Chemical DNA synthesis and recombinant DNA studies

Chemically synthesized DNA has been used in many recombinant DNA studies. These uses have included the total synthesis and cloning of functional genes, the cloning and expression of natural genes, and editing of changing genes by directed mutation.     

DNA gyrase and the supercoiling of DNA

Negative supercoiling of bacterial DNA by DNA gyrase influences all metabolic processes involving DNA and is essential for replication. Gyrase supercoils DNA by a mechanism called sign inversion, whereby a positive supercoil is directly inverted to a negative one by passing a DNA segment through a transient double-strand break. Reversal of this scheme relaxes DNA, and this mechanism also accounts for the ability of gyrase to catenate and uncatenate DNA rings. Each round of supercoiling is driven by a conformational change induced by adenosine triphosphate (ATP) binding: ATP hydrolysis permits fresh cycles. The inhibition of gyrase by two classes of antimicrobials reflects its composition from two reversibly associated subunits. The A subunit is particularly associated with the concerted breakage-and-rejoining of DNA and the B subunit mediates energy transduction. Gyrase is a prototype for a growing class of prokaryotic and eukaryotic topoisomerases that interconvert complex forms by way of transient double-strand breaks.     

2种方法提取日本沼虾基因组DNA

以日本沼虾的尾部肌肉为材料,采用苯酚-氯仿法和氯化钠法提取日本沼虾基因组DNA,并对2种方法提取的结果进行了紫外分光光度、琼脂糖凝胶电泳、PCR扩增等方法的鉴定。结果表明:2种方法提取的日本沼虾基因组DNA浓度分别为3 165.8和396.5ng/μl,OD260/OD280比值分别为1.76和1.63,均能满足分子生物学研究的需要。     

Informational DNA synthesis distinguished from that of nuclear DNA by inhibitors of DNA synthesis

With inhibitors of DNA synthesis used in the presence of ethidium bromide, it has been possible to distinguish between synthesis of informational DNA (I-DNA) and that of nuclear DNA. Hydroxyurea depresses I-DNA synthesis preferentially but does not affect DNA transport between cell compartments. 5-Fluorodeoxyuridine and cytosine arabinoside reduce synthesis of I-DNA to a much lesser degree than that of nuclear DNA.     

Microsome-associated DNA

Deoxyribonucleic acid has been isolated from the microsomes of mouse liver homogenates under conditions designed to prevent or greatly reduce mitochondrial and nuclear contamination. The DNA rapidly incorporates tritiated thymidine, and this, together with its reannealing characteristics after thermal denaturation, shows that it is not mitochondrial or typically nuclear DNA.     

兴安落叶松DNA提取-试剂盒改良法

[目的]寻找一种快速简便的兴安落叶松基因组DNA的提取方法。[方法]对DNA提取试剂盒进行改良,与原试剂盒法(离心柱)进行比较,并用琼脂糖凝胶电泳、蛋白核酸测定和ISSR-PCR对所提取的总DNA进行检测。[结果]试剂盒改良法提取的总DNA纯度和浓度较高,电泳显示其主条带较清晰,无降解现象,A260/A280值为1.75～1.84。[结论]改良后的DNA提取方法更适合兴安落叶松基因组DNA的提取。     

Yeast DNA polymerase epsilon participates in leading-strand DNA replication

Multiple DNA polymerases participate in replicating the leading and lagging strands of the eukaryotic nuclear genome. Although 50 years have passed since the first DNA polymerase was discovered, the identity of the major polymerase used for leading-strand replication is uncertain. We constructed a derivative of yeast DNA polymerase epsilon that retains high replication activity but has strongly reduced replication fidelity, particularly for thymine-deoxythymidine 5'-monophosphate (T-dTMP) but not adenine-deoxyadenosine 5'-monophosphate (A-dAMP) mismatches. Yeast strains with this DNA polymerase epsilon allele have elevated rates of T to A substitution mutations. The position and rate of these substitutions depend on the orientation of the mutational reporter and its location relative to origins of DNA replication and reveal a pattern indicating that DNA polymerase epsilon participates in leading-strand DNA replication.     

Adriamycin-induced DNA damage mediated by mammalian DNA topoisomerase II

Adriamycin (doxorubicin), a potent antitumor drug in clinical use, interacts with nucleic acids and cell membranes, but the molecular basis for its antitumor activity is unknown. Similar to a number of intercalative antitumor drugs and nonintercalative epipodophyllotoxins (VP-16 and VM-26), adriamycin has been shown to induce single- and double-strand breaks in DNA. These strand breaks are unusual because a covalently bound protein appears to be associated with each broken phosphodiester bond. In studies in vitro, mammalian DNA topoisomerase II mediates DNA damage by adriamycin and other related antitumor drugs.     

Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase

A thermostable DNA polymerase was used in an in vitro DNA amplification procedure, the polymerase chain reaction. The enzyme, isolated from Thermus aquaticus, greatly simplifies the procedure and, by enabling the amplification reaction to be performed at higher temperatures, significantly improves the specificity, yield, sensitivity, and length of products that can be amplified. Single-copy genomic sequences were amplified by a factor of more than 10 million with very high specificity, and DNA segments up to 2000 base pairs were readily amplified. In addition, the method was used to amplify and detect a target DNA molecule present only once in a sample of 10(5) cells.