IDENTIFICATION AND CLONING OF A MOBILE TRANSPOSON FROM ASPERGILLUS

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

Description: IDENTIFICATION AND CLONING OF A MOBILE TRANSPOSON FROM ASPERGILLUS FIELD OF THE INVENTION The present invention is directed at the identification, cloning and sequencing of a mobile transposon or transposable element from Aspergillus niger var. awamori. The transposable element, referred to as Vader, is an approximately 440 base pair (bp) element bounded by an inverted repeat sequence of approximately 44 base pairs. The transposable element targets 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 44 bp inverted repeat isolated from Aspergillus niger war. awamori. Also provided are methods for making mutants using the transposable element(s) of the present invention by inactivating genes via insertion of the transposable element(s) into a target gene and, alternatively, to activate or turn on genes using the transposable element(s) as described herein. BACKGROUND OF THE INVENTION It is well know that DNA segments, called transposons, are able to insert into many sites in the genome of their host organisms. 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 a spontaneous 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 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, confirming that Tad was a retrotransposon and that there was a cytoplasmic phase involved in the retrotransposition events. More recently, Cambareri et al. (4) demonstrated that Tad was a LINE-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 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-retrotransposon from Cladosporium fulvum. This transposon was 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. nidulans 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 cloning of a new transposable element from an unstable niaD mutant . This element, FML {Fusarium manner-like), is 1280 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 Magnaporthe grisea. However, in this case the A. nidulans niaD gene which was transformed into M. gnsea by transformation was used as a transposon trap. The element inserted into the niaD gene was shown to belong to a family of M gnsea LTR-retrotransposons, Fos1 (Schull and Hamer, unpublished) and Magi (Farman and Leong, unpublished). The cloned retro-element was 5.6 kb and the target site (AT ATT) was shown to be duplicated. All revertants from this mutant examined had one copy of the LTR Jβft 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 fingerprint patterns of repetitive DNA's which were cloned from the M. grisea 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 TIRs. 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. niger var. awamori. This element, Vader, is present in approximately 15 copies in A. niger and A. niger var. 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, a novel eukaryotic transposable element from Aspergillus niger var. awamori is provided. The novel transposable element is an approximately 440 bp element which comprises a 44 bp inverted repeat sequence at either end of the transposable element. The target for insertion of this novel transposable element 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. Another embodiment of the present invention comprises a fragment of the transposable element comprising a 44 bp inverted repeat sequence found at either terminus of the transposable element from A. niger var. awamori, as well as the use of said 44 bp fragment as a DNA probe useful in isolating and/or cloning transposable elements from other filamentous fungi. Using the inverted repeat of Vader, a transposon from A. niger var. awamori (Tan) was cloned. In a process embodiment of the present invention there are provided methods for gene tagging comprising using the transposable elements of the present invention 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 element to become inserted 5' to a desired gene, the promoter may be activated to drive the expression of the desired gene product. BRIEF DESCRIPTION OF THE DRAWINGS Fig. 1 shows the Southern blot analysis of unstable niaD mutants. Fungal genomic DNA from four niaD mutants and UVK143 were digested with Bglll (sites are 3' of all inserts). Blot probed with 500 bp fragment of Sail digested PCR