Decreased RNA polymerase activity in mammalian zinc deficiency

The activity of DNA-dependent RNA polymerase has been measured in liver nuclei from suckling rats nursed by zinc-deficient dams, or by controls that were either pair-fed or given free access to the diet. In the zinc-deficient pups, the activity of the enzyme did not increase; it fell after the tenth day of life.     

A kinetic framework for a mammalian RNA polymerase in vivo

We have analyzed the kinetics of assembly and elongation of the mammalian RNA polymerase I complex on endogenous ribosomal genes in the nuclei of living cells with the use of in vivo microscopy. We show that components of the RNA polymerase I machinery are brought to ribosomal genes as distinct subunits and that assembly occurs via metastable intermediates. With the use of computational modeling of imaging data, we have determined the in vivo elongation time of the polymerase, and measurements of recruitment and incorporation frequencies show that incorporation of components into the assembling polymerase is inefficient. Our data provide a kinetic and mechanistic framework for the function of a mammalian RNA polymerase in living cells.     

DNA hybridization and evolutionary relationships in three osmunda species

Molecular hybridization techniques have been used to estimate the degree of DNA base sequence homology between some members of the fern genus Osmunda. Under conditions permitting extensive reassociation, measurements of the extent of inter-specific reaction and the thermal stability of the hybrid molecules indicated that O. claytoniana L. (interrupted fern) shares more DNA homology with O. cinnamomea L. (cinnamon fern) than it does with O. regalis L. (royal fern). These findings are in conflict with predictions from a recent analysis of living and fossil specimens by numerical techniques. However, they are consistent with the earlier, more traditional, taxonomic assignments.     

Heterogeneity of murine leukemia virus in vitro DNA; detection of viral DNA in mammalian cells

Kinetic analysis of the reassociation of DNA synthesized in vitro by a murine leukemia virus DNA polymerase revealed two classes of double-stranded product representative of 25 and 100 percent of viral genetic information. The DNA product representing the smaller portion of the viral genome comprised 85 percent of the double-stranded DNA generated in vitro and was extensively duplicated in the genomes of both normal cells and cells containing RNA tumor virus.     

UHRF1 plays a role in maintaining DNA methylation in mammalian cells

Epigenetic inheritance in mammals relies in part on robust propagation of DNA methylation patterns throughout development. We show that the protein UHRF1 (ubiquitin-like, containing PHD and RING finger domains 1), also known as NP95 in mouse and ICBP90 in human, is required for maintaining DNA methylation. UHRF1 colocalizes with the maintenance DNA methyltransferase protein DNMT1 throughout S phase. UHRF1 appears to tether DNMT1 to chromatin through its direct interaction with DNMT1. Furthermore UHRF1 contains a methyl DNA binding domain, the SRA (SET and RING associated) domain, that shows strong preferential binding to hemimethylated CG sites, the physiological substrate for DNMT1. These data suggest that UHRF1 may help recruit DNMT1 to hemimethylated DNA to facilitate faithful maintenance of DNA methylation.     

Mitochondrial DNA mutations, oxidative stress, and apoptosis in mammalian aging

Mutations in mitochondrial DNA (mtDNA) accumulate in tissues of mammalian species and have been hypothesized to contribute to aging. We show that mice expressing a proofreading-deficient version of the mitochondrial DNA polymerase g (POLG) accumulate mtDNA mutations and display features of accelerated aging. Accumulation of mtDNA mutations was not associated with increased markers of oxidative stress or a defect in cellular proliferation, but was correlated with the induction of apoptotic markers, particularly in tissues characterized by rapid cellular turnover. The levels of apoptotic markers were also found to increase during aging in normal mice. Thus, accumulation of mtDNA mutations that promote apoptosis may be a central mechanism driving mammalian aging.     

蓝细菌分离DnaE内含肽的自我剪接研究

【目的】证实蓝细菌Anabaenasp.PCC 7120复制酶中分离的DnaE内含肽的自我剪接活性。【方法】分别将AnabaenaPCC 7120中分离的2个dnaE基因的部分序列克隆到表达载体上,获得重组表达质粒,经超量表达和纯化后,免疫家兔获得相应的抗体;同时,两段分离的dnaE序列被克隆到同一表达载体,构建双重组表达质粒,两段序列具有各自的ORF,但由同一启动子调控。双重组表达质粒经诱导之后,利用以上获得的抗体对含有内含肽的DnaE蛋白在大肠杆菌表达体系中的自我剪接反应进行检测。【结果】在双重组表达质粒的表达产物中,抗体不仅可以分别与对应的蛋白质反应,而且还可以同时识别1条新条带,该条带的表观分子量与成熟DnaE的理论值大致相符。【结论】蓝细菌分离的DnaE内含肽在大肠杆菌表达体系中可以进行自我剪接;本研究中获得的抗体具有较好的特异性,无明显背景干扰,而且效价高,抗体可以稀释1 000倍以上使用,也可以用于相关方面的研究。     

Tet-mediated formation of 5-carboxylcytosine and its excision by TDG in mammalian DNA

The prevalent DNA modification in higher organisms is the methylation of cytosine to 5-methylcytosine (5mC), which is partially converted to 5-hydroxymethylcytosine (5hmC) by the Tet (ten eleven translocation) family of dioxygenases. Despite their importance in epigenetic regulation, it is unclear how these cytosine modifications are reversed. Here, we demonstrate that 5mC and 5hmC in DNA are oxidized to 5-carboxylcytosine (5caC) by Tet dioxygenases in vitro and in cultured cells. 5caC is specifically recognized and excised by thymine-DNA glycosylase (TDG). Depletion of TDG in mouse embyronic stem cells leads to accumulation of 5caC to a readily detectable level. These data suggest that oxidation of 5mC by Tet proteins followed by TDG-mediated base excision of 5caC constitutes a pathway for active DNA demethylation.     

