Kjell Kleppe

Applications of magnetic beads with covalently attached oligonucleotides in hybridization: Isolation and detection of specific measles virus mRNA from a crude cell lysate

A novel, rapid, one-step isolation procedure utilizing oligonucleotides end-attached to magnetic beads (Dynabeads) has been developed. The beads and their captured target nucleic acids were separated after 2 h from the hybridization solution using an external magnet. This procedure was used to isolate measles virus hemagglutinin (H) mRNA from infected cells dissolved in 5 M guanidine thiocyanate. The yield of the specific, undegraded H-mRNA was found to be near the estimated total amount of H-mRNA present in the cells. The magnetic beads were also used for detection of measles virus H-mRNA in a crude cell lysate by sandwich hybridization. The experimental detection limit was 630 amol H-mRNA.

Optimal conditions for hybridization with oligonucleotides: a study with myc-oncogene DNA probes.

We present a study on the refinement of filter-hybridization conditions for a series of synthetic oligonucleotides in the range from 17 to 50 base residues in length. Experimental conditions for hybridization and the subsequent washing steps of the filter were optimized for different lengths of the synthetic oligonucleotides by varying the formamide concentration and washing conditions (temperature and monovalent cation concentration). Target DNA was immobilized to the nitrocellulose filter with the slot blot technique. The sequences of the synthetic oligonucleotides are derived from the third exon of the human oncogene c-myc and the corresponding viral gene v-myc and the G + C content was between 43 and 47%. Optimal conditions for hybridization with a 82% homologous 30-mer and 100% homologous 17-, 20-, 25-, 30-, and 50-mers were found to be a concentration of formamide of 15, 15, 30, 30, 40, and 50%, respectively. Optimal conditions for washing were 0.5X standard sodium citrate (SSC) at 42 degrees C for 2 X 15 min. The melting temperature for these optimal hybridization and washing conditions was calculated to be up to 11 degrees C below the hybridization temperature actually used. This confirms that the duplexes are more stable than expected. The melting points for 17-, 20-, and 30-mers were measured in the presence of 5X SSC and found to be 43, 58, and 60 degrees C, respectively. Competition between double- and single-stranded DNA probes to the target DNA was investigated. The single-stranded DNA probes were about 30- to 40-fold more sensitive than the double-stranded DNA probes.

The effect of caffeine on cell growth and metabolism of thymidine in Escherichia coli

(1) The influence of caffeine on growth and on the metabolism of thymidine was investigated in various E. coli strains. Caffeine caused filamentous growth in all strains investigated. The caffeine effect was reversible. (2) The incorporation of thymidine into DNA was inhibited by caffeine, and the inhibition was most pronounced with bacterial cultures grown overnight in the presence of caffeine before the addition of thymidine. For cells not pretreated with caffeine the inhibitory effect of caffeine decreased with increasing concentrations of thymidine up to about 1 microM whereafter it remained constant. The effect of thymidine concentration on the inhibition was less for bacteria that had grown overnight in the presence of caffeine than for bacteria not pretreated with caffeine. (3) Caffeine inhibited thymidine kinase, but it had no effect on thymidine phosphorylase or thymidine nucleotide kinases. (4) It is suggested that caffeine interferes with uptake of thymidine, conversion of thymidine to dTTP and the DNA synthesis process itself. Filamentous growth could be the result of the inhibition of DNA synthesis.

Affinity of protein HU for different nucleic acids

The binding of protein HU from Escherichia coli to nucleic acids was investigated by affinity chromatography under various conditions, by a nitrocellulose retention assay and by isopycnic centrifugations in metrizamide gradients. The results indicate that HU has a preference for binding to RNA and single‐stranded DNA over double‐stranded DNA. The affinity of HU for supercoiled DNA was also less than that of the corresponding relaxed DNA.

Properties and mechanism of action of eukaryotic 3-methyladenine-DNA glycosylases.

SUMMARY 3-Methyladenine-DNA glycosylase activities have been identified in all eukaryotic cell systems studied. Some of the results from these studies are reviewed here. The enzymes possess molecular weights between 24×103 and 34×103, they have a broad pH optimum at approximately pH8, require double-stranded DNA and act in the absence of any cofactors. The enzyme can excise several different methylated bases from DNA such as 3-methyladenine, 7-methylguanine and 3-methylguanine. The specific activity of this DNA glycosylase in mouse L-cells was found to be a function of the proliferative state of the cell. In vitro quantification of this DNA repair activity in synchronized mouse L-cells suggests that it is regulated within a defined temporal sequence prior to the onset of DNA replication. Using DNA fragments of defined sequences it was observed that the efficiency of removal of the methylated bases is sequence-dependent.

Purification and characterization of the 17 K protein, a DNA-binding protein from Escherichia coli

A basic protein of molecular mass 17 kDa (protein 17 K) which binds to relaxed DNA has been isolated and purified to homogeneity from Escherichia coli cells. The protein behaves as a tetramer in solution and there are 4800 monomers per cell in exponentially growing cells. The amino-acid composition and N-terminal sequence were determined. No effect of the protein on in vitro transcription was observed. The protein was shown to be different from the Ssb protein (Sigal, N. et al. (1972) Proc. Natl. Acad. Sci. USA 69, 3537-3541), protein H1 (Cukier-Kahn et al. (1972) Proc. Natl. Acad. Sci. USA 69, 3643-3647) and the HLP-1 protein (Lathe, R. et al. (1980) Proc. Natl. Acad. Sci. USA 77, 3548-3552).