product of niaD1 and niaD2. DIG- labeled probe concentration was 20 mg/mL prehybridization solution. Wild-type band hybridizes at 2.5 kb while gene with insertion hybridizes at 2.9 kb. Lanes: 1=n/aO392; 2-niaD 587; 3=n aD436; 4=n aD410; 5=UVK143; 6=MW marker III (Boehringer Mannheim). Fig. 2 depicts the mapping of Vader insertions within the niaD gene. The positions of Vader insertions 1-4 are shown relative to the six introns of the structural gene coding region. Because the exact site of insertion for Vader-4 is still unknown, it has been presented using the approximate area of its insertion. Relevant restriction sites are shown using the following letters: E=EcoRI, S=Sall, Sp=Sphl, K=Kpnl, and B=Bglll. Fig. 3 depicts the location of Vader 2 in the niaD gene. The sequence shown here begins at +0.730 kb of niaD. The 2 bp TA duplication is indicated in bold and the 44 bp inverted repeats of Vader are boxed. The flanking region from the DNA sequence of the niaD gene are shown on either side of the Vader insert. Seq. ID No. 1 comprising flanking DNA from the niaD gene, is shown on the left (5') of the Vader insert, ending with the target sequence "TA." Seq. ID No. 2 comprising flanking DNA from the niaD gene, is shown on the right (3 ) of the Vader insert, starting with the target sequence "TA" duplication seen with the Vader insert. Fig. 4 shows Southern blot analysis to determine Vader genomic copy number. Four A. niger var. awamori niaD mutants and UVK143 were digested with EcoRV to completion. EcoRV cuts the Vader sequence once. Hybridization indicates that Vader is present in the genome in more than 14 copies. The hybridizing bands of niaD 392, which are different from the other mutants and UVK143, suggest that the sequence is mobile. Lanes: 1=MW marker III, 2=UVK143, 392. Fig. 5. Southern blot to determine presence of Vader sequence in other fungi. Other filamentous fungi, an industrial production strain and niaD mutant 392, were digested with EcoRV to completion. Low stringency hybridization indicates that sequences homologous to Vader are present in A. nidulans (FGSC A691), A. cinnamomeus, A. phoenicis, A. foetidus, an industrial A. niger strain. Lanes: 1=MW marker III, 2=A. nidulans (FGSC A691), 3= A. cinnamomeus (ATCC# 1027), 4=A versicolor, 5=A. wentii (ATCC# 10593), 6=A. nidulans (FGSC A237), 7=A. phoenicis (ATCC# 11362), 8=A foetidus, 9=an industrial glucoamylase production strain of A. niger (ETC# 2663), 10=A. niger var. awamori niaD mutant 392. Fig. 6. Southern blot to determine Tan (transposon from A. nigeή genomic copy number. Four niaD mutants A. niger var. awamori mutants and UVK143 were digested with EcoR1 to completion. EcoR1 cuts the Tan sequence once. Hybridization indicates that Tan is present as a single copy in the genome. Lanes: 1=MW marker III, 2=UVK143, 3=n aD410, Fig. 7. Southern blots to determine if the inverted repeats of transposable elements Fot1 and Pot2 will hybridize to elements in A. niger var. awamori. Four niaD mutants A. niger var. awamori mutants were digested with EcoR1 to completion. EcoR1 cuts the Tan sequence once. Inverted repeat probes of Tan (Seq. ID No. 5), Fot1 , Pot2 were oligo-tailed using Digoxigenin (Boehringer Mannheim, 1992). Lanes: 1=MW marker III, 2=n/aD436, 3=n aD587. Blots A, B, and C were probed with the labeled inverted repeat probes of Tan, Fot1 and Pot2, respectively. Fig. 8 diagramatically shows the Tan organization where the 2.4 kb element includes 4 inverted repeat sequences, as well as the approximately 440 bp Vader insertion sequence. Due to putative stem loop structure, the complete sequence of Tan in the dashed area between the larger fragment and Vader is unknown (as designated by "N" in the figure and corresponding sequences). Fig. 9 shows the sequence of the Vader insertion (Seq. ID No. 3). Vader was found to be 437 bp in length. The 44 bp inverted repeat of the Vader insert corresponding to Seq. ID No. 4 and Seq. ID No. 5, respectively, 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. Fig. 10 shows the entire DNA sequence of the Tan element (Seq. ID No. 6), as well as the putative amino acid sequence of the transposase coded for by Tan (Seq. ID No. 7). Tan is 2320 bp in length and has a large open reading frame of 1668 bp which encodes for 555 amino acids (Seq. ID No. 7). Tan comprises the sequences of four inverted repeats (underlined) similar to those found in Vader. 