SARS冠状病毒亚基因组RNA的分析和鉴定
发布时间：2018-12-10 22:27
【摘要】：SARS冠状病毒亚基因组RNA的分析和鉴定 冠状病毒的一个重要特点是其独特的转录策略(strategy)——即通过不连续的转录合成一组嵌套(nested)的亚基因组RNA(sgRNA)使基因组3’端基因得到表达。转录过程中,每个亚基因组的合成都涉及到一个不连续的步骤,即一段由基因组5’端编码的前导RNA(leader RNA)与主体序列(body sequence)在不同的转录调节序列(transcription regulatory sequence TRS)相连。前导序列在转录时可以自由交换,因此前导序列和mRNA序列可以来源于两个不同的RNA分子,这一过程发生于亚基因组RNA负链RNA模板合成时。TRS在不连续转录时主体序列与前导序列的融合及不连续转录过程中起关键作用,含有一段高度保守的同一序列(consensus sequence CS)。在严重呼吸综合症(SARS)病毒(Sars-Cov)的感染细胞中,我们使用RT-PCR或Northern blot的方法检测到有12种亚基因组RNA,其可能参与表达3’端12个ORF,其中通过进一步的RT-PCR分析鉴定出2个未报道的亚基因组RNA,命名为2-1,3-1,这些亚基因组RNA集中在Sars-Cov基因组后1/3部分。亚基因组RNA存在于S基因内部,在S基因本身CS(ACGAAC)下游384个碱基处,它的前导序列与主体序列结合位点ACGAGC存在着与CS(ACGAAC)一个位点的错配,。 翻译亚基因组RNA 2-1可以得到一个截短的S蛋白(s’),其N端缺少了143个氨基酸。亚基因组RNA 3—1在3b区,预计可能从mRNA3开始表达,它的存在可能预示着ORF3b是从另外一个mRNA而不是从mRNA3开始表达的。亚基因组RNA 3—1的前导序列与主体序列的结合区AaGAAC在ORF3b起始序列AUG上游10个碱基处,与Sars-Cov冠状病毒CS序列同样存在一个碱基的错配。亚基因组RNA 2-1和3-1都与前导序列TRS有一个碱基的错配,但是与主体序列的TRS都是一致的。对不同亚基因组RNA主体序列和前导序列的结合区测序可以看到连接序列和相关的TRS序列都各有不同,而且在病毒的传代中保持相对的稳定性。亚基因组RNA正链和负链模板的共存和亚基因组RNA保守序列与主体序列保守区一致都可以支持不连续转录发生在从全长基因组RNA模板合成负链RNA时的假说模型。根据这一模型,RNA多聚酶在TRS暂停,然后跳跃
[Abstract]:Analysis and Identification of subgenomic RNA of SARS Coronavirus an important characteristic of Coronavirus is its unique transcription Strategy (strategy), that is, through discontinuous transcriptional synthesis of a set of nested (nested) The 3 '-terminal gene was expressed by subgenomic RNA (sgRNA). In the process of transcription, the synthesis of each subgenome involves a discontinuous step, that is, a leading RNA (leader RNA) encoded by the 5 'end of the genome is linked to the host sequence (body sequence) in different transcriptional regulatory sequences (transcription regulatory sequence TRS). The leading sequence can be exchanged freely during transcription, so the leading sequence and the mRNA sequence can be derived from two different RNA molecules. This process takes place during the synthesis of negative RNA templates of subgenomic RNA. TRS plays a key role in the fusion and discontinuous transcription of the main and leading sequences during discontinuous transcription, and contains a highly conserved (consensus sequence CS). Sequence. In cells infected with severe respiratory syndrome (SARS) virus (Sars-Cov), we used RT-PCR or Northern blot to detect 12 subgenomic RNA, that might be involved in the expression of 12 ORF, at the 3 'end. Among them, two unreported subgenomic RNA, were identified by further RT-PCR analysis as 2-1G 3-1, and these subgenomic RNA were concentrated in the posterior third part of Sars-Cov genome. The subgenomic RNA exists in the S gene. At the 384 bases downstream of the S gene CS (ACGAAC), the leading sequence and the main sequence binding site ACGAGC have a mismatch with the CS (ACGAAC) site. A truncated S protein (S') can be obtained by translating subgenome RNA 2-1 with a N-terminal deficiency of 143 amino acids. The expression of subgenomic RNA 3-1 in 3b region is expected to begin with mRNA3, and its existence may indicate that ORF3b is expressed from another mRNA rather than from mRNA3. The binding region AaGAAC of the leading sequence and the host sequence of subgenomic RNA 3-1 has a mismatch with the CS sequence of Sars-Cov coronavirus at the 10 bases upstream of the ORF3b starting sequence AUG. The subgenomic RNA 2-1 and 3-1 have a base mismatch with the leading sequence TRS, but are consistent with the TRS of the host sequence. By sequencing the binding regions of different subgenomic RNA main and leading sequences, we can see that the linked sequences and the related TRS sequences are different, and the relative stability is maintained in the passage of the virus. The coexistence of positive and negative strands of subgenomic RNA and the conserved sequence of subgenomic RNA can support the hypothesis that discontinuous transcription occurs in the synthesis of negative-stranded RNA from full-length genomic RNA templates. According to this model, RNA polymerase pauses in TRS and then jumps
