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WO2001081598A2 - Familles d'un retrotransposon multiple du champignon asexue candida albicans - Google Patents

Familles d'un retrotransposon multiple du champignon asexue candida albicans Download PDF

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Publication number
WO2001081598A2
WO2001081598A2 PCT/EP2001/004649 EP0104649W WO0181598A2 WO 2001081598 A2 WO2001081598 A2 WO 2001081598A2 EP 0104649 W EP0104649 W EP 0104649W WO 0181598 A2 WO0181598 A2 WO 0181598A2
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WO
WIPO (PCT)
Prior art keywords
retrotransposon
ltr
sequences
sequence
cell
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Application number
PCT/EP2001/004649
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English (en)
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WO2001081598A3 (fr
Inventor
Bart Jozef Maria Nelissen
Marianne Denise De Backer
Walter Herman Mari Luyten
Original Assignee
Janssen Pharmaceutica N.V.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Publication date
Application filed by Janssen Pharmaceutica N.V. filed Critical Janssen Pharmaceutica N.V.
Priority to CA002407386A priority Critical patent/CA2407386A1/fr
Priority to AU2001278421A priority patent/AU2001278421A1/en
Publication of WO2001081598A2 publication Critical patent/WO2001081598A2/fr
Publication of WO2001081598A3 publication Critical patent/WO2001081598A3/fr

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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
    • 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

