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.     

The alpha-amylase gene in Drosophila melanogaster: nucleotide sequence, gene structure and expression motifs.

We present the complete nucleotide sequence of a Drosophila alpha-amylase gene and its flanking regions, as determined by cDNA and genomic sequence analysis. This gene, unlike its mammalian counterparts, contains no introns. Nevertheless the insect and mammalian genes share extensive nucleotide similarity and the insect protein contains the four amino acid sequence blocks common to all alpha-amylases. In Drosophila melanogaster, there are two closely-linked copies of the alpha-amylase gene and they are divergently transcribed. In the 5'-regions of the two gene-copies we find high sequence divergence, yet the typical eukaryotic gene expression motifs have been maintained. The 5'-terminus of the alpha-amylase mRNA, as determined by primer extension analysis, maps to a characteristic Drosophila sequence motif. Additional conserved elements upstream of both genes may also be involved in amylase gene expression which is known to be under complex controls that include glucose repression.     

Study of coronary risk factors related to physical activity in 15 171 men.

Physical activity, both at work and during leisure, was assessed in 15 171 men aged 25-74 years. Heavy leisure activity was associated with lower mean serum cholesterol levels and blood pressure. In men under 60 years the same negative association was also noted between leisure activity and relative weight and cigarette smoking. Different degrees of physical activity at work were not associated with any differences in these risk factors, nor did the level of exercise at work seem to influence the negative association between leisure activity and risk factors. Heavy leisure activity in young and middle-aged men is associated with lower levels of certain coronary risk factors and, therefore, a lower risk of coronary heart disease. The often-reported reduction in coronary morbidity and mortality with physical exercise may not be the direct effect of the exercise itself.     

Does stopping smoking delay onset of angina after infarction?

This study was designed to determine the relation between stopping smoking and angina after infarction in survivors of an acute coronary attack. The study population comprised 408 men aged under 60 who survived a first attack of unstable angina or myocardial infarction by 28 days and were smoking cigarettes at the time of their attack. These patients were followed up for an average of nine years. Three hundred and eighty four were alive at the one year follow up examination, when the presence or absence of angina together with habits of smoking were recorded. The prevalence of angina at one year was 19.5% in the 241 who had stopped smoking cigarettes compared with 32.2% in those who had continued (p less than 0.01). Six years later, however, the prevalence of angina after infarction was the same in the two groups. It is concluded that the onset of angina after infarction can be delayed by stopping smoking cigarettes but that this effect is not maintained in the long term.     

Initial Characterization of Micafungin Pulmonary Delivery via Two Different Nebulizers and Multivariate Data Analysis of Aerosol Mass Distribution Profiles

Pharmaceutical aerosols have been targeted to the lungs for the treatment of asthma and pulmonary infectious diseases successfully. Micafungin (Astellas Pharma US, Deerfield, IL, USA) has been shown to be an effective antifungal agent when administrated intravenously. Pulmonary delivery of micafungin has not previously been reported. In the present pilot study, we characterize the performance of two nebulizers and their potential for delivering micafungin to the lungs as well as the use of multivariate data analysis for mass distribution profile comparison. The concentration of micafungin sodium increased by 21% when delivered by the Acorn II nebulizer and by 20% when delivered by the LC Plus nebulizer, respectively, from the first to the second sampling period. The Acorn II nebulizer delivered a fine particle fraction FPF_5.8 (%<5.8 μm) of 92.5 ± 0.8 and FPF_3.3 (%<3.3 μm) of 82.3 ± 2.1 during the first sampling period. For the LC Plus nebulizer, FPF_5.8 was 92.3 ± 0.1 and FPF_3.3 was 67.0 ± 0.7 during the first sampling period. The mass median aerodynamic diameter (MMAD) increased from 1.67 ± 0.05 to 1.77 ± 0.04 μm (Acorn II nebulizer) and from 2.09 ± 0.01 to 2.20 ± 0.01 μm (Pari LC Plus nebulizer) from the first to the second sampling periods. These changes in MMAD were statistically significant by paired t test. Multivariate data analysis showed that this could be explained systematically by greater drug deposition on stages with larger cutoff sizes and reduced drug deposition on stages with smaller cutoff sizes rather than multimodal deposition or other anomalies in size distribution.     

