Role of histone H3 lysine 27 methylation in X inactivation

The Polycomb group (PcG) protein Eed is implicated in regulation of imprinted X-chromosome inactivation in extraembryonic cells but not of random X inactivation in embryonic cells. The Drosophila homolog of the Eed-Ezh2 PcG protein complex achieves gene silencing through methylation of histone H3 on lysine 27 (H3-K27), which suggests a role for H3-K27 methylation in imprinted X inactivation. Here we demonstrate that transient recruitment of the Eed-Ezh2 complex to the inactive X chromosome (Xi) occurs during initiation of X inactivation in both extraembryonic and embryonic cells and is accompanied by H3-K27 methylation. Recruitment of the complex and methylation on the Xi depend on Xist RNA but are independent of its silencing function. Together, our results suggest a role for Eed-Ezh2-mediated H3-K27 methylation during initiation of both imprinted and random X inactivation and demonstrate that H3-K27 methylation is not sufficient for silencing of the Xi.     

Intersection of the RNA interference and X-inactivation pathways

In mammals, dosage compensation is achieved by X-chromosome inactivation (XCI) in the female. The noncoding Xist gene initiates silencing of the X chromosome, whereas its antisense partner Tsix blocks silencing. The complementarity of Xist and Tsix RNAs has long suggested a role for RNA interference (RNAi). Here, we report that murine Xist and Tsix form duplexes in vivo. During XCI, the duplexes are processed to small RNAs (sRNAs), most likely on the active X (Xa) in a Dicer-dependent manner. Deleting Dicer compromises sRNA production and derepresses Xist. Furthermore, without Dicer, Xist RNA cannot accumulate and histone 3 lysine 27 trimethylation is blocked on the inactive X (Xi). The defects are partially rescued by truncating Tsix. Thus, XCI and RNAi intersect, down-regulating Xist on Xa and spreading silencing on Xi.     

Reactivation of an inactive human X chromosome: evidence for X inactivation by DNA methylation

A mouse-human somatic cell hybrid clone, deficient in hypoxanthine-guanine phosphoribosyltransferase (HPRT) and containing a structurally normal inactive human X chromosome, was isolated. The hybrid cells were treated with 5-azacytidine and tested for the reactivation and expression of human X-linked genes. The frequency of HPRT-positives clones after 5-azacytidine treatment was 1000-fold greater than that observed in untreated hybrid cells. Fourteen independent HPRT-positive clones were isolated and analyzed for the expression of human X markers. Isoelectric focusing showed that the HPRT expressed in these clones is human. One of the 14 clones expressed human glucose-6-phosphate dehydrogenase and another expressed human phosphoglycerate kinase. Since 5-azacytidine treatment results in hypomethylation of DNA, DNA methylation may be a mechanism of human X chromosome inactivation.     

Instability of a 550-base pair DNA segment and abnormal methylation in fragile X syndrome

The fragile X syndrome, a common cause of inherited mental retardation, is characterized by an unusual mode of inheritance. Phenotypic expression has been linked to abnormal cytosine methylation of a single CpG island, at or very near the fragile site. Probes adjacent to this island detected very localized DNA rearrangements that constituted the fragile X mutations, and whose target was a 550-base pair GC-rich fragment. Normal transmitting males had a 150- to 400-base pair insertion that was inherited by their daughters either unchanged, or with small differences in size. Fragile X-positive individuals in the next generation had much larger fragments that differed among siblings and showed a generally heterogeneous pattern indicating somatic mutation. The mutated allele appeared unmethylated in normal transmitting males, methylated only on the inactive X chromosome in their daughters, and totally methylated in most fragile X males. However, some males had a mosaic pattern. Expression of the fragile X syndrome thus appears to result from a two-step mutation as well as a highly localized methylation. Carriers of the fragile X mutation can easily be detected regardless of sex or phenotypic expression, and rare apparent false negatives may result from genetic heterogeneity or misdiagnosis.     

