Elongation of palindromic repetitive DNA by DNA polymerase from hyperthermophilic archaea: a mechanism of DNA elongation and diversification

DNA polymerase from hyperthermophilic bacteria can elongate tandem repetitive oligoDNA with a complete or incomplete palindromic sequence under isothermal conditions by "hairpin elongation". However, the product of the reaction has not yet been sufficiently characterized. Here, I demonstrate that when palindromic repetitive oligoDNA, e.g., (5'AGATATCT3')(6), was added as a "seed" to the DNA synthesis reaction catalyzed by DNA polymerase from the archaea Thermococcus litoralis (Vent polymerase) at 74 degrees C, the product was (5'AGATATCT3')(n). The product itself was palindromic and repetitive, and its motif (unit) sequence was exactly the same as that of the seed oligoDNA. On the other hand, when a pseudopalindrome, which contains a palindrome-breaking nucleotide (underlined), was present in seed oligoDNA, e.g., (5'GATTC3')(6), the product was (5'GATATC3')(n), which had a different motif sequence from that of the seed oligoDNA. When a pseudopalindrome (5'AGATATCA3')(6) was added to the reaction, the products were 5'TATCA . (AGATATCA)(3) . AGATATCT . (TGATATCT)(5) . TGATA3', etc. When 5'AGATATCA . (AGATATCT3')(5) was added, products were 5'TATCT . (AGATATCT)(2).TGATATCT . AGATATCT . AGATATCA . AGATATCT . AGA3', etc., demonstrating the generation of many "mutations" in the product DNA. I conclude that a tandem repetitive sequence is faithfully elongated (amplified) by hyperthermophilic DNA polymerase if it is completely palindromic, but is elongated with many errors if it is incompletely palindromic (pseudopalindromic) or mixed with a pseudopalindrome. The results suggest a protein-catalyzed elongation/diversification mechanism of short repetitive DNAs on the early earth.     

Coupling of glucose response element from L-type pyruvate kinase and G6Pase promoter enhances glucose responsive activity in hepatoma cells

Type 1 diabetes results from the autoimmune destruction of pancreatic β-cells, which leads to severe insulin deficiency. Insulin gene therapy provides an attractive approach to cure diabetes. The critical factor for insulin gene therapy in surrogate cells is to select an appropriate site for insulin expression and a tissue-specific promoter that is responsive to both physiological glucose and insulin concentrations. A novel chimeric promoter, (GIRE)n-G6Pase, consisting of a 1.6 kb glucose 6-phosphatase (G6Pase) promoter and a segment of the regulatory element derived from the L-type pyruvate kinase (L-PK) promoter, was designed to provide strong and tight control of insulin expression in liver. One or three copies of GIRE were linked to the G6Pase promoter, which showed a stronger promoter activity than the G6Pase promoter alone. The chimeric promoter was inhibited by insulin in a dosage-dependent manner and activated by glucose, two features essential for glucose metabolism. The promoter activity is conserved between species and highly specific for liver cells. The construction of a chimeric promoter with stronger and more sensitive responsive activity to glucose and insulin in liver cells could further advance studies in insulin gene therapy. Mr. James Chong was a senior student in the Department of Cell and Molecular Biology, Tulane University. His independent study project was partially overlapped with this study.     

An open issue: the inner mitochondrial membrane (IMM) as a free boundary problem

The inner mitochondrial membrane (IMM) is structured in cristae, which contributes to the best functioning of ions and adenylates exchange between the matrix and the intermembrane space. The central hypothesis of this paper is that the cristae structure favours a minimal mean free path of adenylates between translocation sites (translocase/ANT sites) and metabolic sites (ATPase sites). We propose a mathematical model and then give simulations. Based on simple hypotheses about cristae growth, they show that we can account for the major features of the IMM organization and functioning by minimizing the mean interdistance between ADP/ATP translocation and transformation sites.     

Knockout of the gene (<Emphasis Type="Italic">ste15</Emphasis>) encoding a glycosyltransferase and its function in biosynthesis of exopolysaccharide in <Emphasis Type="Italic">Streptomyces</Emphasis> sp. 139

Streptomyces sp. 139 produces a novel exopolysaccharide (EPS) designated Ebosin which has antagonistic activity for IL-1R in vitro and remarkable anti-rheumatic arthritis activity in vivo. We previously identified a ste (Streptomyces eps) gene cluster consisting of 27 ORFs responsible for Ebosin biosynthesis. The gene product of ste15 shows high homology to known glycosyltransferases (GTFs). To elucidate its function in Ebosin biosynthesis, the ste15 gene was knocked out with a double crossover via homologous recombination. Our analysis of monosaccharide composition for EPS-m produced by the mutant strain Streptomyces sp. 139 (ste15 ⁻) showed that glucose was significantly diminished compared to its natural counterpart Ebosin. This derivative of Ebosin lost the antagonistic activity for IL-1R in vitro and its molecular mass was smaller than Ebosin. These results have demonstrated that the ste15 gene codes for a GTF for glucose, which is functionally involved in Ebosin biosynthesis.     

