棉花枯萎病菌异核体的三个不同核型分离子DNA碱基组成及18S rDNA序列*

从棉花枯萎病菌 (Fusariumoxysporum f.sp .vasinfectum)异核体菌株Ag1 49的菌落角变处分离得到 3个培养特征和致病性差异明显的单孢分离子。分别测定了这 3个分离子的总DNA和核DNA的碱基组成 ,以及它们的 1 8SrDNA的部分序列 ( 1 477个碱基对 )。它们的总DNA (G +C)mol%差异在 0.1 %～ 0.4%之间 ;核DNA (G +C)mol%的差异在0.4%～ 0.8%范围内 ;而 1 8SrDNA的部分序列则完全相     

水稻籼粳亚种间rDNA的ITS(internal transcribed spacer,ITS)序列分析

为探讨水稻亚种间的遗传背景及亲缘关系,运用PCR技术扩增并测序了水稻籼亚种的南京11号,9311、广陆矮4号三个品种和粳亚种的秋光、日本晴、爪哇稻三个品种的完整ITS区（包括5.8S区）。供试材料的5.8S rDNA的长度和G/C含量完全一致。ITSI的长度为193-195bp、G/C含量为72.31%-74.38;ITS2的长度为224-232bp,G/C含量为74.46%-76.86%。序列的相似性为94.4%-99.3%。CLUSTAL.W软件排序及分析表明;1）存在4个信息位点把6个品种分为籼粳两大类群;2）籼稻之间的ITS序列的同源性小于粳稻之间的同源性,由此可见,水稻核糖体DNA的ITS序列的变化规律与其传统的分类具有高度的一致性。     

海绵Pachychalina sp.体内细菌多样性的研究

通过非分离培养分析方法,直接从海绵体内提取细菌总DNA。以样品总DNA为模板进行PCR扩增获得细菌16S rDNA。用16S rDNA限制性酶切片段长度多态性（ARDRA）和测序方法对南海湛江海域海绵Pachychalina sp.体内的细菌多样性进行了研究。在细菌16S rDNA的ARDRA图谱中,大多数克隆的酶切带谱间存在差异;随机挑选22个克隆进行测序得到它们的16S rDNA部分序列,大部分序列属于γproteobacterium和αproteobacterium,但有少数克隆序列与RDP数据库中收录的16S rDNA序列间的相似性极小,不参与系统发育树的构建。研究结果表明海绵Pachychalina sp.体内细菌组成具有丰富的多样性。     

甘肃省冬虫夏草菌的RAPD和ITS序列分析

目的对甘肃省冬虫夏草分离菌株进行鉴定和系统发育研究。方法采用随机扩增多态性DNA（RAPD）分析和核糖体（rDNA）内部转录间隔区（ITS）序列分析。结果共分离到18个菌株,菌株间的遗传相似性系数为0.623～1.000,遗传分化距离为0.000～0.018,每个菌株的序列与中国被毛孢的序列一致性均高于98%且18个菌株（G＋C）%的差别最大值是4.7%。结论 18个分离菌株均为中国被毛孢;多样性分析表明,不同区域样本间的遗传分化较大;同一地点样本间的遗传分化很小;同一材料不同部位获得的菌株在RAPD标记上差异无统计学意义,两技术对相同的供试菌株所反映的遗传分化不尽相同,表明两技术用于冬虫夏草遗传多样性和种类鉴定上各有它们的优势和具体选择。     

细菌分类与鉴定的新热点：16S—23S rDNA间区

随着分子生物学的迅速发展,细菌的分类鉴定亦从传统的表型分类进入到各种基因型分类水平、如（G＋C）mol%、DNA杂交、rDNA指纹图、质粒图谱和16S rDNA序列分析等。rRNA存在于所有细菌中,rRNA基因由保守区和可变区组成,在细菌中高度保守。rRNA基因包含5‘端到3‘端的若干种成分,分别是16S rDNA、间区、23S rDNA、间区和 5S rDNA。16S－23S rDNA间区近年来在细菌系统发育学,特别是相近种和菌 区分和鉴定方面倍受关注。作为细菌分类和鉴定中的一个热点,本文将就16S－23S rDNA间区的一些特性及其胡细菌分类鉴定方面的作用做一简要的介绍。     