DNA甲基化及其生物学功能

DNA甲基化是真核细胞基因组重要修饰方式之一。机体有建立和维持DNA甲基化的机制。DNA甲基化通过与反式因子相互作用或通过改变染色体结构而影响基因的表达,在胚胎发育、X染色体失活、基因组印记等方面起着重要作用。DNA甲基化为哺乳动物的发育、遗传性疾病和肿瘤的发生、生物进化、性状的遗传控制等的研究提供了新的途径。     

Senescence of nickel-transformed cells by an X chromosome: possible epigenetic control

Transfer of a normal Chinese hamster X chromosome (carried in a mouse A9 donor cell line) to a nickel-transformed Chinese hamster cell line with an Xq chromosome deletion resulted in senescense of these previously immortal cells. At early passages of the A9/CX donor cells, the hamster X chromosome was highly active, inducing senescence in 100% of the colonies obtained after its transfer into the nickel-transformed cells. However, senescence was reduced to 50% when Chinese hamster X chromosomes were transferred from later passage A9 cells. Full senescing activity of the intact hamster X chromosome was restored by treatment of the donor mouse cells with 5-azacytidine, which induced demethylation of DNA. These results suggest that a senescence gene or genes, which may be located on the Chinese hamster X chromosome, can be regulated by DNA methylation, and that escape from senescence and possibly loss of tumor suppressor gene activity can occur by epigenetic mechanisms.     

水稻抗白叶枯病新基因的初步定位

【目的】通过与目前国际上已报道的抗白叶枯病基因进行分析比较,推测水稻抗源C4059含有1个新的抗白叶枯病基因,暂命名为Xa36(t)。将水稻抗源C4059的白叶枯病抗性转育到IR24遗传背景下,培育近等基因系并借助分子标记将其抗白叶枯病基因进行定位。【方法】以IR24/C4059的1个F3分离群体为材料,采用分离集团分析法,借助SSR、EST标记对Xa36(t)进行分子标记定位。【结果】找到13个与Xa36(t)连锁的标记,最近的4个标记RM2136、RM7443、RM1233和RM224与目标基因间的遗传距离分别为3.2、3.8、1.9和1.3 cM。其中标记RM2136和RM7443位于染色体近端粒一侧,标记RM1233和RM224位于目标基因的另一侧。【结论】通过分子标记检测,将基因Xa36(t)定位于水稻第11染色体长臂末端附近。     

Genomic sequencing and methylation analysis by ligation mediated PCR

Genomic sequencing permits studies of in vivo DNA methylation and protein-DNA interactions, but its use has been limited because of the complexity of the mammalian genome. A newly developed genomic sequencing procedure in which a ligation mediated polymerase chain reaction (PCR) is used generates high quality, reproducible sequence ladders starting with only 1 microgram of uncloned mammalian DNA per reaction. Different sequence ladders can be created simultaneously by inclusion of multiple primers and visualized separately by rehybridization. Relatively little radioactivity is needed for hybridization and exposure times are short. Methylation patterns in genomic DNA are readily detectable; for example, 17 CpG dinucleotides in the 5' region of human X-linked PGK-1 (phosphoglycerate kinase 1) were found to be methylated on an inactive human X chromosome, but unmethylated on an active X chromosome.     

A chicken transferrin gene in transgenic mice escapes X-chromosome inactivation

Mammalian X-chromosome inactivation involves a coordinate shutting down of physically linked genes. Several proposed models require the presence of specific sequences near genes to permit the spread of inactivation into these regions. If such models are correct, one might predict that heterologous genes transferred onto the X chromosome might lack the appropriate signal sequences and therefore escape inactivation. To determine whether a foreign gene inserted into the X chromosome is subject to inactivation, transgenic mice harboring 11 copies of the complete, 17-kilobase chicken transferrin gene on the X chromosome were used. Male mice hemizygous for this insert were bred with females bearing Searle's translocation, an X-chromosome rearrangement that is always active in heterozygous females (the unrearranged X chromosome is inactive). Female offspring bearing the Searle's translocation and the chicken transferrin gene had the same amount of chicken transferrin messenger RNA in liver as did transgenic male mice or transgenic female mice lacking the Searle's chromosome. This result shows that the inserted gene is not subject to X-chromosome inactivation and suggests that the inactivation process cannot spread over 187 kilobases of DNA in the absence of specific signal sequences required for inactivation.     