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R373
本文编号：2371318
[Abstract]:Analysis and Identification of subgenomic RNA of SARS Coronavirus an important characteristic of Coronavirus is its unique transcription Strategy (strategy), that is, through discontinuous transcriptional synthesis of a set of nested (nested) The 3 '-terminal gene was expressed by subgenomic RNA (sgRNA). In the process of transcription, the synthesis of each subgenome involves a discontinuous step, that is, a leading RNA (leader RNA) encoded by the 5 'end of the genome is linked to the host sequence (body sequence) in different transcriptional regulatory sequences (transcription regulatory sequence TRS). The leading sequence can be exchanged freely during transcription, so the leading sequence and the mRNA sequence can be derived from two different RNA molecules. This process takes place during the synthesis of negative RNA templates of subgenomic RNA. TRS plays a key role in the fusion and discontinuous transcription of the main and leading sequences during discontinuous transcription, and contains a highly conserved (consensus sequence CS). Sequence. In cells infected with severe respiratory syndrome (SARS) virus (Sars-Cov), we used RT-PCR or Northern blot to detect 12 subgenomic RNA, that might be involved in the expression of 12 ORF, at the 3 'end. Among them, two unreported subgenomic RNA, were identified by further RT-PCR analysis as 2-1G 3-1, and these subgenomic RNA were concentrated in the posterior third part of Sars-Cov genome. The subgenomic RNA exists in the S gene. At the 384 bases downstream of the S gene CS (ACGAAC), the leading sequence and the main sequence binding site ACGAGC have a mismatch with the CS (ACGAAC) site. A truncated S protein (S') can be obtained by translating subgenome RNA 2-1 with a N-terminal deficiency of 143 amino acids. The expression of subgenomic RNA 3-1 in 3b region is expected to begin with mRNA3, and its existence may indicate that ORF3b is expressed from another mRNA rather than from mRNA3. The binding region AaGAAC of the leading sequence and the host sequence of subgenomic RNA 3-1 has a mismatch with the CS sequence of Sars-Cov coronavirus at the 10 bases upstream of the ORF3b starting sequence AUG. The subgenomic RNA 2-1 and 3-1 have a base mismatch with the leading sequence TRS, but are consistent with the TRS of the host sequence. By sequencing the binding regions of different subgenomic RNA main and leading sequences, we can see that the linked sequences and the related TRS sequences are different, and the relative stability is maintained in the passage of the virus. The coexistence of positive and negative strands of subgenomic RNA and the conserved sequence of subgenomic RNA can support the hypothesis that discontinuous transcription occurs in the synthesis of negative-stranded RNA from full-length genomic RNA templates. According to this model, RNA polymerase pauses in TRS and then jumps
【学位授予单位】：武汉大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R373
【参考文献】
相关期刊论文 前1条
1 ;The M Protein of SARS-CoV: Basic Structural and Immunological Properties[J];Genomics Proteomics & Bioinformatics;2003年02期
，本文编号：2371318
本文链接：https://www.wllwen.com/yixuelunwen/binglixuelunwen/2371318.html
最近更新
教材专著