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. EXPERIMENTAL Applicants have demonstrated that mobile transposable elements exist in A niger var awamori. The clone element, Vader, is present in approximately fifteen copies in the two 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 SUBSTITUTE SHEET (PULE 26) repeated manipulation of such strains and the possible discarding of aberrant A. nidulans strains displaying genetic instability. It is more difficult to explain why there are very few Vader sequences present in the other Aspergillus species examined. The Vader element shows similarities to transposable elements cloned from the plant pathogens Pot1 from M. grisea (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 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. 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 the Vader element is not closely related to either the previously described Fot1 and Pot2 transposons (see Fig. 7) . 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) 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. Vader has a 44 bp inverted terminal repeat, this indicates that Vader probably transposes through DNA copies. The Vader inverted repeats shown in Fig. 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, is too small (about 440 bp) to encode for an transposase protein. Defective elements that lack transposase activity can transpose in the presence of a transposase encoded by a complete element elsewhere in the genome. For example, the maize Ac transposon encodes a fully functional transposase. However, maize Ds elements, although capable of excision, cannot excise unless a functional Ac element is present (17). These results indicate that for the Vader element to transpose, a transposase activity must be present in the genome of a larger transposon. Recent results using the Vader 44 bp inverted repeat (Seq. ID No. 5) to perform PCR using A. niger var. awamori genomic DNA resulted in the generation of two amplified DNA sequences, one sequence 1.8 kb in length and the second sequence of about 2.2 kb in length. This second sequence, called Tan (Seq. ID No. 6), comprises four inverted repeats similar to those in Vader. Tan is 2322 bp in length and has a large open reading frame (1668 bp) which encodes for a putative transposase comprising 555 amino acids (shown in Seq. ID No. 7). MATERIAL AND METHODS Strains. Spontaneous chlorate resistant mutants were derived from Aspergillus niger var. awamori UVK143, Northern Regional Research Laboratories (NRRL# 3112). The following Aspergillus strains were obtained from the ATCC: A. cinnamomoneus (ATCC# 1027), A. wentii (ATCC# 10593), and A. phoenicis (ATCC# 11362; A. nidulans (FGSC# A237), a nitrate reductase structural gene mutant (n/aD15), and A. nidulans (FGSC# A691), a tryptophan requiring mutant (fφC801), were obtained from Fungal Genetics Stock Center (FGSC). A. versicolor, A. foetidus, and a proprietary A. niger glucoamylase strain are from the Genencor International Inc. culture collection. Escherichia coli strains JM101 (15) and MM294 (16) were used for amplification of plasmids as described (12). o Mutant Selection. Spore suspensions (1 x 10 ) of UVK143 were plated on CM agar (11) containing 600 mM KCIO 3 and 10 mM glutamic acid. Chlorate (KCI0 3 ), a toxic analog of nitrate, allows selection of mutants in the nitrate assimilation pathway by chlorate resistance. The small amount of glutamic acid in the medium reduces space competition between mutant and wild-type spores by inducing the nitrate assimilation pathway, and thus the uptake of the toxic chemical (chlorate) by wild-type spores. Plates were incubated at 37°C until individual colonies of spontaneous mutants could be identified. Single mutants resistant to KCIO 3 were allowed to sporulate on CM plates and spores from these plates were then streaked onto minimal media (11) with various sole nitrogen sources (10 mM): NaN0 3 (nitrate), NaNO 2 (nitrite), hypoxanthine, uric acid or NH 4 CI (ammonium chloride). Each of these compounds are intermediate products of the nitrate assimilation pathway. niaD mutants were identified as those resistant to KCI0 3 and able to grow in the presence of all pathway intermediates, except for NaN0 3 . PCR Amplification. Genomic DNA of A. niger var. awamori niaD mutants and UVK143 was used as template (see Southern 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 No. 