5-Azacytidine-induced reactivation of a herpes simplex thymidine kinase gene

Mouse cells transformed with herpes simplex virus and containing the viral thymidine kinase (TK) gene in an inactive state were treated with 5-azacytidine. The result was the reexpression of the viral TK gene. Two days of exposure to 5-azacytidine followed by 2 days of expression time was sufficient for maximal induction of the TK+ phenotype. The induction of TK expression by 5-azacytidine was concentration-dependent, with maximal induction at 10 micromoles per liter. 5-Azacytidine also inhibited the decay of TK expression in TK+ transformants removed from selective conditions. Analysis of the methylation patterns of the viral TK gene with restriction endonucleases Hpa II and Msp I showed the active gene to be unmethylated, the inactive gene methylated, and the 5-azacytidine-induced gene unmethylated.     

家蚕DNA甲基转移酶功能预测及其在BmNPV感染下的表达特征

【目的】明确家蚕DNA甲基转移酶基因（BmDNMT）生物信息学基本特征及其在家蚕核型多角体病毒（BmNPV）感染前后的表达情况,揭示DNA甲基化在家蚕抗病毒免疫方面的作用机制,为挖掘家蚕抗病毒标志基因和药物靶标蛋白等提供理论依据。【方法】通过OrthoDB、MultiLoc2、SherLoc2、PSORTII、SMART、SWISS-MODEL及Schr?dinger等在线生物信息学分析软件进行BmDNMT蛋白氨基酸同源比对、系统进化分析、亚细胞定位、功能结构域预测、三级结构同源建模及小分子配体对接口袋预测,并采用实时荧光定量PCR分析BmDNMT基因在家蚕感染BmNPV后不同时间不同组织中的时空表达特征。【结果】BmDNMT基因包含BmDNMT1和BmDNMT2,分别位于家蚕8号染色体和11号染色体上,其推导BmDNMT氨基酸序列均与烟草天蛾DNMT氨基酸序列的亲缘关系最近。BmDNMT1蛋白大量存在于细胞核,少量分布在细胞质;而BmDNMT2蛋白大量存在于细胞质,少量分布在细胞核及线粒体。BmDNMT1蛋白的功能结构域多于BmDNMT2蛋白,二者均含有DNA甲基化酶功能结构域,且具...     

Virulence of Xanthomonas oryzae pv.oryzae on Rice Near-lsogenic Lines with Single Resistance Gene and Pyramiding Lines in China

Ninety one isolates of Xanthomonas oryzae pv. Oryzae were collected from different rice growing regions in China and determined for their virulence on 24 rice near-isogenic lines containing single resistance gene and 2-4 genes:IRBB1 (Xa1),IRBB2 (Xa2),IRBB3 (Xa3), IRBB4 (Xa4), IRBB5 (xa5), IRBB7 (Xa7), IRBB8 (xa8), IRBB10 (Xa10), IRBB11 (Xa11),IRBB13 (xa13),IRBB14 (Xa14), IRBB21 (Xa21), IR24 (Xa18), IRBB50 (Xa4 + xa5), IRBB51 (Xa4 + xa13), IRBB52 (Xa4 + Xa21), IRBB53 (xa5 + xa13), IRBB54 (xa5 + Xa21), IRBB55 (xa13 + Xa21),IRBB56 (Xa4 + xa5 + xa13), IRBB57 (Xa4 + xa5 + Xa21), IRBB58 (Xa4 + xa13 + Xa21),IRBB59 (xa5 + xa13 + Xa21) and IRBB60 (Xa4 + xa5 + xa13 + Xa21). The results showed that most isolates were less virulent on lines with more than one genes pyramided than those with single resistance gene. The isolates tested were more virulent on IR24 and IRBB10,less virulent on IRBB5, IRBB7 and IRBB21. Based on interactions between isolates and rice near-isogenic lines, 7 cultivars with single gene (IRBB5, IRBB4, IRBB3, IRBB14, IRBB2, IRBB1 and IR24) were chosen as the differentials, and the tested isolates were classified into 7 virulence groups. The reaction patterns of the 7 groups in order were: RRRRRRR,RRRRRRS, RRRRRSS, RR/SRRSSS, RRRSSSS, RRSSSSS, RSSSSSS. The virulence frequencies were 7.69, 6.59, 14.29, 12.09, 14.29, 28.57 and 16.48％ respectively. The elementary system for races identification has been established in China based on the results. It will be possible to compare with races in other countries, and the results will facilitate the evelopment of rice resistance breeding to bacterial blight in China.     

