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EP0920521A1 - IDENTIFICATION ET CLONAGE D'UN TRANSPOSON MOBILE ISSU D'$i(ASPERGILLUS) - Google Patents

IDENTIFICATION ET CLONAGE D'UN TRANSPOSON MOBILE ISSU D'$i(ASPERGILLUS)

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Publication number
EP0920521A1
EP0920521A1 EP97939558A EP97939558A EP0920521A1 EP 0920521 A1 EP0920521 A1 EP 0920521A1 EP 97939558 A EP97939558 A EP 97939558A EP 97939558 A EP97939558 A EP 97939558A EP 0920521 A1 EP0920521 A1 EP 0920521A1
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European Patent Office
Prior art keywords
dna
gene
vader
transposable element
seq
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EP97939558A
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German (de)
English (en)
Inventor
Maria Genencor International Inc. AMUTAN
Nigel S. Dunn-Coleman
Eini M. Nyyssonen
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Danisco US Inc
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Genencor International Inc
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
    • C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
    • C12N15/815—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts for yeasts other than Saccharomyces

Definitions

  • the present invention is directed at the identification, cloning and sequencing of mobile transposons or transposable elements from Aspergillus niger var. awamori.
  • 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.
  • 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 niger var. awamori.
  • 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-IR to jump in front of (and overexpress) a gene of interest) or to act as an activation marker to, for example, identify new promoters.
  • transposons are a class of DNA sequences that can move from an 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.
  • 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.
  • Schectman (1) 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.
  • 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 cHale 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.
  • niaD nitrate reductase
  • 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, Fort, 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.
  • FML Feusarium mariner-like
  • 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.
  • 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.
  • novel eukaryotic transposable elements from Aspergillus niger var. awamori 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 either terminus and internal IRs.
  • Tan1 comprises a 555 aa open reading frame (ORF) which 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 or Tan1 transposable elements which comprise the 44 or 45 bp (respectively) inverted repeat sequences found at either terminus of the transposable element from A niger var 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.
  • 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).
  • transposable elements of the present invention Vader or Tan1 or any transposable element isolated using the IRs of either
  • methods for gene tagging comprising using the transposable elements of the present invention (Vader 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.
  • the transposable element can be used in activation tagging (to activate or turn on genes) rather than for gene disruption
  • the promoter may be activated to drive the expression of the desired gene product or to turn on cryptic pathways
  • 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 the promoter driving such expression. This may lead to identification of isolation of new strong promoters.
  • Fig. 1 shows the Southern blot analysis of unstable niaD mutants.
  • PCR-amplified genomic niaD gene from four niaD mutants and UVK143f were digested with Bglll (sites are 3' of all inserts).
  • Fig. 2 depicts the mapping of Vader insertions within the niaD gene.
  • Fig. 3 shows Southern blot analysis to determine Vader genomic copy number.
  • Four A. niger var. awamori niaD mutants and UVK143f 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.
  • Fig. 4 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 (32) indicates that sequences homologous to Vader are present in A. nidulans (FGSC A237), A. cinnamomeus, A. phoenicis, A. foetidus, an industrial A. niger strain.
  • 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, 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 Tani element (SEQ ID NO:6) as generated by PCR, as well as the putative amino acid sequence of the transposase coded for by Tani (SEQ ID NO:7).
  • Tani 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). Tani comprises the sequences of four inverted repeats (underlined) similar to those found in Vader.
  • Fig. 9 shows a schematic presentation of Vader and Tani elements.
  • Fig. 10 shows Southern analysis of A. niger var.
  • awamori niaD mutants (/7/aD410, n/ ' aD436, niaD587, niaD392) and the wild-type UVK143f: lane 1 , molecular weight marker III (Boehringer Mannheim); lane 2, UVK143f; lane 3, n/aD410; lane 4, n/a0436; lane 5, niaD5- 87; lane 6, niaD3Q2.
  • This blot was probed for the Vader element (see Fig. 9).
  • this blot (Fig. 10) was superimposed with the blot shown in Fig. 5, one of the illuminated bands from the Vader-probe hybridization overlaid the single band in the ORF-probe hybridization indicating that the Tani element is composed of contiguous ORF and Vader elements.
