To develop a new DNA maker, which could be used in genetic diversity analysis and genetic map construction in plants, IT-ISJ （intron targeted intron-exon splice junction） primer combinations, which were designed acc...To develop a new DNA maker, which could be used in genetic diversity analysis and genetic map construction in plants, IT-ISJ （intron targeted intron-exon splice junction） primer combinations, which were designed according to the intronexon splice junction conserved sequences, were used to construct cotton genetic linkage map in the present study. 49 out of 704 IT-ISJ primer combinations showed polymorphism between upland cotton high quality cultivar Yumian 1 and multiple dominant gene line T586, and the polymorphic primer combinations accounted for 7.0% of total primer combinations. 49 IT-ISJ primer combinations were used to genotype 270 F2：7 recombinant inbred lines developed from （Yumian 1 × T586） F2, and 58 IT-ISJ loci were obtained. 58 IT-ISJ, together with 150 SSR and 8 morphological loci, were used to conduct linkage analysis, and a linkage map including 22 linkage groups and 113 loci （49 IT-ISJ, 62 SSR, and 2 morphological loci） was constructed. The linkage map covered 714.5 cM with an average interval of 6.3 cM between two markers, accounting for 16.1% of cotton genome. The present study demonstrated that the polymorphism of IT-ISJ marker is high, and it could be effectively applied in plant genetic map construction.展开更多
The exon numbers and lengths vary in different eukaryotic species. With increasing completed genomic sequences, it is indispensable to reanalyze the gene organization in diverse eukaryotic genomes. We performed a larg...The exon numbers and lengths vary in different eukaryotic species. With increasing completed genomic sequences, it is indispensable to reanalyze the gene organization in diverse eukaryotic genomes. We performed a large-scale comparative analysis of the exon-intron structure in 72 eukaryotic organisms, including plants, fungi and animals. We confirmed that the exon-intron structure varies massively among eukaryotic genomes and revealed some lineage-specific features of eukaryotic genes. These include a teleost-specific exon-intron structure pattern, relatively small introns and large exons in fungi and algae, and a gradual expansion of introns in vertebrates. Furthermore, the conservation analysis of exon-intron boundaries indicates that several bases near splice site junctions are different in introns with variable length among different species. After comparison, we identified a trend showing increases in intron densities and lengths in diverse species from fungi, plants, invertebrates to vertebrates, while it was the opposite in relation to exon lengths. The statistical properties of eukaryotic genomic organization suggest that genome-specific features are preserved by diverse evolutionary processes, which paves way for further research on the diversification of eukaryotic evolution.展开更多
In this study,three weight vectors L1,L2 and L3 were set.After calculating the probability of three bases in the exons or introns in the genomic DNA of Arabidopsis thaliana,64-dimensional vector P was obtained.Dot pro...In this study,three weight vectors L1,L2 and L3 were set.After calculating the probability of three bases in the exons or introns in the genomic DNA of Arabidopsis thaliana,64-dimensional vector P was obtained.Dot products of P vector and three weight vectors were the feature coordinates for the exons and introns in 3-dimensional phase space.The expression for the interface between the exons and the introns in the genomic DNA of Arabidopsis thaliana in 3-dimensional phase space was established,which could be used to distinguish the exons and the introns in the genomic DNA of Arabidopsis thaliana with an accuracy higher than85%in 3-dimensional phase space.展开更多
Although a great deal of research has been undertaken in the area of the annotation of gene structure, predictive techniques are still not fully developed. In this paper, based on the characteristics of base compositi...Although a great deal of research has been undertaken in the area of the annotation of gene structure, predictive techniques are still not fully developed. In this paper, based on the characteristics of base composition of sequences and conservative of nucleotides at exon/intron splicing site, a least increment of diversity al-gorithm (LIDA) is developed for studying and predicting three kinds of coding exons, introns and intergenic regions. At first, by selecting the 64 trinucleotides composition and 120 position parameters of the four bases as informational parameters, coding exon, intron and intergenic sequence are predicted. The results show that overall predicted accuracies are 91.1% and 88.4%, respectively for A. thaliana and C. ele-gans genome. Subsequently, based on the po-sition frequencies of four kinds of bases in regions near intron/coding exon boundary, initia-tion and termination site of translation, 12 position parameters are selected as diversity source. And three kinds of the coding exons are predicted by use of the LIDA. The predicted successful rates are higher than 80%. These results can be used in sequence annotation.展开更多