小粒野生稻抗白叶枯病新基因的鉴定与初步定位

【目的】将小粒野生稻（Acc. No. 101133）的抗白叶枯病基因导入栽培稻IR24,并对其进行鉴定和分子标记定位,以便应用于育种实践。【方法】以小粒野生稻和栽培稻IR24的BC2F2群体及其F3、F4家系为材料,利用分离集团分析法（BSA）,借助SSR标记对Xa35(t)进行分子标记定位。【结果】通过对抗病基因进行抗谱鉴定和遗传分析,结果表明,该基因对白叶枯病菌株PXO86和PXO99表现感病,而对PXO61、PXO112和PXO339表现抗病,初步将其定位于水稻的第11染色体长臂上,同标记RM144共分离,并位于标记RM7654和RM6293之间,与两标记的遗传距离分别为1.1 cM和0.7 cM。【结论】小粒野生稻（Acc. No. 101133）含有新的抗白叶枯病基因,暂定为Xa35(t)。     

Christmas factor: dosage compensation and the production of blood coagulation factor IX

The amount of factor IX (Christmas factor) for different genotypic classes was determined by means of a variant of the thromboplastin generation test. The mean value for females heterozygous for the Christmas gene was about half the mean values for normal males and for normal homozygous females; means for the latter two groups were about equal. This dosage compensation is interpreted as evidence in support of Lyon's hypothesis, according to which one X chromosome is inactive in mammalian females.     

鸡传染性支气管炎病毒HN99株N基因在大肠杆菌中的表达及产物免疫活性检测

鸡传染性支气管炎病毒HN99株核蛋白(N)基因PCR产物经BamHⅠ、HindⅢ双酶切,克隆入大肠杆菌原核表达载体pMAL-p2X,构建重组表达质粒pMAL-p2X-N,转化大肠杆菌TB1并进行诱导表达。通过pMALTM融合蛋白纯化系统对表达产物进行非变性纯化,将纯化的N蛋白按常规方法免疫新西兰大白兔,制备兔抗鸡传染性支气管炎病毒N蛋白的多克隆抗体,并用ELISA法检测其生物活性。结果表明,表达的重组核蛋门纯化后出现2条带,相对分子质量分别约为92和82 ku,与Western blot出现的2条蛋白带一致;纯化的重组蛋白经裂解因子作用后,出现2条蛋白带,相对分子质量分别为45和35 ku,与预期结果相符合;制备的多克隆抗体可与不同鸡传染性支气管炎病毒株发生反应,与亲本毒株的反应性略高于与其他毒株的反应性。以上结果表明,原核表达的可溶性N蛋白具有高度的生物活性,有望作为新的基因工程抗原用于鸡传染性支气管炎病毒的群特异性诊断。     

分子标记辅助选择改良杂交水稻的白叶枯病抗性

分子标记辅助选择技术以其对目标基因快速而精确的选择为回交育种提供了非常有效的工具。华中农业大学作物遗传改良实验室运用分子标记辅助的回交育种方法,将广谱高抗白叶枯病基因Ｚａ２１迅速导入到杂交水稻的两个优良恢复系明灰６３和６０７８之中,成功地完成了其抗生改良,并通过配组而达到了改良杂交水稻抗性的目的。本研究所运用的分子标记辅助的水稻回交育种程序是：通过一次杂交、二次回交改良杂交水稻抗性的目的。本研究所