The Effect of Oxidised Nicotinamide

soluble fraction from infected cells, whereas preimmune immunoglobulin G did not.
These results are consistent with there being an important role for the DNA-binding
protein in adenovirus-DNA replication as suggested by Van der Vliet et al. (1977).
Further studies are required to characterize the products of DNA synthesis in vitro,
and the host-cell enzymes and virus proteins involved in this process.
Ensinger, M. J. & Ginsberg, M. S. (1972) J. Virol. 10, 328-339
Philipson, L., Pettersson, U. & Lindberg, U. (1975) Molecular Biology of Adenouiruses,
pp. 11-16, Springer-Verlag, Vienna and New York
Russell, W. C. & Blair, G. E. (1977) J. Gen. Virol. 34, 19-35
Weingartner. B., Winnacker, E. L., Tolun, A. & Pettersson, U. (1976) Cell 9, 259-268
Wilhelm, J., Brison, O . , Kedinger, C. & Chambon, P. (1976) J. Virol. 19, 61-81
Van der Vliet, P. C., Zandberg, J. & Jansz, H. S. (1977) Virology 80, 98-110
The Effect of Oxidised Nicotinamide-Adenine Dinucleotide on Ribonucleic
Acid Synthesis in Isolated Nuclei from Baby-Hamster Kidney Cells
Department of Biochemistry, University of Aberdeen,
Marischal College, Aberdeen AB9 1 AS, Scotland, U.K.
Several studies on mammalian cell metabolism have established that there is a
relationship between the intracellular concentration of NAD+ and the growth state of the
cell (Wintzerith et al., 1961; Ferris & Clark, 1971; Schwartz et al., 1974). This
relationship led to the idea, originally proposed by Morton (1958), that NAD+ may
control cellular growth. The way in which this could occur is not readily understandable
in terms of the classic role of NAD+ as a cofactor in biological redox reactions.
There are, however, reactions in which NAD+ participates not as a cofactor but as a
substrate. In particular, it is now appreciated that the ADP-ribose moiety of NAD+
may be covalently bound to proteins (Sugimura, 1973; Hilz & Stone, 1976;-Shall et al.,
In mammalian cells the enzyme that catalyses this reaction (ADP-ribosylation) is
localized in the nucleus, the site of NAD+ biosynthesis. The mammalian enzyme
[poly(ADP-ribose) polymerase] can also catalyse the addition of further ADP-ribose
units (by a 2’-1’ glycosidic linkage), forming the homopolymer poly(ADP-ribose).
Poly(ADP-ribose) polymerase modifies all classes of nuclear protein in uitro (Rickwood
et al., 1977). Thus it seems unlikely that this modification reaction will have a unique
role in cellular metabolism. It does, however, suggest that this modification reaction
may be the link between the intracellular NAD+ concentration and the growth state
of the cell.
If we accept this hypothesis, then it seems likely that ADP-ribosylation of nuclear
proteins will be involved in the three major cellular growth processes, i.e. DNA,
RNA and protein synthesis. In the case of DNA and RNA synthesis it is possible
to test this involvement directly. Isolated nuclei from mammalian cells are capable of
synthesizing DNA and RNA in vitro (Kidwell & Mueller, 1969; Zylber &Penman, 1971).
A possible experimental approach therefore would be to examine the effect of NAD+
on DNA and RNA synthesis in isolated nuclei.
The effect of NAD+ on DNA synthesis in isolated nuclei has been extensively studied
(Burzio & Koide, 1970; Roberts et al., 1973; Burzio et al., 1975; Suhadolnik et al., 1977).
On the other hand there are few reports dealing specifically with the involvement of
ADP-ribosylation in RNA synthesis. Miiller & Zahn (1976) reported that the activity
of the endogenous RNA polymerase in isolated nuclei from quail oviduct is inhibited
by the addition of NAD+. They also provided evidence for a covalent linkage between
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ADP-ribose and RNA polymerase I. In the present communication we describe the
effect of ADP-ribosylation of nuclear proteins on RNA synthesis in isolated nuclei from
BHK (baby-hamster kidney) cells.
Previous work (Furneaux & Pearson, 1977) has established that NAD+ added
to nuclei isolated from BHK cells is rapidly incorporated into acid-insoluble material.
This acid-insoluble material was shown to be composed of ADP-ribose. To examine
the effect of NAD+ on RNA synthesis in isolated nuclei it is necessary to analyse its
metabolic fate in more detail. Extraction of the acid-soluble fraction, after incubation of
NAD+ with isolated nuclei, and analysis by PEI [poly(ethyleneimine)]~ellulose t.1.c.
revealed no other acid-soluble metabolites.
Analysis of the acid-insoluble material by Cs2SO4-density-gradient centrifugation
(containing 6 ~ - u r e aand 0.4~-guanidiniumchloride to disrupt non-covalent binding)
revealed the association of radioactive residues and nuclear proteins. Hydrolysis with
NaOH removed the ADP-ribose residues from nuclear protein. These results strongly
suggest that the ADP-ribose residues are covalently bound to nuclear proteins.
RNA synthesis in isolated nuclei was followed by the incorporation of [3H]UTP
into acid-insoluble material. We have found that the effect of NAD+ on RNA synthesis
in these nuclei depends on the ionic strength of the incubation medium. Under
conditions of low ionic strength, NAD+ inhibits RNA synthesis, whereas at high ionic
strength it stimulates RNA synthesis. Addition of nicotinamide (a powerful inhibitor
of ADP-ribosylation) prevents the inhibition. This demonstrates the effects induced
by NAD+ are due to ADP-ribosylation of nuclear proteins and not to any non-specific
changes in incubation conditions. With our method of isolation, nuclei from BHK
cells were found to contain two RNA polymerase activities (Cooper & Keir, 1975).
