DNA methylation status of H19 and Xist genes in lungs of somatic cell nuclear transfer bovines

In somatic cell nuclear transfer (SCNT) technologies,the donor cell’s nuclei need to be epigenetically reprogrammed for embryonic development. The incomplete reprogramming of donor cell nuclei has been implicated as a primary reason for the low efficiency of SCNT. DNA methylation is a major epige-netic modification of the genome that regulates crucial aspects of genome function,including estab-lishment of genomic imprinting. In order to make sure whether the DNA methylation reprogramming is efficient in SCNT animals,we analyzed the DNA methylation status of two imprinting genes,H19 and Xist,in lungs of deceased SCNT bovines that died within 48 h of birth using bisulfite sequencing analysis. Our findings demonstrated that cloned bovines showed significantly lower DNA methylation of H19 than controls (P<0.05),and three tested CpGs sites (1,2,3) exhibited unmethylation in one cloned bovine (9C3); however,Xist showed similar DNA methylation levels between clones and con-trols,and both showed hypermethylation (96.11% and 86.67%).     

F-MSAP： A practical system to detect methylation in chicken genome

By replacing radiation with fluorescent system in the technique of methylation sensitive amplified polymorphism （MSAP） and optimizing reaction conditions, a modified technique to detect DNA methylation called F-MSAP （fluorescent labeled methylation sensitive amplified polymorphism） was developed. In the present study, cytosine methylation patterns of genomic DNA were investigated in two inbred chickens and their F1 hybrids. Three types of methylation patterns were observed in each individual, namely fully methylated, hemi-methylated or not methylated types. The average incidence of methylation was approximately 40%. The percentage that the F1 hybrid individual inherits the methylation for any given sites from either/both parent amounted to 95%, while the percentage of altered methylation patterns in F1 individual was only 5%, including 14 increased and 12 decreased methylation types, demonstrating that F-MSAP was highly efficient for large-scale detection of cytosine methylation in chicken genome. Our technique can be further extended to other animals or plants with complex genome and rich in methylation polymorphism.     

Speciation- induced heritable cytosine methylation changes in polyploid wheat

To study possible epigenetic changes accompanying polyploid speciation, genomic DNA from natural polyploid wheats and their putative diploid progenitors were digested with a pair of isoschizomers Hpa II / Msp I and hybridized to 21 different types of low-copy DNA sequences. It was found that cytosine methylation changes were abundant in natural polyploid wheats after their speciation. The hybridization of the same set of sequences to a synthetic hexaploid wheat along with its parental lines indicated that the extensive DNA methylation changes already existed in the early generations (S5, S6 and Sy) of this plant. Moreover, the high similarity of the changed restriction fragment length polymorphism (RFLP) patterns among three randomly chosen individual plants suggested that the methylation changes occurred even earlier, and/or were of a nonrandom nature. The changed patterns were stably inherited in the three successive selfed generations. Though methylation changes are probably a genome-wide occurrence, they appeared to be confined to the specific types of DNA sequences. The possible implications of the rapid and extensive cytosine methylation changes for several attributes of allopolyploid genome evolution, such as genetic diploidization and gene diversification, are discussed .     

Aberrant DNA methylation in cloned ovine embryos

By using the approach of immunofluorescence staining with an antibody against 5-methylcytosine （5MeC）, the present study detected the DNA methylation patterns of cloned ovine embryos. The embryos derived from in vitro fertilization were also examined for reference purpose. The results showed that： （1） during the preimplantation development, cloned embryos displayed a similar demethylation profile to the fertilized embryos; that is, the methylation level decreased to the lowest at 8-cell stage, and then increased again at morulae stage. However, methylation level was obviously higher in cloned embryos than in stage-matched fertilized embryos, especially at 8-cell stage and afterwards; （2） at blastocyst stage, the methylation pattern in cloned embryos was different from that in fertilized embryos. In cloned blastocyst, inner cell mass （ICM） exhibited a comparable level to trophectoderm cells （TE）, while in in-vitro fertilized blastocyst the methylation level of ICM was lower than that of TE, which is not consistent with that reported by other authors. These results indicate that DNA methylation is abnormally reprogrammed in cloned embryos, implying that aberrant DNA methylation reprogramming may be one of the factors causing cloned embryos developmental failure.     

Imprinting in plants

Genomic imprinting leads to the differential expression of parental alleles after fertilization. Imprinting appears to have evolved independently in mammals and flowering plants to regulate the development of nutrient-transfer placental tissues. In addition, the regulation of imprinting in both mammals and flowering plants involves changes in DNA methylation and histone methylation, thus suggesting that the epigenetic signals that regulate imprinting have been co-opted in these distantly related species.     

