大鼠衰老过程中脑细胞DNA与c—Ha—ras原癌基因的甲基化

用Ｍｓｐ Ｉ／ＨｐａⅡ酶解电泳法和高效液相色谱（ＨＰＬＣ）两种方法进行比较,研究了不同年龄大鼠的肝、脑细胞基因组ＤＮＡ的甲基化程度。从酶解电泳图谱可观察到,肝、脑细胞基因组ＤＮＡ甲基化在青年鼠和老年鼠之间没有差异。但用具有高分辨率的高效液相色谱测量ＤＮＡ中５－ｍＣ的含量时发现,老年鼠脑细胞ＤＮＡ甲基化程度较青年鼠的下降６２％,而肝细胞ＤＮＡ甲基化程度在老年鼠与青年鼠之间并没有显著差异。这此结果提示     

人淋巴细胞CD2基因5’侧翼序列的克隆和鉴定

本文报告以ＣＤ２ｃＤＮＡ５’端的片段作探针,从人Ｔ淋巴细胞基因组文库筛选阳性重组克隆,经限制性内切酶降解和Ｓｏｕｔｈｅｒｎ杂交分析,证明其中一个阳性克隆的插入片段中含ＣＤ２基因５’侧翼顺序。经插入片段的亚克隆、限制性内切酶图谱及ＤＮＡ序列分析,鉴定出一含转录起始点及其上游序列的４．０ｋｂ片段。将此片段中含转录起始点和两个ＤＮ（ａｓｅ）Ⅰ高敏感位点的２．５ｋｂ片段定向克隆到以虫萤光素酶为报告基因的表达载体ｐＭＧ３中,并用限制性内切酶对此２．５ｋｂ片段作不同程度缺失,构成一系列突变子。这些重组的表达质粒转染人ＪｕｒｋａｔＴ细胞后,以瞬时表达实验分析各突变子驱动虫萤光素酶基因的表达,结果发现在ＣＤ２基因５’上游具有很弱的启动子活性,初步测定该启动子位于－１．２ｋｂ～－９８ｂｐ域。ＣＤ２基因具有弱启动子、强增强子的特点与Ｔ细胞表面其它抗原分子基因是相似的。     

麦迪霉素4″—O—丙酰基转移酶（mpt）基因结构的研究

对含有麦迪霉素４″－Ｏ－丙酰基转移酶（ｍｐｔ）基因的ＢａｍＨＩ－ＢａｍＨＩ８．０ｋｂ的ＤＮＡ片段进行限制性酶切分析,绘制出了含有２１个酶切位点的限制性酶切图谱。以含有碳霉素异戊酰基转移酶基因（ＣａｒＥ）的２．４ｋｂ ＤＮＡ片段为探针,经Ｓｏｕｔｈｅｒｎ ｂｌｏｔ分子杂交,将ｍｐｔ定位于一个ＥｃｏＲＩ－ＥｃｏＲＩ－ＰｓｔＩ３．０ｋｂ的ＤＮＡ片段上,将该片段克隆至大肠杆菌／链霉菌穿梭质粒载体ｐＷＨＭ３     

大鼠4.5S RNAs的cDNA克隆与序列分析

利用ＲＴ－ＰＣＲ方法,首次从大鼠肝脏细胞总ＲＮＡ中扩增出４．５Ｓ ＲＮＡｓ的ｃＤＮＡ。该ｃＤＮＡ被克隆到ｐＧＥＭ３Ｚｆ（＋）质粒上,经酶切电泳鉴定,然后测序。与报道的小鼠和仓鼠４．５Ｓ ＲＮＡｓ序列进行了比较研究,并对该分子的结构特点进行了初步分析。     

CAR－bacillus菌亚单位核糖核酸RNA限制性内切酶图谱的研究

利用生物体中编码亚单位核糖核酸ＲＮＡ（ｓｍａｌｌｓｕｂｕｎｉｔｒｉｂｏｓｏｍａｌ　ＲＮＡ,ＳＳＵｒＲＮＡ）的ＤＮＡ序列为引物,经ＰＣＲ法扩增到ＣＡＲ－ｂａｃｉｌｌｕｓ的大约１．５ｋｂ的ＳＳＵｒＲＮＡ序列,采用ＨｉｎｄⅡ、ＨｉｎｆⅠ、ＥｃｏＴ１４,ＨａｅⅢ和ｘｈｏⅠ等５种限制性内切酶进行酶切电泳分析,同时比较了来源于小鼠的ＣＢＭ株及来源于大鼠的ＣＢＲ株,未发现两者有差异。     