  • Figs. 11A and 11D show the nucleotide sequence (genomic copy) of Tani (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 the sequence listing as "N.”
  • the figure shows the DNA sequence as 2324 base pairs, excluding the unknown nucleotides indicated by "?” in the figure.
  • nucleotide bases are designated as adenine (A); thymine (T); guanine (G) ; and cytosine (C).
  • A adenine
  • T thymine
  • G guanine
  • C cytosine
  • Applicants have isolated two transposable elements from A niger var. awamori.
  • the cloned 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.
  • 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.
  • Fot1 this transposon does not excise precisely.
  • 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.
  • Pot2 is a functional element.
  • a homology search made at the nucleotide level gave a strong 60.7% homology between Tani and a 1230 bp overlap to the A. oryzae agdA gene coding for an ⁇ - glucosidase (33).
  • This homology search revealed that the last 1.2 kb of a total of 5.2 kb of the ⁇ -glucosidase sequence submitted to GenBank is, in fact, part of a novel transposon, hereinafter called Tao1 (transposon Aspergillus oryzae), which also belongs to the Fot1 family. Only the 5' half of the Tao1 element is included in the GenBank sequence, thus, for the lack of comparison, the exact size of the inverted repeat cannot be determined.
  • Tao1 would hybridize to a probe comprising Tani 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 Tani or Vader may be used to isolate other transposable elements from filamentous fungi.
  • 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 can be without long terminal repeats such as the Drosphilia I element (for a review see (16)).
  • retrotransposons can have long terminal repeats such as the Drosphilia copia element.
  • 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
  • Tani This element, called Tani (SEQ ID NO:6), compnses four inverted repeats (SEQ ID NOS:1, 2, 15 and 16 from 5' to 3', respectively) similar to those in Vader and has a unique organization IR-ORF-IR-IR-Vader-IR Tani 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.
  • transposable element(s) with certain Aspergillus species
  • transposable elements are believed to be quite useful in the development 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 stabilize the mutations by, e g., a two transposons system (23, 24 and 25).
  • 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 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., o//C) between the transcription and translation initiation sites.
  • This disrupted hygromycin phosphotransferase cassette is flanked by marker genes - or alternatively 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 I containing these elements will be transferred to A. nidulans and transformants expressing the two 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 II, 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 Tani) in pHELP, which can be monitored by activation of the hygromycin resistance gene. Tani is not cloned into the vector in full length to disrupt its mobility.
  • Vector II 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 benomyl R , acetamidase or ⁇ -glucuronidase (GUS).
  • GUS ⁇ -glucuronidase
  • the target gene for mutagenesis should be one with 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 acid protease gene. It has been shown previously for A. niger that disruption of this one protease is sufficient to abolish halo formation almost completely on skim milk plates.
  • transposon tagging is that the mutants produced can be identified by subsequent isolation of the mutated gene.
  • PCR methods developed for genomic walking are, e.g., “Inverse PCR” (27 and 28), “Vectorette PCR” (29) and “Panhandle PCR” (30).
  • transposon tagging system 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.
  • 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.
  • Vader and Tani elements were isolated from Aspergillus niger var. awamori UVK143f, derived from Northern Regional Research Laboratories (NRRL) #3112.
  • E. co// JM101 [F' traD36 lacf A(lacZ)M15 proA * B * IsupE thi A(lac-proAB)] and Epicurian coli SURE 2 (Stratagene Cloning Systems, La Jolla, CA) were used for propagation of Vader and Tani subclones, respectively.
  • Spontaneous chlorate resistant mutants were derived from Aspergillus niger var. 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 (n/aD15), and A. nidulans (FGSC #A691), a tryptophan requiring mutant (rrpC801), 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. p
  • niaD mutants 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): NaNO 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 KCIO 3 and able to grow in the presence of all pathway intermediates, except for NaNO 3 .
  • the reaction mixture contained 4 mM MgSO 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). Confirmation of Excision Foot Print by PCR Amplification and Sequencing.
  • Template DNA from n/aD436 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'- ATATGAATTCCTTCTTGACTTCCCCGGAAC-3' (SEQ ID NO:11) and NiaD5 (position
  • 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 using the Qiaex DNA gel extraction kit (Qiagen) and subcloned as blunt-ended inserts into EcoRV cut pSL1180 (Pharmacia Biotech).
