Roy H. Burdon

The in vivo methylation of DNA in mouse fibroblasts

Abstract Studies on the in vivo methylation of DNA occurring in cultured mouse fibroblasts (Strain L929) treated with hydroxyurea have suggested that the only DNA base to be methylated, namely cytosine, is methylated after the initial synthetic stage of DNA replication and is a process which proceeds efficiently only in the presence of a continual supply of nascent DNA. Experiments with synchronised cell cultures demonstrated that cytosine methylation occurred within an hour of the synthesis of DNA in the S phase of the cell cycle. Additional ‘chase’ experiments showed that the 5-methylcytosine of DNA so formed is stable, no evidence being found for its demethylation, deamination or excision from the polymer.

Methylation of mosquito DNA

Cells of the mosquito Aedes albopictus have 5.6 pg DNA/cell. This DNA is 58 mol% (A + T) and has about 0.03 mol% each of 5-methylcytosine and 6-methylaminopurine. The DNA is largely degraded by the restriction enzyme HpaII showing the virtual absence of the sequence CMeCGG. Small amounts of enzymic activity recovered largely in non-nuclear fractions transfer methyl groups from S-adenosylmethionine to cytosine and adenine.

Intracellular location and molecular characteristics of tumour cell transfer RNA precursors

Abstract A 3 H-labelled cytoplasmic RNA fraction which behaves as a precursor of transfer RNA has been partially purified from Krebs 2 ascites tumour cells incubated with [ 3 H]guanosine using a cold phenol extraction technique followed by gel filtration on Sephadex G100. Heat treatment of the precursor tRNA molecules, at 60 °C in the presence of Mg 2+ at pH 7.8, served to increase their retention by the gel, suggesting that precursor tRNA molecules might have a molecular conformation different from that of mature tRNA molecules. However their behaviour on Sephadex G100 before and after formaldehyde treatment suggested the further possibility that they are somewhat longer than mature tRNA molecules. Examination of labelled RNA from various cytoplasmic fractions indicated that although precursor tRNA molecules are found in the soluble portion of the cytoplasm (cell sap) rather than associated with mitochondrial or microsomal components, there was no obvious intimate association with any soluble cell sap proteins.

Human heat shock gene expression and the modulation of plasma membrane Na+, K+-ATPase activity.

The potential of hyperthermia in human cancer treatment has been recognised for some time [ 1,2], although a practical problem is the development of ‘thermotolerance’ [3]. Brief hyperthermic treatment of HeLa cells (45°C; 10 min) followed by an obligatory ‘development’ period of 2 h at 37°C results in the maximal induction of specific heat shock proteins (HSPs) at Mr 100 000,72 000-74 000 and 37 000 [4]. The proteins at 100 000 Mr and 37 000 Mr are encoded by single mRNA species while the 72 OOO74 000 Mr group are coded for by 7 mRNAs which show some sequence homologies but have widely differing sizes (1.9-6.3 kilobasepairs) [ 5,7]. In all cases the mRNAs are bimorphic, existing in poly(Af) or poly(A-) forms [5]. Northern blot analyses using cloned cDNA sequences encoding the 72 000-74 000 Mr group [7] confirm experiments with actinomycin D [4] suggesting control of gene expression at the transcriptional level. High Mr mRNA precursors at 15.8 kilobasepairs do not appear in nuclei until l-2 h. in a partial recovery of activity. Evidence is presented to suggest HSP gene expression involvement in the modulation of Na*,K+-ATPase activity in HeLa cells.

Heat shock causes diverse changes in the phosphorylation of the ribosomal proteins of mammalian cells

When HeLa cells or BHK cells were subjected to heat shock at 42°C (for 2 h) or 45°C (for 10 min) there was extensive dephosphorylation of ribosomal protein S6. Concomitantly ribosomal protein L14, which is not significantly phosphorylated in normal cells, became phosphorylated, as did a non‐structural protein of M r = 27 000, associated with the ribosomes. The latter effects were not prevented by cycloheximide or actinomycin D. When cells shocked at 45°C for 10 min were returned to 37°C for 2 h there was rephosphorylation of ribosomal protein S6 and dephosphorylation of the 27 kDa protein, but not of ribosomal protein L14.