人巨细胞病毒的分子克隆及其特异性DNA探针的制备

从人巨细胞病毒（ＨＣＭＶ）培养物中提取ＨＣＭＶ并抽提其ＤＮＡ,经限制性内切酶ＢａｍＨＩ完全消化后,与质粒ｐＢｌｕｅｓｃｒｉｐｔ－ＳＫ重组建立了ＨＣＭＶ的ＤＮＡ文库,从此文库。中随机筛选出两个重组质粒（ｐＣＭＶ－１和ｐＣＭＶ－２）,用ＢａｍＨＩ分析证明其中所含的病毒ＤＮＡ片段的大小分别为１．０ｋｂ和７．５ｋｂ,将这两种质粒大量扩增纯化后,用光生物素进行标记作为探针,证明其只与ＨＣＭＶ反应,与正常人细胞ＤＮＡ及Ⅰ型和Ⅱ型单纯疤疹病毒ＤＮＡ无交叉反应。     

曼氏血吸虫发育过程中的基因组DNA变异

曼氏血吸虫（Ｓｃｈｉｓｔｏｓｏｍａｍａｎｓｏｎｉ）和日本血吸虫（Ｓ．ｊａｐｏｎｉｃｕｍ）成虫和曼氏血吸虫尾蚴基因组ＤＡＮ经限制性内切酶ＢａｍＨＩ消化后,分别与＾３２ｐ－ｄＣＴＰ标记的来源于核糖体ＤＮＡ的ＰＳＭＨＣＲ５、ＰＳＭＨＣＲ４ＰＳＭ８８９探针杂交,曼我血吸虫尾蚴和成虫在ＰＳＭＨＣＲ４杂交带型的１．６－２．８ｋｂ之间,存在有明显不同的次杂带;而ＰＳＭＨＣＲ５和ＰＳＭ８８９的杂交带型,无明显差异     

BstNI同功酶限制—修饰系统基因的表达检测和定位分析

鉴定了Ｅ．ｃｏｌｉ ＨＢ１０１和ＪＭ１１０的部分遗传标记,作为受体菌分别用于ＢｓｔＮＩ同功酶限制－修饰系统中限制性内切酶（Ｒ）基因和甲基化酶（Ｍ）基因表达的检测。用外切酶Ⅲ单向删切含Ｒ－Ｍ基因的ＤＮＡ片段,获得２３个缺失突变亚克隆。通过检测各亚克隆表达的Ｒ酶和Ｍ酶活性,将Ｒ和Ｍ基因分别定位在距克隆位点ＰｓｔＩ和０．２→１．４ｋｂ和１．５→３．３ｋｂ范围内。分析表明：该系统属于Ⅱ类限制－修饰系统,两     

马立克氏病病毒（MDV）814株B抗原基因的克隆及部分序列分析

提取感染鸡胚成纤维细胞ＭＤＶ－Ｉ弱毒株８１４病毒ＤＮＡ为模板,根据ＲＢＩＢ株ｇＢ基因５′及３′两端核苷酸离列设计引物,利用ＰＣＲ技术扩增了我国ＭＤＶ－Ｉ弱毒株８１４ｇＢ基因（２．９ｋｂ）将扩增片段平末端克隆到载体ｐＢｌｕｅｓｃｒｉｐｔＳＫ中ＥｃｏＲＶ位点,经ＢａｍＨＩ,ＨｉｎｄＩＩＩ酶切鉴定得到不同插入方向的重组质粒。构建圹增片段的酶切图谱及部分序列分析证明与ＲＢＩＢ株ｇＢ基因无差异,显示了极高的     

