The design of single-stranded nucleic acid knots

A general strategy is described for the synthesis of single-stranded nucleic acid knots. Control of nucleic acid sequence is used to direct the formation of secondary structures that produce the target topology. The key feature of the strategy is the equation of a half-turn of double helical DNA or RNA with a node in a knot. By forming nodes from complementary DNA sequences, it appears possible to direct the assembly of any simple knot. Stabilization of individual nodes may be achieved by constructing them from long regions containing both B-DNA and Z-DNA. Control over the braiding of DNA that acts as a link between node-forming domains can be realized by condensing the nodes into well-defined DNA structures, such as extended domains of linear duplex, branched junctions, antijunctions or mesojunctions. Further topological control may be derived from the pairing of linker regions to complementary single-stranded molecules, thereby preventing them from braiding in an undesirable fashion.     

A BC200-derived element and Z-DNA as structural markers in annexin I genes: Relevance to Alu evolution and annexin tetrad formation

We have identified two types of structural elements in genomic DNA for annexin I that provide physical evidence of genetic events leading to conserved changes in gene structure. The sequence upstream of the transcribed region in human annexin I contained a rare, Alu-like repetitive element with flanking direct repeats, probably derived from the active BC200 gene via germline retroposition. Nucleotide substitutions in this BC200 insert relative to the 7SL gene and its absence in rodent annexins I identified it as a recent primate pseudogene. Phylogenetic analysis showed that the BC200 gene represents a new clade of primate Alu evolution that branched near the time of appearance of the progenitor to the free left Alu monomer, FLAM-C. Separate analysis identified a Z-DNA motif in pigeon annexin I intron 7 that may represent the vestigial recombination site involved in primordial assembly of the annexin tetrad. These distinct structural features in annexin I genes provide insight into the evolution of Alu repeats and the mechanism of annexin tetrad formation.     

Three distinct regulatory elements comprise the upstream promoter region of the nopaline synthase gene

Summary Fine deletion mutants were generated in the upstream control region of the nopaline synthase (nos) promoter to define the position and role of upstream regulatory elements. The results indicated that the 8 bp sequence (CAGAAACC) at -106/-113 and its inverted repeat (GGTTTCTG) at -140/-147 are important for promoter function. The downstream element appears more important than the upstream element since deletion of the former reduced promoter activity more significantly than deletion of the latter. Deletion of the element alone, however, did not abolish promoter function, whereas, deletion of the 10 bp potential Z-DNA-forming (Z) element located between the repeat elements nullified promoter activity. Therefore, it appears that the Z element is an essential upstream regulator and the repeated elements are upstream modulators of the nos promoter. These elements are functionally distinct since alteration of stereospecificity or insertion of short oligonucleotides between the elements did not significantly influence promoter activity. These regulatory elements were unable to function from 200 bp upstream of the CCAAT-TATA box region.     

The Structural Organization of Oligonucleosomes

Abstract

We have used electric birefringence to study the structure of oligonucleosomes and to show the influence of histone H1 depletion on their conformation in solution. Measurements are made at low ionic strength on monodisperse samples containing up to 8 nucleosomes. For each oligomer, having H1 or not, the analysis of both relaxation and orientation times gives information about the particle's orientation mechanism through the ratio r of permanent over induced dipole terms. For native oligomers, the data confirm the previous finding of a discontinuity in hydrodynamic behavior between pentamer and heptamer: the rotational times are multiplied by 10 and r increases from 0.2 to 0.7 showing the appearance of a non-negligible contribution of a permanent dipole to the orientation mechanism. We suggest a model for the hexanucleosome at low ionic strength and discuss its implications for the higher-order structure of chromatin.

The treatment for H1 depletion abolishes the transitions in electro-optical properties: the value of r remains constant, r=0.15, and both rotational times increase progressively with the number of nucleosomes in the chain. That reflects an important unfolding of oligonucleosomal structure which we attributed to the unwinding of DNA tails and internucleosomal segments. The disc planes of nucleosomes become closely parallel to the nucleosomal chain axis.     

左手螺旋Z-DNA的研究 Ⅴ 环境因子对Oligo-d(G-C)_6构象的影响

脱氧寡聚核苷酸ｄ（Ｇ－Ｃ）６是迄今所报道的同类脱氧多聚核苷酸中能形成左手螺旋ＤＮＡ（Ｚ－ＤＮＡ）的最短序列。环境因子如ＰＨ,温度和离子的类别与浓度对ｄ（Ｇ－Ｃ）６在溶液中的Ｚ－构象的形成和Ｂ－、Ｚ－构象的相对稳定性有着明显的影响。在合适的条件下,ｄ（Ｇ－Ｃ）６在溶液中可呈现Ｂ－构象区,Ｚ－构象相对稳定区和Ｂ－、Ｚ－构象跃迁过渡区。     

Chemical-induced formation of BZ-junction with base extrusion

The crystal structure of BZ-junction reveals that left-handed Z-DNA stabilized by Z-DNA binding domain (Zα) is continuously stacked to right-handed B-DNA with AT bases’ extrusion in the junction site. However, this structure might not fully represent the BZ-junction in solution due to the possibility of the junction formation either by crystal packing or Zα interaction. Therefore, we investigated BZ-junction in solution with chemical Z-DNA inducers using CD and 2-aminopurine base-extrusion assay. We confirmed the formation of Z-DNA and BZ-junction with base-extrusion by chemical Z-DNA inducers. However, neither typical Z-DNA nor base-extrusion could be detected with some inducers such as spermine, suggesting that the energy barrier for the formation of the BZ junction might vary depending on the Z-DNA induction conditions.     

Cloning and sequencing of a cDNA encoding ascorbate peroxidase fromArabidopsis thaliana

A cDNA clone encoding ascorbate peroxidase (AP, EC 1.11.1.11) was isolated from a phage gt11 library of cDNA fromArabidopsis thaliana by immunoscreening with monoclonal antibodies against the enzyme, and then sequenced. The cDNA insert hybridized to a 1.1 kb poly(A)⁺ RNA from leaves ofA thaliana. Genomic hybridization suggests that the cDNA obtained here corresponds to a single-copy gene. The N-terminal amino acid sequence ofArabidopsis AP was determined by protein sequencing of the immunochemically purified enzyme, and proved to be homologous to the N-terminal amino acid sequence of the chloroplastic AP of spinach. The predicted amino acid sequence of the mature AP ofA. thaliana, deduced from the nucleotide sequence, consists of 249 amino acid residues, which is 34% homologous with cytochromec peroxidase of yeast, but less homologous with other plant peroxidases. Amino acid residues at the active site of yeast cytochromec peroxidase are conserved in the amino acid sequence ofArabidopsis AP. The poly(dG-dT) sequence, which is a potential Z-DNA-forming sequence, was found in the 3 untranslated region of the cDNA.     

Immunochemical evidence for the presence of 5mC, 6mA and 7mG in human, Drosophila and mealybug DNA

We have reported that production and characterization of antibodies highly specific to 5-methyl-cytosine (5mC) and the development of a sensitive immunochemical method for the detection of 5mC in DNA [FEBS Lett. (1982) 150, 469]. Extension of this method to two other modified bases, 6-methyladenine (6mA) and 7-methylguanine (7mG), is reported here. By use of this immunochemical approach, we are able to detect 5mC, 6mA and 7mG in human and Drosophila DNA and confirm their presence in the DNA of two mealybug species.