SARS冠狀病毒亞基因組RNA的分析和鑒定
發(fā)布時(shí)間：2018-12-10 22:27
【摘要】：SARS冠狀病毒亞基因組RNA的分析和鑒定 冠狀病毒的一個(gè)重要特點(diǎn)是其獨(dú)特的轉(zhuǎn)錄策略(strategy)——即通過不連續(xù)的轉(zhuǎn)錄合成一組嵌套(nested)的亞基因組RNA(sgRNA)使基因組3’端基因得到表達(dá)。轉(zhuǎn)錄過程中,每個(gè)亞基因組的合成都涉及到一個(gè)不連續(xù)的步驟,即一段由基因組5’端編碼的前導(dǎo)RNA(leader RNA)與主體序列(body sequence)在不同的轉(zhuǎn)錄調(diào)節(jié)序列(transcription regulatory sequence TRS)相連。前導(dǎo)序列在轉(zhuǎn)錄時(shí)可以自由交換,因此前導(dǎo)序列和mRNA序列可以來源于兩個(gè)不同的RNA分子,這一過程發(fā)生于亞基因組RNA負(fù)鏈RNA模板合成時(shí)。TRS在不連續(xù)轉(zhuǎn)錄時(shí)主體序列與前導(dǎo)序列的融合及不連續(xù)轉(zhuǎn)錄過程中起關(guān)鍵作用,含有一段高度保守的同一序列(consensus sequence CS)。在嚴(yán)重呼吸綜合癥(SARS)病毒(Sars-Cov)的感染細(xì)胞中,我們使用RT-PCR或Northern blot的方法檢測到有12種亞基因組RNA,其可能參與表達(dá)3’端12個(gè)ORF,其中通過進(jìn)一步的RT-PCR分析鑒定出2個(gè)未報(bào)道的亞基因組RNA,命名為2-1,3-1,這些亞基因組RNA集中在Sars-Cov基因組后1/3部分。亞基因組RNA存在于S基因內(nèi)部,在S基因本身CS(ACGAAC)下游384個(gè)堿基處,它的前導(dǎo)序列與主體序列結(jié)合位點(diǎn)ACGAGC存在著與CS(ACGAAC)一個(gè)位點(diǎn)的錯(cuò)配,。 翻譯亞基因組RNA 2-1可以得到一個(gè)截短的S蛋白(s’),其N端缺少了143個(gè)氨基酸。亞基因組RNA 3—1在3b區(qū),預(yù)計(jì)可能從mRNA3開始表達(dá),它的存在可能預(yù)示著ORF3b是從另外一個(gè)mRNA而不是從mRNA3開始表達(dá)的。亞基因組RNA 3—1的前導(dǎo)序列與主體序列的結(jié)合區(qū)AaGAAC在ORF3b起始序列AUG上游10個(gè)堿基處,與Sars-Cov冠狀病毒CS序列同樣存在一個(gè)堿基的錯(cuò)配。亞基因組RNA 2-1和3-1都與前導(dǎo)序列TRS有一個(gè)堿基的錯(cuò)配,但是與主體序列的TRS都是一致的。對不同亞基因組RNA主體序列和前導(dǎo)序列的結(jié)合區(qū)測序可以看到連接序列和相關(guān)的TRS序列都各有不同,而且在病毒的傳代中保持相對的穩(wěn)定性。亞基因組RNA正鏈和負(fù)鏈模板的共存和亞基因組RNA保守序列與主體序列保守區(qū)一致都可以支持不連續(xù)轉(zhuǎn)錄發(fā)生在從全長基因組RNA模板合成負(fù)鏈RNA時(shí)的假說模型。根據(jù)這一模型,RNA多聚酶在TRS暫停,然后跳躍
[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
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2005
【分類號】：R373
本文編號：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
【學(xué)位授予單位】：武漢大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2005
【分類號】：R373
【參考文獻(xiàn)】
相關(guān)期刊論文 前1條
1 ;The M Protein of SARS-CoV: Basic Structural and Immunological Properties[J];Genomics Proteomics & Bioinformatics;2003年02期
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