用PCR扩增和克隆鸡传染性贫血病毒全基因组DNA

根据已发表的序列,分别在鸡贫血病毒（ＣＡＶ）环形基因组ＤＮＡ（全长２．３ｋｂ）的ＥｃｏＲＩ位点和ＢａｍＨＩ位点的两侧选择适当序列合成两对引物,用ＰＣＲ技术,从斑点杂交检测到病毒核酸的ＣＡＶ感染的ＭＤＣＣＲＰ１细胞基因组ＤＮＡ中,分别扩增出包含ＥｃｏＲＩ和ＢａｍＨＩ分割开的病毒基因组两部分（１．５ｋｂ和０．８ｋｂ）约１．５ｋｂ和约１．２５ｋｂ的两个片段。再将其中相应序列拼接克隆进ｐＵＣ１８载体,获得包含ＣＡＶ全基因组序列ＤＮＡ片段的克隆质粒ｐＣＡＶ２．４。酶切分析表明,该质粒具有预期的ＢａｍＨＩ位点、ＰｓｔＩ位点、ＨｉｎｄⅢ位点,而预期的ＥｃｏＲＩ位点消失。重组质粒插入ＤＮＡ片段的两端序列分析表明,质粒ｐＣＡＶ２．４是包含ＣＡＶ全基因组序列的重组质粒,插入ＤＮＡ片段序列中的ＥｃｏＲＩ位点序列发生了一个碱基突变。     

Analysis of the methylation pattern of c-Ha-ras oncogene in human prostatic cancer

To determine the methylation pattern of c-Ha-ras oncogene in prostatic tissue and to identify possible changes of methylation associated with cancer, high molecular weight DNA was extracted from 7 normal and 6 carcinomatous human prostates. Analysis of the samples was performed by cleaving DNA with the restriction endonucleases Msp I, Hpa II and Cfo I, and by Southern hybridizing the DNA digests with the 32P-labelled c-Ha-ras (pT24-C3) probe. Several discrete fragments were obtained with Hpa II and Cfo I digestion while the Msp I pattern showed fewer and smaller bands. Therefore, c-Ha-ras appears to be partially methylated. While a considerable polymorphism of the sequence 5'-CCGG-3' was observed at several Msp I sites in all cases, no significant differences could be evidenced in the methylation patterns of normal and neoplastic prostatic DNA samples extracted and purified from each patient.     

人hsp90β基因近端上游CpG位点甲基化状态的研究

真核细胞基因组ＤＮＡ的甲基化主要发生在ＣｐＧ二核苷酸对的胞嘧啶环上（ｍ５Ｃ）［１,２］．在真核细胞基因组ＤＮＡ内,约７０％的ＣｐＧ位点发生了甲基化．ＣｐＧ位点在基因组ＤＮＡ内并不是均匀分布的,多数聚集在一些基因的５’端．大量的实验证据表明,真核基因５...     

Methylation of DNA of the brain and liver of young and old rats.

In vitro methylation of purified DNA and chromatin-DNA in nuclei of the liver and brain of young (18 week) and old (120 week) female rats has been studied using 3H-SAM as the -CH3 group donor. Incorporation of -CH3 group is higher in old liver and brain, but it is far higher in the latter. 5 mC is 11% lower in the old brain, but there is no difference in the liver. Methylation by Hpa II methylase does not show any difference in the incorporation of -CH3 group into DNA of the liver of the two ages. However, its incorporation is lower in the old brain. Methylation by Msp I methylase causes slightly higher incorporation of -CH3 groups in the old brain. This shows a higher percentage of unmethylated external cytosines in the 5'-CCGG-3' sequences. On the contrary, methylation by Eco RI methylase is considerably higher in the old brain. These studies show alterations in the methylation status of the DNA during ageing which may cause changes in the expression of genes.     

基于MSAP和SSR标记的高粱甲基化连锁群LGC、LGD的构建

以高粱(Sorghum bicolor(L.)Moench)品种‘B_2V_4’和‘1383-2’杂交获得的F_2群体为材料,通过SSR和MSAP标记检测高粱基因组差异,构建其甲基化遗传连锁群。结果显示,高粱甲基化连锁群LGC含有3个SSR标记和23个甲基化标记,覆盖高粱基因组44.3 cM;甲基化连锁群LGD含有4个SSR标记和8个甲基化标记,覆盖高粱基因组46.2 cM。LGC上甲基化位点仅来源于EcoRⅠ/MspⅠ酶切组合,而LGD上有来源于EcoRⅠ/MspⅠ和EcoRⅠ/HpaⅡ两种酶切组合的甲基化位点。在LGC连锁群Xtxp 69附近检测到一个密集的甲基化位点区域。研究结果表明MSAP标记可以快速检测植物基因组甲基化差异,适用于构建甲基化连锁群。     

Methylation of DNA and its modulation by estrogen in the uterus of aging rats.

