Kernel development plays an important role in determining kernel size in maize.Here we present the cloning and characterization of a maize gene,nitrate transporter1.5(NRT1.5),which controls small kernel phenotype by p...Kernel development plays an important role in determining kernel size in maize.Here we present the cloning and characterization of a maize gene,nitrate transporter1.5(NRT1.5),which controls small kernel phenotype by playing an important role in kernel development.A novel recessive small kernel mutant miniature2-m1(mn2-m1)was isolated from self-pollinated progenies of breeding materials.The mutant spontaneously showed small kernel character arresting both embryo and endosperm development at an early stage after pollination.Utilizing 21 polymorphic SSR markers,the mn2-m1 locus was limited to a 209.9-kb interval using 9176 recessive individuals of a BC1 segregating population from mn2-m1/B73.Only one annotated gene was located in this 209.9 kb region,Zm00001 d019294,which was predicted to encode nitrate transporter1.5(NRT1.5).Allelism tests confirmed that mn2-m1 was allelic to miniature2-m2(mn2-m2)and miniature2-710 B(mn2-710 B).The mn2-m1 and mn2-m2 alleles both had nucleotide deletions in the coding region resulting in premature termination,and the mn2-710 B allele had some missence mutations.Subcellular localization showed that Miniature 2(MN2)is localized in the plasma membrane.Quantitative real-time PCR(qRT-PCR)analysis revealed that the expression of MN2 and some genes involved in the basal endosperm transfer layer(BETL)and embryo surrounding region(ESR)development were affected in mn2-m1 seeds.These results suggested that MN2 plays an important role in maize seed development.展开更多
AIMTo investigate the role of non-structural open reading frame 1 “Y-domain” sequences in the hepatitis E virus (HEV) life cycle.METHODSSequences of human HEV Y-domain (amino acid sequences 216...AIMTo investigate the role of non-structural open reading frame 1 “Y-domain” sequences in the hepatitis E virus (HEV) life cycle.METHODSSequences of human HEV Y-domain (amino acid sequences 216-442) and closely-related viruses were analyzed in silico. Site-directed mutagenesis of the Y-domain (HEV SAR55) was carried out and studied in the replicon-baculovirus-hepatoma cell model. In vitro transcribed mRNA (pSK-GFP) constructs were transfected into S10-3 cells and viral RNA replicating GFP-positive cells were scored by flow cytometry. Mutant virions’ infectivity was assayed on naïve HepG2/C3A cells.RESULTSIn silico analysis identified a potential palmitoylation-site (C<sub>336</sub>C<sub>337</sub>) and an α-helix segment (L<sub>410</sub>Y<sub>411</sub>S<sub>412</sub>W<sub>413</sub>L<sub>414</sub>F<sub>415</sub>E<sub>416</sub>) in the HEV Y-domain. Molecular characterization of C<sub>336</sub>A, C<sub>337</sub>A and W<sub>413</sub>A mutants of the three universally conserved residues showed non-viability. Further, of the 10 consecutive saturation mutants covering the entire Y-domain nucleotide sequences (nts 650-1339), three constructs (nts 788-994) severely affected virus replication. This revealed the indispensability of the internal sequences but not of the up- or downstream sequences at the transcriptional level. Interestingly, the three mutated residues corresponded to the downstream codons that tolerated saturation mutation, indicating their post-translational functional/structural essentiality. In addition, RNA secondary structure prediction revealed formation of stable hairpins (nts 788-994) where saturation mutation drastically inhibited virion infectivity.CONCLUSIONThis is the first demonstration of the critical role of Y-domain sequences in HEV life cycle, which may involve gene regulation and/or membrane binding in intracellular replication complexes.展开更多
The p27Kip1 is a cell cycle repressor protein that regulates primarily the cell cycle transition from G1 to S phase and hence the DNA replication is in the S phase and cell division in the M phase. Expression of p27Ki...The p27Kip1 is a cell cycle repressor protein that regulates primarily the cell cycle transition from G1 to S phase and hence the DNA replication is in the S phase and cell division in the M phase. Expression of p27Kip1 protein has dual roles for both cancer prevention and promotion. For example, numerous nutritional and chemopreventive anti-cancer agents specifically increase the expression of p27Kip1 protein without directly affecting the expression of any other cell cycle regulatory proteins. On the other hand, pro-cancer agents (like glucose, insulin and other growth factors frequently seen in obesity and/or diabetes) specifically decrease the expression of p27Kip1 protein without directly affecting the expression of any other cell cycle regulatory proteins. Unlike expression of any other cell cycle regulatory proteins, expression of p27Kip1 protein is very unusual. The mRNA of p27Kip1 has a very long and unusual 5’-untranslated region (from -575 to -1 in human). It appears that the 5’-untranslated region of p27Kip1 mRNA forms two alternative secondary structures. One increases the expression of p27Kip1 protein when anti-cancer agents are added and another decrease the expression of p27K1p1 when pro-cancer agents are added. For this short concept proposal, Dr. Albert Einstein’s “visualized thought experiments (German: Gedanken experiment)” were used as a fundamental tool for understanding how either anti- or pro-cancer agents bring the primary structure of the 5’-untranslated region of p27Kip1 mRNA into two alternative secondary structures, thereby either increasing or decreasing, respectively, the translation initiation of p27Kip1 protein.展开更多
