基因机器:自动合成DNA

在实验室里把遗传物质的四种材料连接成合成的顺序已经是一项熟练的工艺。一位有机化学家和几位技师精心设计逐步合成几微克单一基因的片段不要化几个月的时间。现在可用电脑系统自动合成DNA,并且使这种工艺进入工业化生产。     

DNA damage: air-breaks?

Cells deficient in repairing DNA double-strand breaks have an increased level of spontaneous chromosomal aberrations. Modulating the level of molecular oxygen and its reactive metabolites demonstrates that oxygen metabolism is a major source of genomic instability.     

基因机器:自动合成DNA

在实验室里把遗传物质的四种材料连接成合成的顺序已经是一项熟练的工艺。一位有机化学家和几位技师精心设计逐步合成几微克单一基因的片段不要化几个月的时间。现在可用电脑系统自动合成DNA,并且使这种工艺进入工业化生产。     

基因工程:重组体DNA研究

重组体脱氧核糖核酸(DNA)研究是人为地将一小段DNA(基因)嵌入另一种DNA分子中,成为DNA杂交分子。然后使含有此杂交分子的宿主进行繁殖。这段外来的基因随着杂交分子的复制而增加其模板本。所以称此过程为分子繁殖。这种技术在基因的结构和功能的研究中有非常重要的作用,而在应用方面(医学、农业、工业、环境卫生等)都有广阔的发展前景,因而又有基因工程之称。本文介绍了重组体DNA研究的技术概要,它在基础理论研究中的价值和发展,以及它在医学卫生、工农业等方面的实用可能。最后介绍了关于这项研究所引起的安全问题的争论。     

DNA Dynamics: Bubble ‘n’ Flip for DNA Cyclisation?

A recent demonstration of the facile in vitro formation of DNA microcircles of fewer than 100 base pairs throws new light on the basis of DNA flexibility.     

D-loop mutations in mitochondrial DNA: link with mitochondrial DNA depletion?

Clinical presentation of the patients with mitochondrial DNA depletion is quite diverse and is suggestive of genetic heterogeneity. Autosomal recessive inheritance of the disease appears likely, thus implying the nuclear origin of the disease. This has been demonstrated recently in large families with neonatal presentation of the disease. Here, we report upon a family with one child having a late-onset disease associated with severe mitochondrial DNA depletion. The presence of mitochondrial alterations in the muscle of the patient's mother prompted us to extensively analyse the mitochondrial DNA in the family. We found mitochondrial DNA multiple deletions, but also three heteroplasmic point mutations of the D-loop region, two of which (T119C and T408A) affect conserved regions involved in the mtDNA replication process. These mutations were non-randomly distributed in the maternal lineage and, for one of them, among single muscle fibres. Involvement of the mitochondrial DNA in its own depletion appears therefore possible. It may act in close relationship with a hypothetical modified nuclear factor.     

Wheat DNA polymerase CI: a homologue of rat DNA polymerase β

We have previously described a low-molecular-weight DNA polymerase (52 kDa) from wheat embryo: DNA polymerase CI (pol CI). This enzyme shares some biochemical properties with animal DNA polymerase (pol ). In this report, we analyse pol CI in wheat embryo germination. Immunodetection and measurement of the enzyme activity show that wheat pol CI remains at a constant level during germination, whereas dramatic changes of the replicative DNA polymerase A and B activities were previously reported. We observe that the level of pol CI in physiological conditions (embryo germination and dividing cell culture) is in agreement with a pol -type DNA polymerase. By microsequencing of the electroblotted 52 kDa polypeptide, we determined the sequence of a dodecapeptide from the N-terminal region. A comparative analysis of the N-terminal pol CI peptide with some mammalian pol sequences shows a clear homology with helix 1 of the N-terminal ssDNA domain (residues 15 to 26) of the rat pol . Thus, the helical structure of this region should be conserved in the wheat peptide. This represents the first evidence of a partial primary structure of a -type DNA polymerase in plants.     