The effect of estrogen on methylation of DNA from the uteri of young (20 weeks) and old (96 weeks) female Wistar rats has been examined by isoschizomeric restriction enzymes and HPLC analysis. In vitro methylation of DNA is significantly higher in the uteri of young rats as compared to old ones. This is reduced by estrogen to greater extent in young than in old age. Furthermore, the digestion of DNA with EcoRI+Msp I shows a distinct 1.2 kb band only in young control. Such band is absent in old control and estrogen-treated sets of both age groups. The HPLC data further reveal that the level of 5-methyl cytosine is high in young and decreases by nearly 18% in old. Estrogen lowers the level of 5-methyl cytosine by 8% in young but shows no effect in the old. Such age-dependent changes in the methylation of DNA brought by estrogen in the rat uterus attribute to alterations in gene expression during aging.     

Absence of cytosine methylation at C-C-G-G and G-C-G-C sites in the rDNA coding regions and intervening sequences of Drosophila and the rDNA of other insects.

Cytosine residues in C-G dinucleotides are frequently methylated in eukaryote DNA. In DNA of the dinoflagellate C. cohnii, the sequence C-MeC-G-G apparently renders Hpa II (C-C-G-G) incapable of digesting whole cell DNA in general, and rDNA in particular. Msp I, which also recognizes C-C-G-G but cleaves irrespective of methylation, degrades C. cohnii DNA and produces rDNA segments of 10.2 to 1.4 kb. We have applied this Hpa II/Msp I test to unfractionated DNA, and to rDNA and the rDNA intervening sequence of Drosophila virilis embryos and adults. There is no evidence of C-MeC-G-G sequences in either developmental stage of this species. Absence of G-MeC-G-C from coding and intervening sequences of rDNA was shown in comparisons of Hha I (G-C-G-C) cleavage patterns of unfractionated DNA and cloned (unmodified) segments of rDNA. Comparisons of Hpa II and Msp I cleavage products of DNA from the house fly, the flesh fly and a bumblebee also revealed no internal cytosine methylation in the sequence C-C-G-G. Because amounts of McC in C-G dinucleotides vary greatly among species, from apparent nonexistence to substantial proportions, no inference may yet be drawn about the role of such base modifications in DNA.     

The methylation status of DNA derived from potato plants recovered from slow growth

The in vitro conservation of potato using tissue culture medium supplemented with the growth retardant mannitol causes morphological changes in the propagated material. These culture conditions seem to have an affect on the DNA extracted from the regenerated plants, when it is digested by the methylation sensitive restriction enzymes Hpa II/Msp I and Eco RII/Bst NI, compared to the control material. In most of these plants, there appears to be preferential methylation of nuclear domains that contain Eco RII/Bst NI recognition sites in contrast to those that contain Hpa II/Msp I sites. The refractory nature of the isolated DNA to these restriction enzymes was attributed to hypermethylation of genomic DNA and the ribosomal RNA genes. These findings indicate that methylation of DNA sequences may be an adaptive response to conditions of high osmotic stress. The importance of these results for the conservation of potato germplasm and international exchange is discussed.     

Absence of global genomic cytosine methylation pattern erasure during medaka (Oryzias latipes) early embryo development

Two techniques were used to analyze global genomic 5-methyl cytosine methylation at CCGG sites of medaka embryo DNA. DNA was labeled by incorporation of microinjected radiolabeled deoxynucleotide into one-cell embryos. After Hpa II or Msp I digestion the radiolabeled DNA was fractionated in agarose gels and the distribution of label quantified throughout each sample lane to detect differences in fragment distribution. Alternately isolated DNA was digested with Hpa II or Msp I and the resulting generated termini end-labeled. The end-labeled digestion products were then analyzed for fragment distribution after gel fractionation. These techniques proved to be extremely sensitive, allowing comparison of genomic DNA methylation values from as few as 640 fish cells. The data suggest that in medaka embryos the vast majority (>90%) of genomic DNA is methylated at CCGG sites. Furthermore, these data support the conclusion that the extent of methylation at these sites does not change or changes very little during embryogenesis (from 16 cells to the hatchling). These data argue against active demethylation, or loss of methylation patterns by dilution, during the developmental stages between the one cell zygote and gastrulation. From a comparative viewpoint, these data may indicate that mammals and fishes methylate and demethylate their genomes in very different manners during development.