Nature of recombination involved in excision and rearrangement of human repetitive DNA

An alphoid-like human repetitive DNA of the Sau3A family is present extrachromosomally and in the chromosomes. In the chromosomes, the DNA is located on chromosome 11 but related sequences are present in chromosome 17. We characterized the nature of the recombination involved in the excision of the extrachromosomal DNA from chromosome 11. The results show that the recombination occurs both between the homologous subunits and between the heterologous subunits with only a 70 to 80% sequence homology among them, suggesting that a DNA structure other than a sequence homology mediates the recombination process. The same type of recombination is responsible for the rearrangement of the related sequences in chromosome 17.     

Conservation and Periodicity of DNA Bend Sites in Eukaryotic Genomes

DNA bend sites appear every 680 bp on average in the human -and ß-globin gene regions. Although most of theirmolecular nature has not been unraveled, a potential bend coresequence A₂N₈A₂N₈A₂ (A/A/A) and its complementary T₂N₈T₂N₈T₂(T/T/T) appeared preferentially either in or very close to mostof the bend sites, whereas other combinations of A₂ and T₂ dinucleotides,A/T/T + A/A/T, T/T/A + T/A/A and A/T/A + T/A/T, did not. Thedistances between any two of the core sequences in the entireß-globin locus showed a strong bias to a length of701–800 bp and multiples thereof, suggesting that thereis periodicity throughout the locus. This bias was not foundfor other combinations of A₂ and T₂. Again, this periodicitywas identified in many eukaryotic genes, whereas the tendencywas absent in mRNAs and prokaryotic as well as viral genomes.     

Locations of local anesthetic dibucaine in model membranes and the interaction between dibucaine and a Na+ channel inactivation gate peptide as studied by 2H- and 1H-NMR spectroscopies.

To study the molecular mechanisms of local anesthesia, locations of local anesthetic dibucaine in model membranes and the interactions of dibucaine with a Na+ channel inactivation gate peptide have been studied by 2H- and 1H-NMR spectroscopies. The 2H-NMR spectra of dibucaine-d9 and dibucaine-d1, which are deuterated at the butoxy group and at the 3 position in its quinoline ring, respectively, have been observed in multilamellar dispersions of the lipid mixture composed of phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine. 2H-NMR spectra of deuterated palmitic acids incorporated, as a probe, into the lipid mixture containing cholesterol have also been observed. An order parameter, SCD, for each carbon segment was calculated from the observed quadrupole splittings. Combining these results, we concluded that first, the butoxy group of dibucaine is penetrating between the acyl chains of lipids in the model membranes, and second, the quinoline ring of dibucaine is located at the polar region of lipids but not at the hydrophobic acyl chain moiety. These results mean that dibucaine is situated in a favorable position that permits it to interact with a cluster of hydrophobic amino acids (Ile-Phe-Met) within the intracellular linker between domains III and IV of Na+ channel protein, which functions as an inactivation gate. To confirm whether the dibucaine molecule at the surface region of lipids can really interact with the hydrophobic amino acids, we synthesized a model peptide that includes the hydrophobic amino acids (Ac-GGQDIFMTEEQK-OH, MP-1), the amino acid sequence of which corresponds to the linker part of rat brain type IIA Na+ channel, and the one in which Phe has been substituted by Gln (MP-2), and measured 1H-NMR spectra in both phosphate buffer and phosphatidylserine liposomes. It was found that the quinoline ring of dibucaine can interact with the aromatic ring of Phe by stacking of the rings; moreover, the interaction can be reinforced by the presence of lipids. In conclusion, we wish to propose that local anesthesia originates from the pi-stacking interaction between aromatic rings of an anesthetic molecule located at the polar headgroup region of the so-called boundary lipids and of the Phe in the intracellular linker between domains III and IV of the Na+ channel protein, prolonging the inactivated state and consequently making it impossible to proceed to the resting state.     

Protection of DNA sequences by triplex-bridge formation.

We have demonstrated that the DNA sequence between two triplex-forming polypurine.polypyrimidine (Pu.Py) tracts was protected from DNA modifying enzymes upon formation of triplex DNA structures with an oligodeoxyribonucleotide in which two triplex-forming Pu or Py tracts were placed at the termini (triplex-bridge formation). In model experiments, when two triplex structures were formed between double-stranded DNA with the sequence (AG)17-(N)18-(T)34, and an oligodeoxyribonucleotide, (T)34-(N)18-(GA)17, not only the Pu.Py tracts but also the 18 bp non-Pu.Py sequence in the duplex DNA between the tracts was protected from restriction enzymes, HpaII methylase and DNase I. This protection occurred only when both of the Pu.Py tracts were involved as triplexes. The length of the tracts could be as short as 21 bp, while the difference in length between the non-Pu.Py sequences on the duplex and the oligodeoxyribonucleotide should be within 10 nucleotides. The efficiency of protection was enhanced in the presence of a cationic detergent, cetyltrimethylammonium bromide, during triplex formation. Protection was also observed with another type of the triplex bridge formed between (G)34 and (T)34 tracts with an oligodeoxyribonucleotide, (T)34-(N)20-(G)34. These findings suggest that the protection of specific DNA sequences from enzymes by triplex-bridge formation can be applied to any DNA sequence by placing it between two triplex-forming sequences.     

