Further purification of the DNA methylase activity from the nuclear supernatant fractions was performed by (NH4)2S04 precipitation followed by chromatography on DEAE-cellulose and hydroxyapatite. When the properties of these more extensively purified DNA methylase preparations were examined, differences between the transformed and the non-transformed line were again found. Whereas concentrations of 0.1~-NaCItend to inhibit the ability of the BHK-21/ C13-cell preparation to modify E. coli DNA, such a salt concentration stimulated the BHK-21/PyY-cell preparation. Examination of the pattern of oligonucleotide sequencesmethylated by the BHK-21/PyY-cell preparation were notably different from the characteristicpattern for vertebrate produced by the BHK-21/C13-cell preparation. Only in the presence of 0.1 M-NaCI (i.e. at maximum stimulation) is the BHK-21BYcell preparation able to ,producea pattern similar to that characteristicof the BHK-21/ C13-cell preparation. However, whether such differences in enzymic properties can be related to the presence or absence of the polyoma genome as part of the BHK-cell genome must await further experimentation. Browne, M. J., Turnbull, J. F. & Burdon, R.H. (1976)Biuchem. Suc. Truns.4,1130-1131 Nass, M.M.K.(1973)J.Muf. Bid. 80, 155-175 Rubery, E.D.& Newton, A. A. (1973)Biuchim. Biuphys. Actu 324,2436

The Relationship Between the Activity of Ribonuclease H and Synthesis of Deoxyribonucleic Acid in Synchronously Growing BHK-211C13Cells PATRICIA M. DUFF and HAMISH M. KEIR Department of Biochemistry, University of Aberdeen, Marischal College, Aberdeen AB9 IAS, Scotland, U.K.

BHK-21/C13 cells grown in culture contain four ribonucleases H distinct from any mitochondria1 ribonuclease H. Ribonuclease H activity was separated into two fractions, HA and HB, by chromatography of a cell extract on phosphocellulose. On DEAE-Sephadex A25, ribonuclease HA activity was separated into two fractions, HA1 and HA2. By gel filtration on Sephadex G-100 or on Sepharose 6B,ribonuclease HB activity was separated into two fractions, HB1 and HB2 (Cooper et al., 1974). By using these methods, ribonucleases HA1, HA2, HB1 and HB2 were purified 99-fold, 85-fold, 20-fold and 320-fold respectively. The enzymes had distinct properties and were classified as true ribonucleases H by their abilities to degrade only the RNA component of a RNA-DNA hybrid. Since the enzymes require a RNA-DNA hybrid as substrate, they have been assigned a postulated biological role in eukaryotic cells either in the regulation of transcription or in the replication of DNA for the removal of RNA primers. The suggested role in replication of DNA was supported by the observation that ribonucleases HB1 and HB2 co-fractionate with BHR-cell DNA-dependent DNA polymerases a and B respectively. Indirect evidence for a function(s) of ribonuclease H in the replication of DNA in uiuo may be adduced if a positive correlation between enzyme activity and the rate of DNA synthesis in the cells exists. BHK-21/C13 cells were grown in culture to the resting or quiescent (Go)state by the method of serum depletion (Biirk, 1970; Craig et al., 1975). DNA synthesis was monitored by pulse-labelling the cells with [3H]thymidine.The cells were in the Gocondition after 4 days in the ‘low-serum’ medium; by this time the rate of synthesis of DNA had declined almost to zero. In general, after an initial increase, the activity of ribonuclease H appeared to decreaseonly slightly as the cells entered the quiescent state. However, after separation and purification of the four ribonuclease H species, it was found that the activity ofribonucleaseHA1 had decreased 13-fold relative to its activity in exponentially growing cells. Although there was very little change in the activities of 1977


568th MEETING, ABERDEEN Table 1. Ribonuclease H activity in Go-and S-phase cells

Ribonuclease H activity was measured as a ratio of the maximum pmol of ['HIUMP residues rendered acid-soluble by alkaline hydrolysis of the substrate used in the assay (BHK-cell RNA-DNA hybrid made enzymically by using [3H]UTp). This ratio was expressed as a fraction of a unit of activity. The total enzyme activity was the activity of each enzyme purified from Go cells or S-phase cells and totalled for the four enzymes. The number of quiescent cells harvested was 3 . 3 4 ~lo8 and that of S-phase cells was 2.415 x 108. The activity per 106 cells was the total enzyme activity per cell number harvested at quiescence or S-phase. The S/Goratio was the activity per 106 cells for each enzyme at S-phase relative to that at Go-state. Cells in s-phase

Cells in Go-state I

Eazyme Ribonuclease HA1


Total enzyme activity 42.17 515.68 68.48 226.54



Total Activity/106 enzyme cells activity 510.3 0.126 273.0 1.544 100.16 0.205 174.38 0.678


Activity/lP cells 2.113 1.130 0.415 0.722

Ratio S/Go 16.8 0.75 2.024 1.065

ribonucleases HA2, HB1 and HB2, comparison of the profiles of gel filtration on Sepharose 6B of HBl from exponentially growing and quiescent cells revealed (a)less than the usual heterodisperse distribution, (b) a peak of activity corresponding to enzyme of higher molecularweight, and (c)the absenceof activity of DNA polymerase a. On stimulationof the cells to enter a synchronouswave of DNA synthesis(by addition of serum; Craig et al., 1975),the activities of all four ribonucleaseH species increasedand subsequently decreased in parallel with the rate of synthesis of DNA. An increase in the synthesisof DNA was first detected 8h after the serum stimulusand the maximum rate of synthesis was observed at 16-17h. At this point, mid-S-phase, the cells were harvested and extracted, the ribonucleaes H purified, and the activity of each enzyme compared with that in quiescent cells (Table 1). A correlation was observed between the activity of ribonuclease HA1 and the rate of DNA synthesis in uiuo, since the enzyme activity increased 17-fold to the peak of DNA synthesis in cells in S-phase. The activity of ribonuclease HB1 increased only 2-fold; however, on examination of the elution profiles of the enzyme from Sepharose 6B, it was found that the S-phase profile correspondedto that of exponentially-growingcells and not to that from Go-phasecells. The activities of ribonucleases HA2 and HB2 are not obviously related to the rate of DNA synthesis in the cells. We acknowledge with thanks the support given to this work by the Cancer Research Campaign and by Emeritus Principal Sir Edward Wright, University of Aberdeen. Btirk, R. R. (1970) Exp. Cefl Res. 63,309-316 Cooper, R. J., Duff, P. M., Olivier, A., Craig, R. K. & Keir, H. M. (1974) FEBSLett.45,3843 Craig, R. K., Costello, P. A. & Keir, H. M. (1975) Biochern. J. 145,233-240

Vol. 5

C13 cells [proceedings].

676 BIOCHEMICAL SOCIETY TRANSACTIONS Further purification of the DNA methylase activity from the nuclear supernatant fractions was performed by (NH4...
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