A microsatellite-enriched library of plateau pika （Ochotona curzoniae） was constructed according to the strong affinity between biotin and streptavidin. Firstly, genomic DNA was fragmented by ultrasonication, which ...A microsatellite-enriched library of plateau pika （Ochotona curzoniae） was constructed according to the strong affinity between biotin and streptavidin. Firstly, genomic DNA was fragmented by ultrasonication, which is a major improvement over traditional methods. Linker-ligated DNA fragments were hybridized with biotinylated microsatellite probes, and then were subjected to streptavidin-coated magnetic beads. PCR amplification was performed to obtain double-stranded DNA fragments containing microsatellites. Ligation and transformation were carried out by using the pGEM-T Vector System I and Escherichia coli DH10B competent cells. Sequencing results showed that 80.2% of clones contained microsatellite repeat motif. Several modifications make this protocol time-efficient and technically easier than the traditional ones; particularly, composition and relative abundance of microsatellite repeats in plateau pika genome were truly represented through the optimized PCR conditions. This method has also been successfully applied to construct microsatellite-enriched genomic libraries of Chinese hamster （Cricetulus griseus） and small abalone [Haliotis diversicolor （Reeve）] with high rates of positive clones, demonstrating its feasibility and stability.展开更多
Agrocybe cylindracea,an edible mushroom,is widely cultivated for its abundance of nutrients and flavor,and many of its metabolites are reported to have beneficial roles,such as medicinal effects on tumors and chronica...Agrocybe cylindracea,an edible mushroom,is widely cultivated for its abundance of nutrients and flavor,and many of its metabolites are reported to have beneficial roles,such as medicinal effects on tumors and chronical illnesses.However,the lack of genomic information has hindered further molecular studies on this fungus.Here,we present a genome assembly of A.cylindracea together with comparative genomics and pathway analyses of Agaricales species.The draft,generated from both next-generation sequencing(NGS)and single-molecule real-time(SMRT)sequencing platforms to overcome high genetic heterozygosity,is composed of a56.5 Mb sequence and 15,384 predicted genes.This mushroom possesses a complex reproductive system,including tetrapolar heterothallic and secondary homothallic mechanisms,and harbors several hydrolases and peptidases for gradual and effective degradation of various carbon sources.Our pathway analysis reveals complex processes involved in the biosynthesis of polysaccharides and other active substances,including B vitamins,unsaturated fatty acids,and N-acetylglucosamine.RNA-seq data show that A.cylindracea stipes tend to synthesize carbohydrate for carbon sequestration and energy storage,whereas pilei are more active in carbon utilization and unsaturated fatty acid biosynthesis.These results reflect diverse functions of the two anatomical structures of the fruiting body.Our comprehensive genomic and transcriptomic data,as well as preliminary comparative analyses,provide insights into the molecular details of the medicinal effects in terms of active compounds and nutrient components.展开更多
Histone H3 lysine 4 trimethylation （H3K4me3） is well known to occur in the promoter region of genes for transcription activation. How- ever, when investigating the H3K4me3 profiles in the mouse cerebrum and testis, ...Histone H3 lysine 4 trimethylation （H3K4me3） is well known to occur in the promoter region of genes for transcription activation. How- ever, when investigating the H3K4me3 profiles in the mouse cerebrum and testis, we discovered that H3K4me3 also has a significant enrichment at the 3＇ end of actively transcribed （sense） genes, named as 3＇-H3K4me3. 3＇-H3K4me3 is associated with ~15% of pro- tein-coding genes in both tissues. In addition, we examined the transcriptional initiation signals including RNA polymerase II （RNAPII） binding sites and Y-CAGE-tag that marks transcriptional start sites. Interestingly, we found that 3＇-H3K4me3 is associated with the ini- tiation of antisense transcription. Furthermore, 3＇-H3K4me3 modification levels correlate positively with the antisense expression levels of the associated sense genes, implying that 3＇-H3K4me3 is involved in the activation of antisense transcription. Taken together, our findings suggest that H3K4me3 may be involved in the regulation of antisense transcription that initiates from the 3＇ end of sense genes. In addition, a positive correlation was also observed between the expression of antisense and the associated sense genes with 3＇-H3K4me3 modification. More importantly, we observed the 3＇-H3K4me3 enrichment among genes in human, fruitfly and Arabidopsis, and found that the sequences of 3＇-H3K4me3-marked regions are highly conserved and essentially indistinguishable from known promoters in ver- tebrate. Therefore, we speculate that these 3＇-H3K4me3-marked regions may serve as potential promoters for antisense transcription and 3＇-H3K4me3 appear to be a universal epigenetic feature in eukaryotes. Our results provide a novel insight into the epigenetic roles of H3K4me3 and the regulatory mechanism of antisense transcription.展开更多
文摘A microsatellite-enriched library of plateau pika （Ochotona curzoniae） was constructed according to the strong affinity between biotin and streptavidin. Firstly, genomic DNA was fragmented by ultrasonication, which is a major improvement over traditional methods. Linker-ligated DNA fragments were hybridized with biotinylated microsatellite probes, and then were subjected to streptavidin-coated magnetic beads. PCR amplification was performed to obtain double-stranded DNA fragments containing microsatellites. Ligation and transformation were carried out by using the pGEM-T Vector System I and Escherichia coli DH10B competent cells. Sequencing results showed that 80.2% of clones contained microsatellite repeat motif. Several modifications make this protocol time-efficient and technically easier than the traditional ones; particularly, composition and relative abundance of microsatellite repeats in plateau pika genome were truly represented through the optimized PCR conditions. This method has also been successfully applied to construct microsatellite-enriched genomic libraries of Chinese hamster （Cricetulus griseus） and small abalone [Haliotis diversicolor （Reeve）] with high rates of positive clones, demonstrating its feasibility and stability.