基于16S rDNA和COⅠ基因序列探讨四种溞类的系统关系和分类地位

为了检验已记录的4个溞属种类（(隆线溞Daphnia carinata、透明溞D. hyalina、蚤状溞D. pulex和大型溞D. magna）的系统分类, 用试剂盒法分别提取4种溞类的基因组DNA。利用特异性引物, 通过PCR扩增了4种溞属种类的16S rDNA和COⅠ基因部分序列, 并与来自GenBank中每个种类相似度较高的同种序列进行分析。结果表明, 基于16S rDNA和COⅠ基因, 4个溞属种类的平均种间相似度分别为85.56%和80.67%, 碱基中A+T含量均明显高于G+C含量。基于COⅠ基因, 巢湖隆线溞与来自GenBank上的拟同形溞（(Daphnia similoides AB549199）)相似度为99%, 分歧度为0.4%0.9%。就16S rDNA和COⅠ基因而言, 巢湖透明溞更接近于盔形溞（Daphnia galeata）。基于16S rDNA和COⅠ基因构建的NJ树和贝叶斯树, 两者的主要分枝基本一致。结果暗示, 巢湖隆线溞与拟同形溞为一个种, 透明溞应属于盔形溞。由于缺乏核基因的研究, 有关巢湖溞属的分类地位还需进行更深入的探讨。       

一株毒死蜱降解细菌的分离鉴定及其在土壤修复中的应用

从蔬菜大棚土壤中分离到一株能以毒死蜱为唯一碳源和能源生长的菌株DSP3,该菌在含毒死蜱（100mg/L）的酵母膏和蛋白胨与同样毒死蜱含量而无酵母膏蛋白胨的无机盐培养基中,18d对毒死蜱的降解率分别为986%和762%;在土壤实验中20d对毒死蜱（100mg/kg）的降解率接近100%,加入DSP3菌在蔬菜大棚新鲜土壤中能有效促进毒死蜱在土壤中的降解。根据生理生化特征、16S rDNA序列分析、（G+C）mol%测定和DNA同源性分析,将菌株DSP3鉴定为粪产碱杆菌（Alcaligenes faecalis）。     

海绵Pachychalina sp.体内古菌多样性非培养技术分析

采用非分离培养分析方法,即16S rDNA限制性酶切片段长度多态性（ARDRA）和测序方法对南海湛江海域海绵Pachychalina sp.体内的古菌多样性进行了研究。从海绵体内直接提取古菌总DNA。以样品总DNA为模板,用古菌16S rDNA通用引物进行PCR扩增获得16S rDNA,回收、纯化16S rDNA产物并克隆到TVector。进行第二次PCR扩增反应,且对扩增产物进行ARDRA。在古菌16S rDNA的ARDRA图谱中,大多数克隆的酶切带谱上存在差异;随机挑选8个克隆子进行测序,获得古菌16S rDNA的部分序列,并对16S rDNA序列进行聚类分析构建了系统进化树,结果发现海绵体内的古菌主要属于Methanogenium organophilum、Methanoplanus petrolearius等古菌类。但它们与目前数据库中收录的古细菌间的相似性均不超过90%,它们极有可能是一些新的古菌。     

海绵Pacnychalina sp.体内古菌多样性非培养技术分析

采用非分离培养分析方法,即16S rDNA限制性酶切片段长度多态性(ARDRA)和测序方法对南海湛江海域海绵Pachychalina sp.体内的古菌多样性进行了研究.从海绵体内直接提取古菌总DNA.以样品总DNA为模板,用古菌16S rDNA通用引物进行PCR扩增获得16S rDNA,回收、纯化16S rDNA产物并克隆到T-Vector.进行第二次PCR扩增反应,且对扩增产物进行ARDRA.在古菌16S rDNA的ARDRA图谱中,大多数克隆的酶切带谱上存在差异;随机挑选8个克隆子进行测序,获得古菌16S rDNA的部分序列,并对16S rDNA序列进行聚类分析构建了系统进化树,结果发现海绵体内的古菌主要属于Methanogenium organophilum、Methanoplanus petrolearius等古菌类.但它们与目前数据库中收录的古细菌间的相似性均不超过90%,它们极有可能是一些新的古菌.     