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 mixture contained 4 mM MgS0 4 . 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). To determine if other sequences exist which recognize the inverted repeats of Vader, a single primer (IR1) was synthesized for use with A. niger genomic DNA. The sequence of IR1 is 5'-ATA-TGA-ATT-CAC-GTA-ATC-AAC-GGT-CGG-ACG-GGC-CAC-ACG-GTC-AGG- CGG-GCC-ATC-3' (Seq. ID No. 10). Reaction mix included: Vader mutants and UVK143 fungal genomic DNA, Vent (exo') DNA polymerase buffer, 100 pmol IR1 primer, 250 mM each of dATP, dCTP, dTTP and dGTP, DMSO, and 4 units Taq DNA polymerase. The amplifications were conducted as follows: 1 cycle of 10 min. at 94°C, 30 cycles of 1 min. at 94°C, 1 min. at 55°C, 1 min. at 72°C and one cycle of 15 min. at 72°C. Reactions were pooled, run and extracted as in the niaD reactions. Template DNA from n/aD436, derived from a purified colony grown in the presence of KCI0 3 , 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): δ'-ATATGAATTCCTTCTTGACTTCCCCGGAAC-S' (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. Estimation of niaD Mutant Reversion Frequency. Spores from niaD mutants n/aD392, n aD410, n/aD436 and niaD587 were streaked onto minimal media containing NaN0 3 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 chlorate (KCIO 3 ) and incubated to confluency at 37°C. Ten-fold dilution series of spore suspensions (in 0.8% NaCI-0.25% Tween 80) of n/ ' aD392, n/aD410, n aD436, n/aD587 and UVK143 wild-type spores were plated on minimal media with nitrate (10 mM) 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 KCIO 3 in order to reduce reversion of niaD back to the wild-type during cultivation. DNA (10 μg) 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 μg) 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 membrane (Boehringer Mannheim) by capillary action. The DNA probe for the niaD gene was derived from the PCR product (UVK143 DNA template amplified with primers NiaD1 (Seq. ID No. 8) and NiaD2 (Seq. ID No. 9)), which was digested with Sail, resulting in a 528 bp probe fragment. The probe for the insertion element, Vader, was derived from a PCR reaction in which n/aD436 DNA was used as a template. This PCR product was purified and digested with Sail and Sphl and subcloned into the vector pUC19. This subclone was digested with Seal and Xbal to yield a 236 bp fragment which was used for estimation of the copy number of Vader sequences in the genomes of various fungi. A DNA labeling and detection kit (Geniusl , 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 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 of DNA from other Aspergillus sp.) was conducted using hybridization buffer with 25% formamide at 37°C. Washes were performed as in stringent wash protocol. Nitrate Reductase Assays. Nitrate reductase assays were performed as described in Dunn-Coleman, et al. (18). DNA analysis and Sequence Determination. Sequences were determined using dye Deoxy terminators and Taqr 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 sequences were analyzed and compared to those in GenBank, EMBL and Prot-Swiss using the Genetics Computer Group, Inc. software package (Madison, Wl). Example 1 Isolation of Spontaneous High Frequency Reverting niaD Mutants of A. niger var. awamori Assuming that niaD mutants which arise from the insertion of a transposable 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 Conidiaplated No. Wild-Type Frequency Mutant No. x 10 3 Colonies x lO -4 n aD392 2.9 27 93 n aD436 3.7 164 443 n aD587 18.9 12 6.3 There appeared to be two classes of niaD mutants which reverted at high frequency. The niaD mutants n/aD436 and n aD392 reverted at high frequency, while mutants π/aD410 and niaD5B7 yielded smaller numbers of revertant colonies. The level of nitrate reductase activity was determined by revertant colonies isolated from two niaD mutants. Nitrate reductase activity was detected in all six n/aD410 mutants In the case of n/ ' aD436 revertants, nitrate reductase activity could be detected in 3 of 6 revertants analyzed. Example 2 Cloning of