Ala Tyr Ser Phe Val Lys Arg His Glu Ser Leu Arg Thr Arg Phe Ala Arg Arg Leu Asn Tyr Gln Arg Ala Lys Met Glu Asp Pro Glu Val Ile Lys Asp Trp Phe Lys Arg Val Gln Glu Val ~le Gln Glu Tyr Gly Ile Ser Ser Asp Asp Ile Tyr Asn Phe Asp Glu ~hr Gly Phe Ala Met Gly Met Ile Ala Thr Tyr Lys Val Val Thr Ser Ser Gln Arg Ala Gly Arg Pro Ser Leu Val Gln Pro Gly Asn Arg Glu Trp Val Thr Ala Ile Glu Cys Ile Arg Ser Asn Gly Glu Val Leu Pro Ser Thr Leu Ile Phe Lys Gly Lys Thr His Leu Lys Ala Trp Tyr Glu ~ly Gln Ser Ile Pro Pro Thr Trp Arg Phe Glu Val Ser Asp Asn Gly ~rp Thr Thr Asp Lys Ile Gly Leu Arg Trp Leu Gln Lys His Phe Ile Pro Leu Ile Arg Gly Lys Ser Val Gly Lys Tyr Ser Leu Leu Val Leu Asp Gly His Gly Ser His Leu Thr Pro Glu Phe Asp Gln Ser Cys Ala CA 02262~19 1999-02-03 Glu Asn Glu Val Ile Pro Ile Cys Met Pro Ala His Ser Ser His Leu Leu Gln Pro Leu Asp Val Gly Cys Phe Ser Val Leu Lys Arg Thr Tyr Gly Gly Met Val Gln Lys Gln Met Gln Tyr Gly Arg Asn His Ile Asp Lys Leu Asp Phe Leu Glu Val Tyr Pro Lys Ala His Gln Cys Ala Leu Ser Lys Ser Asn Ile Ile Ser Gly Phe Arg Ala Thr Gly Leu Val Pro Leu Asp Pro Asp Gln Val Leu Ser Arg Leu His Ile Arg Leu Lys Thr Pro Pro Thr Pro Asp Ser Gln Ser Ser Gly Ser Val Leu Gln Thr Pro His Asn Ile Lys His Leu Leu Lys His Pro Lys Ser Val Glu Arg Leu 420 425 q30 Leu Arg Lys Arg Gln Ala Ser Pro Thr Ser Pro Thr Asn Ser Thr Leu Arg Gln Leu Leu Lys Gly Cys Glu Leu Ala Ile Thr Asn Ser Ile Ile Leu Ala Lys Glu Asn Ala Glu Leu Arg Ala Ser His Glu Lys Gln Leu Pro Lys Arg Lys Arg Ser Arg Lys Gln Val Ile Tyr Thr Glu Gly Thr Thr Val Glu Glu Ala Gln Arg Ala Ile Gln Glu Val Glu Glu Val Gln Asn Asp Glu Asp Ile Glu Val Glu Pro Gln Ser Gln Tyr Thr Glu Thr Pro Ser Arg Ala Pro Pro Arg Cys Ser Asn Cys Phe Asn Ile Gly His Arg Arg Thr Gln Cys Ser Lys Pro Pro Thr Asn (2) INFORMATION FOR SEQ ID NO: 15:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 44 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA

(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 15:

(2) INFORMATION FOR SEQ ID NO: 16:

CA 02262~l9 l999-02-03 (i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 45 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ID NO: 16:

(2) INFORMATION FOR SEQ ID NO: 17:
(i) SEQUENCE CHARACTERISTICS:
(A) LENGTH: 44 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA
(xi) SEQUENCE DESCRIPTION: SEQ ~D NO: 17:

Claims (20)

What is Claimed

1. A transposable element isolated from Aspergillus niger var. awamori comprising a DNA fragment of about 2.3 kb which comprises SEQ ID NO: 1.

2. The transposable element of Claim 1 comprising the DNA sequence of SEQ ID
NO:13 or variations thereof.

3. A fragment of the transposable element of Claim 1 comprising part or all of the DNA
sequence selected from the group consisting of SEQ ID NOS:1 and 16 or variationsthereof.