Population genetic structure of Vitex negundo (Verbenaceae) in Three-Gorge Area of the Yangtze River: The riverine barrier to seed dispersal in plants

Previous studies have demonstrated that large rivers can influence inter- and intra-specific gene flow for many animals. The effects of large rivers on the genetics of plant populations have focused on either hydrochoric impacts of water current on gene flow or genetic differentiation among populations from different watersheds. Few studies have explicitly tested the barrier effects on plant gene flow across banks of large rivers, especially their relative effects on pollen and seed dispersals. The Yangtze River (Changjiang River), one of the major rivers of the world, provides an excellent model to evaluate the impacts of rivers on gene flow in plants. Using RAPD (random amplified polymorphic DNA) and cpDNA (chloroplast DNA) markers, we investigated the genetic structure of 10 populations of Vitex negundo in two regions of Three-Gorge Area along the Yangtze River. Each region contained two populations on the north bank, two on the south bank and one island population along the river. The analyses indicated low RAPD between banks, and similar or a little higher differentiation between populations within the same bank. In contrast, a large proportion of chloroplast polymorphism was ascribed to among-bank variation but much lower cpDNA differentiation was among populations within the same bank. These results indicate that the Yangtze River represents a general barrier to the dispersal of seeds but not to the movement of pollen in V. negundo. The cpDNA genetic distances or differentiations between the island populations and those on either bank of the river are intermediate to those between the banks across the river, implying that the islands in the Yangtze River may serve as a stepping-stone for seed dispersal. Our results suggest that large rivers may serve as a general barrier, not only for the movement of animals, but also for the dispersal of plants, which should be of great significance for the conservation of biodiversity around the rivers.     

Direct quantification of gene expression using capillary electrophoresis with laser-induced fluorescence

Quantification of gene expression provides valuable information regarding the response of cells or tissue to stimuli and often is accomplished by monitoring the level of messenger RNA (mRNA) being transcribed for a particular protein. Although numerous methods are commonly used to monitor gene expression, including Northern blotting, real-time polymerase chain reaction, and RNase protection assay, each method has its own drawbacks and limitations. Capillary electrophoresis with laser-induced fluorescence (CE-LIF) can reduce protocol time, eliminate the need for radioactivity, and provide superior sensitivity and dynamic range for quantification of RNA. In addition, CE-LIF can be used to directly determine the amount of an RNA species present, something that is difficult and not normally accomplished using current methods. Gene expression is detected using a fluorescently labeled riboprobe specific for a given RNA species. This direct approach was validated by analyzing levels of 28S RNA and also used to determine the amount of discoidin domain receptor 2 mRNA in cardiac tissue.     

Crystal structure of an aerobic FMN-dependent azoreductase (AzoA) from Enterococcus faecalis

The initial critical step of reduction of the azo bond during the metabolism of azo dyes is catalyzed by a group of NAD(P)H dependant enzymes called azoreductases. Although several azoreductases have been identified from microorganisms and partially characterized, very little is known about the structural basis for substrate specificity and the nature of catalysis. Enterococcus faecalis azoreductase A (AzoA) is a highly active azoreductase with a broad spectrum of substrate specificity and is capable of degrading a wide variety of azo dyes. Here, we report the crystal structure of the AzoA from E. faecalis determined at 2.07 A resolution with bound FMN ligand. Phases were obtained by single wavelength anomalous scattering of selenomethionine labeled protein crystals. The asymmetric unit consisted of two dimers with one FMN molecule bound to each monomer. The AzoA monomer takes a typical NAD(P)-binding Rossmann fold with a highly conserved FMN binding pocket. A salt bridge between Arg18 and Asp184 restricts the size of the flavin binding pocket such that only FMN can bind. A putative NADH binding site could be identified and a plausible mechanism for substrate reduction is proposed. Expression studies revealed azoA gene to be expressed constitutively in E. faecalis.     

VEGF promotes tumorigenesis and angiogenesis of human glioblastoma stem cells

There is increasing evidence for the presence of cancer stem cells (CSCs) in malignant brain tumors, and these CSCs may play a pivotal role in tumor initiation, growth, and recurrence. Vascular endothelial growth factor (VEGF) promotes the proliferation of vascular endothelial cells (VECs) and the neurogenesis of neural stem cells. Using CSCs derived from human glioblastomas and a retrovirus expressing VEGF, we examined the effects of VEGF on the properties of CSCs in vitro and in vivo. Although VEGF did not affect the property of CSCs in vitro, the injection of mouse brains with VEGF-expressing CSCs led to the massive expansion of vascular-rich GBM, tumor-associated hemorrhage, and high morbidity, suggesting that VEGF promoted tumorigenesis via angiogenesis. These results revealed that VEGF induced the proliferation of VEC in the vascular-rich tumor environment, the so-called stem cell niche.