  • Opportunistic fungal infections in immunocompromised hosts represent an increasingly common cause of mortality and morbidity (Fisher Hoch and Hutwagner, 1995; Groll et al, 1998).
  • Several factors have contributed to the rise of opportunistic infections (Shaberg et al, 1991 ; Fox, 1993): the expansion of severely ill and/or immunocompromised populations, such as extremely low birth weight infants; patients with HIV infection, with profound and prolonged chemotherapy-induced neutropenia, with immunosuppression after transplantation, or with severe burns; the increase in invasive medical procedures, such as extensive surgery; and the use of prosthetic devices and vascular catheters together with treatment with corticosteroids and broad-spectrum antibiotics (Bodey, 1988; Groll et al, 1998).
  • the most prevalent opportunistic infection in HIV-infected patients is caused by the species of Candida.
  • the most frequently isolated and abundant fungus in clinical isolates is Candida albicans, a commensal yeast of the va
  • C.albicans has come to be a model pathogenic fungus in which to study dimorphism, to analyze virulence, or to identify new fungal targets.
  • a main limitation, however, is its molecular genetic manipulation, which, because of its diploidy and lack of sexual cycle, is difficult.
  • the genetic manipulation of C. albicans is therefore much less advanced than that of S. cerevisiae, a closely related non-pathogenic yeast and the favorite microbial eukaryotic organism for genetic studies.
  • Initial experiments to elucidate the biological function of a putative fungal target gene typically include its inactivation by deletion of the open reading frame.
  • albicans ORFs would be a daunting task.
  • Several methods have been developed to allow “en masse” disruption of genes in S. cerevisiae (“mass-murder”, Fairhead et al., 1998; “genetic footprinting”, Smith et al., 1995).
  • the “mass-murder” method was used to delete multiple contiguous ORFs simultaneously (Fairhead et al., 1998). This strategy could in principle also be used for primary phenotypic screening in C. albicans but a second round of inactivation is required to obtain homozygous disruptions. More powerful techniques have been developed for S.
  • Ty elements These retrotransposons, collectively referred to as Ty elements (Boeke and Sandmeyer, 1991 ), are all of the long terminal repeat (LTR) class and as such they resemble the vertebrate retroviruses in their genomic organisation and replication cycle.
  • the most well-characterised Ty element is Ty1 which is composed of two ⁇ 330-bp LTRs (delta elements) flanking an ⁇ 5.3-kb internal domain. The element replicates via the reverse transcription of a genomic mRNA into a double-stranded DNA copy followed by the insertion of this DNA into a new site within the host genome.
  • the proteins required for the reverse transcription and integration reactions are encoded by two long open reading frames (ORFs) in the internal region while the LTRs contain the promoter and polyadenylation signals which direct the synthesis of the full-length transcript. Recombinations occasionally occur between the two LTRs of a retrotransposon. This has the effect of deleting the internal region and one LTR of the element but leaving behind one isolated, or 'solo', LTR. Full-length retrotransposons and also the solo LTRs are often found flanked by short (4-5 bp) direct repeats. These are duplications of the target site sequence which are formed during the insertion process. Of the S.
  • Ty1 , Ty2 and Ty3 are known to be functional (Curcio et al., 1988; Hansen and Sandmeyer, 1990). Full-length, apparently uncorrupted, Ty4 elements are present but they have not yet been shown to be active (Hug and Feldmann, 1996). No functional Ty5 elements are known in S. cerevisae, though some retain significant amounts of internal coding sequence and functional Ty5s have been found in the closely related species S. paradoxus (Voytas and Boeke, 1992; Zou et al., 1996). A recent survey of the complete genome sequence of S.
  • LTR2 is a non- LTR retrotransposon reverse transcriptase identified by Chibana et al. (1998) during their mapping project. Retrotransposons can alter the arrangement of their host genomes in a number of ways (Finnegan, 1989).
  • retrotransposon families can influence gene structure by inserting into a coding sequence; the regulation of genes adjacent to retrotransposon insertions can be altered due to the presence of strong promoter/enhancer elements in the LTRs; and major chromosomal rearrangements can occur when a recombination occurs between two copies of a retrotransposon present at different locations in the genome.
  • retrotransposon families can dramatically increase the size of the genome. For instance, a relatively small number of retrotransposon families are estimated to make up at least 50% of the maize genome (SanMiguel et ai, 1996). To understand further the contribution of retrotransposons to the genomic organisation of C. albicans a search for additional retrotransposon sequences was conducted. Here an additional 18 families of C.
  • albicans retrotransposons are described. Some of these families may still be functional, but for most no evidence of full-length elements was found - just solo LTRs and LTR fragments remain. There are, therefore, at least 23 families of retrotransposons in C. albicans, most of which are no longer functional. This is substantially more than in S. cerevisiae and may be related to C. albicans being asexual and an obligate diploid. Rearrangements associated with these elements may have played a significant role in establishing the present-day organisation of the C. albicans genome. OBJECT OF THE INVENTION
  • LTR long terminal repeat
  • the invention provides one of sixty long terminal repeat (LTR) - retrotransposons, isolated from Candida albicans, ranging in size from about 400 to about 12100 b.p.
  • LTR long terminal repeat
  • the LTR-retrotransposons may be flanked by 4- or 5- bp duplications of the target sequence.
  • a primer binding site complementary to an internal portion of the initiator methionine tRNA and preferably upstream of the right LTR is a polyporine tract.
  • Also provided is a method of introducing DNA into the genome of a cell which method comprises introducing a transposon element comprising a nucleotide sequence encoding a desired protein located between long terminal repeat sequences having the sequences illustrated by any one of retrotransposons 39-99 of the present application, which element is such that it can insert into the genome of said cell in the presence of an appropriate integration factor.
  • said integration factor comprises an integrase which preferably is itself included in said transposable element and which integrase is derived from the POL region of said pCal retrotransposon.
  • the transposable element for introducing a desired DNA sequence into the genome of the cell also forms part of the present invention.
  • the transposable element preferably comprises an internal domain for receiving a nucleotide sequence encoding a desired protein flanked by two long terminal repeat regions having the sequences identified by any one of retrotransposons 39-99.
  • the transposable element may advantageously be included in a DNA transfer system comprising said transposable element, which is capable of integrating into the genome of said cell in the presence of an appropriate integration factor and, said integration factor.
  • the transposable element comprises an open reading frame encoding said integration factor which is an integrased protein, which preferably is encoded by a nucleotide sequence within the POL region of the retrotransposon from above.
  • the nucleotide sequences may have at least 60% similarity with the sequences shown herein or may hybridise under conditions of standard stringency to the nucleotide sequences shown herein or may be a functional fragment of either the nucleotide sequence, the nucleotide sequence with 60% similarity or the nucleotide sequence that hybridizes under conditions of standard stringency.
  • the nucleotide sequences Preferably have 70%, more preferably 90% and most preferably 95% similarity with the sequences shown herein.
  • the invention also provides an expression vector including any of the above mentioned retrotransposons or fragments thereof.
  • the expression vector maybe used to transform the cell into which the DNA is to be introduced.
  • the aforementioned retrotransposons may be used in a gene disruption system or in a gene discovery system.
  • the invention also provides a retroviral-like carrier system comprising any of the above-mentioned retrotransposons.
  • the invention also provides a transformation and expression system for fungi/yeast (preferably Candida) comprising any of the aforementioned retrotransposons.
  • the invention also provides cells containing the nucleic acid including transposable elements and retrotransposons according to the invention. The cells may be contacted with a desired compound to identify its affect of the phenotype of the ceil conferred by expression of the protein encoded by the nucleotide sequence provided in the transposable element.
  • the invention also provide the linear or circular, double stranded DNA copy of the retrotransposon.
  • Also provided by the present invention is a method of assigning a function to a nucleotide sequence comprising inserting said sequence between the long term repeat or repeat sequences of the transposable element and introducing it into said cell and monitoring it for the presence of an altered phenotype of said cell compared to a cell which has not had said nucleotide sequence introduced therein.
  • the invention also provides a retrotransposon selected from the group comprising: a) a nucleic acid sequence as described in this specification and numbered 39-99; b) a nucleic acid sequence with at least 60% similarity with the LTR and POL region of sequence of a); c) a nucleic acid sequence that hybridizes under conditions of standard stringency to the nucleotide sequence of a); and d) a functional fragment of a), b) or c).
  • a retrotransposon selected from the group comprising: a) a nucleic acid sequence as described in this specification and numbered 39-99; b) a nucleic acid sequence with at least 60% similarity with the LTR and POL region of sequence of a); c) a nucleic acid sequence that hybridizes under conditions of standard stringency to the nucleotide sequence of a); and d) a functional fragment of a), b) or c).
  • the nucleic acid sequence comprises two long terminal direct repeats flanking a series of genes in the order gag (group antigen), pol (polyprotein) where the pol sequence comprises an aspartic protease, an integrase and a reverse transcriptase/RNAseH.
  • Figure 1 shows the identification of potential LTR sequence regions done by a dotplot of the original LTR query sequences.
  • Figure 2. shows the C.albicans retrotransposon sequences retrieved by means of SRS from the EMBL nucleotide sequence database.
  • Figure 3 shows retrieved retrotransposon polyproteins gag, and reverse transcriptase sequences.
  • Figure 4. shows a PHRAP assembly of retrotransposon sequences.
  • Figure 5 shows homologous sequences and corresponding retrotransposon sequences.
  • Known retrotransposons are given in plain text, new and/or updated retrotransposons are given in bold italics.
  • Figure 6. shows the sixty (60) new retrotransposon sequences.
  • Figure 7. shows the full nucleotide sequences of the sixty novel retrotransposons.
  • the general strategy was to update known sequences and secondly to search for new sequences.
  • LTR long terminal direct repeat
  • a non-redundant data set was constructed by excluding these redundant sequences from the original data set, resulting in a total of 25 sequences.
  • a second, more sensitive, non-redundant dataset was constructed that only contained the LTR sequences
  • AF050215 comprises AF007776, the POL protein sequences 013308 and O74209 were nearly identical, and only 013308 was used.
  • Retrotransposons 01-38 that were based on the query sequences used in the original patent application, were put in the original patent application update
  • Figure 6 shows the sixty (60) novel retrotransposons found. Their length in base pairs
  • (bp) ranges from 508 to 12084 b.p.
  • the LTR region of each retrotransposon comprises LTR sampi, LTR cricron, LTR upsilon, LTR iota, LTR koppa, LTR pi, LTR psi, LTR rho, LTR eta, LTR phi, LTR chi, LTR episemon, LTR nu, LTR mu.
  • Retrotransposon 39 includes the Tca8 reverse transcriptase (pol) gene.
  • the invention provides a number of LTR retrotransposons present in C. albicans. Such retrotransposons may be useful in methods for introducing DNA into the genome of a cell.

