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Unusual retrotransposon from the yeast Candida albicans

a retrotransposon, yeast technology, applied in the field of new retrotransposon, can solve the problems of systemic infections in immuno-compromised patients, ineffective reverse transcription, and high cost of treatmen

Inactive Publication Date: 2006-06-15
JANSSEN PHARMA NV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The invention provides an isolated and purified retrotransposon with a copy number of between 40-150 per genome, preferably (50-100). The retrotransposon is isolated from fungi or yeast, preferably Candida. The retrotransposon has a linear or circular double stranded DNA form and includes a nucleotide sequence that can integrate into the genome of a cell and cause gene disruption. The invention also provides a transformation and expression system for fungi / yeast using the retrotransposon. The invention also provides a method of introducing DNA into a cell using the retrotransposon. The retrotransposon can be used in gene disruption and gene discovery systems. The invention also provides a retroviral-like carrier system for fungi / yeast. The invention also provides a nucleic acid encoding the retrotransposon and a vector for its introduction into cells. The cells can be contacted with a desired compound to identify its effect on the phenotype of the cell.

Problems solved by technology

Although it is commonly found as a harmless commensal organism, inhabiting mucosal membranes and the digestive tract, it can cause superficial infections, such as oral thrush, in otherwise healthy people and can cause severe, often fatal, systemic infections in immuno-compromised patients.
Reverse transcription is generally very inefficient; greater than 10% of cellular mRNA can be retrotransposon RNA yet the dsDNA form is not usually detectable by Southern blotting.

Method used

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  • Unusual retrotransposon from the yeast Candida albicans
  • Unusual retrotransposon from the yeast Candida albicans
  • Unusual retrotransposon from the yeast Candida albicans

Examples

Experimental program
Comparison scheme
Effect test

example 1

Cloning and Mapping

[0186] Some uncut genomic DNA prepared from Candida albicans strain hOG1042 was analysed on an agarose gel and a distinct band running at about 6.5 kb was found (FIG. 1). Such a band had never previously been reported from any Candida strain or species. To analyse this feature the band was extracted from an agarose gel and tested to see if it could be cut with restriction enzymes. A number of enzymes cut the band into smaller fragments which indicated that it was made up of double-stranded DNA. At this point the band was named pCal (plasmid of Candida albicans). The restriction digests allowed the construction of a simple restriction map of pCal. This work revealed that pCal was linear, with a Pst1 site about 1 kb from one end, an EcoR1 site about 1 kb from the opposite end and an Asp718 site near the middle. To permit further analysis the fragments of pCal produced with Asp7118 were cloned into the Asp7118 site of pUC19. Five clones were isolated and each was f...

example 2

Nucleotide Sequence of pCal

[0187] The five plasmids containing the pCal fragments were all sequenced from both ends in the hope of finding an identifiable feature which would provide an insight into the nature of pCal. The first remarkable features to be found were 280 bp direct repeats. The existence of these direct repeats suggested that pCal was likely to be a retrotransposon. As no other retrotransposon had ever been found existing at a high copy number in a free, linear, dsDNA form we determined the complete sequence of pCal. Therefore, the three clones of pCal carrying the Pst1 site and one of the two clones carrying the EcoR1 site were completely sequenced. In addition a region of pCal spanning the central Asp718 site used in the cloning was amplified by PCR and each strand was sequenced. This analysis confirmed that there was only one Asp718 site and that therefore the clones that we had of each half of pCal truly represented adjacent fragments.

[0188] Assembly of the 6426...

example 3

The open reading frames

[0192] Two long open reading frames were found in pCal, the first 972 bp (324aa) and the second 4728 bp (1576aa) long. Conserved motifs from the four pol proteins—protease, integrase, reverse transcriptase and RNase H—were identified in the second ORF. The order of these motifs (as listed above) places pCal within the Ty1 / copia group of retrotransposons. The pCal motifs are shown compared to those of other Ty1 / copia elements in FIG. 4. No conserved motifs were found in the first ORF but it is similar in size and position to the gag genes of other retroelements. Retroelement gag genes in general are known to be extremely variable and it is not uncommon for no identifiable conserved features to be present.

[0193] Unlike other retrotransposons, the gag and pol ORFs of pCal are in the same phase separated only by a UGA termination codon. This arrangement is similar to what has been found for mammalian type C retroviruses such as Moloney murine leukemia virus (MM...

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Abstract

TCa2 is a TyI / copia retrotransposon from the pathogenic yeast Candida albicans. In contrast to other retrotransposons it can appear as an abundant, extrachromosomal double-stranded DNA molecule, called pCal. The invention relates to the isolation and characterisation of TCa2 and pCal together with its uses for inducing random mutagenesis in a genome, as a component of a transposable element and of an expression vector.

Description

RELATED APPLICATIONS [0001] This is a divisional application of U.S. application Ser. No. 09 / 430,590, filed Oct. 29, 1999, which claims priority to U.S. application Ser. No. 60 / 106,342, filed Oct. 30, 1998. The foregoing applications all documents cited therein (“the application-cited documents”) and all documents referenced or cited in the application-cited documents are hereby incorporated herein by reference. In addition all documents cited herein (“herein cited documents”) and all documents cited or referenced in herein cited documents are likewise incorporated herein by reference.FIELD OF INVENTION [0002] The invention relates to a novel retrotransposon. The novel retrotransposon is from the yeast Candida albicans. In particular, the invention relates to a retrotransposon pCal which belongs to the TyI / copia group. INTRODUCTION [0003]Candida albicans is an asexual yeast species which is the major fungal pathogen of humans. Although it is commonly found as a harmless commensal or...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N15/74C12N1/18
CPCC12N15/815C12N15/905C12N2800/90
Inventor POULTER, RUSSELLLUYTEN, WALTER HERMANDE BACKER, MARIANNENELISSEN, BART
Owner JANSSEN PHARMA NV
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