These correspond to the RNA polymerase I and I1 isolated by other workers. In
general, RNA polymerase I is most active under conditions of low ionic strength,
whereas RNA polymerase I1 is most active under conditions of high ionic strength.
Thus it seems likely that ADP-ribosylation of nuclear proteins inhibits RNA polymerase
I activity and stimulates RNA polymerase I1 activity. We have studied the inhibition
in more detail. To distinguish between the two enzymes, a-amanitin, a specific
inhibitor of RNA polymerase I1 (Kedinger et al., 1971) was added to the incubation
.-> 1 .o
x 0.5
Time (min)
Fig. 1 . Effect of preincubating nuclei with N A D + on the subsequent activity of partially
purified RNA polymerase I
Activity of RNA polymerase I isolated from control nuclei; 0,activity of RNA
polymerase I isolated from nuclei previously incubated with NAD+.
medium. Under low-ionic-strength conditions and in the presence of a-amanitin
(l,ug/ml) the inhibition of RNA synthesis by NAD+ was increased. This inhibition may
be due either to the ADP-ribosylation of the DNA-binding proteins that control the
transcription of the DNA template or to the direct modification of the RNA polymerase
itself. To distinguish between these two possibilities, nuclei were incubated in the
presence and absence of NAD+. The RNA polymerases were then extracted from the
control and NAD+-treated nuclei and assayed with exogenous DNA template in the
presence of a-amanitin. The result shown in Fig. 1 suggests that the inhibition is due to the
ADP-ribosylation of RNA polymerase I.
We thank the Medical Research Council for support, and acknowledge the skilled technical
assistance of Mrs. Alison Blair.
Burzio, L. & Koide, S . S . (1970) Biochem. Biophys. Res. Commun. 40, 1013-1020
Burzio, L., Reich, L. & Koide, S . S . (1975) Proc. SOC.Exp. Biol. Med. 149,932-938
Cooper, R. J. & Keir, H. M. (1975) Biochem. J. 145, 539-516
Ferris, G. M. & Clark, J. B. (1971) Biochem. J. 121, 655-662
Furneaux, H. M. & Pearson, C. K. (1977) Biochem. Soc. Trans. 5, 745-747
Hilz, H. & Stone, P. (1976) Rev. Physiol. Biochem. Pharmacol. 76, 1-58
Kedinger, C., Nuret, P. & Chambon, P. (1971) FEBSLett. 15, 169-174
Kidwell, W. E. & Mueller, G. C. (1969) Biochem. Biophys. Res. Commun. 36, 756-763
Morton, R. K. (1958) Nature (London) 181, 540-542
Miiller, W. E . G. & Zahn, R. K. (1976) Mol. Cell. Biochem. 12, 147-159
Rickwood, D., MacGillivray, A. J. & Whish, J. D. (1977) Eur. J . Biochem. 7 9 , 589-598
Roberts, J. H., Stark, P. & Smulson, M. (1973)Biochem. Biophys. Res. Commun. 52,43-50
Schwartz, J. P., Passoneau,J. V., Johnson, G. S . &Pastan,I. (1974)J . Biol. Chem.249,4138-4143
Shall, S . , Goodwin, P., Halldorsson, H., Khan, G., Skidmore, C. & Tsopanakis, C. (1977)
Biochem. SOC.Symp. 42, 103-116
Sugimura, T. (1973) Prog. Nucleic Acid Res. Mol. Biol. 13, 127-151
Suhadolnik, R. J., Baur, R., Lictenwaler, D. M., Uematsu, T., Roberts, J. H., Sudhakar, S .
& Smulson, M. (1977) J. Biol. Chem. 252, 4134-4144
Wintzerith, M., Klein, N., Mandel, L. & Mandel, P. (1961) Nature (London) 191,467-469
Zylber, E. A. & Penman, S . (1971) Proc. Natl. Acad. Sci. V.S.A. 68, 2861-2865
Oestrogen-Induced Changes in the Population of Uterine
Polyribosomal Polyadenylated RNA
Institute of Biochemistry, University of Glasgow, Glasgow G12 SQQ, ScotIand, U.K.
Complementary DNA, prepared from polyribosomal polyadenylated uterine RNA,
has been used to study oestrogen-induced changes in the mRNA population of rat
uterus. Uterine polysomes were prepared as previously described (Merryweather &
Knowler, 1977) and polysomal RNA was extracted as follows: polysomal pellets were
suspended in 2ml of 0.1 M-buffer (0.1 M-NaCI, 0.001 M-EDTA,0.01 M-Tris/HCI, pH7.5,
and 0.2% sodium dodecyl sulphate) containing 200pg of proteinase K [Boehringer
Corp. (London), London W.5, U.K.] and incubated at 37°C for 30min. Then 2ml of
phenol/chloroform/3-methylbutan-l-ol(1 : 1 :0.1, by vol.) was added and vortexed
briefly. RNA was extracted on a mechanical shaker for lOmin at room temperature.
The phases were separated by centrifugation at 20000g for 6min at 4°C. The
phenol and interphase were subjected to two further extractions with 0.1 M-Tris/HCl
buffer, pH9.0, containing 1 mg of bentonite/ml and 1 % (w/v) sodium dodecyl
sulphate. The combined aqueous phases were centrifuged at 20000g for 15min to remove
bentonite and subsequently precipitated with 2vol. of ethanol at -20°C overnight. The
polysomal RNA was washed twice with ethanol and dried in a gentle stream of N2.
Polysomal RNA (0.9-lmg) was then chromatographed on 1.5-2ml bed volume of
poly(U)-Sepharose 4B [Pharmacia (G.B.), London W.5, U.K.] and poly(A)+ RNA
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