Liposome-mediated DNA transfer in eukaryotic cells: Dependence of the transfer efficiency upon the type of liposomes used and the host cell cycle stage

The pBR322 plasmid containing the sequence encoding β-lactamase, the enzyme conferring resistance to ampicillin, was encapsulated in liposomes of different phospholipid composition and incubated with synchronized cells. In mitotic cells as compared to cells synchronized in G₁, twice as many exogeneous DNA molecules were found associated with the cell nuclear DNA, when fluid, neutral liposomes were used. These liposomes are taken up by the cells mainly via endocytosis. When fluid, negatively charged liposomes were used as carriers about the same number of exogeneous DNA molecules were found associated with the nuclear DNA both in mitotic and in G₁-synchronized cells. The efficiency for gene transfer of liposomes entering the cells by different mechanisms was further studied and expressed both by the fraction of the radioactive plasmid associated with the nuclear DNA and by the level of the β-lactamase activity detected in the transfected cells. It appears that liposomes entering the cells mainly via an energy-dependent mechanism are more efficient for this type of DNA transfer.     

Replicative DNA synthesis in permeable fibroblasts from normal individuals and ataxia-telangiectasia patients

Replicative DNA synthesis in normal human fibroblasts was inhibited by 50% when they were X-irradiated (8 Gy) and made permeable 30 min later, whereas only a slight inhibition (20%) was observed in similarly treated ataxia-telangiectasia cells. Treatment of irradiated normal cells with caffeine (2 mM) before permeabilization reversed the inhibitory effects of X-rays, buf caffeine had no effect on DNA synthesis in permeable ataxia-telangiectasia cells. Diadenosine tetraphosphate (0.1 mM) did not affect DNA synthesis in permeable normal fibroblasts.     

Role of 3-methyladenine-DNA glycosylase in host-cell reactivation of methylated T7 bacteriophage

Purified T7 phage, treated with methyl methanesulfonate, was assayed on four Escherichia coli K12 host cells: (1) AB1157, wild-type; (2) PK432-1, lacking 3-methyladenine-DNA glycosylase (tag); (3) NH5016, lacking apurinic endonuclease VI (xthA); (4) p3478, lacking DNA polymerase I (polA), the latter three strains being deficient in enzymes of the base excision repair pathway. For inactivation measured immediately after alkylation, phage survival was lowest on strains PK432-1 and p3478; for delayed inactivation, measured after partial depurination of alkylated phage, survival was much lower on strain p3478 than on PK432-1. These results demonstrate the important role played by 3-methyladenine-DNA glycosylase in the survival of methylated T7 phage. Quantitative analysis of the data, using the results of Verly et al. (Verly, W.G., Crine, P., Bannon, P. and Forget, A. (1974) Biochim. Biophys. Acta 349, 204–213) to correlate the dose with the number of methyl groups introduced into phage DNA, revealed that 5–10 3-methyladenine residues per T7 DNA constituted an inactivation hit for the tag mutant. Thus, 3-methyladenine may be as toxic a lesion as an apurinic site.     

Interaction of aromatic ionen oligomers with DNA

A novel polycationic ionen was synthesized and fractionated on carboxymethyl-Sephadex using a salt gradient in 7M urea. A series of oligomers of discrete length were characterised by ultraviolet spectra. The ultraviolet spectra of oligomers revealed a new band centred at 232.5 nm which was probably due to exciton splitting. Thermal denaturation studies indicated both stabilization of the helix conformation and a higher degree of cooperativity in the melting of DNA (oligomers)n complex as compared to native calf thymus DNA. Ionen oligomers exhibited large extrinsic Cotton effect at 232.5 nm which could be attributed to exciton interaction.     