Differentiation and adaptation epigenetic networks: Translational research in gastric carcinogenesis

There are several kinds of epigenetic networks in the human body including the cell differentiation epigenetic network(DiEN) and the host adaptation epigenetic network(AdEN).DiEN networks are static and cell/tissue-specific.AdEN networks are variable and dependent upon environmental factors.DiEN and AdEN alterations can respectively serve as biomarkers for different kinds of diseases.Cancer is a consequence of accumulated pathophysiological adaptations of tissue stem cells to exposure of environmental carcinogens.Cancer cells are de-differentiated cells that obtain the capacity of unrestricted proliferation,local invasion,and distant migration/metastasis.Both DiEN and AdEN changes can be observed in cancer tissues.Alterations of DNA methylation are the most stable epigenetic modifications and can be sensitively detected in a small cell population.These advantages make DNA methylation the optimal biomarkers for detection of initiated cells in precancerous lesions and metastasis stem cells in cancer tissues.It has been proven that p16 methylation can be used as a diagnostic biomarker to determine malignant potential of epithelium dysplasia in many organs including the stomach.In a large-scale validation study on the DNA methylome of gastric carcinomas(GC),the methylation status of more than 90 CpG islands has been analyzed by DHPLC.Furthermore,GFRA1 demethylation and methylation of SRF and ZNF382 are frequent events during gastric carcinogenesis and consistently correlate to GC metastasis and overall survival of GC patients from China,Japan,and Korea,respectively.In a population study,it has been demonstrated that gradual increasing of plasma miR-211 and other miRNA levels may be an early risk predictor for GC development.     

Analysis of DNA methylation variation in wheat genetic background after alien chromatin introduction based on methylation-sensitive amplification polymorphism

During the process of alien germplasm introduced into wheat genome by chromosome engineering, extensive genetic variations of genome structure and gene expression in recipient could be induced. In this study, we performed GISH （genome in situ hybridization） and AFLP （amplified fragment length polymorphism） on wheat-rye chromosome translocation lines and their parents to detect the identity in genomic structure of different translocation lines. The results showed that the genome primary structure variations were not obviously detected in different translocation lines except the same 1RS chromosome translocation. Methylation sensitive amplification polymorphism （MSAP） analyses on genomic DNA showed that the ratios of fully-methylated sites were significantly increased in translocation lines （CN12, 20.15%; CN17, 20.91%; CN18, 22.42%）, but the ratios of hemimethylated sites were significantly lowered （CN12, 21.41%; CN17, 23.43%; CN18, 22.42%）, whereas 16.37% were fully-methylated and 25.44% were hemimethylated in case of their wheat parent. Twenty-nine classes of methylation patterns were identified in a comparative assay of cytosine methylation patterns between wheat-rye translocation lines and their wheat parent, including 13 hypermethylation patterns （33.74%）, 9 demethylation patterns （22.76%） and 7 uncertain patterns （4.07%）. In further sequence analysis, the alterations of methylation pattern affected both repetitive DNA sequences, such as retrotransposons and tandem repetitive sequences, and low-copy DNA.     

Molecular mechanism of the inhibitory effect of methylation outside the recognition sequence of restriction endonuclease Pvu Π on its cleavage activity

In 1991, we found that methylation outside the PvuⅡ recognition sequence could partially inhibit its cleavage activity. To clarify the molecular mechanism, three plasmids with different methylation states were constructed. Then, together with the original one, four plasmids were digested with different amounts of PvuⅡ. Results show that methylation on both sites results in 90% inhibition; moving the methylated site one base further away decreases the inhibitory effect to about 30%; with the adjacent dam methylation site eliminated, the inhibitory effect disappears. The data suggest that the inhibition of cleavage activity caused by outside methylation is not "all or none", and the degree of inhibition is dependent on the position and the number of methylated bases.     

DNA methylation status of H19 and Xist genes in lungs of somatic cell nuclear transfer bovines

In somatic cell nuclear transfer （SCNT） technologies, the donor cell＇s nuclei need to be epigenetically reprogrsmmed for embryonic development. The incomplete reprogramming of donor cell nuclei has been Implicated as s primary reason for the low efficiency of SCNT. DNA methylstion is s major epigenetic modification of the genome that regulates crucial aspects of genome function, including establishment of genomic imprinting. In order to make sure whether the DNA methylstion reprogramming is efficient in SCNT animals, we analyzed the DNA methylstion status of two imprinting genes, H19 and Xist, in lungs of deceased SCNT bovines that died within 48 h of birth using bisulfite sequencing analysis. Our findings demonstrated that cloned bovines showed significantly lower DNA methylstion of H19 than controls （P〈0.05）, and three tested CpGs sites （1, 2, 3） exhibited unmethylstion in one cloned bovine （9C3）; however, Xist showed similar DNA methylation levels between clones and controis, and both showed hypermethylstion （96.11% and 86.67%）.     

