Inhibition of DNA Synthesis but not of Poly-dAT Synthesis by the Arabinose Analogue of Cytidine <Emphasis Type="Italic">in vitro</Emphasis>

Kimball and Wilson¹ reported that the arabinose analogue of cytidine (ara-C) inhibited DNA polymerase in a crude extract prepared from Ehrlich ascites cells. Furth and Cohen² observed cytosine arabinoside triphosphate (ara-CTP) inhibited DNA polymerase in extracts from either calf thymus or bovine lymphosarcoma tissue, although these investigators³ had already found no effect of ara-CTP on DNA polymerase from Escherichia coli. The inhibition in both of these cases could be substantially reversed by dCTP; but incorporation of the arabinose nucleotide (ara-CMP) into DNA could not be unequivocally demonstrated. Graham and Whitmore⁴ reported the incorporation of ara-C into DNA in vivo and the inhibition of a DNA polymerase from L cells by ara-CTP. They found that ara-CMP was initially incorporated into small DNA strands but subsequently appeared in long strands. Momparler⁵ has presented evidence that, in vitro, ara-C incorporation was limited to the 3′-hydroxyl end of DNA chains. Such incorporation might be expected to block further chain elongation but this expectation was not supported by the evidence presented by Graham and Whitmore.     

Endopeptidase Activity of Phage λ-Endolysin

JACOB and Fuerst^1,2 demonstrated the presence of a bacteriolytic enzyme (λ-endolysin) in the induced cultures of lysogenic Escherichia coli K12 (λ). The enzyme was later identified as the product of gene R; of phage λ³ which is involved in bacterial lysis at the end of a latent period. The enzyme is apt to form spheroplast-like structures in E. coli² and one would therefore expect its substrate to be murein.     

The diminution of heterochromatic chromosomal segments in Cyclops (Crustacea,Copepoda)

The chromosomes of Cyclops divulsus, C. furcifer, and C. strenuus, like those of several other Copepods, undergo a striking diminution of chromatin early in embryogenesis. The process is restricted to the presumptive soma cells and occurs at the 5th cleavage in C. divulsus, at the 6th and 7th in C. furcifer, and at the 4th in C. strenuus. The eliminated chromatin derives from the excision of heterochromatic chromosome segments (H-segments). Their chromosomal location is different in the three investigated species: Whereas in C. divulsus and C. furcifer the H-segments form large blocks — exclusively terminal in the former and terminal as well as kinetochoric in the latter — the germ line heterochromatin in C. strenuus is scattered all along the chromosomes. Extensive polymorphism exists with respect to the length of the terminal H-segments in C. furcifer, and with respect to the overall content of heterochromatin in the chromosomes of C. strenuus. In a local race of C. strenuus an extreme form of dimorphism has been found which is sex limited: females as a rule are heterozygous for an entire set of large (heterochromatin-rich), and a second set of small chromosomes in their germ line. Males are homozygous for the large set. In the first three cleavage divisions the H-polymorphism is solely expressed through differences of chromosome length. Following diminution the differences between homologous have disappeared. Feulgen cytophotometry demonstrates that in the three species the 1C DNA value for the germ line, as measured in sperm, is about twice that measured in somatic mitoses (germ line/soma C-values in picograms of DNA: C. strenuus 2.2/0.9, C. furcifer 2.9/1.44, C. divulsus 3.1/1.8). — The data imply that chromatin diminution is based on a mechanism which allows specific DNA segments, regardless of their location and size, to be cut out from the chromosomes without affecting the structural continuity of the remaining DNA. This mechanism may be analogous to that of prokaryotic DNA excision.     

Persistence and spread of a chloramphenicol resistance-mediating plasmid in antigenic types of Escherichia coli, pathogenic for piglets

All chloramphenicol-resistant Escherichia coli strains isolated from piglets in the State veterinary Serum Laboratory, Copenhagen, in 1974-1975 harbored plasmids of IncFII group with largely the same resistance markers. Two strains from 1978 carried plasmids with similar characters. Restriction enzyme analysis of DNA from these plasmids with restriction endonucleases EcoRI, BglII, and PstI shows that the Cm plasmids are extremely closely related; but the patterns obtained (particularly from PstI digests) enable the classification of the plasmids into groups. These bear a strong relation to time and place of isolation so that plasmids isolated on the same farm belong to the same group even when their host strains are of different antigenic types. It is concluded that these plasmids have evolved from a single plasmid.     

Protection of foreign DNA against host-controlled restriction in bacterial cells

Summary Foreign Flac plasmid DNA which is introduced into potentially restricting E. coli recipient cells can be protected from restriction by preinfecting the recipient cells with UV-inactivated T3 or T7 bacteriophages which express the ocr gene function. The recipient cells survive and are able to replicate themselves as well as the newly acquired plasmid.     

