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PCal, a highly unusual Ty1/copia retrotransposon from the pathogenic yeast Candida albicans.

ABSTRACT: Retrotransposons are mobile genetic elements. They can transpose via the reverse transcription of mRNA into double-stranded DNA (dsDNA) followed by the insertion of this dsDNA into new sites within the host genome. The unintegrated, linear, dsDNA form of retrotransposons is usually very rare. We report here the isolation of a retrotransposon from Candida albicans which is unusual in this respect. This element, which we have named pCal, was first identified as a distinct band when uncut C. albicans DNA was examined on an agarose gel. Sequence analysis of the cloned element revealed that it is a retrotransposon belonging to the Ty1/copia group. It is estimated that pCal produces 50 to 100 free, linear, dsDNA copies of itself per cell. This is a much higher level of expression than even that of the system in which Ty1 is expressed behind the highly active GAL1 promoter on a high-copy-number plasmid (about 10 copies per cell). Another unusual feature of pCal is that its Pol enzymes are likely to be expressed via the pseudoknot-assisted suppression of an upstream, in-phase stop codon, as has been shown for Moloney murine leukemia virus.

SUBMITTER: Matthews GD

PROVIDER: S-EPMC179655 | biostudies-other | 1997 Nov

REPOSITORIES: biostudies-other

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pCal, a highly unusual Ty1/copia retrotransposon from the pathogenic yeast Candida albicans.

Matthews G D GD Goodwin T J TJ Butler M I MI Berryman T A TA Poulter R T RT

Journal of bacteriology 19971101 22

Retrotransposons are mobile genetic elements. They can transpose via the reverse transcription of mRNA into double-stranded DNA (dsDNA) followed by the insertion of this dsDNA into new sites within the host genome. The unintegrated, linear, dsDNA form of retrotransposons is usually very rare. We report here the isolation of a retrotransposon from Candida albicans which is unusual in this respect. This element, which we have named pCal, was first identified as a distinct band when uncut C. albica ...[more]

PMID: 9371461

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Project description:BACKGROUND: Retrotransposons make a significant contribution to the size, organization and genetic diversity of their host genomes. To characterize retrotransposon families in the grapevine genome (the fourth crop plant genome sequenced) we have combined two approaches: a PCR-based method for the isolation of RnaseH-LTR sequences with a computer-based sequence similarity search in the whole-genome sequence of PN40024. RESULTS: Supported by a phylogenic analysis, ten novel Ty1/copia families were distinguished in this study. To select a canonical reference element sequence from amongst the various insertions in the genome belonging to each retroelement family, the following screening criteria were adopted to identify the element sequence with: (1) perfect 5 bp-duplication of target sites, (2) the highest level of identity between 5' and 3'-LTR within a single insertion sequence, and (3) longest, un-interrupted coding capacity within the gag-pol ORF. One to eight copies encoding a single putatively functional gag-pol polyprotein were found for three families, indicating that these families could be still autonomous and active. For the others, no autonomous copies were identified. However, a subset of copies within the presumably non-autonomous families had perfect identity between their 5' and 3' LTRs, indicating a recent insertion event. A phylogenic study based on the sequence alignment of the region located between reverse transcriptase domains I and VII distinguished these 10 families from other plant retrotransposons. Including the previously characterized Ty1/copia-like grapevine retrotransposons Tvv1 and Vine 1 and the Ty3/gypsy-like Gret1 in this assessment, a total of 1709 copies were identified for the 13 retrotransposon families, representing 1.24% of the sequenced genome. The copy number per family ranged from 91-212 copies. We performed insertion site profiling for 8 out of the 13 retrotransposon families and confirmed multiple insertions of these elements across the Vitis genus. Insertional polymorphism analysis and dating of full-length copies based on their LTR divergence demonstrated that each family has a particular amplification history, with 71% of the identified copies being inserted within the last 2 million years. CONCLUSION: The strategy we used efficiently delivered new Ty1/copia-like retrotransposon sequences, increasing the total number of characterized grapevine retrotrotransposons from 3 to 13. We provide insights into the representation and dynamics of the 13 families in the genome. Our data demonstrated that each family has a particular amplification pattern, with 7 families having copies recently inserted within the last 0.2 million year. Among those 7 families with recent insertions, three retain the capacity for activity in the grape genome today.