A rapid and accurate method was used to identify the exon intron boundaries in a novel zinc finger gene ZNF191. Genomic DNAs containing the sequence of ZNF191 cDNA were digested with proper restriction enzymes and th...A rapid and accurate method was used to identify the exon intron boundaries in a novel zinc finger gene ZNF191. Genomic DNAs containing the sequence of ZNF191 cDNA were digested with proper restriction enzymes and then ligated to an annealed bubble linker. The ligation product was used as the template to carry out PCR with the primers of ZNF191 cDNA and the bubble linker. Then the PCR products were sequenced to determine the exon intron boundaries. The results show that the zinc finger gene(ZNF191) has 4 exons and 3 introns. It was further confirmed by the sequencing data of genomic DNA of the gene ZNF191.展开更多
Spinal muscular atrophy(SMA)is an autosomal recessive hereditary neuromuscular disease.Exon 7 and 8 of survival of motor neuron 1(SMN1)gene or only exon 7 homology deletion leads to the failure to produce a full-lengt...Spinal muscular atrophy(SMA)is an autosomal recessive hereditary neuromuscular disease.Exon 7 and 8 of survival of motor neuron 1(SMN1)gene or only exon 7 homology deletion leads to the failure to produce a full-length SMN gene.The copy number of SMN2 gene with high homology of SMN1 affects the degree of disease and was the target gene for targeting therapy,in which splicing silencer in intron 7 was the key to suppress the inclusion of exon 7.In this study,we projected to use CRISPR/Case 9 for the targeted editing of intronic-splicing silencer(ISS)sequence to promote the inclusion of SMN2 exon 7 and increase the production of SMN2 full-length(FL)gene expression.It happens that there was a protospacer adjacent motif(PAM)at one end of the ISS sequence according to the design of sgRNA.The recombinant vector of sgRNA HSMN2 CRISPR/Case 9 was constructed and transfected into HEK293 cells.Sequencing results showed that the ISS sequence could be edited accurately and targeting in the predicted direction,in which deleting small fragments,inserting small amounts and mutation.Quantitative analysis of RT-PCR products by restriction enzyme of DdeI digestion showed that the FL of SMN2 increased by 8%(P<0.05).In the primary cultured chondrocytes of SMA mice,in which sgRNA HSMN2 CRISPR/Case9 recombinant vector transfection could increase the SMN2 FL gene by 23%(P<0.05)and significantly improve SMN protein levels(P<0.05).CRISPR/Case 9 is an effective tool for gene editing and therapy of hereditary diseases,but it is rarely reported in the treatment of SMA diseases.This study shows that CRISPR/Case 9 was first used for the precision target of ISS sequence editing,which can effectively promote the production of SMN2 FL gene expressions,in which there was an important clinical reference value.展开更多
Pentatricopeptide repeat(PPR)proteins are a large group of eukaryote-specific RNA-binding proteins that play pivotal roles in plant organelle gene expression.Here,we report the function of PPR21 in mitochondrial intro...Pentatricopeptide repeat(PPR)proteins are a large group of eukaryote-specific RNA-binding proteins that play pivotal roles in plant organelle gene expression.Here,we report the function of PPR21 in mitochondrial intron splicing and its role in maize kernel development.PPR21 is a typical P-type PPR protein targeted to mitochondria.The ppr21 mutants are arrested in embryogenesis and endosperm development,leading to embryo lethality.Null mutations of PPR21 reduce the splicing efficiency of nad2 intron 1,2,and 4 and impair the assembly and activity of mitochondrial complex I.Previous studies show that the P-type PPR protein EMP12 is required for the splicing of identical introns.However,our protein interaction analyses reveal that PPR21 does not interact with EMP12.Instead,both PPR21 and EMP12 interact with the small MutS-related(SMR)domain-containing PPR protein 1(PPR-SMR1)and the short P-type PPR protein 2(SPR2).PPR-SMR1 interacts with SPR2,and both proteins are required for the splicing of many introns in mitochondria,including nad2 intron 1,2,and 4.These results suggest that a PPR21-(PPR-SMR1/SPR2)-EMP12 complex is involved in the splicing of nad2 introns in maize mitochondria.展开更多
Linear mRNA vaccines are constrained by exonuclease susceptibility and instability,leading to compromised antigen expression.Circular RNA(circRNA) lacking canonical 5' and 3' untranslated regions demonstrates ...Linear mRNA vaccines are constrained by exonuclease susceptibility and instability,leading to compromised antigen expression.Circular RNA(circRNA) lacking canonical 5' and 3' untranslated regions demonstrates intrinsic exonuclease resistance.Current circularization strategies face three principal limitations:chemical methods produce non-native 2',5'-phosphodiester bonds;ribozyme-mediated approaches are restricted to RNA fragments shorter than 500 nucleotides;the Anabaena Group I intron system retains immunogenic exon sequences.In contrast,the self-splicing Group I intron ribozyme from Tetrahymena enables precisely controlled circularization through autonomous structural rearrangement,yielding exonfree constructs.Through optimized purification protocols,historical scalability challenges are systematically addressed.This Perspective establishes the mechanistic rationale and therapeutic superiority of this engineered RNA circularization platform.展开更多