海绵Pachychalina sp.体内细菌多样性的研究

通过非分离培养分析方法,直接从海绵体内提取细菌总DNA。以样品总DNA为模板进行PCR扩增获得细菌16S rDNA。用16S rDNA限制性酶切片段长度多态性(ARDRA)和测序方法对南海湛江海域海绵Pachychalina sp．体内的细菌多样性进行了研究。在细菌16S rDNA的ARDRA图谱中,大多数克隆的酶切带谱间存在差异;随机挑选22个克隆进行测序得到它们的16S rDNA部分序列,大部分序列属于γ-proteobacterium和α-proteobacterium,但有少数克隆序列与RDP数据库中收录的16S rDNA序列间的相似性极小,不参与系统发育树的构建。研究结果表明海绵Pachychalina sp.体内细菌组成具有丰富的多样性。     

Nuclear location of mammalian DNA polymerase activities.

Nuclei were isolated from monolayer cultures of mouse and human cells using a nonaqueous procedure of cell fractionation in which lyophilized cells were homogenized and centrifuged in 100% glycerol. In previous work we have shown that the nuclear pellet and cytoplasmic supernatant fraction contained 10% or less of the nucleic acids characteristic of the other cell fraction. Aqueous extracts made from fresh cultures and from nonaqueous material at each step of the fractionation procedure were assayed fro DNA polymerase activity. Activities were normalized to DNA contents of extracted material. Specific activity was preserved quantitatively through freezing and drying the cells, but was found to be unstable in glycerol suspensions with approximate half-lives and 1 h at 23 degrees and 4 h at 0-4 degrees. Activities were relatively stable at -25 degrees, however, so that by homogenizing only 15 min at 4 degrees and centrifuging at -25 degrees we preserved approximately 85% of the specific activity of fresh cultures in the nonaqueous nuclear fraction. Sedimentation analyses showed that the nuclear fraction contained both DNA polymerase-alpha and-beta in approximately the proportions expected if all polymerase activities were confined to the nucleus in living cells. DNA polymerase-alpha was found to be more unstable in glycerol suspensions than DNA polymerase-beta. Nuclear location of both activities was found in exponential cultures and in 3T3 mouse cultures synchronized in the G1 and S phases of the cell division cycle. We found no evidence for cytoplasmic factors affecting nuclear polymerase activities. We have concluded that the two major DNA polymerases are nuclear although one, DNA polymerase-alpha, frequently is present as a weakly bound nuclear protein.     

Determination of ploidy levels in Ipheion uniflorum (R. C. Graham) Rafin (Liliaceae)

In this study, chromosome number and ploidy levels of Ipheion uniflorum cv. "Wisley Blue" (spring starflower) were determined. In meristematic root tip cells, chromosome number was found as 2n = 12 and 4n = 24. The ratios of diploid and tetraploid cells were found as 80.74% and 19.26%, respectively. In differentiated root tissues and mature leaf tissues ploidy levels were analysed by flow cytometry and polysomaty were found in both organs. In differentiated root tissues, ploidy levels were found as 2C, 4C, 8C and 16C DNA. In root tissues percentages of 2C, 4C, 8C and 16C nuclear DNA content were observed as 57.2%, 33.1%, 2.47% and 7.23%, respectively. In mature leaf tissues, ploidy levels were determined 2C, 4C, 8C and 16C DNA. In this tissue the frequency of 4C DNA was found very higher (74.3%) and 2C DNA content was determined as 19.2%. In mature leaf tissue, 8C and 16C nuclear DNA contents were observed as 2.72% and 3.78%, respectively. When nuclear DNA contents in leaves and roots were compared, an apparent difference in 2C and 4C DNA contents was found.     

