Plasmid rescue - a tool for reproducible recovery of genes from transfected mammalian cells?

Summary The efficient rescue of plasmids containing the thymidine kinase gene (tk) of Herpes simplex virus type I from genetically transformed mouse cells by transformation of bacteria is described. Rescued plasmids contain insertions of calf DNA used as a carrier in the transfection but usually lack portions of plasmid DNA. Deletions generally concern the region spanning from around the PvuII site of pBR322 to within the tetracycline resistance coding sequence, whereas the extent of tk sequence deletion varies, depending on the site of its integration (BamHI or PvuII) into the plasmid. Modelling the rescue process by transformation of bacteria with a mixture of original plasmids and sheared mouse cell DNA clearly demonstrates that deletions are caused by the presence of the mammalian DNA and they probably occur during re-transformation of bacteria before the onset of tetracycline gene expression. Plasmids lacking the Tc^r region are reproducibly rescuable without deletion. Methods for reproducible re-isolation of transferred genes from mammalian cells are discussed.     

Chemical accessibility of the 4.5S RNA in spinach chloroplast ribosomes.

We have examined the accessibility to diethylpyrocarbonate of spinach chloroplast 4.5S ribosomal RNA when free and when it is part of the ribosomal structure. The modifications in free 4.5S RNA were found mostly in single-stranded regions of the secondary structure model proposed in our previous paper (Kumagai, I. et al. (1982) J.B.C. 257, 12924-28): adenines at positions 17, 19, 33, 36, 54, 55, 60, 64, 68, 72, 77, 86 and 87 were identified as the reactive residues. On the other hand, in 4.5S RNA in 70S ribosomes or 50S subunits, adenine 33 was exclusively modified, and its reactivity was much higher than in free 4.5S RNA. This highly accessible A33 of spinach 4.5S RNA is located within a characteristic seven nucleotide sequence, which is found in the 4.5S rRNAs from spinach, tobacco and a fern but deleted in 4.5S RNAs from maize and wheat.     

Tryptophan-dependent indoleacetic-acid biosynthesis from indole,demonstrated by double-labelling experiments

Summary ³H-serine+¹⁴C-indole were administered to tips of sterily-grown pea seedlings and of non-sterile oat coleoptiles. The Try and IAA produced were extracted and purified by paper chromatography, and their ³H/¹⁴C ratios were determined. The ³H/¹⁴C ratio of IAA was lower than the ³H/¹⁴C ratio of Try. However, the same decrease of the IAA ³H/¹⁴C ratio was found when ³H, ¹⁴C-Try was supplied instead of ³H-serine+¹⁴C-indole. This result supports the view that Try is the native IAA precursor and that no significant bypass from indole to IAA exists in the plant material used.Abbreviations IAA indole-3-acetic acid - IPyA indole-3-pyruvic acid - Try tryptophan     

Mikrospektralphotometrische Untersuchungen zum Speichermechanismus des kationischen Farbstoffes Chrysoidin durch die lebende und tote Pflanzenzelle

Wie aus Elektrophorese- und spektralphotometrischen Untersuchungen hervorgeht, liegt der kationische Farbstoff Chrysoidin G, je nach dem pH-Wert der wäßrigen Farblösungen, als I-, II-, III- und IV-wertiges Kation und elektroneutrales Farbbasenmolekül vor. Von physiologischer Bedeutung ist nur das I-wertige Kation und das Farbbasenmolekül. Die Unabhängigkeit der Absorptionsmaxima wäßriger Farbstofflösungen mit konstantem pH-Wert von der Farbstoffkonzentration deutet darauf hin, daß Chrysoidin keine Assoziate bildet. In organischen Lösungsmitteln ergibt Chrysoidin G je nach dem Grad der Polarität des Solvens und dem pH-Wert der wäßrigen Phase bei Ausschüttelungs-versuchen unterschiedliche Absorptionskurven. Natriumnucleinat bedingt eine negative Metachromasie; die jeweilige Lage des Maximums wird von der Natriumnucleinatkonzentration bestimmt. Rutin übt keinen wahrnehmbaren Einfluß auf das Absorptionsspektrum aus. Nach einer Vitalfärbung von Oberepidermiszellen der Schuppenblätter von Allium cepa mit Chrysoidin G zeigen das diffus gefärbte Plasma und die darin auftretenden gelben Kugeln übereinstimmende Absorptionsspektren mit einem breiten Bandenmaximum bei ? 420 nm. Der lebende Zellkern färbt sich nicht. Der gefärbte volle Zellsaft der Unterepidermis besitzt ein Maximum bei ? 448 nm. Aus der Lage der Absorptionsmaxima und dem Verlauf der Absorptionskurven kann geschlossen werden, daß die Färbung des lebenden Plasmas auf eine Anreicherung des einwertigen Kations und des Farbbasenmoleküls in polaren Lipoiden beruht, während es sich bei der Färbung des fixierten Zellkerns um eine Bindung des Chrysoidins an Nucleinsäuren handelt. Die Vitalfärbung des vollen Zellsaftes mit Chrysoidin G ist nicht auf den Gehalt der Vakuolen an Flavonolen zurückzuführen, sondern hängt vermutlich vom pH-Wert des Zellsaftes ab.     