基金the National Natural Science Foundation of China (30370898,30571187, 30871556)National High Tech Research and Development Program of China (2006AA10Z1D3,2006AA100105)
文摘To develop a new DNA maker, which could be used in genetic diversity analysis and genetic map construction in plants, IT-ISJ （intron targeted intron-exon splice junction） primer combinations, which were designed according to the intronexon splice junction conserved sequences, were used to construct cotton genetic linkage map in the present study. 49 out of 704 IT-ISJ primer combinations showed polymorphism between upland cotton high quality cultivar Yumian 1 and multiple dominant gene line T586, and the polymorphic primer combinations accounted for 7.0% of total primer combinations. 49 IT-ISJ primer combinations were used to genotype 270 F2：7 recombinant inbred lines developed from （Yumian 1 × T586） F2, and 58 IT-ISJ loci were obtained. 58 IT-ISJ, together with 150 SSR and 8 morphological loci, were used to conduct linkage analysis, and a linkage map including 22 linkage groups and 113 loci （49 IT-ISJ, 62 SSR, and 2 morphological loci） was constructed. The linkage map covered 714.5 cM with an average interval of 6.3 cM between two markers, accounting for 16.1% of cotton genome. The present study demonstrated that the polymorphism of IT-ISJ marker is high, and it could be effectively applied in plant genetic map construction.
文摘The exon numbers and lengths vary in different eukaryotic species. With increasing completed genomic sequences, it is indispensable to reanalyze the gene organization in diverse eukaryotic genomes. We performed a large-scale comparative analysis of the exon-intron structure in 72 eukaryotic organisms, including plants, fungi and animals. We confirmed that the exon-intron structure varies massively among eukaryotic genomes and revealed some lineage-specific features of eukaryotic genes. These include a teleost-specific exon-intron structure pattern, relatively small introns and large exons in fungi and algae, and a gradual expansion of introns in vertebrates. Furthermore, the conservation analysis of exon-intron boundaries indicates that several bases near splice site junctions are different in introns with variable length among different species. After comparison, we identified a trend showing increases in intron densities and lengths in diverse species from fungi, plants, invertebrates to vertebrates, while it was the opposite in relation to exon lengths. The statistical properties of eukaryotic genomic organization suggest that genome-specific features are preserved by diverse evolutionary processes, which paves way for further research on the diversification of eukaryotic evolution.
基金Supported by Eleventh Five-Year Development Planning For Instructional Science in Hubei Province(2006B131)
文摘In this study,three weight vectors L1,L2 and L3 were set.After calculating the probability of three bases in the exons or introns in the genomic DNA of Arabidopsis thaliana,64-dimensional vector P was obtained.Dot products of P vector and three weight vectors were the feature coordinates for the exons and introns in 3-dimensional phase space.The expression for the interface between the exons and the introns in the genomic DNA of Arabidopsis thaliana in 3-dimensional phase space was established,which could be used to distinguish the exons and the introns in the genomic DNA of Arabidopsis thaliana with an accuracy higher than85%in 3-dimensional phase space.
文摘Although a great deal of research has been undertaken in the area of the annotation of gene structure, predictive techniques are still not fully developed. In this paper, based on the characteristics of base composition of sequences and conservative of nucleotides at exon/intron splicing site, a least increment of diversity al-gorithm (LIDA) is developed for studying and predicting three kinds of coding exons, introns and intergenic regions. At first, by selecting the 64 trinucleotides composition and 120 position parameters of the four bases as informational parameters, coding exon, intron and intergenic sequence are predicted. The results show that overall predicted accuracies are 91.1% and 88.4%, respectively for A. thaliana and C. ele-gans genome. Subsequently, based on the po-sition frequencies of four kinds of bases in regions near intron/coding exon boundary, initia-tion and termination site of translation, 12 position parameters are selected as diversity source. And three kinds of the coding exons are predicted by use of the LIDA. The predicted successful rates are higher than 80%. These results can be used in sequence annotation.
文摘A rapid and accurate method was used to identify the exon intron boundaries in a novel zinc finger gene ZNF191. Genomic DNAs containing the sequence of ZNF191 cDNA were digested with proper restriction enzymes and then ligated to an annealed bubble linker. The ligation product was used as the template to carry out PCR with the primers of ZNF191 cDNA and the bubble linker. Then the PCR products were sequenced to determine the exon intron boundaries. The results show that the zinc finger gene(ZNF191) has 4 exons and 3 introns. It was further confirmed by the sequencing data of genomic DNA of the gene ZNF191.