黄花烟草和枸杞属间原生质体融合再生杂种小苗

利用改良的PEG方法以2%的频率诱导黄花烟草原生质体和经IOA失活的枸杞原生体融合。在获得的250块愈伤组织中,有5块为形态上与枸杞愈伤组织类似。对这5块愈伤组织的过氧化物酶和酯酶同工酶分析结果表明,其中的2块(记作Hy2和Hy5)为对称程度较高的体细胞核杂种,其它3块为不对称核杂种或细胞质杂种。细胞学观察指出,Hy2和Hy5的平均染色体数目分别为98.2和59.3。此外,两个杂种的几乎所有中期细胞均含有结构变异的染色体。以小麦rDNA为探针的Southern杂交结果表明,Hy2和Hy5均含有双亲DNA特异片段。从Hy5再生出了形态上介于双亲之间的小苗。对再生苗叶片的酯酶同工酶分析指出,这些小苗含有双亲的特征酶带。     

DNA binding properties of the nuclear matrix and individual nuclear matrix proteins. Evidence for salt-resistant DNA binding sites

The DNA binding characteristics of the rat nuclear matrix were investigated. A saturable and temperature-dependent, salt-resistant DNA binding to the nuclear matrix was discovered, with 70-80% of total bound DNA resistant to extraction with high concentrations of salt at 37 degrees C, compared to less than 5% at 0 degrees C. The initial binding of DNA to nuclear matrix is sensitive to salt concentration, indicating a transition to a salt-resistant binding state. The nuclear matrix shows a preference for single-stranded DNA, both in saturation and competition assays, with little binding of RNA or double-stranded DNA. Further competition studies show a preference for matrix-attached DNA probably involving predominantly AT-rich sequences, while a specific sequence defined previously as a matrix-attached region (MAR; Cockerill, P. N., and Garrard, W. T. (1986) Cell 46, 273-282) only showed preference for a limited number of the total matrix binding sites. These results and estimates from saturation data of approximately 150,000 single-stranded DNA binding sites per matrix lead us to propose that the nuclear matrix contains different classes of DNA binding sites, each with a separate sequence specificity. Binding of DNA to individual matrix polypeptides separated on sodium dodecyl sulfate-polyacrylamide gels and transferred to nitrocellulose blots was also temperature-dependent, salt-resistant, and showed a preference for binding DNA over RNA and nuclear matrix DNA over total genomic DNA. Subnuclear fractionation experiments further demonstrated that the nuclear matrix is enriched in the subset of higher molecular weight (greater than 50,000) DNA binding proteins of isolated nuclei and correspondingly depleted of the lower molecular weight ones. Of the approximately 12 major proteins separated on nonequilibrium two-dimensional gels, 7 were identified as specific DNA binding proteins including lamins A and C (but not B), and the internal nuclear matrix proteins, matrins D, E, F, G, and 4.     

Temperature dependent phosphorylation of [H]thymidine and its incorporation into DNA by KB cells

The incorporation of [³H]TdR into DNA by KB cells cooled to 4 °C falls rapidly to about 1–2% of that of controls held at 37 °C. The amounts of four enzymes involved in TdR metabolism: TdR kinase, thymidylate kinase, cytoplasmic DNA polymerase, and nuclear DNA polymerase, never fall below 50% of those in the control cells even after 12 h at 4 °C. The activities of these enzymes were measured in vitro at different temperatures and it was found that whereas the two kinases retained appreciable activity at low temperature, the DNA polymerase activities were severely inhibited. Cultures of cells rewarmed to 37 °C after 12 h at 4 °C immediately re-started incorporation of labelled TdR into DNA, showing that sufficient enzyme activity for normal functioning had been preserved during the cold period.     

Nuclear protein content and Ki-67 immunoreactivity in nonneoplastic and neoplastic thyroid cells.

The nuclear DNA content in thyroid tumor cells has been shown to be closely related to the malignant potential of the neoplasm. Besides DNA, nuclear protein (NP) constitutes the major mass of the nucleus. The NP content may vary significantly in relation to the proliferative stage in growing as compared to growth-arrested cells. The increase in NP content associated with the transition from G0 to G1 occurs before the onset of DNA synthesis and may be used to assess growth activity. The nuclear DNA and NP contents were analyzed in 90 nonneoplastic lesions and 75 benign and 62 malignant thyroid tumors. All nonneoplastic specimens were euploid, and 1 of 90 was growth activated. In the group of benign tumors, 59 were euploid, and 16 were aneuploid. Among these there were 5 (9%) of 59 and 6 (38%) of 16 growth-activated specimens, respectively. In the group of malignant tumors 57 of 62 were classified as euploid, and in this group 12 (21%) showed growth activity according to the NP content. Five of 62 were aneuploid, and 3 (60%) of these 5 tumors were growth activated. Evaluation of the growth activity by means of monoclonal antibody Ki-67 was performed on a subgroup of 32 thyroid specimens, both nonneoplastic and neoplastic lesions. Ki-67 immunoreactivity was observed in 0-1.1% cells of 6 nonneoplastic lesions, in 0-3.1% cells of 14 benign cells and in 0.2-3.9% cells of 12 malignant thyroid tumors. Growth activity, as reflected by the NP/DNA ratio and Ki-67 immunoreactivity, appears to be low both in nonneoplastic thyroid lesions and thyroid tumors.     