Principles and applications of luminescence spectrometry coupled to liquid chromatographic techniques

An overview is presented of the physicochemical basis of luminescence, and its application to the detection of chemicals (drugs, biomedically important compounds, environmentally active substances) in liquid chromatographic systems.     

IL-1 and IL-4 modulate IL-1 receptor expression in a murine T cell line

The combination of IL-1 and IL-4 stimulates the proliferation of certain murine T cell populations. Although this effect has been best characterized for a number of murine type 2 Th cell (Th2) clones, the mechanism(s) by which these cytokines effect this response is unclear. We have examined the effects of IL-1 and IL-4 on IL-1R expression by MD10 cells, and IL-1-responsive murine T cell line. These cells bear specific IL-1R, which bind human and murine IL-1 alpha and -beta. The measured apparent IL-1R dissociation constant ranged from 41 to 255 pM using 125I-HrIL-1 alpha. Cross-linking studies demonstrated two different 125I-HrIL-1 alpha binding complexes having Mr of 70,000 and 130,000 to 156,000. When removed from passage conditions and placed in non-growth factor-supplemented media, MD10 IL-1R expression spontaneously increased two- to fourfold over the first 11 to 12 h of culture followed by a decline. This phenomenon is partially inhibitable by cycloheximide suggesting that protein synthesis is involved. In agreement with other reports, HrIL-1 alpha down-regulated the expression of its own receptor with an ED50 of between 1 and 10 pM HrIL-1 alpha for this effect. In most experiments, low amounts of HrIL-1 alpha (1.0, 0.1 pM) significantly augmented IL-1R expression. Scatchard analysis of data obtained with all HrIL-1 alpha treatment conditions showed that the effects were due to a change in receptor number, not affinity. Significantly, purified murine IL-4 (MpIL-4) augmented MD10 IL-1R expression in both a time- and dose-dependent fashion. In the presence of 50 U/ml MpIL-4, MD10 IL-1R expression increased two- to threefold after 24 h without a change in receptor affinity. When MpIL-4 (50 U/ml) and various amounts of HrIL-1 alpha (.01-1000 pM) were co-added, the down-regulatory effect of high levels of HrIL-1 alpha was significantly antagonized. When added to cultures after 24 h of HrIL-1 alpha (100 pM) treatment, MpIL-4 reversed the IL-1R down-regulatory effect induced by high levels of HrIL-1 alpha. Finally, when combined in MD10 proliferation assays, MpIL-4 synergistically enhanced the proliferation of MD10 cells treated with suboptimal levels of HrIL-1 alpha.(ABSTRACT TRUNCATED AT 400 WORDS)     

Crystallographic studies of 3-ketoacylCoA thiolase from yeast Saccharomyces cerevisiae

Good diffracting crystals of 3-ketoacylCoA thiolase (EC 2.3.1.16) from yeast Saccharomyces cerevisiae have been obtained. The crystals diffract to at least 2.4 A. The space group of these crystals is P2(1)2(1)2(1), with cell dimensions a = 71.8 A, b = 93.8 A and c = 119.9 A. There is one dimer per asymmetric unit.     

Effects of overexpression of Pkn2, a transmembrane protein serine/threonine kinase, on development of Myxococcus xanthus.

Pkn2 is a putative transmembrane protein serine/threonine kinase required for normal development of Myxococcus xanthus. The effect of Pkn2 overexpression on development of M. xanthus was examined by expressing pkn2 under the control of a kanamycin promoter. Pkn2 was clearly detected by Western blot (immunoblot) analysis in the overexpression strain (the PKm/pkn2 strain) but could not be detected in the wild-type strain. Overexpressed Pkn2 was located almost exclusively in the membrane fraction, suggesting that Pkn2 is a transmembrane receptor-type protein Ser/Thr kinase. The PKm/pkn2 strain formed fruiting bodies more slowly than the wild-type strain, in contrast to a Pkn2 deletion strain, the delta pkn2 strain, which developed faster than the wild-type strain. However, spore production was reduced in both the PKm/pkn2 and delta pkn2 strains. These data suggest that Pkn2 functions as a negative regulator for fruiting-body formation and that the proper level of Pkn2 is necessary for maximum myxospore yield.