4. An isolated transposase coded for by the transposable element of Claim 1.

5. The transposase of Claim 4 comprising the amino acid sequence of SEQ ID NO:14.

6. A method of isolating a transposable element from a filamentous fungus, comprising the steps of:
(a) hybridizing fungal DNA under low stringency conditions to a probe, wherein the probe comprises part or all of one of the DNA fragments of Claim 3; and (b) isolating fungal DNA which hybridizes to said probe.

7. The method of Claim 6 wherein the probe comprises an imperfect direct repeat within the DNA sequence selected from the group consisting of SEQ ID NOS:1 and 16.

8. A method of isolating a transposable element from a filamentous fungus genomic library, the method comprising probing said library with an ORF-specific probe and isolating DNA which hybridizes to said ORF-specific probe.

9. A method of isolating a transposable element from a filamentous fungi, the method comprising, (a) subjecting fungal DNA to polymerase chain reaction amplification using part or all of one of the DNA fragments of Claim 3 as a primer, thereby generating amplified DNA sequences;
(b) isolating the amplified DNA sequences, and (c) optionally identifying said amplified DNA sequence.

10. A transposable element isolatable by the method of Claim 6, 7, 8 or 9.

11. A method of isolating activation sequences comprising:
(a) inserting a marker gene within the inverted repeats of a transposable element of Claim 1 to form a modified marker gene having the structure IR-marker-IR;
(b) inserting the modified marker gene into a DNA target;
(c) selecting for expression of the modified marker; and (d) isolating DNA upstream of said modified marker gene in said DNA target, which DNA upstream of said modified marker gene comprises an activation sequence driving expression of said modified marker gene.

12. A method for inactivating a gene in a host cell wherein said gene encodes a gene product, the method comprising:
(a) transforming a host cell with a genetic element to create a transformed host cell, wherein the genetic element comprises DNA for the gene and a transposable element of Claim 1 inserted within the DNA; and (b) selecting for the transformed host cells which are deficient in the gene product.

13. A method for activating a desired gene in a host cell, the method comprising:
(a) inserting a regulatory gene within the inverted repeats of a transposable element of Claim 1 to form a modified regulatory gene having the structure IR-regulatory gene-IR;
(b) inserting the modified regulatory gene in DNA comprising the desired gene toform a DNA construct containing the modified regulatory gene upstream of said desired gene;
(c)transforming the host cell with the DNA construct; and (d) selecting for transformants having enhanced expression of said desired gene.

14. A transposable element isolated from Aspergillus oryzae comprising a DNA
fragment of at least about 1.2 kb.

15. The transposable element of Claim 14 comprising an inverted repeat DNA
sequence of SEQ ID NO:17 or a variation thereof.

16. A fragment of the transposable element of 14 comprising part of all of the DNA
sequence of SEQ ID NO:17 or a variation thereof.

17. A method of isolating a transposable element from a filamentous fungus, comprising the steps of:
(a) hybridizing fungal DNA under low stringency conditions to a probe, wherein the probe comprises part or all of one of the DNA fragments of Claim 16;
and (b) isolating fungal DNA which hybridizes to said probe.

18. A method of isolating activation sequences comprising:
(a) inserting a marker gene within the inverted repeats of a transposable element of Claim 10 to form a modified marker gene having the structure IR-marker-IR;
(b) inserting the modified marker gene into a DNA target;
(c) selecting for expression of the modified marker; and (d) isolating DNA upstream of said modified marker gene in said DNA target, which DNA upstream of said modified marker gene comprises an activation sequence driving expression of said modified marker gene.

19. A method for inactivating a gene in a host cell wherein said gene encodes a gene product, the method comprising:
(a) transforming a host cell with a genetic element to create a transformed host cell, wherein the genetic element comprises DNA for the gene and a transposable element of Claim 10 inserted within the DNA; and (b) selecting for the transformed host ceils which are deficient in the gene product.

20. A method for activating a desired gene in a host cell, the method comprising:
(a) inserting a regulatory gene within the inverted repeats of a transposable element of Claim 10 to form a modified regulatory gene having the structure IR-regulatory gene-IR;
(b) inserting the modified regulatory gene in DNA comprising the desired gene toform a DNA construct containing the modified regulatory gene upstream of said desired gene;
(c) transforming the host cell with the DNA construct; and (d) selecting for transformants having enhanced expression of said desired gene.