Behaviour of DNA,protein and acid hydrolases in response to thyroxine in isolated tail tips ofXenopus-larvae

Summary Analysis of factors influencing survival of tail tips ofXenopus larvae in vitro has shown that prevention of infection by antibiotic pretreatment of donor tadpoles and amputated tips is most critical. In tail tips exceeding not more than 1/3 of the body length, maintenance of N-content and regenerative capacity are superior in Niu-Twitty and Steinberg's saline than in Holtfreter's solution, all media being supplemented by 0.04 % sulfathiazole. Addition of nutrient ingredients to saline (glucose, Parker's medium 199, serum protein) does not improve viability of tail explants.In isolated tail tips 2.5×10^–7M L-thyroxine (T₄) induces involution in vitro, resulting in losses of about 85% in DNA and 70% of protein respectively after 12 days of treatment. A significant decrease in DNA occurs after three days, and for protein after 6 days of hormone treatment, when tail fins are almost fully resorbed.Statistical analysis of the regression curves for decrease in DNA and protein, produced by different concentrations of T₄, indicates the presence of an upper threshold (2.5×10^–7M) and a lower threshold (2.5×10^–8M) of sensitivity to hormone. Intermediate concentrations affect the latency time required for onset of DNA and protein loss, lengthening it at lower concentration.In tail tips exposed to 2.5×10^–7M T₄ a concurrent rise in activity of cathepsins, DNase and acid phosphatase has been demonstrated. The specific activities of these acid hydrolases are significantly higher in T₄-treated tails after four days of hormone administration, this response proceeding detectable loss in protein by two days. Extent and duration in the rise of activity are characteristic for each enzyme, the increase in both specific and total activities being highest for cathepsins, intermediate for DNase and lowest for acid phosphatase.

---

Zusammenfassung Die Untersuchung der für das Überleben von isolierten Schwanzspitzen vonXenopus-Larven wesentlichen Kulturbedingungen hat ergeben, daß die Verhütung von Infektionen durch Vorbehandlung der Kaulquappen und der amputierten Schwanzspitzen für den Erfolg entscheidend ist. Schwanzspitzen, deren Länge 1/3 der Körperlänge der Kaulquappen nicht überschreitet, zeigen einen geringeren Verlust an N-haltigen Stoffen und ein besseres Regenerationsvermögen in Niu-Twitty- und Steinberglösung gegenüber der Lösung nach Holtfreter, obwohl alle Medien 0,04% Sulfathiazol enthalten. Zusatz von Nährstoffen (Glucose, Parker's Medium 199, Serumprotein) ergeben keine Verbesserung der Überlebensfähigkeit.In isolierten Schwanzspitzen bewirken 2,5×10^–7M L-Thyroxin (T₄) die Rückbildung in vitro, wobei im Verlaufe von 12 Tagen der DNS-Gehalt um 85% und derjenige an Protein um 70% sinkt. Der DNS-Verlust ist bereits nach 3 Tagen signifikant, während eine signifikante Abnahme an Protein erst nach 6 Tagen Hormonbehandlung nachgewiesen werden kann, wenn die Flossensäume bereits resorbiert sind.Aufgrund der statistischen Analyse der für den DNS- und Proteinverlust in Abhängigkeit von verschiedenen T₄-Konzentrationen ermittelten Regressionsgeraden ergeben sich folgende Aussagen: 2,5×10^–7 M repräsentiert die obere und 2,5×10^–8 M T₄ die untere Empfindlichkeitsschwelle. Innerhalb dieses Bereiches zeigt die Latenzzeit für den Beginn der DNS -und Proteinabnahme, nicht aber die Geschwindigkeit des Involutionsprozesses eine Konzentrationsabhängigkeit, indem die Latenzzeit mit fallender T₄-Konzentration zunimmt.Unter der Einwirkung von 2,5×10^–7 M T₄ nimmt in den Schwanzspitzen die Aktivität von Kathepsinen, DNase und saurer Phosphatase gleichzeitig zu. Die spezifische Aktivität dieser sauren Hydrolasen ist bereits nach 4 Tagen Hormonbehandlung signifikant erhöht, während ein signifikanter Proteinverlust erst am 6. Tag der Behandlung nachgewiesen werden kann. Ausmaß und Dauer der Aktivitätszunahme sind für jedes Enzym charakteristisch; die Zunahme der spezifischen und Gesamtaktivität ist am größten bei den Kathepsinen, etwas geringer bei der DNase und am niedrigsten bei der sauren Phosphatase.

---

---

These studies were carried out within the scope of a project supported by the Schweizerischer Nationalfonds zur Förderung der wissenschaftlichen Forschung under the direction of Prof. B. Weber.

---

The author would like to acknowledge the advice and encouragement of Prof. R. Weber in all phases of these studies, and the assistance of Prof. S. Rosin with the statistical techniques. Further, the technical assistance of Miss A. Rohner, Miss B. Fahrer, Mr. J. Zbären, and Mr. T. Wyler is gratefully acknowledged.