Senecavirus A(SVA)has a positive-sense,single-stranded RNA genome.Its 5´untranslated region harbors an internal ribosome entry site(IRES),comprising 10 larger or smaller stem-loop structures(including a pseudokno...Senecavirus A(SVA)has a positive-sense,single-stranded RNA genome.Its 5´untranslated region harbors an internal ribosome entry site(IRES),comprising 10 larger or smaller stem-loop structures(including a pseudoknot)that have been demonstrated to be well conserved.However,it is still unclear whether each stem-loop subdomain,such as a single stem or loop,is also highly conserved.To clarify this issue in the present study,a set of 29 SVA cDNA clones were constructed by site-directed mutagenesis(SDM)on the IRES.The SDM-modified scenarios included:(1)stem-formed complementary sequences exchanging with each other;(2)loop transversion;(3)loop transition;and(4)point mutations.All cDNA clones were separately transfected into cells for rescuing viable viruses,whereas only four SVAs of interest could be recovered,and were genetically stable during 20 passages.One progeny grew significantly slower than the other three did.The dual-luciferase reporter assay showed that none of the SDM-modified IRESes significantly inhibited the IRES activity.Our previous study indicated that a single motif from any of the ten stem structures,if completely mutated,would cause the failure of virus recovery.Interestingly,our present study revealed three stem structures,whose individual complementary sequences could exchange with each other to rescue sequence-modifying SVAs.Moreover,one apical loop was demonstrated to have the ability to tolerate its own full-length transition,also having no impact on the recovery of sequence-modifying SVA.The present study suggested that not every stem-loop structure was strictly conserved in its conformation,while the full-length IRES itself was well conserved.This provides a new research direction on interaction between the IRES and many factors.展开更多
The 2.2 kb EcoRI fragment containing the chloroplast pshA gene from millet (Setaria italica) has been cloned and the nucleotide sequence of the 5’-noncoding region has been determined. The 5’-flanking region is foun...The 2.2 kb EcoRI fragment containing the chloroplast pshA gene from millet (Setaria italica) has been cloned and the nucleotide sequence of the 5’-noncoding region has been determined. The 5’-flanking region is found to contain prokaryote-like promoter structures: compared with prokaryotic promoters, the“-35”box (TTGACA) shows 100% homology while, in the ‘-10’box (TATACT), one different nucleotide is found. In addition, between these two boxes, there is a consensus sequence“TATATA”.just as in prokaryotic promoters. All these results indicate that millet psbA promoter has both prokaryotic and eukaryotic characteristies. The mRNA leader region of millet pshA gene is 87 bp, the same length as sorghum. However, an additional CTATTT sequence is found as compared with rice and an additional TTTT, as with wheat, barley and rye. So the differences between C3 and C4 plants may be universal in the family of Gramineae. Furthermore, computer analysis shows that a small stem-loop structure might be formed in pshA mRNA leader region in these six plants. The above-mentioned additional CTATTT sequence happens to be just located within the stem-loop structure, thus leading to different sizes of the stem-loops among these six species. It is likely that this small secondary structure may have some effect on the expression and regulation of the psbA gene.展开更多
猪繁殖与呼吸综合征(porcine reproductive and respiratory syndrome,PRRS)是世界养猪业的一个巨大的威胁。近年来,此病给各国养猪业造成了巨大的经济损失。本文立足于反向遗传操作技术,介绍了调控PRRS病毒粒子的感染性、基因组RNA复...猪繁殖与呼吸综合征(porcine reproductive and respiratory syndrome,PRRS)是世界养猪业的一个巨大的威胁。近年来,此病给各国养猪业造成了巨大的经济损失。本文立足于反向遗传操作技术,介绍了调控PRRS病毒粒子的感染性、基因组RNA复制、亚基因组mRNA转录以及翻译等重要过程的顺式调控元件的一级序列和高级结构的最新研究进展。展开更多
基金This work was supported by the National Natural Science Found ation of China(32273000)the Qingdao Demonstration Project for People-benefit from Science and Techniques,China(23-2-8-xdny-14nsh and 24-2-8-xdny-4-nsh)+1 种基金the National Program of Undergraduate Innovation and Entrepreneurship,China(202310435039)the Open Project Fund of State Key Laboratory of Microbial Technology,China(M2023-03)。
文摘Senecavirus A(SVA)has a positive-sense,single-stranded RNA genome.Its 5´untranslated region harbors an internal ribosome entry site(IRES),comprising 10 larger or smaller stem-loop structures(including a pseudoknot)that have been demonstrated to be well conserved.However,it is still unclear whether each stem-loop subdomain,such as a single stem or loop,is also highly conserved.To clarify this issue in the present study,a set of 29 SVA cDNA clones were constructed by site-directed mutagenesis(SDM)on the IRES.The SDM-modified scenarios included:(1)stem-formed complementary sequences exchanging with each other;(2)loop transversion;(3)loop transition;and(4)point mutations.All cDNA clones were separately transfected into cells for rescuing viable viruses,whereas only four SVAs of interest could be recovered,and were genetically stable during 20 passages.One progeny grew significantly slower than the other three did.The dual-luciferase reporter assay showed that none of the SDM-modified IRESes significantly inhibited the IRES activity.Our previous study indicated that a single motif from any of the ten stem structures,if completely mutated,would cause the failure of virus recovery.Interestingly,our present study revealed three stem structures,whose individual complementary sequences could exchange with each other to rescue sequence-modifying SVAs.Moreover,one apical loop was demonstrated to have the ability to tolerate its own full-length transition,also having no impact on the recovery of sequence-modifying SVA.The present study suggested that not every stem-loop structure was strictly conserved in its conformation,while the full-length IRES itself was well conserved.This provides a new research direction on interaction between the IRES and many factors.
文摘The 2.2 kb EcoRI fragment containing the chloroplast pshA gene from millet (Setaria italica) has been cloned and the nucleotide sequence of the 5’-noncoding region has been determined. The 5’-flanking region is found to contain prokaryote-like promoter structures: compared with prokaryotic promoters, the“-35”box (TTGACA) shows 100% homology while, in the ‘-10’box (TATACT), one different nucleotide is found. In addition, between these two boxes, there is a consensus sequence“TATATA”.just as in prokaryotic promoters. All these results indicate that millet psbA promoter has both prokaryotic and eukaryotic characteristies. The mRNA leader region of millet pshA gene is 87 bp, the same length as sorghum. However, an additional CTATTT sequence is found as compared with rice and an additional TTTT, as with wheat, barley and rye. So the differences between C3 and C4 plants may be universal in the family of Gramineae. Furthermore, computer analysis shows that a small stem-loop structure might be formed in pshA mRNA leader region in these six plants. The above-mentioned additional CTATTT sequence happens to be just located within the stem-loop structure, thus leading to different sizes of the stem-loops among these six species. It is likely that this small secondary structure may have some effect on the expression and regulation of the psbA gene.
文摘猪繁殖与呼吸综合征(porcine reproductive and respiratory syndrome,PRRS)是世界养猪业的一个巨大的威胁。近年来,此病给各国养猪业造成了巨大的经济损失。本文立足于反向遗传操作技术,介绍了调控PRRS病毒粒子的感染性、基因组RNA复制、亚基因组mRNA转录以及翻译等重要过程的顺式调控元件的一级序列和高级结构的最新研究进展。
