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Identification and characterization of polyubiquitin gene from cDNA library of aspergillus fumigatus.

ABSTRACT: Aspergillus fumigatus (Afu) causes allergic and invasive forms of diseases in humans. In order to identify genes relevant for pathogenesis, a total of 235 cDNA clones were randomly selected and sequenced from cDNA library of Afu. One of the partially sequenced cDNA clones was homologous to polyubiquitin. Sequencing of the complete cDNA clone showed an open reading frame of 912 bases. Comparison with genomic sequence of Afu using BlastN program, revealed that polyubiquitin gene comprises of 992 bases and contains one intron of 80 bases. The recombinant expression of fusion protein showed an approximately molecular weight of 43-kDa on SDS-PAGE. The translation product of the cDNA sequence showed four tandem repeats of 76 amino acid residues in a single polyubiquitin protein and showed 100% identity with polyubiquitin protein sequences of S. cerevisiae, N. crassa, C. albicans, S. pombe, and M. grisae. Polyubiquitin gene is known to play important role in a variety of cellular processes and recently have been implicated in fungal pathogenesis. Identification of polyubiquitin gene of Afu has opened up scope to study its role in understanding Aspergillus biology and pathogenesis.

SUBMITTER: Shankar J

PROVIDER: S-EPMC3454157 | biostudies-other | 2005 Jan

REPOSITORIES: biostudies-other

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Identification and characterization of polyubiquitin gene from cDNA library of aspergillus fumigatus.

Shankar Jata J Madan Taruna T Basir Seemi Farhat SF Sarma P Usha PU

Indian journal of clinical biochemistry : IJCB 20050101 1

Aspergillus fumigatus (Afu) causes allergic and invasive forms of diseases in humans. In order to identify genes relevant for pathogenesis, a total of 235 cDNA clones were randomly selected and sequenced from cDNA library of Afu. One of the partially sequenced cDNA clones was homologous to polyubiquitin. Sequencing of the complete cDNA clone showed an open reading frame of 912 bases. Comparison with genomic sequence of Afu using BlastN program, revealed that polyubiquitin gene comprises of 992 b ...[more]

PMID: 23105524

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Project description:UnlabelledThe mating efficiency of 50 Aspergillus fumigatus isolates from both clinical and environmental sources was analyzed. Forty isolates completed the sexual cycle in 4 weeks with variable levels of fertility designated high, medium, or low. Two opposite-mating-type strains exhibiting the highest fertility, AFB62 (MAT1-1), isolated from a case of invasive aspergillosis, and AFIR928 (MAT1-2), isolated from the environment, were chosen as the supermater pair. Single cleistothecia obtained from a cross of the two strains harbored a minimum of 1 × 10(4) ascospores. The viability of ascospores increased with the age of the fruiting body, 17% at 4 weeks and reaching 95% at 20 weeks. AFB62 and AFIR928 were equally virulent in two different murine models, despite differences in their sources. High recombination frequencies were observed when the closely linked genes alb1 (AFUA_2G17600) and abr2 (AFUA_2G17530) were used as genetic markers. Comparative genome hybridization analyses revealed that only 86 genes (ca. 0.86% of the genome) are significantly diverged between AFB62 and AFIR928. The high fertility in a relatively short period, combined with a high degree of virulence and a high recombination frequency, demonstrates that the mating pair AFB62 and AFIR928 provides an excellent tool for genetic studies of A. fumigatus.ImportanceAspergillus fumigatus is a heterothallic fungal pathogen that causes life-threatening infections in immunocompromised hosts. Although heterothallism facilitates genetic study via recombinational analysis, previous work showed that a 6-month incubation period is required for the completion of sexual reproduction in this species. Such a long incubation period impedes progress in genetic research. To discover a highly fertile (supermater) pair that can complete the sexual cycle in a considerably shorter period, we screened 50 strains collected from various geographic regions for mating efficiency. We identified a highly virulent pair of supermaters that can be an invaluable tool for genetic study.