Habitat-induced reciprocal transformation in the root phenotype of Oriental ginseng is associated with alteration in DNA methylation

Oriental ginseng is an important medicinal plant that grows in 2 major forms or ecotypes, wild and domesticated. Each form differs conspicuously in root phenotype, but can be converted from one type to another by habitat. Here we show that the habitat-induced transformation of ginseng root phenotype was accompanied by alteration in cytosine methylation at a large number of 5′-CCGG-3′ sites detected by the methylation-sensitive polymorphism (MSAP) marker. The collective CG and CHG methylation levels of all 4 landraces of the domesticated form were significantly lower than those of the wild form. Interestingly, artificially transplanted ginseng plants recreated in both directions the methylation levels (at least in CHG) of their natural counterparts. The methylation differences between the 2 ginseng ecotypes were validated at 2 isolated MSAP loci bearing homology to a 5S rRNA gene or a copia retrotransposon. Our results implicate a link between epigenetic variation and habitat-induced phenotypic flexibility in Oriental ginseng.     

Patterns of DNA cytosine methylation between haploids and corresponding diploids in rice

In higher plant, about 30% cytosines are methy-lated[1], among which about 90% methylated sites lie in CpG dinucleotide and CpNpG trinucleotide[2]. The me-thylated DNA has inducing and epigenetic effects on cell biological procedures such as gene differen…     

高活力水稻种子萌发过程中DNA甲基化变化的MSAP分析

DNA甲基化是基因组DNA的一种主要表观遗传修饰形式,是调节基因组功能的重要手段。本实验采用MSAP方法分析,旨在研究高活力水稻种子萌发过程中的甲基化与去甲基化变化的规律,为研究种子的老化机理和种子的长期保存提供依据。结果表明:水稻种子萌发过程中,同时发生了甲基化与去甲基化作用,且去甲基化作用先于甲基化作用发生。发生去甲基化可能与基因活化有关,发生甲基化可能与组织特异性有关。     

MSAP在水稻害虫白背飞虱中的应用研究

甲基化敏感的扩增多态性分析方法 (MSAP)是在扩增片段长度多态性技术基础上建立起来的主要用来检测样品基因组DNA甲基化情况的一种方法,特别适用于全基因组范围内检测CCGG位点的胞嘧啶甲基化情况,在高等动物和许多植物上已广泛应用,但尚未有在昆虫中应用该方法的报道。从DNA提取、酶切基因组DNA用量、酶切和接头连接时间、电泳图谱显影方法等方面对MSAP方法进行了改进,并在水稻重要害虫白背飞虱上得到成功应用。这为将来在其它昆虫中使用该方法研究基因组DNA甲基化情况提供了一定借鉴和指导价值。且通过比较白背飞虱雌雄性别间和长短翅型间的MSAP图谱,发现雌雄性别间和长短翅型间的基因组DNA甲基化式样都存在明显的差异,说明DNA甲基化可能参与了性别和翅型分化的调节。     

PEG-6000引发对苦荞种子萌发及幼苗生长的影响

为探讨PEG-6000溶液对苦荞种子萌发的影响,采用浓度为10%、20%、25%、30%的PEG-6000溶液对川荞1号种子进行12 h,24 h,36 h处理,以测定川荞1号种子的发芽率、发芽势、芽鲜重、芽干重与芽长,并计算其简单活力指数.结果表明:浓度为25%的PEG-6000溶液引发可以有效提高苦荞种子的发芽率,其显著性明显高于其他浓度,且在此浓度下处理24 h的苦荞种子发芽率显著高于其他处理;不同的引发时间均可以提高苦荞种子的发芽率,引发24 h比引发12 h和36h的苦荞种子发芽能力高,但差异不显著.     

不同外源激素处理对长白松种子发芽能力的影响

选用不同浓度吲哚丁酸、萘乙酸及赤霉素浸种1,2 h,对长白松种子进行预处理,以清水浸种2 h为对照,试验分析预处理后长白松种子的萌发状况.结果表明:长白松种子经不同外源激素处理后发芽率、发芽势明显提高.其中,以100 mg/L的IBA浸种2 h长白松种子的发芽效果最好,发芽率为53.66%,发芽势为12.33%,发芽指数为38.15.     

不同预处理对薜荔种子发芽的影响(Ⅰ)

研究了薜荔种子的萌发特性,以及不同浸种时间、不同浓度的HNO3、KNO3以及GA溶液对薜荔种子发芽的影响.结果表明:不同地区不同植株薜荔种子的各项发芽指标差异显著;浸种时间与种子发芽势之间存在相关性,适宜浓度的HNO3、KNO3以及GA溶液对薜荔种子发芽有促进作用.