DNA- and RNA-binding proteins of chromatin from Escherichia coli

The different proteins present in chromatin of Escherichia coli have been analyzed by a variety of techniques. The chromatin was isolated using a previously published procedure (Sjåstad, K., Fadnes, P., Krüger, P.G. Lossius, I. and Kleppe, K. (1982) J. Gen. Microbiol. 128, 3037) and solubilized by the action of micrococcal nuclease or DNAase I. The DNA-protein and RNA-protein complexes thus obtained were purified by sucrose gradient centrifugation and isopycnic gradient centrifugation in metrizamide in low ionic strength. The protein: DNA ratio of the DNA-protein complexes was estimated from the latter method and found to be approx. 1.75. The protein components were analyzed further by one- and two-dimensional gel electrophoresis. Approx. 15 major polypeptides were detected in the DNA-protein complex, whereas 10 were present in the RNA-protein complex. The majority of the polypeptides in both complexes had acidic isoelectric pH. The polypeptides in the two complexes differed markedly and only two polypeptides, having molecular weights of 57,000 and 37,000, respectively, were found to be common in both complexes. In agreement with earlier studies, the basic protein HU was not present in the DNA-protein complex. Affinity studies of the proteins from chromatin using DNA- and RNA-Sepharose columns in general confirmed the above conclusions. The two-dimensional gel electrophoretic patterns of the proteins in the different complexes were compared with those of proteins in the inner and outer membranes. Only one of the major polypeptides present in the inner membrane, having a molecular weight of 57,000, was enriched in the DNA-protein complex.

Isolation, Properties and Nucleolytic Degradation of Chromatin from Escherichia coli

A new procedure has been developed for the isolation of the chromosome complex, termed chromatin, from Escherichia coli. The bacteria were subjected to low ionic strength and T4 lysozyme, followed by detergent treatment analogous to that employed for the isolation of eukaryotic chromosomes. The chromatin was an insoluble viscous material which contained approximately equal amounts of DNA and RNA. The protein content of the chromatin was almost three times greater than the nucleic acid content. Electron microscopy revealed that the chromatin was highly condensed, having multiple loops and beaded structures with various diameters. The chromatin could be completely solubilized by both micrococcal nuclease and DNAase I, whereas RNAase had no effect. The initial degradation by micrococcal nuclease resulted in the production of a DNA-protein particle, sedimentation coefficient 10S, and an RNA-protein complex of 24S. Further degradation led to a decrease in sedimentation coefficient of the DNA-protein complex, but not of the RNA-protein particle. The peak size of the DNA of the initial DNA-protein particle was approximately 2400 bp. The action of micrococcal nuclease also resulted in the production of several discrete RNA species of various sizes. Several low molecular weight proteins (12000-27000) were found in the DNA-protein complex. The DNA-binding protein HU was present in the undigested chromatin; varying amounts of HU were, however, detected in the DNA-protein and RNA-protein particles.

Mechanism of inhibition of thymidine kinase from Escherichia coli by caffeine

The influence of caffeine on chromosome structure and DNA repair processes has been well documented. For a comprehensive review on caffeine, see [ 11. Caffeine has also been noted to have an effect on the incorporation of thymidine into DNA both in eukaryotic and in prokaryotic cells [2-41. In Escherichia coli, caffeine may affect both uptake, conversion of thymidine to dTTP and the DNA synthesis process itself [S]. Thymidine kinase (EC 2.7.1.21), an important enzyme in the conversion of thymidine to dTTP, was shown inhibited by caffeine [S]. Here we detail the mechanism of caffeineinduced inhibition of thymidine kinase from E. coli. Caffeine was found to be a competitive inhibitor of thymidine kinase, competing with the substrate ATP for its binding site.

Effect of Methyl Methanesulphonate on the Nucleoid Structure of Escherichia coli

Incubation of a strain of Escherichia coli K12 with 25 mM-methyl methanesulphonate (MMS) for 1 h changed the sedimentation coefficient of the nucleoids from 1600S to 850S. When isolated nucleoids were treated with MMS under identical conditions in vitro there was no change in the sedimentation coefficient. Alkaline sucrose-gradient centrifugation of DNA from cells treated with 25 mM-MMS for 1 h indicated that there were approximately 100 breaks plus apurinic sites per chromosome. Titration with ethidium bromide of nucleoids from MMS-treated cells showed that almost all supercoiling had been lost, suggesting that the breaks plus apurinic sites consisted mostly of breaks. Further experiments showed that the apurinic sites were probably created by non-enzymic depurination and that little non-enzymic strand breakage had occurred. The depurinated sites thus created could then serve as substrates for the apurinic-specific endonucleases of the cell, with the result that strand breakage occurred. MMS treatment did not cause any changes in the DNA:RNA ratio of the nucleoids. Removal of MMS followed by a period of incubation resulted in a decrease in the number of breaks plus apurinic sites and an increase in the sedimentation coefficient of the nucleoids. After 2 h incubation in MMS-free medium the sedimentation coefficient of the nucleoids from MMS-treated cells was the same as that of the control; the supercoiling was also partially restored. The effect of MMS on two MMS-sensitive mutants of E. coli, one a polA and the other a recA mutant, was also studied. In both cases MMS caused complete collapse of the nucleoid structure.