a Vader Element To determine if an insertion sequence was located within the niaD gene, two pπmers were synthesized. The first primer, niaDI (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) in the niaD gene. For Southem 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 Sail digestion fragment of the 500 bp PCR product generated using the niaDI (Seq. ID No. 8) and nιaD2 (Seq. ID No. 9) oligomeric probes (see Fig. 1). The probe hybridized to a 2.5 kb fragment with wild-type DNA (lane 5). In the case of the niaD mutants 410 (lane 1), 436(lane 3) and 392 (lane 4), 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 n aD587, 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 KCI0 3 to favor growth of the niaD mutant and not reverent 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 Example 3 Determination of Vader Copy Number To determine the Vader copy number a 236 bp Sca1-Xba1 internal fragment of Vader-2 (cloned from the mutant n aD436) was hybridized to EcoRV cleaved genomic DNA There is only one EcoRV site within the Vader transposon (Fig 2) Southern blot analysis indicated that there are approximately fifteen copies of Vader sequences in the genome of A niger var awamori (Fig 4). The Vader sequences were integrated at identical genomic locations in the three niaD mutants, 410, 436 and 587 However, in the n/aD392 mutant, Vader sequences were located in five different locations compared to the three niaD 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 could be detected. Although some of the hybridization patterns appeared to be identical, clear differences could be seen between A. niger var. awamori and A. niger. Example 4 Isolation of Vader in Other Fungal Species In an attempt to determine if this transposable element was found in other filamentous fungi, genomic Southem blot analysis was performed using the 236 bp fragment (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 (n/aD15), and FGSC# A237, a tryptophan-requiring mutant (frpC801). No hybridization signals could be visualized with strain A691 , and a single strong hybridization signal could be detected with strain A691. 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. Similariy, only 2 hybridization signals could be detected in A. foetidus and A. phoenicis, and 1 hybridization signal was detected in A. cinnamomeus. No hybridizations could be detected in A. wentii and A. versicolor. In addition, no hybridization signals could be detected with Humicola grisea var. thermoidea, Neurospora crassa and Trichoderma reesei (results not shown). These results indicate that the Vader element is most commonly found in A. niger var. awamori and A. niger. Example 5 Excision of the Vader Element Part of the niaD gene from n aD436 containing the Vader element was amplified using PCR. The PCR amplification resulted in the expected 1200 bp fragment of the Vader element flanked by niaD 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 n/aD436 and n/aD410 were assayed for their nitrate reductase activity (18), a spectrum of activities were detected (results not shown). Example 6 Insertional Inactivation/Gene Tagging 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, KCIO 3 . It is possible that one of the reasons heterologous protein production in fungi is lower than that of homologously produced protein using the same promoter is that the heterologous protein is being degraded by the cell. If there are genes whose products are responsible for degrading/sequestering 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 Tan 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 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 the transposon Tan located on the replication vector pHELP (20) (as per Scheme 1 below) Once the strain has been transformed a second time, a spore suspension is made from transformants Genencor International, Inc. has shown that the pHELP plasmid is not found in spores (i e , when spores are made, the pHELP plasmid is excluded from the spore) (unpublished) Making a spore suspension will then "freeze" the transposon in the genome, i e , it will not be able to move again after the spore isolation phase because the transposase required for excision is now missing from the cell 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 for cloning strategies (See generally (19) ) — To — Scheme 1 Vectors Useful in Gene Tagging -pHELP VADER Tan Selectable Marker for Transformation (linearized version) -pHELP Tan Selectable Marker for Transformation (linearized version) Scheme 1 : The Vader component in these two vectors will have a unique DNA sequence inserted into it between the inverted repeats to allow it's cloning from strains which already have copies of Vader present in it. Example 7 Elevation of Gene Expression Using Transposons A reason that heterologous protein production is lower than expected in fungi is presumed to be that genes essential for foreign (heterologous) gene production are NOT 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 Tan gene is inactivated by gene disruption. This strain is used to express a heterologous protein whose expression can be easily visualized, such as chymosin (US Patent 5,364,770) using the following vector: pHELP VADER** Tan Selectable Marker for Transformation (linearized version) "Detail of VADER: -IVR Regulatable Promoter Such as Glucoamylase IVR- One of the many integration events possible will be the integration of the Vader element 5' to a gene, which if elevated in activity by using a very strong promoter such as glucoamylase, will enhance the secretion of the heterologous protein (i.e. , chymosin) For example, see Scheme 2 below. Scheme 2 5 1 VADER** Gene Required for Secretion 3' > Elevated Transcription of Genes 3' to VADER Spore suspensions will be isolated from transformants. As previously discussed, the spore isolation step results in the elimination of the pHELP (20) vector from the strain and "freezes" the transposon integrated into the genome. The spores are then plated out and transformants which make more chymosin characterized. Then, using conventional cloning strategies, the gene 3' to Vader can be cloned. (See generally (19), incoφorated herein by reference.) 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, I.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 Letter 41A: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 Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Plainview, NY, pp. 1.11-1.85 13. Timberiake, W.E. & Barnard, E.C. (1981) Cell 26:29-37. 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. Natl Acad. Sci. USA 81 :3829-3835 18. Dunn-Coleman, N.S., Tomsch, A.D. & Garrett, R.H. (1981) Molec. Gen. Getet. 182:234-239 19. Walden, R. & Schell, J. (1994) Agro-Food-Industry-Hi-Tech, Nov/Dec:9-12 20. Gems, D.H., Johnstone, I.L & ClutteΦuck, A.J. (1991) Gene 98:61-67 — IB — SEQUENCE LISTING (1) GENERAL INFORMATION: (i) APPLICANT: Amutan, Maria Dunn-Coleman, Nigel Nyyssonen, Eini M. (ii) TITLE OF INVENTION: Identification of and Cloning a Mobxle Transposon from Aspergillus (iii) NUMBER OF SEQUENCES: 12 (iv) CORRESPONDENCE ADDRESS: (A) NAME: Genencor International, Inc. (B) STREET: 180 Kimball Way (C) CITY: So. San Francisco (D) STATE: CA (E) COUNTRY: USA (F) POSTAL CODE (ZIP) : 94080 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: Floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: Patentin Release #1.0, Version #1.25 (EPO) (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: US (B) FILING DATE: March 21, 1995 (C) CLASSIFICATION: (viii) ATTORNEY/AGENT INFORMATION: (A) NAME: Horn, Margaret A. (B) REGISTRATION NUMBER: 33,401 (C) REFERENCE/DOCKET NUMBER: GC270 (xi) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: (415) 742-7536 (B) TELEFAX: (415) 742-7217 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 15 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 1: GAGGAAAGCC AAGTA 15 (2) INFORMATION FOR SEQ ID NO: 2: (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 16 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 2: TATGTTCTGC ATGGAG 16 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 437 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: ACGTAATCAA CGGTCGAACG GGCCACACGG TCAGGCGGGC CATCCTGAAA TCCCATATAA 60 AAGATGTCTT GGGGATTCTA TTATATATCA ACCAGTACTA CTTCTATGAA GCTCTAACTT 120 TGTAGATAGT TATATATATA AGAATAAGTA TTCCATGAAT TTTTCAGATT TTAGAATTTT 180 TACTTTGATA ATGAAACCAG ATTCTTATAT AAAACATATA AATACAGATA TTGTAATATG 240 ATAAGTCCAT AAGTAAAAGT ATATTCATTT TTAGAAGGTA TATAGATATT ATTTATATTA 300 TTTAAAATCT ATATAGAAGA AATCTAATTC TTCTAGACCT GGATGGTAGA GATATATTAT 360 GTTTAAAAAG ATATCTTTTG