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Analysis of transcripts from predicted open reading frames of Musca domestica salivary gland hypertrophy virus.

ABSTRACT: The Musca domestica salivary gland hypertrophy virus (MdSGHV) is a large dsDNA virus that infects and sterilizes adult houseflies. The transcriptome of this newly described virus was analysed by rapid amplification of cDNA 3'-ends (3'-RACE) and RT-PCR. Direct sequencing of 3'-RACE products revealed 78 poly(A) transcripts containing 95 of the 108 putative ORFs. An additional six ORFs not amplified by 3'-RACE were detected by RT-PCR. Only seven of the 108 putative ORFs were not amplified by either 3'-RACE or RT-PCR. A series of 5'-RACE reactions were conducted on selected ORFs that were identified by 3'-RACE to be transcribed in tandem (tandem transcripts). In the majority of cases, the downstream ORFs were detected as single transcripts as well as components of the tandem transcripts, whereas the upstream ORFs were found only in tandem transcripts. The only exception was the upstream ORF MdSGHV084, which was differentially transcribed as a single transcript at 1 and 2 days post-infection (days p.i.) and as a tandem transcript (MdSGHV084/085) at 2 days p.i. Transcriptome analysis of MdSGHV detected splicing in the 3' untranslated region (3'-UTR) and extensive heterogeneity in the polyadenylation signals and cleavage sites. In addition, 23 overlapping antisense transcripts were found. In conclusion, sequencing the 3'-RACE products without cloning served as an alternative approach to detect both 3'-UTRs and transcript variants of this large DNA virus.

SUBMITTER: Salem TZ

PROVIDER: S-EPMC2887563 | biostudies-literature | 2009 May

REPOSITORIES: biostudies-literature

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Analysis of transcripts from predicted open reading frames of Musca domestica salivary gland hypertrophy virus.

Salem Tamer Z TZ Garcia-Maruniak Alejandra A Lietze Verena-U VU Maruniak James E JE Boucias Drion G DG

The Journal of general virology 20090304 Pt 5

The Musca domestica salivary gland hypertrophy virus (MdSGHV) is a large dsDNA virus that infects and sterilizes adult houseflies. The transcriptome of this newly described virus was analysed by rapid amplification of cDNA 3'-ends (3'-RACE) and RT-PCR. Direct sequencing of 3'-RACE products revealed 78 poly(A) transcripts containing 95 of the 108 putative ORFs. An additional six ORFs not amplified by 3'-RACE were detected by RT-PCR. Only seven of the 108 putative ORFs were not amplified by either ...[more]

PMID: 19264592

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Project description:BackgroundPlasmodium falciparum causes the deadliest form of malaria, which remains one of the most prevalent infectious diseases. Unfortunately, the only licensed vaccine showed limited protection and resistance to anti-malarial drug is increasing, which can be largely attributed to the biological complexity of the parasite's life cycle. The progression from one developmental stage to another in P. falciparum involves drastic changes in gene expressions, where its infectivity to human hosts varies greatly depending on the stage. Approaches to identify candidate genes that are responsible for the development of infectivity to human hosts typically involve differential gene expression analysis between stages. However, the detection may be limited to annotated proteins and open reading frames (ORFs) predicted using restrictive criteria.MethodsThe above problem is particularly relevant for P. falciparum; whose genome annotation is relatively incomplete given its clinical significance. In this work, systems proteogenomics approach was used to address this challenge, as it allows computational detection of unannotated, novel Open Reading Frames (nORFs), which are neglected by conventional analyses. Two pairs of transcriptome/proteome were obtained from a previous study where one was collected in the mosquito-infectious oocyst sporozoite stage, and the other in the salivary gland sporozoite stage with human infectivity. They were then re-analysed using the proteogenomics framework to identify nORFs in each stage.ResultsTranslational products of nORFs that map to antisense, intergenic, intronic, 3' UTR and 5' UTR regions, as well as alternative reading frames of canonical proteins were detected. Some of these nORFs also showed differential expression between the two life cycle stages studied. Their regulatory roles were explored through further bioinformatics analyses including the expression regulation on the parent reference genes, in silico structure prediction, and gene ontology term enrichment analysis.ConclusionThe identification of nORFs in P. falciparum sporozoites highlights the biological complexity of the parasite. Although the analyses are solely computational, these results provide a starting point for further experimental validation of the existence and functional roles of these nORFs.

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Analysis of transcripts from predicted open reading frames of Musca domestica salivary gland hypertrophy virus.

Publications

Analysis of transcripts from predicted open reading frames of Musca domestica salivary gland hypertrophy virus.

Similar Datasets

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