DNA甲基化起始的原动力:sidRNAs

正DNA胞嘧啶甲基化(5m C)是一种重要的表观遗传修饰,它对于调控基因表达,沉默转座子和维持基因组的稳定等具有重要作用.相对于动物中DNA的甲基化主要以CG形式存在,植物中DNA的甲基化有3种类型:CG,CHG和CHH(H代表A,T或C)甲基化.这3种类型甲基化修饰的从头建立都必须由     

DNA structure: what's in charge?

DNA structure is well known to be sensitive to hydration and ionic strength. Recent theoretical predictions and experimental observations have raised the idea of the intrusion of monovalent cations into the minor groove spine of hydration in B-form DNA. To investigate this further, extensions and further analysis of molecular dynamics (MD) simulations on d(CGCCGAATTCGCG), d(ATAGGCAAAAAATAGGCAAAAATGG) and d(G(5)-(GA(4)T(4)C)(2)-C(5)), including counterions and water, have been performed. To examine the effective of minor groove ions on structure, we analyzed the MD snapshots from a 15 ns trajectory on d(CGCGAATTCGCG) as two subsets: those exhibiting a minor groove water spine and those with groove-bound ions. The results indicate that Na(+) at the ApT step of the minor groove of d(CGCCGAATTCGCG) makes only small local changes in the DNA structure, and these changes are well within the thermal fluctuations calculated from the MD. To examine the effect of ions on the differential stability of a B-form helix, further analysis was performed on two longer oligonucleotides, which exhibit A-tract-induced axis bending localized around the CpG step in the major groove. Plots of axis bending and proximity of ions to the bending locus were generated as a function of time and revealed a strong linear correlation, supporting the idea that mobile cations play a key role in local helix deformations of DNA and indicating ion proximity just precedes the bending event. To address the issue of "what's in charge?" of DNA structure more generally, the relative free energy of A and B-form d(CGCGAATTCGCG) structures from MD simulations under various environmental circumstances were estimated using the free energy component method. The results indicate that the dominant effects on conformational stability come from the electrostatic free energy, but not exclusively from groove bound ions per se, but from a balance of competing factors in the electrostatic free energy, including phosphate repulsions internal to the DNA, the electrostatic component of hydration (i.e. solvent polarization), and electrostatic effects of the counterion atmosphere. In summary, free energy calculations indicate that the electrostatic component is dominant, MD shows temporal proximity of mobile counterions to be correlated with A-track-induced bending, and thus the mobile ion component of electrostatics is a significant contributor. However, the MD structure of the dodecamer d(CGCGAATTCGCG) is not highly sensitive to whether there is a sodium ion in the minor groove.     

DNA structure: cations in charge?

Recent X-ray diffraction, NMR spectroscopy and molecular mechanics results suggest that monovalent cations selectively partition into the minor groove of AT-tracts in DNA. These observations are consistent with DNA deformation by electrostatic collapse around areas of uneven cation density. This model predicts the occurrence of known DNA deformations, such as AT-tract bending and changes in the minor-groove width.     

CpG DNA:一种新型免疫佐剂

美国依阿华大学医学院Ｋｒｉｅｇ［１］等报道,一种含有胞嘧啶鸟嘌呤二核苷酸（ＣｐＧ）的ＤＮＡ片段是一种强烈的非特异性免疫刺激剂。这种ＣｐＧＤＮＡ可作用于多种免疫细胞。用含ＣｐＧ序列的细菌ＤＮＡ可诱导小鼠９５％的Ｂ细胞进入细胞增殖周期并分泌ＩｇＭ、ＩＬ－...     

Silent DNA: speaking RNA language?

The sequence of silent DNA in the human genome (intergenic spacers, introns and synonymous codon positions of protein-coding genes) was found here to have the higher thermostability of corresponding RNA/RNA and RNA/DNA duplexes as compared with randomized sequence. This difference increased with elevation of GC content. The revealed effect was not due to correlation of RNA/RNA and RNA/DNA thermostabilities with thermostability of the DNA/DNA duplex, which, on the contrary, was lower than in the randomized sequence and lagged behind the elevation of GC content. The same picture was observed in the genomes of other warm-blooded vertebrates but not in the lower organisms. This finding suggests that RNA-RNA and RNA-DNA interactions could be involved in the putative function of silent DNA.     