Project description:In the last few years, increasing numbers of viruses infecting fungi have been identified. In this study, we used an in silico approach for the analysis of deep RNA sequencing data in order to discover and characterize putative genomic ssRNA or dsRNA mycovirus sequences in Aspergillus fumigatus. RNA sequencing reads of A. fumigatus strains were mapped against the A. fumigatus Af293 reference genome. Unmapped reads were collected for de novo assembly. Contigs were analyzed by Blastx comparison with a mycovirus protein database. Assembled viral genomes were used as template for remapping of RNA sequencing reads. In total, deep RNA sequencing results from 11 A. fumigatus strains were analyzed for the presence of mycoviral genomic RNAs. In 9 out of 11 strains, putative mycoviral RNA genomes were identified. Three strains were infected with two different mycovirus species. Two strains were infected with Aspergillus fumigatus polymycovirus type-1 (AfuPmV-1). Four strains contained fully recovered genomic RNA of unknown narna-like viruses designated as Aspergillus fumigatus narnavirus-1 and Aspergillus fumigatus narnavirus-2 (AfuNV-1 and AfuNV-2). Both viruses showed 38% amino acid sequence identity to Beihai narna-like virus-21. Three strains contained partially recovered genomic RNA of an unknown narna-like virus. Two strains contained fully recovered genomic RNAs of an unknown partitivirus designated as Aspergillus fumigatus partitivirus-2 (AfuPV-2) which showed 50% amino acid sequence identity to Alternaria alternata partitivirus-1. Finally, one strain contained fully recovered genomic RNA of an unknown mitovirus designated as Aspergillus fumigatus mitovirus-1 (AfuMV-1) which showed 34% amino acid sequence identity to Sclerotina sclerotiorum mitovirus. In silico analysis of deep RNA sequencing results showed that a majority of the A. fumigatus strains used here were infected with mycoviruses. Four novel A. fumigatus RNA mycoviruses could be identified: two different Aspergillus fumigatus narna-like viruses, one Aspergillus fumigatus partitivirus, and one Aspergillus fumigatus mitovirus.

Project description:Invasive aspergillosis, largely caused by Aspergillus fumigatus, is responsible for a growing number of deaths among immunosuppressed patients. Immunosuppressants such as FK506 (tacrolimus) that target calcineurin have shown promise for antifungal drug development. FK506-binding proteins (FKBPs) form a complex with calcineurin in the presence of FK506 (FKBP12-FK506) and inhibit calcineurin activity. Research on FKBPs in fungi is limited, and none of the FKBPs have been previously characterized in A. fumigatus. We identified four orthologous genes of FKBP12, the human FK506 binding partner, in A. fumigatus and designated them fkbp12-1, fkbp12-2, fkbp12-3, and fkbp12-4. Deletional analysis of the four genes revealed that the ?fkbp12-1 strain was resistant to FK506, indicating FKBP12-1 as the key mediator of FK506-binding to calcineurin. The endogenously expressed FKBP12-1-EGFP fusion protein localized to the cytoplasm and nuclei under normal growth conditions but also to the hyphal septa following FK506 treatment, revealing its interaction with calcineurin. The FKBP12-1-EGFP fusion protein didn't localize at the septa in the presence of FK506 in the cnaA deletion background, confirming its interaction with calcineurin. Testing of all deletion strains in the Galleria mellonella model of aspergillosis suggested that these proteins don't play an important role in virulence. While the ?fkbp12-2 and ?fkbp12-3 strains didn't show any discernable phenotype, the ?fkbp12-4 strain displayed slight growth defect under normal growth conditions and inhibition of the caspofungin-mediated "paradoxical growth effect" at higher concentrations of the antifungal caspofungin. Together, these results indicate that while only FKBP12-1 is the bona fide binding partner of FK506, leading to the inhibition of calcineurin in A. fumigatus, FKBP12-4 may play a role in basal growth and the caspofungin-mediated paradoxical growth response. Exploitation of differences between A. fumigatus FKBP12-1 and human FKBP12 will be critical for the generation of fungal-specific FK506 analogs to inhibit fungal calcineurin and treat invasive fungal disease.

Dataset Information

Identification and characterization of polyubiquitin gene from cDNA library of aspergillus fumigatus.

Publications

Identification and characterization of polyubiquitin gene from cDNA library of aspergillus fumigatus.

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