DNA,RNA, protein and DNases in developing rat cerebellum: Effects of early postnatal nutritional deprivation

The effect of early postnatal undernutrition and subsequent rehabilitation on wet weight, DNA, RNA, protein and the activities of acid and alkaline DNases in the cerebellar region of rat brain was studied. The cerebellar region was found to be affected significantly during early undernutrition. Further, earlier the initiation of nutritional rehabilitation the better was the recovery and in some cases timely nutritional rehabilitation resulted in better than normal biochemical composition of the brain. The specific activities of acid and alkaline DNases were not affected by early undernutrition. However, the total activities of these enzymes were significantly low in undernourished rats (R1₁₅ and R₂₁) Rehabilitation of these deprived groups upto 150 days resulted in higher amounts of these enzymes as compared to those of age-matched controls. It is concluded that the two DNases, are synthesized in a preferential manner during rehabilitation, It is further concluded that cerebellar region, in terms of development schedule and response to imposed calorie restriction, is intermediary between grey and white matter regions.     

Analysis of bovidae genomes: Arrangement of repeated and single copy DNA sequences in bovine,goat and sheep

Approximately 43–60% of the total genome in bovine, goat and sheep consisted of interspersed repeated and single copy DNA sequences. Most of the interspersed repeated DNA sequences were 1500–2400 nucleotide pair long while a minor portion was more than 4000 nucleotide pair long in goat and sheep and 3200 nucleotide pair long in bovine. About 1/3rd of single copy sequence were interspersed and their length was in the range of 1000–1500 nucleotide pairs.     

The effect of gyrase inhibitors and cyclic AMP on induction and glucose repression of the 6-hydroxy-nicotine oxidases in Arthrobacter oxidans

The induction by d,l-nicotine of the enantiozymes 6-hydroxy-L-nicotine oxidase and 6-hydroxy-D-nicotine oxidase in Archrobacter oxidans was differently affected by the inhibitors of Escherichia coli gyrase, novobiocin and nalidixic acid. These compounds inhibited 6-hydroxy-L-nicotine oxidase induction slightly, but led to an increase in the level of 6-hydroxy-D-nicotine oxidase activity. Furthermore, the specific repression by glucose of 6-hydroxy-D-nicotine oxidase synthesis was not abolished by the addition of cAMP but by that of novobiocin.Abbreviations 6-HDNO 6-hydroxy-D-nicotine oxidase - 6-HLNO 6-hydroxy-L-nicotine oxidase - cAMP cyclic 3,5-adenosine monophosphate - Enzymes Adenylate cyclase - ATP pyrophosphate-lyase (cyclizing) (EC 4.6.1.1) - cAMP-phosphodiesterase 3:5-cyclic-nucleotide 5-nucleotido-hydrolase (EC 3.1.4.17) - DNA gyrase DNA topoisomerase II (EC 5.99) - DNA polymerase deoxynucleosidetriphosphate: DNA desoxynucleotidyl-transferase (EC 2.7.7.7) - 6-hydroxy-L-nicotine oxidase 6-hydroxy-L-nicotine: oxygen oxidoreductase (EC 1.5.3.5) - 6-hydroxy-D-nicotine oxidase 6-hydroxy-D-nicotine: oxygen oxidoreductase (EC 1.5.3.6) - -lactamase penicillin amido--lactamhydrolase (EC 3.5.2.6) - nicotine dehydrogenase nicotine: (acceptor)6-oxidoreductase (hydroxylating) (EC 1.5.99.4)     

Cloning of alkaline phosphatase isozyme gene (iap) of Escherichia coli

In Escherichia coli three major alkaline phosphatase isozymes are formed by molecular conversions depending on physiological conditions. A chromosomal gene, iap, is responsible for alkaline phosphatase isozyme conversion and is assumed to code for a proteolytic enzyme removing the arginine residue(s) from the N-terminal position of alkaline phosphatase subunits. A chromosomal fragment which complemented the Iap^? phenotype was cloned into pBR322 by a shotgun method. Transducing phage λiap was constructed in vitro from the chromosomal fragment containing the iap gene and λtna DNA. The integration site of the phage on chromosome was identified as the iap locus by PI transduction, which meant that the cloned chromosomal DNA contained authentic iap gene.The restriction map of the hybrid plasmid was constructed. Based upon this information, several iap deletion plasmids as well as smaller iup⁺ plasmids were constructed. Analysis of the phenotypes conferred by these plasmids enabled us to locate iap gene within a 2-kb segment of the cloned DNA.The cells carrying the iap⁺ plasmid showed very efficient isozyme conversion even in medium containing arginine, an inhibitor for the isozyme conversion. This indicates overproduction of the iap gene product.