Trading places: how do DNA polymerases switch during translesion DNA synthesis?

The replicative bypass of base damage in DNA (translesion DNA synthesis [TLS]) is a ubiquitous mechanism for relieving arrested DNA replication. The process requires multiple polymerase switching events during which the high-fidelity DNA polymerase in the replication machinery arrested at the primer terminus is replaced by one or more polymerases that are specialized for TLS. When replicative bypass is fully completed, the primer terminus is once again occupied by high-fidelity polymerases in the replicative machinery. This review addresses recent advances in our understanding of DNA polymerase switching during TLS in bacteria such as E. coli and in lower and higher eukaryotes.     

UvrAB activity at a damaged DNA site: is unpaired DNA present?

To study the activity of the Escherichia coli UvrA and UvrB nucleotide excision repair proteins during the formation of the pre-incision complex at a damaged DNA site, we used substrates with modifications around a single 2-(acetylamino)fluorene (AAF) lesion. Based on the release of AAF-containing oligonucleotides from a single-stranded DNA circle, we conclude that during interaction with our substrates UvrAB introduces changes in DNA which are localized at the lesion and are limited to 1-3 bp. Since these changes might include a denaturation of DNA at the lesion site and, consequently, a bubble structure might be present in a pre-incision complex, we studied incision activity of UvrABC excinuclease on substrates with 1-4 unpaired bases next to an AAF adduct. Opening more than one base on either or both sides of the lesion caused a significant decrease in the incision activity of UvrABC, but did not change the position of the incision sites. We conclude that the UvrAB action leading to a pre-incision complex does not include the formation of a bubble intermediate generated by extensive denaturation of base pairs.     

Molecular recognition of plant DNA: Does it differ from conventional animal DNA?

The recognition mechanism of DNA with small drugs/ligands is an important field of research from pharmacological point of view. Such studies are ample with DNAs extracted from animal cells, but are rare for those extracted from plant cells. However, such a study is strongly demanding for the formulation of pesticides and other agrochemicals. In this contribution, for the first time, we report the interaction of two well-known DNA binder ethidium bromide (EB) and Hoechst 33258 (H33258) with two genomic DNAs extracted from the leaves of Ricinus communis L. (castor bean) and Mangifera indica (mango) using steady-state and picosecond-resolved fluorescence spectroscopy. The purity of the extracted DNAs is confirmed from gel electrophoresis and optical absorption studies. As evidenced from the circular dichroism (CD) measurements the DNAs retain physiologically relevant B forms. The well-known DNA intercalator EB has been found to show an additional electrostatic mode of binding with the DNAs, which is not present in the conventional animal DNAs. The binding affinity of EB is found to be even weaker for the DNA extracted from M. indica compared to that in R. communis L. On the other hand, the binding affinity of H33258 with the plant DNAs is found to be comparable to that of animal DNAs. The difference in interaction could be rationalized from the possible differences in the base sequences.     

DNA profile of the spore of blastocladiella emersonii: Evidence for γ-particle DNA

Summary Procedures were developed for isolating DNA from zoospores of Blastocladiella emersonii, critical steps being the use of non-frozen cells, enzymatic elimination of polysaccharide, and a combined treatment with T₁RNAse and pancreatic RNAse for complete removal of RNA. Methods for isolating -particles from spores were also established. Extraction of DNA directly from purified -particles substantiated previous assumptions that they contain DNA. Heat denaturation and preparative CsCl equilibrium sedimentation studies of whole spore DNA revealed the presence of four distinct components, I–IV, with buoyant densities of 1.731, 1.715, 1.705, and 1.687 g/cm³. The major species appeared to be nuclear chromatin. Nucleolar and mitochondrial sites were tentatively assigned to species II and III, respectively. Evidence was presented that species IV resided in -particles. Some possible roles for -particle DNA were discussed.Abbreviations EGTA ethyleneglycol-bis (B-aminoethyl ether)-N,N-tetracetic acid - EDTA ethylenediaminetetracetic acid - TCA trichloroacetic acid - SDS sodium dodecyl sulfate - SSC 0.15 M NaCl and 15 mM Na citrate