Protection against acute paraquat toxicity by ambroxol

An expression vector for G-CSF, pASLB3-3, was constructed and introduced into Namalwa KJM-1 cells (Hosoi et al., 1988), and cells resistant to 100 nM of methotrexate (MTX) were obtained. Among them, the highest producer, clone SC57, was selected and the productivity of this clone was further characterized. The maximal production of G-CSF was at the most 1.8 g/ml/day using a 25 cm² tissue culture flask, even though the cell number was above 7×10⁵ cells/ml. The limiting factors at high density were analyzed as the deficiency of nutrients, such as glucose, cysteine and serine, and pH control. The depression of specific G-CSF productivity per cell under the batch culture conditions was overcome by using a perfusion culture system, Biofermenter^TM (Sato, 1983) with modifications of nutrients supplementation by a dialysis membrane and/or dissolved oxygen (DO) supplementation by microsilicone fibers. ITPSGF medium was modified to elevate concentrations of amino acids and glucose by 2.0- and 2.5-times, respectively. Under the control of pH at 7.4 and DO at 3 ppm, the specific G-CSF productivity was not depressed even at high cell density (above 1×10⁷ cells/ml), and the amount of G-CSF reached 41 g/ml. These results indicated the possibility of finding the optimum culture conditions for the production of recombinant proteins by Namalwa KJM-1 cells.Abbreviations ABTS 2,2-Azino-di-(3-ethylbenzothiazoline)-6-sulfonic acid - BSA Bovine Serum Albumin - BSA-PBS Phosphate-buffered Saline without Ca²⁺ and Mg²⁺ containing Bovine Serum Albumin - dhfr Dihydrofolate Reductase - DO Dissolved Oxygen - G-CSF Granulocyte Colony-stimulating Factor - HEPES 4-(2-Hydroxyethyl)-1-piperazineethansulfonic Acid - IFN Interferon - MTX Methotrexate - PBS(-) Phosphate-buffered saline without Ca²⁺ and Mg²⁺ - Tween-PBS Phosphate-buffered saline without Ca²⁺ and Mg²⁺ containing 0.05% of Tween 20     

Influence of clay minerals on sorption of bacteriolytic enzymes

Myxobacteria presumably produce extracellular bacteriolytic enzymes when they are growing in soil. In order to study their ecological significance, adsorption experiments were performed with lytic enzymes produced byMyxococcus virescens in casitone media. Different soils as well as montmorillonite and kaolinite can rapidly adsorb the bacteriolytic but not the proteolytic enzymes. About 1 gm of montmorillonite per liter of cell-free culture solution is enough for the adsorption of 97% of the bacteriolytic enzymes. The adsorption per unit weight is about 100 times greater on montmorillonite than on kaolinite. About 40% of the adsorbed enzymes can be eluted with solutions of high pH or high ionic strength. The only desorbed bacteriolytic enzyme is the alanyl-∈-N-lysine endopeptidase.     

Staining of Some Specific Regions of Human Chromosomes,particularly the Secondary Constriction of No. 9

SEVERAL procedures have been described recently which produce specific patterns of differential staining in human chromosomes^1–9. Techniques which involve DNA denaturation and reannealing reveal deeply stained areas on centromere and secondary constriction regions which have been equated with constitutive heterochromatin⁹.     

The mode of action of thidiazuron: auxins,indoleamines, and ion channels in the regeneration of <Emphasis Type="Italic">Echinacea purpurea</Emphasis> L.

The biochemical mechanisms underlying thidiazuron (TDZ)-induced regeneration in plant cells have not been clearly elucidated. Exposure of leaf explants of Echinacea purpurea to a medium containing TDZ results in undifferentiated cell proliferation and differentiated growth as mixed shoot organogenesis and somatic embryogenesis. The current studies were undertaken to determine the potential roles of auxin, indoleamines, and ion signaling in the dedifferentiation and redifferentiation of plant cells. E. purpurea leaf explants were found to contain auxin and the related indoleamine neurotransmitters, melatonin, and serotonin. The levels of these endogenous indoleamines were increased by exposure to TDZ associated with the induction of regeneration. The auxin-transport inhibitor 2,3,5-triiodobenzoic acid and auxin action inhibitor, p-chlorophenoxyisobutyric acid decreased the TDZ-induced regeneration but increased concentrations of endogenous serotonin and melatonin. As well, inhibitors of calcium and sodium transport significantly reduced TDZ-induced morphogenesis while increasing endogenous indoleamine content. These data indicate that TDZ-induced regeneration is the manifestation of a metabolic cascade that includes an initial signaling event, accumulation, and transport of endogenous plant signals such as auxin and melatonin, a system of secondary messengers, and a concurrent stress response.