基金the project of “Cooperation with Localities and Industries”,Gansu,China(2012HZ-02)。
文摘Agrocybe cylindracea,an edible mushroom,is widely cultivated for its abundance of nutrients and flavor,and many of its metabolites are reported to have beneficial roles,such as medicinal effects on tumors and chronical illnesses.However,the lack of genomic information has hindered further molecular studies on this fungus.Here,we present a genome assembly of A.cylindracea together with comparative genomics and pathway analyses of Agaricales species.The draft,generated from both next-generation sequencing(NGS)and single-molecule real-time(SMRT)sequencing platforms to overcome high genetic heterozygosity,is composed of a56.5 Mb sequence and 15,384 predicted genes.This mushroom possesses a complex reproductive system,including tetrapolar heterothallic and secondary homothallic mechanisms,and harbors several hydrolases and peptidases for gradual and effective degradation of various carbon sources.Our pathway analysis reveals complex processes involved in the biosynthesis of polysaccharides and other active substances,including B vitamins,unsaturated fatty acids,and N-acetylglucosamine.RNA-seq data show that A.cylindracea stipes tend to synthesize carbohydrate for carbon sequestration and energy storage,whereas pilei are more active in carbon utilization and unsaturated fatty acid biosynthesis.These results reflect diverse functions of the two anatomical structures of the fruiting body.Our comprehensive genomic and transcriptomic data,as well as preliminary comparative analyses,provide insights into the molecular details of the medicinal effects in terms of active compounds and nutrient components.
基金supported by Knowledge Innovation Program of the Chinese Academy of Sciences (Grant No.KSCX2-EW-R-01-04)Natural Science Foundation of China (Grant No. 90919024 and 30900831)the National Basic Research Program (973 Program) from the Ministry of Science and Technology of China (GrantNo. 2011CB944100)
文摘Histone H3 lysine 4 trimethylation （H3K4me3） is well known to occur in the promoter region of genes for transcription activation. How- ever, when investigating the H3K4me3 profiles in the mouse cerebrum and testis, we discovered that H3K4me3 also has a significant enrichment at the 3＇ end of actively transcribed （sense） genes, named as 3＇-H3K4me3. 3＇-H3K4me3 is associated with ~15% of pro- tein-coding genes in both tissues. In addition, we examined the transcriptional initiation signals including RNA polymerase II （RNAPII） binding sites and Y-CAGE-tag that marks transcriptional start sites. Interestingly, we found that 3＇-H3K4me3 is associated with the ini- tiation of antisense transcription. Furthermore, 3＇-H3K4me3 modification levels correlate positively with the antisense expression levels of the associated sense genes, implying that 3＇-H3K4me3 is involved in the activation of antisense transcription. Taken together, our findings suggest that H3K4me3 may be involved in the regulation of antisense transcription that initiates from the 3＇ end of sense genes. In addition, a positive correlation was also observed between the expression of antisense and the associated sense genes with 3＇-H3K4me3 modification. More importantly, we observed the 3＇-H3K4me3 enrichment among genes in human, fruitfly and Arabidopsis, and found that the sequences of 3＇-H3K4me3-marked regions are highly conserved and essentially indistinguishable from known promoters in ver- tebrate. Therefore, we speculate that these 3＇-H3K4me3-marked regions may serve as potential promoters for antisense transcription and 3＇-H3K4me3 appear to be a universal epigenetic feature in eukaryotes. Our results provide a novel insight into the epigenetic roles of H3K4me3 and the regulatory mechanism of antisense transcription.