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  • Life Sciences & Earth Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biomedical Technology (AREA)
  • Organic Chemistry (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Mycology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
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  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
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Abstract

L'invention concerne les numéros 39-99 du rétrotransposon à LTR du Candida qui peuvent être utiles pour l'introduction d'ADN dans le génome d'une cellule. Ils peuvent également être utilisés pour associer une fonction à une séquence d'acides aminés.
PCT/EP2001/004649 2000-04-26 2001-04-25 Familles d'un retrotransposon multiple du champignon asexue candida albicans WO2001081598A2 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
CA002407386A CA2407386A1 (fr) 2000-04-26 2001-04-25 Familles d'un retrotransposon multiple du champignon asexue candida albicans
AU2001278421A AU2001278421A1 (en) 2000-04-26 2001-04-25 Multiple retrotransposon families in candida albicans

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IB0000729 2000-04-26
IBPCT/IB00/00729 2000-04-26

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WO2001081598A2 true WO2001081598A2 (fr) 2001-11-01
WO2001081598A3 WO2001081598A3 (fr) 2002-08-08

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022198014A1 (fr) * 2021-03-19 2022-09-22 Flagship Pioneering Innovations Vi, Llc Compositions à base de transposons ltr et procédés

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1988003169A1 (fr) * 1986-10-27 1988-05-05 Whitehead Institute For Biomedical Research Amplification de genes utilisant de retrotransposons
IL142831A0 (en) * 1998-10-30 2002-03-10 Janssen Pharmaceutica Nv An unusual retrotransposon from the yeast candida albicans

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022198014A1 (fr) * 2021-03-19 2022-09-22 Flagship Pioneering Innovations Vi, Llc Compositions à base de transposons ltr et procédés

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CA2407386A1 (fr) 2001-11-01
AU2001278421A1 (en) 2001-11-07

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