西双版纳尚勇自然保护区野生印支虎及其三种主要有蹄类猎物种群现状调查

中国野生印支虎及其猎物种群状况的野外实地研究一直处于空白。本研究使用足迹鉴别法、粪堆计数法,首次对西双版纳尚勇自然保护区野生印支虎种群数量现状及该区域内的虎猎物种群状况进行了调查研究。结果显示:2004 ～ 2009 年间,确认西双版纳保护区存在3 只成年印支虎个体(2 雌1 雄),西双版纳尚勇保护区拥有比较丰富的有蹄类种群,其中虎的主要猎物:水鹿平均密度为7.63 (7.40 ～ 9.23)只/ km² ;赤麂平均密度为17. 39 (11.33 ～24.94)只/ km² ,野猪平均密度为10.26 (7.69 ～ 14.51) 只/ km² ,该区域虎猎物生物量为1 715. 74 kg/ km² 。本研究还探讨了该区域印支虎种群的保护前景以及中国境内开展虎种群调查的适用办法等。     

A DNA vaccine against tuberculosis based on the 65 kDa heat-shock protein differentially activates human macrophages and dendritic cells

Background

Application of plasmid DNA for immunization of food-producing animals established new standards of food safety. The addition of foreign products e.g. pDNA into the food chain should be carefully examined to ensure that neither livestock animals nor consumers develop unpredicted or undesirable side-effects.

Methods

A quantitative real-time PCR (QRTPCR) methodology was developed to study the biodistribution and persistence of plasmid DNA vaccine pDNAX (pVAX-Hsp60 TM814) in mice and beef cattle. The linear quantification range and the sensitivity of the method was found to be 10 – 10⁹ copies per reaction (500 ng/gDNA) and 3 copies per reaction, respectively.

Results

Persistence of pDNAX in mice muscle tissue was restricted to injection site and the amount of pDNAX showed delivery formulation dependent (naked pDNA, electroporation, cationic liposome complexes) and mouse age-dependent clearance form injection site but pDNAX was still detectable even after 365 days. The QRTPCR analysis of various muscle tissue samples of vaccinated beef bulls performed 242–292 days after the last revaccination proved that residual pDNAX was found only in the injection site. The highest plasmid levels (up to 290 copies per reaction) were detected in the pDNAX:CDAN/DOPE group similarly to mice model. No pDNA was detected in the samples from distant muscles and draining lymph nodes.

Conclusion

Quantitative real-time PCR (QRTPCR) assay was developed to assess the residual pDNA vaccine pVAX-Hsp60 TM814 in mice and beef cattle. In beef cattle, ultra low residual level of pDNA vaccine was only found at the injection site. According to rough estimation, consumption of muscles from the injection site represents almost an undetectable intake of pDNA (400 fg/g muscle tissue) for consumers. Residual plasmid in native state will hardly be found at measurable level following further meat processing. This study brings supportive data for animal and food safety and hence for further approval of pDNA vaccine field trials.     

DNA bend sites in the human beta-globin locus: evidence for a basic and universal structural component of genomic DNA.

Here we summarize the DNA bend sites in a 66-kb region of the human beta-globin locus. A total of 98 sites were mapped by circular permutation assay along the locus with an average interval of 679.2 +/- 229.6 bp between them. The distribution of the bend sites indicated that although the most frequent distance was about 650-700 bp, there appeared to be preferences at 300-400, 500-550, 800-850, 1,000-1,050, and 1,150-1,200 bp, indicating that these distances are multimers of a 170-bp basic unit. DNA bend sites in the globin-encoding regions indicated that most of their locations relative to the cap sites were conserved during evolution. Insertion of Alu and L1 sequences that occurred at various times and changed the distances of the sites was corrected for the epsilon-, psi beta-, and delta-globin genes. The only exception of the conservation was observed at the duplication junctions of the two gamma-globin genes, which occurred 25-35 MYA. Among the 75 A/A/A (A2N8A2N8A2) sequences found in the 51 bend sites, 59 sequences from 47 sites showed bending profiles by oligonucleotide-based assay. All of these sites were included in the sites predicted by computer analysis based on the distribution of AA and TT dinucleotides. These lines of evidence suggest that these DNA bend sites are one of the basic structural components universally present in genomic DNA.     

Altered expression of neprilysin family members in the pituitary gland of sleep-disturbed rats, an animal model of severe fatigue

Alterations of the expression of some peptidases in the pituitary gland of a fatigued rat model were identified. Rats were kept in a cage filled with water to a height of 1.5 cm to disturb deep sleep. After 24-h sleep disturbance, expression of neutral endopeptidase 24.11 (neprilysin) mRNA was increased in the intermediate lobe of the pituitary gland, whereas the mRNA expression of another family member, damage-induced neuronal endopeptidase, which is normally expressed in a subgroup of anterior pituitary cells, was significantly suppressed. These alterations were demonstrated by RT-PCR, northern blotting and in situ hybridization. Other family members, such as neprilysin 2 and endothelin converting enzyme-1, did not show any change in mRNA expression. An increase of neprilysin mRNA expression was not seen in any other tissues of the sleep-disturbed rats. The enzymatic activity of neprilysin was also increased in the pituitary. The augmentation of neprilysin expression and activity was prolonged as long as the sleep disturbance continued (up to 5 days), and returned to the basal level when rats were allowed to sleep freely. These results suggest that peptide processing and degradation in the pituitary may be an influential factor in fatigued states such as sleep disturbance.