Deoxyadenylate-rich and deoxyguanylate-rich regions in mammalian DNA

Abstract The presence of deoxyadenylate-rich and deoxyguanylate-rich regions in mammalian DNA has been demonstrated by hybridization with 3 H-labelled poly(U) and 3 H-labelled poly(C). For hamster BHK-21/C13 cells, the dA-rich regions are up to 130 nucleotides long and comprise up to 0.4% of the DNA. Those dA-rich regions which comprise 0.13% of the DNA contain 2 to 6% of bases other than adenine. The dG-rich regions, in which 10 to 30% of the bases are other than guanine, are less than 40 nucleotides long and are present at a level of about 0.1% of the DNA. Exhaustive digestion of the hybrids with RNAase enables detection of deoxyhomopolymeric regions in the DNA, poly (dA) sequences of an average size of about 30 nucleotides long accounting for 0.008% of the DNA, and poly(dG) sequences, 17 nucleotides long, comprising 0.0016% of the DNA. Both dA-rich and dG-rich regions are found in DNA sequences with a wide variety of base composition. Extensive shearing of the DNA is required to produce some enrichment for dA-rich sequences in the (A + T)-rich fraction, although dG-rich sequences are slightly enriched in the (G + C)-rich fraction of even unsheared DNA. The buoyant density of hybrid molecules was found to be significantly greater than that of unhybridized DNA only when highly sheared DNA was used. These findings suggest that the dA-rich and dG-rich regions have a widespread distribution throughout DNA molecules. In situ hybridization studies with 3 H-labelled poly(U) further suggest that the dA-rich regions are not localized to any particular chromosome or to any specific region of the chromosomes. Analysis of DNA from a number of different species has shown that, in general, the dA-rich and dG-rich regions are present at a much higher level in mammalian DNA than in bacterial, bacteriophage or mammalian virus DNA. Possible functions of these unusual deoxynucleotide sequences are discussed.

The incorporation of uridine 5'-triphosphate into ribonucleic acid by enzyme fractions from Ehrlich ascites carcinoma cells.

Abstract Fractionation of extracts of Ehrlich ascites carcinoma cells with ammonium sulphate yields two fractions which catalyse the incorporation of uridine 5′-triphosphate labelled with 32P or 14C into polyribonucleotide. With one of these, Fraction A, the incorporation of uridine triphosphate is greatly increased by the addition of a mixture of the 5′-triphosphates of adenosine, guanosine and cytidine and in these conditions most of the incorporated radioactivity is distributed in internucleotide linkages. Fraction B catalyses less extensive incorporation of uridine 5′-triphosphate, much of which is located terminally. This reaction is unaffected by the addition of a mixture of the 5′-triphosphates of adenosine, guanosine and cytidine, but is greatly stimulated by a mixture of amino acids. Both systems are inhibited by inorganic pyrophosphate.

Non‐polyadenylated mRNAs from eukaryotes

The investigation of eukaryotic mRNA has focussed on molecules that possess a 3’-terminal poly(A) seg ment [l-3] since they can be isolated easily by virtue of their high affinity for oligo(dT)-cellulose [4,5], poly(U)-Sepharose [6,7] or Millipore filters [8]. The possible functions of the poly(A) tracts in terms of nuclear and cytoplasmic events have been extensively reviewed [2,3,10-131. Recently, however, a considerable body of evidence has accumulated suggesting the presence of mRNA molecules lacking poly(A) (poly(A)mRNA) as judged by their failure to bind to Millipore filters [8], oligo(dT)cellulose [14-161 or poly(U)-Sepharose [6,17]. The purpose of this short review is to assess present knowledge of these poly(A)mRNAs.

Eukaryotic DNA methylation

Abstract The evidence for a role of 5-methylcytosine in the control of transcription is examined and some possible additional functions are considered.

Hyperthermia, Na+K+ATPase and lactic acid production in some human tumour cells

When HeLa cells are exposed to brief heat shock at 45 degrees C there is a reduction in the cellular level of Na+K+ATPase. Return of the cells to the normal growth temperature of 37 degrees C leads to a partial restoration of enzyme activity. The pattern of this recovery of activity suggests that it may be associated with the induction of heat shock proteins. Indeed other means of heat shock protein induction such as continuous heat treatment at 42 degrees C, or treatment of cells at 37 degrees C with sodium arsenite, leads to elevated levels of Na+K+ATPase activity and alterations in the kinetic properties of the enzyme. Continuous hyperthermia at 42 degrees C led to increased lactate production which could be blocked with ouabain suggesting that effects on Na+K+ATPase activity could partly influence glycolysis. A number of other human and hamster cells also showed increased lactate production at 42 degrees C and also an inhibition of lactate production by ouabain. Whilst incubation of HeLa cells with cyanide had little effect on glycolysis at 37 degrees C elevation of the temperature to 42 degrees C (or 45 degrees C), in the presence of cyanide, impaired glycolysis. The possible role in this phenomenon, of an unusual oxygen-sensitive isoenzyme of lactate dehydrogenase, expressed in human cancers, is discussed.