TATAGTATTA CCAGATGGCC CGCCTGACCG TGTGGCCCGT 420 CCGACCGTTG ATTACGT 437 (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 44 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: ACGTAATCAA CGGTCGAACG GGCCACACGG TCAGGCGGGC CATC 44 (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 44 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: GATGGCCCGC CTGACCGTGT GGCCCGTCCG ACCGTTGATT ACGT 44 (2) INFORMATION FOR SEQ ID NO: 6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 2315 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 6: ACGTAATCAA CGGTCGGACG GGCCACACGG TCAGGCGGGC CATCCCTTCG AAAACACCAC 60 CTTGAATCAC CTACCCGAGG CTTTTCAACC ACCACAAATG CCACCAAAAG CATCTATCCC 120 ATCAAAATCG CAGGTGGAGC AGGAAGGCAG GATTCTTCTT GCCATTGAAG CTATTCAGAA 180 AGGCCAAATC ACTAGTATTC GTGAAGCAGC GCGTGTTTAT GACGTCGCTC GAACTACTCT 240 CCAGGCTCGA TTATCTGGAC GTGTTTTCGC TAAAAATATG ACCAACGCAC GTCAAAAATT 300 GTCAAATAAT GAAGAGGAAT CGCTTGTTAA ATGGATCCTA TCTCTAGATA AGCGAGGAGC 360 AAGCCCCCGG CCACTTGATA TCAGAGATAT GGCTAATTTG ATTATCTCTA AACGAGGTTA 420 TTCAACTGTT GAACAAGTAG GCATCAACTG GGCTTATAGC TTTGTTAAAC GCCACGAATC 480 CCTACGAACT CGATTTGCTA GACGACTCAA CTATCCAAGA GCTAAAATGG AGGATCCTGA 540 AGTTATAAAA GACTGGTTCC AACGCGTACA GGAAGTTATT CAAGAGTACG GGATCTCATC 600 AGATGATATA TACAATTTCG ATGAAACAGG GTTTGCTATG GGAATGATTG CTACATATAA 660 AGTAGTAACT AGTTCCCAGA GGGCAGGTCG GCCGTCCCTA GTTCAACCAG GGAATCGGGA 720 ATGGGTCACT CCAATTGAGT GTATTCGCTC TAATGGAGAG GTTCTACCTT CGACCCTGAT 780 CTTTAAAGGC AAAACACATC TAAAGGCATG GTATGAAGGT CAATCTATTC CTCCTACCTG 840 GAGATTTGAA GTCAGTGATA ATGGTTGGAC TACTGATAAA ATTGGACTTC GATGGCTTCC 900 AAAACACTTC ATTCCCTTGA TTAGAGGCAA ATCAGTAGGC AAATATAGCC TCCTAGTCCT 960 CGATGGCCAC GGTAGTCATT TGACACCTGA ATTCGACCAA TCCTGTGCTG AAAATGAGGT 1020 TATACCTATT TGTATGCCAG CTCATTCGTC CCATCTACTT CAGCCTCTTG ATGTTGGTTG 1080 TTTTAGTGTG CTTAAACGCA CGTACGGAGG CATGGTTCCC AAGCAGATGC AATACGGCCG 1140 CAATCATATC GACAAGCTTG ACTTCTTAGA GGTCTATCCT AAAGCTCACC AGTGTGCTTT 1200 ATCAAAGTCG AATATAATCA GTGGTTTTAG AGCAACAGGT CTTGTTCCTC TAGATCCTGA 1260 TCAAGTGCTT TCTCGACTCC ATATTCGCTT GAAAACACCA CCAACCCCGG ATAGCCAGTC 1320 AAGTGGCTCA GTGCTTCAAA CACCACATAA TATAAAACAC CTTTTGGAGC ATCCAAAATC 1380 AGTGGAACGC CTACTTCGGA AACGGCAAGC AAGTCCAACT TCACCTACAA ACTCTACACT 1440 ACGTCAGCTT CTCAAAGGGT GTGAACTAGC AATAACAAAC TCAATCATAC TGGCTAAGGA 1500 GAATGCGGAA TTACGTGCTA GCCATGAAAA GCAACTACCA AAGAGGAAGC GTTCAAGGAA 1560 GCAGGTGATC TATACAGAAG GCACTACCGT TGAAGAGGCC CAGAGAGCTA TACAGGAAGT 1620 GGAAGAGGTG CAGAATGATG AAGATATTGA GGTTGAACCC CAATCTCAAT ATACGGAGAC 1680 CCCCTCGCGC GCGCCTCCAC GCTGCAGTAA TTGCTTCAAT ATAGGCCACC GACGTACACA 1740 GTGTTCTAAA CCACCTACTA ATTAGTTAGA TAGCTGTTTT TACAAGCATT TATGTTGATT 1800 TAGAGGCCTC ATTTGGATCA TATCGGGTAA TCCTACCGGG AGATGGCCCG CCTGACCGTG 1860 TGGCCCGCCC GACCGTTGAT TACGTNNNNN ACGTAATCAA CGGTCGGACG GGCCCCCCGG 1920 TCCGGCGGGC CATCTGGTAA TACTATACCA AAGATATCTT TTTAAACATA ATATATCTCT 1980 ACCATCCAGG TCTAGGAGAA TTAGATTTCT TCTATATAGA TTTTAAATAA TATAA-VTAAT 2040 ATCTATATAC CTTCTAAAAA TGAATATACT TTTACTTATG GACTTATCAT CTGTATTTAT 2100 ATGTATTATA TAAGAATCTG GTTTCATTAT CAAAGTAAAA ATTCTAAAAT CTGAAAAATT 2160 CATGGAATAC TTATTCTTAT ATATATAACT ATCTACAAAG TTAGAGCTTC ATAGAAGTAG 2220 TACTGGTTGA TATATAATAG AATCCCCAAG ACATCTTTTA TATGGGATTT CAGGATGGCC 2280 GCCGACCGTG TGGCCCGTCC GACCGTTGAT TACGT 2315 (2) INFORMATION FOR SEQ ID NO: 7: (i) SEQUENCE CHARACTERISTICS: (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 lie Pro Ser Lys Ser Gin Val Glu Gin Glu 1 5 10 15 Gly Arg lie Leu Leu Ala lie Glu Ala lie Gin Lys Gly Gin lie Thr 20 25 30 Ser lie Arg Glu Ala Ala Arg Val Tyr Asp Val Ala Arg Thr Thr Leu 35 40 45 Gin Ala Arg Leu Ser Gly Arg Val Phe Ala Lys Asn Met Thr Asn Ala 50 55 60 Arg Gin Lys Leu Ser Asn Asn Glu Glu Glu Ser Leu Val Lys Trp lie 65 70 75 80 Leu Ser Leu Asp Lys Arg Gly Ala Ser Pro Arg Pro Leu Asp lie Arg 85 90 95 Asp Met Ala Asn Leu lie lie Ser Lys Arg Gly Tyr Ser Thr Val Glu 100 105 110 Gin Val Gly lie Asn Trp Ala Tyr Ser Phe Val Lys Arg His Glu Ser 115 120 125 Leu Arg Thr Arg Phe Ala Arg Arg Leu Asn Tyr Pro Arg Ala Lys Met 130 135 140 Glu Asp Pro Glu Val He Lys Asp Trp Phe Gin Arg Val Gin Glu Val 145 150 155 160 He Gin Glu Tyr Gly He Ser Ser Asp Asp He Tyr Asn Phe Asp Glu 165 170 175 Thr Gly Phe Ala Met Gly Met He Ala Thr Tyr Lys Val Val Thr Ser 180 185 190 Ser Gin Arg Ala Gly Arg Pro Ser Leu Val Gin Pro Gly Asn Arg Glu 