Project description:Candida albicans is the leading fungal pathogen of humans, causing life-threatening disease in immunocompromised individuals. Treatment of candidiasis is hampered by the limited number of antifungal drugs whose efficacy is compromised by host toxicity, fungistatic activity, and the emergence of drug resistance. We previously established that the molecular chaperone Hsp90, which regulates the form and function of diverse client proteins, potentiates resistance to the azoles in C. albicans and in the model yeast Saccharomyces cerevisiae. Genetic studies in S. cerevisiae revealed that Hsp90's role in azole resistance is to enable crucial cellular responses to the membrane stress exerted by azoles via the client protein calcineurin. Here, we demonstrate that Hsp90 governs cellular circuitry required for resistance to the only new class of antifungals to reach the clinic in decades, the echinocandins, which inhibit biosynthesis of a critical component of the fungal cell wall. Pharmacological or genetic impairment of Hsp90 function reduced tolerance of C. albicans laboratory strains and resistance of clinical isolates to the echinocandins and created a fungicidal combination. Compromising calcineurin function phenocopied compromising Hsp90 function. We established that calcineurin is an Hsp90 client protein in C. albicans: reciprocal co-immunoprecipitation validated physical interaction; Hsp90 inhibition blocked calcineurin activation; and calcineurin levels were depleted upon genetic reduction of Hsp90. The downstream effector of calcineurin, Crz1, played a partial role in mediating calcineurin-dependent stress responses activated by echinocandins. Hsp90's role in echinocandin resistance has therapeutic potential given that genetic compromise of C. albicans HSP90 expression enhanced the efficacy of an echinocandin in a murine model of disseminated candidiasis. Our results identify the first Hsp90 client protein in C. albicans, establish an entirely new role for Hsp90 in mediating resistance to echinocandins, and demonstrate that targeting Hsp90 provides a promising therapeutic strategy for the treatment of life-threatening fungal disease.

Project description:BackgroundThe pathogenic yeast Candida albicans can proliferate in environments with different carbon dioxide concentrations thanks to the carbonic anhydrase CaNce103p, which accelerates spontaneous conversion of carbon dioxide to bicarbonate and vice versa. Without functional CaNce103p, C. albicans cannot survive in atmospheric air. CaNce103p falls into the β-carbonic anhydrase class, along with its ortholog ScNce103p from Saccharomyces cerevisiae. The crystal structure of CaNce103p is of interest because this enzyme is a potential target for surface disinfectants.ResultsRecombinant CaNce103p was prepared in E. coli, and its crystal structure was determined at 2.2 Å resolution. CaNce103p forms a homotetramer organized as a dimer of dimers, in which the dimerization and tetramerization surfaces are perpendicular. Although the physiological role of CaNce103p is similar to that of ScNce103p from baker's yeast, on the structural level it more closely resembles carbonic anhydrase from the saprophytic fungus Sordaria macrospora, which is also tetrameric. Dimerization is mediated by two helices in the N-terminal domain of the subunits. The N-terminus of CaNce103p is flexible, and crystals were obtained only upon truncation of the first 29 amino acids. Analysis of CaNce103p variants truncated by 29, 48 and 61 amino acids showed that residues 30-48 are essential for dimerization. Each subunit contains a zinc atom in the active site and displays features characteristic of type I β-carbonic anhydrases. Zinc is tetrahedrally coordinated by one histidine residue, two cysteine residues and a molecule of β-mercaptoethanol originating from the crystallization buffer. The active sites are accessible via substrate tunnels, which are slightly longer and narrower than those observed in other fungal carbonic anhydrases.ConclusionsCaNce103p is a β-class homotetrameric metalloenzyme composed of two homodimers. Its structure closely resembles those of other β-type carbonic anhydrases, in particular CAS1 from Sordaria macrospora. The main differences occur in the N-terminal part and the substrate tunnel. Detailed knowledge of the CaNce103p structure and the properties of the substrate tunnel in particular will facilitate design of selective inhibitors of this enzyme.

Dataset Information

PCal, a highly unusual Ty1/copia retrotransposon from the pathogenic yeast Candida albicans.

Publications

pCal, a highly unusual Ty1/copia retrotransposon from the pathogenic yeast Candida albicans.

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