一种基于模板DNA的PCR扩增难易预测方法

借助赋予四种碱基A,G,C,T四个三维空间中的点(0,0,1),(0,1,0),(-1,-1,0),(1,0,0),将一条DNA序列转化为一个二十四维向量。在此基础上利用Fisher线性判别法对人类基因组中189条基因片段进行PCR扩增难易预测,识别率达到了90%。     

Mammalian DNA polymerase: separation of binding from incorporation of deoxyribonucleoside triphosphates

An assay for the binding of deoxyribonucleoside triphosphate with an enzyme-DNA complex has been developed. This binding requires active enzyme and magnesium ion, takes place equally with native or denatured DNA, and may proceed in the absence of demonstrable DNA synthesis. The binding reaction appears to be specific for deoxyribonucleoside triphosphates, and studies on competition indicate that one active site accommodates the four common triphosphates.     

Antigenic material: persistence in hypersensitive cells

Radioactivity derived from injections of tritiated tetanus toxin was observed in lymphocytes and macrophages for as long as 9 months after injection into immunized mice. These labeled cells became highly vacuolated and attracted eosinophils and lymphocytes when the animals were reinjected with specific antigen.     

Three distinct genes in human DNA related to the transforming genes of mammalian sarcoma retroviruses

Southern blot hybridization was used to identify human and other vertebrate DNA sequences that were homologous to cloned DNA fragments containing the oncogenic nucleic acid sequences of three different type C mammalian retroviruses (simian sarcoma virus, the Snyder-Theilen strain of feline sarcoma virus, and the Harvey strain of murine sarcoma virus). Each onc gene counterpart has a single genetic locus, which probably contains non-onc intervening sequences. The human DNA sequences may represent genes important to cell growth or cell differentiation, or both. Their identification and isolation may allow elucidation of their role in these processes and in neoplasias.     

Stable RNA/DNA hybrids in the mammalian genome: inducible intermediates in immunoglobulin class switch recombination

Although it is well established that mammalian class switch recombination is responsible for altering the class of immunoglobulins, the mechanistic details of the process have remained unclear. Here, we show that stable RNA/DNA hybrids form at class switch sequences in the mouse genome upon cytokine-specific stimulation of class switch in primary splenic B cells. The RNA hybridized to the switch DNA is transcribed in the physiological orientation. Mice that constitutively express an Escherichia coli ribonuclease H transgene show a marked reduction in RNA/DNA hybrid formation, an impaired ability to generate serum immunoglobulin G antibodies, and significant inhibition of class switch recombination in their splenic B cells. These data provide evidence that stable RNA/DNA hybrids exist in the mammalian nuclear genome, can serve as intermediates for physiologic processes, and are mechanistically important for efficient class switching in vivo.     

Individual Antigenic Specificity of Isolated Antibodies

Antisera produced against certain isolated human antibodies showed clear antigenic differences between these antibodies. Individual antigenic specificity was demonstrated for two anti-A antibodies, one anti-dextran, and one anti-levan antibody. Failure to produce specific antisera in other instances appeared to be correlated with a greater heterogeneity of the antibody population used as antigen.     

Structural basis of transcription: separation of RNA from DNA by RNA polymerase II

The structure of an RNA polymerase II-transcribing complex has been determined in the posttranslocation state, with a vacancy at the growing end of the RNA-DNA hybrid helix. At the opposite end of the hybrid helix, the RNA separates from the template DNA. This separation of nucleic acid strands is brought about by interaction with a set of proteins loops in a strand/loop network. Formation of the network must occur in the transition from abortive initiation to promoter escape.     

Antigenic competition: cellular or humoral

The injection of one antigen into mice inhibited the response to a second when 1 to 10 days separated the two injections. When the same type of inhibition was attempted in gamma-irradiated mice reconstituted with normal spleen cells, the inhibition was greater in mice receiving 50 million spleen cells than in those receiving 10 million. The results are interpreted as favoring a humoral mechanism of inhibition.     

Antigenic diversity in the human malaria parasite Plasmodium falciparum

Monoclonal antibodies against blood forms of Plasmodium falciparum were used to demonstrate considerable antigenic diversity in this species. Different isolates were distinguished by their ability to react with certain antibodies, and most of the antibodies reacted specifically with merozoites, schizonts, or both. The distribution of different antigenic types appeared not to be related to geographic origin. Serological typing with monoclonal antibodies extends the range of methods for identification of different strains of this malaria parasite.