195 200 205 Trp Val Thr Pro He Glu Cys He Arg Ser Asn Gly Glu Val Leu Pro 210 215 220 Ser Thr Leu He Phe Lys Gly Lys Thr His Leu Lys Ala Trp Tyr Glu 225 230 235 240 Gly Gin Ser He Pro Pro Thr Trp Arg Phe Glu Val Ser Asp Asn Gly 245 250 255 Trp Thr Thr Asp Lys He Gly Leu Arg Trp Leu Pro Lys His Phe He 260 265 270 Pro Leu He Arg Gly Lys Ser Val Gly Lys Tyr Ser Leu Leu Val Leu 275 280 285 Asp Gly His Gly Ser His Leu Thr Pro Glu Phe Asp Gin Ser Cys Ala 290 295 300 Glu Asn Glu Val He Pro He Cys Met Pro Ala His Ser Ser His Leu 305 310 315 320 Leu Gin Pro Leu Asp Val Gly Cys Phe Ser Val Leu Lys Arg Thr Tyr 325 330 335 Gly Gly Met Val Pro Lys Gin Met Gin Tyr Gly Arg Asn His He Asp 340 345 350 Lys Leu Asp Phe Leu Glu Val Tyr Pro Lys Ala His Gin Cys Ala Leu 355 360 365 Ser Lys Ser Asn He He Ser Gly Phe Arg Ala Thr Gly Leu Val Pro 370 375 380 Leu Asp Pro Asp Gin Val Leu Ser Arg Leu His He Arg Leu Lys Thr 385 390 395 400 Pro Pro Thr Pro Asp Ser Gin Ser Ser Gly Ser Val Leu Gin Thr Pro 405 410 415 His Asn He Lys His Leu Leu Glu His Pro Lys Ser Val Glu Arg Leu 420 425 430 Leu Arg Lys Arg Gin Ala Ser Pro Thr Ser Pro Thr Asn Ser Thr Leu 435 440 445 Arg Gin Leu Leu Lys Gly Cys Glu Leu Ala He Thr Asn Ser He He 450 455 460 Leu Ala Lys Glu Asn Ala Glu Leu Arg Ala Ser His Glu Lys Gin Leu 465 470 475 480 Pro Lys Arg Lys Arg Ser Arg Lys Gin Val He Tyr Thr Glu Gly Thr 485 490 495 Thr Val Glu Glu Ala Gin Arg Ala He Gin Glu Val Glu Glu Val Gin 500 505 510 Asn Asp Glu Asp He Glu Val Glu Pro Gin Ser Gin Tyr Thr Glu Thr 515 520 525 Pro Ser Arg Ala Pro Pro Arg Cys Ser Asn Cys Phe Asn He Gly His 530 535 540 Arg Arg Thr Gin Cys Ser Lys Pro Pro Thr Asn 545 550 555 (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 (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: CCAACCGAGT CCTCAGTATA GAC 23 (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 (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 9: CAACGCTTCA TAGGCGTCCA GATC 24 (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 (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 10: ATATGAATTC ACGTAATCAA CGGTCGGACG GGCCACACGG TCAGGCGGGC CATC 54 (2) INFORMATION FOR SEQ ID NO: 11: (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: DNA (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 11: ATATGAATTC CTTCTTGACT TCCCCGGAAC 30 (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 (genomic) (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 12: ATATAAGCTT GTCACTGGAC GACATTTCAG 30

Claims: WHAT IS CLAIMED IS: 1. A transposable element isolated from Aspergillus niger var. awamori. 2. A transposable element of Claim 1 comprising the DNA sequence of Seq. ID No. 3. 3. A fragment of the transposable element of Claim 1 comprising a forty-four base pair inverted repeat. 4. The fragment of Claim 3 comprising the DNA sequence of Seq. ID No. 4 or Seq. ID No. 5. 5. A method for isolating a transposable element from a filamentous fungi, the method comprising using as a probe one of the fragments of Claim 4. 6. A transposable element isolated by the method of Claim 5. 7. The transposable element of Claim 1 comprising the DNA sequence of Seq. ID No. 6. 8. A putative transposase coded for by the transposable element of Claim 7. 9. The transposase of Claim 8 comprising the amino acid sequence of Seq. ID No. 7. 10. A method of inactivating a gene, the method comprising transforming into a host cell containing said gene, a transposable element targeted at a TA sequence in the gene to be inactivated, said transposable element being inserted into said gene to be inactivated and disrupting gene expression from said gene. 11. A method of activating a gene, the method comprising transforming into a host cell containing said gene, a transposable element having inserted therein a desired DNA sequence capable of regulating said gene to be activated, and targeting said transposable element at a TA sequence in the genome of the host cell, said transposable element being inserted into the genome of the host cell in a manner to regulate the expression of the activated gene product.

Inventors: AMUTAN MARIA, DUNN-COLEMAN NIGEL S, NYYSSONEN EINI M, AMUTAN, MARIA, DUNN-COLEMAN, NIGEL, S., NYYSSONEN, EINI, M.
Applicants: GENENCOR INT, GENENCOR INTERNATIONAL, INC.
Application year: 1996
Main classification: C12N15
IPC codes: C12N9, C12N15, C12R1