Single-molecule studies of the effect of template tension on T7 DNA polymerase activity

T7 DNA polymerase catalyses DNA replication in vitro at rates of more than 100 bases per second and has a 3'-->5' exonuclease (nucleotide removing) activity at a separate active site. This enzyme possesses a 'right hand' shape which is common to most polymerases with fingers, palm and thumb domains. The rate-limiting step for replication is thought to involve a conformational change between an 'open fingers' state in which the active site samples nucleotides, and a 'closed' state in which nucleotide incorporation occurs. DNA polymerase must function as a molecular motor converting chemical energy into mechanical force as it moves over the template. Here we show, using a single-molecule assay based on the differential elasticity of single-stranded and double-stranded DNA, that mechanical force is generated during the rate-limiting step and that the motor can work against a maximum template tension of approximately 34 pN. Estimates of the mechanical and entropic work done by the enzyme show that T7 DNA polymerase organizes two template bases in the polymerization site during each catalytic cycle. We also find a force-induced 100-fold increase in exonucleolysis above 40 pN.     

Cloning and expression of the essential gene for poly(A) polymerase from S. cerevisiae.

Poly(A) polymerase is essential for the maturation of messenger RNA, adding tracts of adenosine residues to the 3' end of precursor RNA generated by endonucleolytic cleavage. This mechanism of mRNA 3' processing seems to be similar in yeast and in higher eucaryotes, although there are differences in the recognition signals in the pre-mRNA. Here we describe the cloning of the gene for yeast poly(A) polymerase. The enzyme is encoded by a single and essential gene located near the centromere on the left arm of chromosome 11. Poly(A) polymerase purified from recombinant Escherichia coli has the same physical and biochemical properties as the yeast enzyme. The yeast poly(A) polymerase shares features of sequence with its mammalian homologue.     

Radioimmunoassay of an invertebrate peptide hormone — the crustacean neurosecretory hyperglycemic hormone

Summary Antibodies against hyperglycemic hormone (CHH) ofCarcinus were raised in rabbits by injection of extract from sinus glands which contain high concentrations of CHH. The antiserum neutralizes the biological activity of CHH and binds¹²⁵J-CHH. A RIA for CHH was established and was used to measure the hormone content of sinus glands.Dedicated to Prof. M. Gersch, Jena, on the occasion of his 70th birthday.This study was supported by the Deutsche Forschungsgemeinschaft (Ke 206/2).We thank S. Grabig. E. schmid and B. Reichwein for expert technical assistance, and Dr D. Breitig and G. Kegel for critical discussions.     

Retrovirus transfer of a bacterial gene into mouse haematopoietic progenitor cells

The haematopoietic system is made up of a hierarchy of cells with different developmental, functional and proliferative capacities. Although cellular diversity appears to arise from the commitment and maturation of stem cells, the molecular basis for this differentiation process is unknown. The introduction of cloned DNA sequences into haematopoietic progenitor cells would provide a novel approach for studying this differentiating in vivo system. One laboratory has reported DNA-mediated transfer of genes into mouse bone marrow cells. However, retroviruses offer a number of advantages over DNA-mediated gene transfer procedures, including high efficiency infection of a wide range of cell types in vitro and in vivo, stable and low copy integration into the host chromosome, and a defined integrated provirus structure. For these reasons recombinant DNA techniques have been utilized to construct high efficiency retrovirus vectors expressing foreign genes. We demonstrate here, using such a retrovirus vector, the transfer of a dominant selectable drug-resistance gene into defined classes of mouse haematopoietic progenitor cells. These observations should facilitate the development of molecular genetic approaches to fundamental and clinical problems in haematopoiesis.     

Interaction of cortisol and epinephrine in the regulation of leucine kinetics in man

To assess the interaction of the two major stress hormones epinephrine and cortisol in the regulation of leucine kinetics in man, epinephrine (50 ng/kg/min) was infused either alone or in combination with cortisol (2 micrograms/kg/min) into two groups of 6 postabsorptive normal male subjects during 180 min. Plasma leucine concentrations decreased by 28% (p less than 0.05) from baseline during epinephrine treatment (plasma levels 515 pg/ml); this was due to a decrease of leucine appearance (determined by 1-13C-leucine infusions) by 23% (p less than 0.025); leucine oxidation decreased by 29% (p less than 0.05). However, when plasma cortisol concentrations were elevated to supraphysiological levels (16.3 mumol/l) during epinephrine administration, the decreases of leucine plasma concentrations, appearance and oxidation were abolished. Plasma glucose and FFA concentrations were similarly elevated during both kinds of treatment. Since leucine appearance represents a measurement of total body protein breakdown and leucine disappearance into non-oxidative pathways reflects protein synthesis, the data indicate that plasma epinephrine concentrations during severe stress exert a protein anabolic effect in man which may counteract catabolic properties of elevated plasma cortisol.     

Antagonismus zwischen Heparin und 5-Hydroxytryptamin (Serotonin)in vitro

Summary In the isolated rat colon and uterus, heparin caused inhibition or elimination of contractions occurring after the administration of 5-hydroxytryptamine. In contrast to this, neither Tyrode's solution, nor glycogen, nor dextran produced any change in the response of these organs to 5-hydroxytryptamine.     

Phyllosilicates on Mars and implications for early martian climate

The recent identification of large deposits of sulphates by remote sensing and in situ observations has been considered evidence of the past presence of liquid water on Mars. Here we report the unambiguous detection of diverse phyllosilicates, a family of aqueous alteration products, on the basis of observations by the OMEGA imaging spectrometer on board the Mars Express spacecraft. These minerals are mainly associated with Noachian outcrops, which is consistent with an early active hydrological system, sustaining the long-term contact of igneous minerals with liquid water. We infer that the two main families of hydrated alteration products detected-phyllosilicates and sulphates--result from different formation processes. These occurred during two distinct climatic episodes: an early Noachian Mars, resulting in the formation of hydrated silicates, followed by a more acidic environment, in which sulphates formed.