5-Methylcytosine content of nuclear DNA during chemical hepatocarcinogenesis and in carcinomas which result.

The 5-methylcytosine content of nuclear DNA from nuclear hepatocellular tissues was determined during various phases of hepatic regeneration and carcinogenesis. DNA from premalignant nodules and primary hepatocellular carcinomas induced by exposure to acetylaminofluorene, as well as PHC induced by diethylnitrosamine was undermethylated by 20%, 45%, and 32.5% respectively. Since a 12.5% hypomethylation occurred during the DNA synthetic phase of hepatic regeneration, the effect of cell proliferation on DNA-methylation in malignancies was examined in transplantable hepatocellular carcinomas. The DNA from two transplantable hepatocellular carcinoma lines was less methylated than predicted rates of cell division in these tumors. This finding suggested that an aberration in endogenous DNA methylation may occur during neoplastic transformation.     

The DNA polymerases of Chinese hamster cells. Subcellular distribution and properties of two DNA polymerases.

We have fractionated homogenates of Chinese hamster cells grown in tissue culture, and found that >80% of those cells' DNA-dependent DNA polymerase appears localized in the soluble cytoplasm. The Chinese hamster cytoplasmic DNA polymerase is very similar to DNA polymerases from several mammalian sources: it is large and heterogeneous (165,000–200,000 daltons), sensitive to sulfhydryl-blocking reagents and absolutely requires double stranded templates containing free 3′-OH primers. Two distinct species of DNA polymerase also have been isolated from purified Chinese hamster nuclei. One nuclear DNA polymerase appeared to be identical to DNA polymerase found in the cells' soluble cytoplasm. The second polymerase, comprising 1.5–3% of the total DNA polymerase activity, was found only in nuclear extracts. That enzyme is resistant to sulfhydryl-blocking reagents and has an apparent molecular weight of 49,000. The data discussed in this report suggest that Chinese hamster cells, like other mammalian cell types, possess at least two DNA-dependent DNA polymerases that might participate in replicative DNA biosynthesis.     

Reassociation and dissociation of cytoplasmic membrane-associated DNA

The relationship between nuclear DNA and cytoplasmic membrane-associated DNA, extracted from a human lymphocyte cell line, was examined by DNA-DNA reannealing and by dissociation of renatured molecules. Up to 2% of the total cellular DNA is found in the cytoplasm as cytoplasmic membrane-associated DNA and of this 2%, approximately 70% is comprised of repeated sequences. These sequences are homologous to only about 4% of the repeated sequences of nuclear DNA. The repeat fraction of cytoplasmic membrane-associated DNA consists of sequences which are only moderately repeated. The number of copies in the average “family” could range from about 1500 copies to as few as 25 copies. A small rapidly reannealing portion of cytoplasmic membrane-associated DNA (C₀t < 4 × 10^?3) appears to consist of sequences derived from a single “family”.About 30% of cytoplasmic membrane-associated DNA reassociates slowly with a $\text{C}{}_0\text{t}\text{1}\text{2}$ value of 223 (unique cytoplasmic membrane-associated DNA). This fraction has homology with about 11% of the unique sequences of nuclear DNA. However, unique cytoplasmic membrane-associated DNA comprises only about 0·6% of the total cellular DNA. If it is assumed that each cell has the same amount of cytoplasmic membrane-associated DNA, homology with 11% of the unique sequences of nuclear DNA suggests that different cells may have different unique nucleotide sequences in the cytoplasm.