Characterization of RNA primers synthesized by the human breast cancer cell DNA synthesome

We previously reported on the purification and characterization of a functional multi‐protein DNA replication complex (the DNA synthesome) from human cells and tissues. The synthesome is fully competent to carry‐out all phases of the DNA replication process in vitro. In this study, DNA primase, a component of the synthesome, is examined to determine its activity and processivity in the in vitro synthesis and extension of RNA primers. Our results show that primase activity in the P4 fraction of the synthesome is 30‐fold higher than that of crude cell extracts. The synthesome synthesizes RNA primers that are 7–10 ribonucleotides long and DNA primers that are 20–40 deoxyribonucleotides long using a poly(dT) template of exogenous single‐stranded DNA. The synthesome‐catalyzed RNA primers can be elongated by E. coli DNA polymerase I to form the complementary DNA strands on the poly(dT) template. In addition, the synthesome also supports the synthesis of native RNA primers in vitro using an endogenous supercoiled double‐stranded DNA template. Gel analysis demonstrates that native RNA primers are oligoribonucleotides of 10–20 nt in length and the primers are covalently link to DNA to form RNA‐primed nascent DNA of 100–200 nt. Our study reveals that the synthesome model is capable of priming and continuing DNA replication. The ability of the synthesome to synthesize and extend RNA primers in vitro elucidates the organizational and functional properties of the synthesome as a potentially useful replication apparatus to study the function of primase and the interaction of primase with other replication proteins. J. Cell. Biochem. 106: 798–811, 2009. © 2009 Wiley‐Liss, Inc.     

Four new diterpenoid alkaloids from Delphiniumpentagynum

Four new diterpenoid alkaloids, dihydrogadesine, 14-acetyldihydrogadesine, pentagynine and dihydropentagynine, were isolated and identified in Delphinium pentagynum.     

Identifying non-sperm particles during flow cytometric physiological assessment: a simple approach

Flow cytometry is now being used more frequently to determine sperm functional characteristics during semen assessment for artificial insemination. With this methodology, viable and potentially functional cells are detected as unstained events differentiated from non-sperm events through their light-scattering characteristics. However, it can be shown mathematically that identification of sperm on the basis of light scatter leads to significant overestimation of unstained viable cells and underestimation of responding cells in tests of sperm function (subpopulations expressing different fluorescence patterns). We have developed a simple and cost-efficient flow cytometric approach for identifying non-sperm particles that can be carried out in parallel with functional assessments. Our method is based on the sperm's osmotic intolerance. Diluted in water, lethal osmotic shock causes major damage to the cell membranes, and all sperm will stain with propidium iodide (PI). Particulate material which is not PI-positive can then be quantitatively evaluated by FACS analysis and the results substituted in mathematical equations to provide true values for sperm counts and subpopulations. In practical tests, the percentage of non-sperm particles determined by this technique was closely comparable to the figure obtained either by SYBR14^®/PI staining or by PI/CFDA staining. As well as being valuable with respect to tests of sperm function, the procedure is also suitable for obtaining accurate sperm counts during routine semen evaluation.     

Comparison of the structures of the native form of rat liver 5S rRNA and yeast tRNAphe: small-angle and wide-angle X-ray scattering study

The small-angle and wide-angle X-ray scattering of tRNA^phe (yeast) and ribosomal 5S RNA (rat liver) in solution have been analysed and compared. tRNA^phe in solution is folded into a compact L-shaped structure similar to its structure in crystals. The geometry of the secondary structure of the double helical regions is also equivalent to the A-form in the crystalline state. Despite differences between the molar mosses of 5S rRNA (40 000 g mol^?1) and tRNA^phe (25 000 g mol^?1), and the fact that the 5S rRNA molecule is more anisometric than the tRNA^phe molecule, there are many structural similarities. The geometrical parameters of the secondary structure of double helical regions in both RNA molecules are almost identical; the mean rise per base pair is about 0.253–0.28 nm and the mean turn angle is about 32.5–33.5. Identical cross-sectional radii of gyration, R_sq,1 ≈ 1.16 nm and R_sq,2 = 0.92 nm, identical molar mass per unit length, , and a mean thickness of the molecules D ≈ 1.65 nm suggest a similar, nearly coplanar organization of isolated, double helical arms. Furthermore, there are compact regions in the central parts of both molecules, which are the sites of tertiary interactions in the tRNA^phe molecule and are a potential site of tertiary interactions in the SS rRNA molecule for stabilization of the complicated L-shape of the two molecules. Both molecules have a pseudo-twofold axis,w hich may play a role in recognition for binding of specific proteins.     