基金supported by the National Key Research and Development Program of China(2017YFD0101204)the National Natural Science Foundation of China(31701443)+1 种基金the Agricultural Science and Technology Innovation Project of the Shandong Academy of Agricultural Sciences,China(CXGC2017B01)the Natural Science Foundation of Shandong Province,China(ZR2016CB52)。
文摘Kernel development plays an important role in determining kernel size in maize.Here we present the cloning and characterization of a maize gene,nitrate transporter1.5(NRT1.5),which controls small kernel phenotype by playing an important role in kernel development.A novel recessive small kernel mutant miniature2-m1(mn2-m1)was isolated from self-pollinated progenies of breeding materials.The mutant spontaneously showed small kernel character arresting both embryo and endosperm development at an early stage after pollination.Utilizing 21 polymorphic SSR markers,the mn2-m1 locus was limited to a 209.9-kb interval using 9176 recessive individuals of a BC1 segregating population from mn2-m1/B73.Only one annotated gene was located in this 209.9 kb region,Zm00001 d019294,which was predicted to encode nitrate transporter1.5(NRT1.5).Allelism tests confirmed that mn2-m1 was allelic to miniature2-m2(mn2-m2)and miniature2-710 B(mn2-710 B).The mn2-m1 and mn2-m2 alleles both had nucleotide deletions in the coding region resulting in premature termination,and the mn2-710 B allele had some missence mutations.Subcellular localization showed that Miniature 2(MN2)is localized in the plasma membrane.Quantitative real-time PCR(qRT-PCR)analysis revealed that the expression of MN2 and some genes involved in the basal endosperm transfer layer(BETL)and embryo surrounding region(ESR)development were affected in mn2-m1 seeds.These results suggested that MN2 plays an important role in maize seed development.
基金Supported by the Deanship of Scientific Research at King Saud University,Riyadh,No.RG-1435-053
文摘AIMTo investigate the role of non-structural open reading frame 1 “Y-domain” sequences in the hepatitis E virus (HEV) life cycle.METHODSSequences of human HEV Y-domain (amino acid sequences 216-442) and closely-related viruses were analyzed in silico. Site-directed mutagenesis of the Y-domain (HEV SAR55) was carried out and studied in the replicon-baculovirus-hepatoma cell model. In vitro transcribed mRNA (pSK-GFP) constructs were transfected into S10-3 cells and viral RNA replicating GFP-positive cells were scored by flow cytometry. Mutant virions’ infectivity was assayed on naïve HepG2/C3A cells.RESULTSIn silico analysis identified a potential palmitoylation-site (C<sub>336</sub>C<sub>337</sub>) and an α-helix segment (L<sub>410</sub>Y<sub>411</sub>S<sub>412</sub>W<sub>413</sub>L<sub>414</sub>F<sub>415</sub>E<sub>416</sub>) in the HEV Y-domain. Molecular characterization of C<sub>336</sub>A, C<sub>337</sub>A and W<sub>413</sub>A mutants of the three universally conserved residues showed non-viability. Further, of the 10 consecutive saturation mutants covering the entire Y-domain nucleotide sequences (nts 650-1339), three constructs (nts 788-994) severely affected virus replication. This revealed the indispensability of the internal sequences but not of the up- or downstream sequences at the transcriptional level. Interestingly, the three mutated residues corresponded to the downstream codons that tolerated saturation mutation, indicating their post-translational functional/structural essentiality. In addition, RNA secondary structure prediction revealed formation of stable hairpins (nts 788-994) where saturation mutation drastically inhibited virion infectivity.CONCLUSIONThis is the first demonstration of the critical role of Y-domain sequences in HEV life cycle, which may involve gene regulation and/or membrane binding in intracellular replication complexes.
文摘The p27Kip1 is a cell cycle repressor protein that regulates primarily the cell cycle transition from G1 to S phase and hence the DNA replication is in the S phase and cell division in the M phase. Expression of p27Kip1 protein has dual roles for both cancer prevention and promotion. For example, numerous nutritional and chemopreventive anti-cancer agents specifically increase the expression of p27Kip1 protein without directly affecting the expression of any other cell cycle regulatory proteins. On the other hand, pro-cancer agents (like glucose, insulin and other growth factors frequently seen in obesity and/or diabetes) specifically decrease the expression of p27Kip1 protein without directly affecting the expression of any other cell cycle regulatory proteins. Unlike expression of any other cell cycle regulatory proteins, expression of p27Kip1 protein is very unusual. The mRNA of p27Kip1 has a very long and unusual 5’-untranslated region (from -575 to -1 in human). It appears that the 5’-untranslated region of p27Kip1 mRNA forms two alternative secondary structures. One increases the expression of p27Kip1 protein when anti-cancer agents are added and another decrease the expression of p27K1p1 when pro-cancer agents are added. For this short concept proposal, Dr. Albert Einstein’s “visualized thought experiments (German: Gedanken experiment)” were used as a fundamental tool for understanding how either anti- or pro-cancer agents bring the primary structure of the 5’-untranslated region of p27Kip1 mRNA into two alternative secondary structures, thereby either increasing or decreasing, respectively, the translation initiation of p27Kip1 protein.
