Analysis of transcripts from predicted open reading frames of Musca domestica salivary gland hypertrophy virus
Analysis of transcripts from predicted open reading frames of Musca domestica salivary gland hypertrophy virus
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DOI：
10.1099/vir.0.009613-0
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发表时间：
2009-05-01
影响因子：
3.8
通讯作者：
Boucias, Drion G.
中科院分区：
文献类型：
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作者：
Salem, Tamer Z.;Garcia-Maruniak, Alejandra;Boucias, Drion G.
家蝇唾液腺肥大病毒(MdSGHV)是一种感染成年家蝇并使其绝育的大型双链DNA病毒。用3‘端快速扩增(3’-RACE)和RT-PCR方法分析了新病毒的转录组。对3‘-RACE产物的直接测序显示，78个Poly(A)转录本包含108个可能的ORF中的95个。RT-PCR检测到6个3‘-RACE未扩增的ORF。在108个可能的ORF中，只有7个未经3‘-RACE或RT-PCR扩增。在选择的ORF上进行了一系列的5‘-RACE反应，这些ORF被3’-RACE鉴定为串联转录(串联转录)。在大多数情况下，下游ORF被检测到是单个转录本，也是串联转录本的组成部分，而上游ORF仅在串联转录本中被发现。唯一的例外是上游的ORF MdSGHV084，它在感染后1天和2天(P.I.)被差异转录为单个转录本。和2天P.I.的串联转录本(MdSGHV084/085)。MdSGHV的转录组分析检测到3‘非翻译区(3’-UTR)的剪接，并在多聚腺苷信号和切割位点上存在广泛的异质性。此外，还发现了23个重叠的反义转录本。总之，在不进行克隆的情况下对3‘-RACE产物进行测序是检测这种大型DNA病毒3’-UTRs和转录变异体的另一种方法。
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.