Alkaloids from the leaves of Strychnos wallichiana

The leaves of Strychnos wallichiana Steud. ex. DC. from Bangladesh contain icajine and novacine as their major alkaloids. Smaller amounts of strychnine, brucine, pseudostrychnine, pseudobrucine, N-methyl-sec.-pseudo-β-colubrine, 14-hydroxyicajine, strychnine N-oxide, and brucine N-oxide are also present. The new bases 14 hydroxynovacine and icajine N-oxide have been isolated.     

Use of anin situ labeling technique for the determination of seasonal14C distribution in Ponderosa pine

A¹⁴C labeling apparatus was developed to permit the labeling of four-year-old Ponderosa pine with¹⁴CO₂ in the field. The labeling system is a completely closed canopy system with¹⁴CO₂ monitored by a GM tube ratemeter apparatus. The level of¹⁴CO₂ corresponding to ambient levels is monitored by a microloggercomputer which controls a¹⁴CO₂ generating system. The generated¹⁴CO₂ is mixed in the canopy by circulating the atmosphere with 12V diaphram pumps. The portable system requires little operator attention. At approximately monthly intervals over a one-year period two four-year-old Ponderosa pine trees were labeled for three to five days using this labeling apparatus. After an assimilate distribution period, one tree was excavated and analyzed for¹⁴C distribution. During late spring and early summer most of the carbon assimilated (>60%) was found in the active growing tips and new needles, with little being allocated to the roots (<10%) or woody material (<20%). During mid to late fall there was an increase in root labeling along with an increase in carbon going to woody material. Over the winter period, most of the fixed carbon (65%) resided in the older leaves. The early spring labeling period showed another pulse of root labeling along with some labeling of woody tissues.     

Protein Turnover and Growth of the Rat Brain from the Foetus to Old Age

Growth of the rat brain was studied between 16 days of foetal life and old age (105 weeks). Developmental changes in cerebral RNA, DNA, and protein contents are described. The age-related decline in brain growth rates correlates with progressive decreases in the fractional rates of protein synthesis (from 58 to 6.8% per day) and breakdown (from 36.4 to 4.1% per day).     

Mediation of high-temperature injury by roots and shoots during reproductive growth of wheat

Abstract Previous studies suggest that high temperature stress on wheat (Triticum aestivum L.) involves root processes and acceleration of monocarpic senescence. Physiological changes in wheat roots and shoots were investigated to elucidate their relationship to injury from elevated temperatures after anthesis. Plants were grown under uniform conditions until 10 d after anthesis, when shoot/root regimes of 25°C/25°C, 25°C/35°C, 35°C/25°C and 35°C/35°C were imposed. Growth and senescence of shoots and grain were influenced more by root temperatures than by shoot temperatures. High root temperatures increased activities of protease and RNasc enzymes, and loss of chlorophyll, protein and RNA from shoots, whereas low root temperatures had opposite effects. High root temperatures appeared to induce shoot senescence directly. High shoot temperatures probably disrupted root processes, including export of cytokinins, and induced high leaf protease activity, senescence and cessation of grain development. The authors concluded that responses of wheat to high temperatures, whether of roots or shoots, are manifested as acceleration of senescence and may be mediated by roots during grain development.