基金Nantong Science and Technology Program,grant number(JC2018090)the Practice Innovation Training Program Projects for the Jiangsu College Students,grant number(201810304028z)the Scientific Innovation Research of College Graduates in Jiangsu Province,grant number(KYCX18-2415)。
文摘Spinal muscular atrophy(SMA)is an autosomal recessive hereditary neuromuscular disease.Exon 7 and 8 of survival of motor neuron 1(SMN1)gene or only exon 7 homology deletion leads to the failure to produce a full-length SMN gene.The copy number of SMN2 gene with high homology of SMN1 affects the degree of disease and was the target gene for targeting therapy,in which splicing silencer in intron 7 was the key to suppress the inclusion of exon 7.In this study,we projected to use CRISPR/Case 9 for the targeted editing of intronic-splicing silencer(ISS)sequence to promote the inclusion of SMN2 exon 7 and increase the production of SMN2 full-length(FL)gene expression.It happens that there was a protospacer adjacent motif(PAM)at one end of the ISS sequence according to the design of sgRNA.The recombinant vector of sgRNA HSMN2 CRISPR/Case 9 was constructed and transfected into HEK293 cells.Sequencing results showed that the ISS sequence could be edited accurately and targeting in the predicted direction,in which deleting small fragments,inserting small amounts and mutation.Quantitative analysis of RT-PCR products by restriction enzyme of DdeI digestion showed that the FL of SMN2 increased by 8%(P<0.05).In the primary cultured chondrocytes of SMA mice,in which sgRNA HSMN2 CRISPR/Case9 recombinant vector transfection could increase the SMN2 FL gene by 23%(P<0.05)and significantly improve SMN protein levels(P<0.05).CRISPR/Case 9 is an effective tool for gene editing and therapy of hereditary diseases,but it is rarely reported in the treatment of SMA diseases.This study shows that CRISPR/Case 9 was first used for the precision target of ISS sequence editing,which can effectively promote the production of SMN2 FL gene expressions,in which there was an important clinical reference value.
基金supported by the National Natural Science Foundation of China(32072126 and 32230075)the Shandong Provincial Natural Science Foundation(ZR2019MC005).
文摘Pentatricopeptide repeat(PPR)proteins are a large group of eukaryote-specific RNA-binding proteins that play pivotal roles in plant organelle gene expression.Here,we report the function of PPR21 in mitochondrial intron splicing and its role in maize kernel development.PPR21 is a typical P-type PPR protein targeted to mitochondria.The ppr21 mutants are arrested in embryogenesis and endosperm development,leading to embryo lethality.Null mutations of PPR21 reduce the splicing efficiency of nad2 intron 1,2,and 4 and impair the assembly and activity of mitochondrial complex I.Previous studies show that the P-type PPR protein EMP12 is required for the splicing of identical introns.However,our protein interaction analyses reveal that PPR21 does not interact with EMP12.Instead,both PPR21 and EMP12 interact with the small MutS-related(SMR)domain-containing PPR protein 1(PPR-SMR1)and the short P-type PPR protein 2(SPR2).PPR-SMR1 interacts with SPR2,and both proteins are required for the splicing of many introns in mitochondria,including nad2 intron 1,2,and 4.These results suggest that a PPR21-(PPR-SMR1/SPR2)-EMP12 complex is involved in the splicing of nad2 introns in maize mitochondria.
基金supported by the National Key Research&Development Program of China(Nos.2021YFC2302400,2021YFA1201000,2023YFC2606004)the Fundamental Research Funds for the Central Universities(No.2022CX01013)。
文摘Linear mRNA vaccines are constrained by exonuclease susceptibility and instability,leading to compromised antigen expression.Circular RNA(circRNA) lacking canonical 5' and 3' untranslated regions demonstrates intrinsic exonuclease resistance.Current circularization strategies face three principal limitations:chemical methods produce non-native 2',5'-phosphodiester bonds;ribozyme-mediated approaches are restricted to RNA fragments shorter than 500 nucleotides;the Anabaena Group I intron system retains immunogenic exon sequences.In contrast,the self-splicing Group I intron ribozyme from Tetrahymena enables precisely controlled circularization through autonomous structural rearrangement,yielding exonfree constructs.Through optimized purification protocols,historical scalability challenges are systematically addressed.This Perspective establishes the mechanistic rationale and therapeutic superiority of this engineered RNA circularization platform.