  • niaD Mutant Reversion Frequency Spores from niaD mutants n/aD392, ⁇ /aD410, n/aD436 and niaD587 were streaked onto minimal media containing NaNO 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.
  • KCIO 3 600 mM potassium chlorate
  • 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 ⁇ g) 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 ⁇ g) 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 (UVK143f 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 SphI 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 (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 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 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 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).
  • the Tani probe was prepared by digesting Tani with HindlW and Stu ⁇ resulting in a 650 bp fragment corresponding to the 3' end of the transposase coding region (ORF-probe in Fig. 9).
  • the Vader element was digested with Xba ⁇ and Seal to yield a 236 bp fragment to be used for recognition of internal Vader sequence in Southern analysis (Vader-probe in Fig. 9).
  • 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.
  • 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.
  • niaD mutants There appeared to be two classes of niaD mutants which reverted at high frequency.
  • the niaD mutants rw ' aD436 and niaD392 reverted at high frequency, while mutants n/aD410 and niaD587 yielded smaller numbers of revertant colonies.
  • 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.
  • UVK143f wild-type 100 niaD436 (niaD mutant) ND 1
  • niaDI SEQ ID NO:8
  • niaD2 SEQ ID NO:9
  • 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 revealed an approximately 440 bp insertion (Vader) in the niaD gene.
  • 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 niaD2 (SEQ ID NO:9) oligomeric probes.
  • the probe hybridized to a 2.5 kb fragment with wild-type DNA (lane 5, Fig. 1).
  • the niaD mutants 410 (lane 1 , Fig. 1), 436(lane 3, Fig. 1) and 392 (lane 4, Fig.
  • Vader Copy Number To determine the Vader copy number a 236 bp Scal-Xbal 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. 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.
  • Example 4 Isolation of Vader in Other Fungal Species
  • genomic Southern 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 ( ⁇ /aD15), and FGSC #A237, a tryptophan-requiring mutant ( rpC801). No hybridization signals could be visualized with strain A691, and a single strong hybridization signal could be detected with strain A237.
  • Example 6 Isolation of Tani
  • 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. awamori 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.
  • the larger 2.3 kb fragment had a unique organization, IR-ORF-IR-IR-Vader-IR, with a total of four inverted repeats (Figs. 9 and 11).
  • 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.
  • 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.
  • niger var. awamori 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 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 Tani.
  • a genomic clone of the Tani element (2.3 kb) was isolated from a partial genomic library. Restriction enzymes, which were shown not to have any recognition sites in the PCR-amplified Tani , were used separately and in combinations in Southern analysis of the genomic DNA. A double digestion with BglW and Xhol resulted in a relatively short, 4.5 kb, fragment which hybridized with the ORF-specific probe (data not shown). Genomic DNA fragments cleaved by BglU and Xhol and between 4 kb and 5 kb in size were cloned into pSP73 (Promega). The correct clone containing the Tani element was isolated by colony hybridization using the ORF-specific probe.
  • 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.
  • a strain is constructed using gene disruption, which lacks the Tani 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.)
  • 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).)
  • Example 8 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.
  • a strain is constructed in which the native Tani 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).
  • a second transformation is made with Vader and Tani , 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.
  • this beneficial gene is activated and such integrant colonies can be screened for, e.g., increased halo size (chymosin).
  • the activated gene can be cloned using the transposon sequences as a marker for cloning strategies.
  • MOLECULE TYPE DNA
  • SEQUENCE DESCRIPTION SEQ ID NO: 5:
  • ATCAAAGTCG AATATAATCA GTGGTTTTAG AGCAACAGGT CTTGTTCCTC TAGATCCTGA 1260
  • MOLECULE TYPE DNA (genomic)
  • ATCAAAGTCG AATATAATCA GTGGTTTTAG AGCAACAGGT CTTGTTCCTC TAGATCCTGA 1260

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Abstract

L'invention concerne des éléments transposables isolés à partir d'Aspergillus. Des fragments comprenant la (les) séquence(s) répétée(s) inverse(s) des éléments transposables, ces fragments pouvant être utiles en tant que sondes pour isoler des éléments transposables parmi d'autres champignons filamenteux.
EP97939558A 1996-08-26 1997-08-25 IDENTIFICATION ET CLONAGE D'UN TRANSPOSON MOBILE ISSU D'$i(ASPERGILLUS) Withdrawn EP0920521A1 (fr)

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