“Pleiotypic Response”

A hypothesis has been developed to relate stringent control in bacteria to a set of interactions involved in the regulation of growth of transformed and untransformed mammalian cells.     

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.     

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     

Mutations within an intron and its flanking sites: Patterns of novel polypeptides generated by mutants in one segment of the cob-box region of yeast mitochondrial DNA

Summary We have undertaken a systematic examination of the polypeptides accumulating in thirteen (out of 23) mutants in the intron cluster box7 and its flanking clusters box2 and box9 of the cob-box (cytochrome b) region of the mitochondrial genome of Saccharomyces cerevisiae. We have subjected these polypeptides to fingerprint analysis, both sequential and in parallel, with two proteases in order to disclose sequence homologies and differences between the different novel polypeptides themselves, and between them and the wild type product of the gene, i.e. apocytochrome b. One of our aims has been to establish the existence of possible correlations between the nature of the novel polypeptides and the fine structure genetic map of that segment of the mitochondrial genome.Our results show that all box7 mutants accumulate the following set of polypeptides not seen in wild type cells: a) a characteristic set of large polypeptides consisting of three species: p56, p42 and p35 or p34.5; b) a polypeptide p23; c) a much shorter fragment (of which the apparent molecular weight varies from 12.5 to 13, according to the mutation) with the exception of two mutants; d) in addition, the majority accumulate in varying amounts a polypeptide p30 closely related to but not identical with apocytochrome b.Moreover only two box7 mutants accumulate a polypeptide in the range of mobilities corresponding to 25–27 Kd (referred to as class p26) while such a polypeptide is seen in all box9 and box2 mutants examined with one exception in box2.Only one mutant in box2 resembles box7 mutants with respect to criterion a), and no box2 or box9 mutants resemble box7 mutants with respect to criterion c); criteria b) and d) appear to apply equally well to mutants in all three clusters.Fingerprint analysis, carried out with polypeptides p56, p42, p35, p34.5, p30, p26, p23, discloses that a) The polypeptides belonging to the same class of mobility exhibit very similar if not identical sequences in various cases. b) These polypeptide classes, except p56, p42 and p26, share considerable sequence homologies with wild type apocytochrome b, perhaps encompassing 50% or more of the wild type sequences. b) Polypeptides belonging to the classes p42 and p26 exhibit less extensive but nevertheless significant homologies with the wild type sequence. c) Sequences in polypeptides belonging to class p56 are virtually indistinguishable from ones in cytochrome oxidase subunit I.The inferences from these findings are 1) one gene can produce a multiplicity of polypeptide products that share a common sequence at the promoter-proximal (N-terminal) portion, but diverge beyond these regions of homology. 2) Both the multiplicity of products in single mutants, and the protein structure found, argue against the divergent segments to be due to frameshift terminations, and instead suggest that the novel products are consequences of mRNA processing defects (excision and/or ligation) at and near intron regions. 3) Mutations at edges and the center of an intron can generate similar polypeptide patterns, i.e. produce analoguous mRNA processing defects. 4) Mutations in exons, at their boundary with introns, can produce polypeptide patterns indistinguishable from those at the neighbouring intron; they diverge and eventually become typically exonlike in mutants localized at increasing distances from the boundary. 5) Taken together these findings argue that pre-mRNA processing extends beyond the boundaries of the intron proper and that certain exonsequences participate in excision and ligation. 6) Accumulated pre-mRNAs, resulting from defects in splicing can be translated. 7) Product p56 is formally analogous to p23, as a faulty but highly conserved partial product of the wild type protein, the former of Cox I (oxi3 gene), the latter of the cob-box gene proper. Therefore both genes may utilize identical RNA processing elements which are affected by box7 mutations. 8) A small amount of product similar to p56 may accumulate even in some wild types but not in others. This observations suggests that in certain nuclear backgrounds RNA processing may be more error-prone than in others.Publication No. XXXX from the Department of Chemistry, Indiana UniversitySupported by Research Grant GM 12228 from the National Institute of General Medical Sciences, National Institutes of Health; recipient of Research Career Award K06 05060 from the same Institute.Supported by Délégation Générale à la Recherche Scientifique et Technique grant n⁰ 78-70341     

In vitro translation of messenger ribonucleic acid from sporulating and nonsporulating strains of Bacillus subtilis.

An Escherichia coli translation system supplemented with ribonucleic acid from sporulating Bacillus subtilis produces unique polypeptides which are missing among translation products of ribonucleic acid from six early sporulation mutants.     

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.