10. D N A A N D R N A S Y N T H E S I S
325
In spite of the last results, the use of thymidine uptake alone to
assess D N A synthesis does need caution, particularly where no synthesis
is indicated. Thymidine is not a normal D N A metabolite and some
cells may be incapable of utilizing it; this may be so for certain leukaemic
cells in culture (Gavosto, Maraini and Pileri, 1960a, b ) , as was pointed
out by Bianchi (1962) who has, in fact, demonstrated a very low level of
thymidine kinase in leukaemic cells.
With the aid of rat-fibroblast cultures partially synchronized with
respect to mitosis, Harris (1959a) has attempted to follow the relative
timing of thymidine incorporation into the nucleolus and chromatin
separately. He has concluded that nucleolar incorporation precedes
chromatin incorporation during interphase in his material. However,
this does not appear to be the case in randomly-growing cultures of the
five types of cells I have investigated (see above), where the nucleolar
incorporation of thymidine during interphase closely parallels the
timing of incorporation into the chromatin (Seed, unpublished experiments) .
An interesting exception to the nuclear localization of thymidine is
given by the autoradiographic experiments of Chevremont and co-workers (Chevremont, Chevremont-Comhaire and Baeckeland, 1959a, b ) .
In these experiments, chick-heart cells in culture were treated with an
acid DNAase. After the treatment, it was observed that the mitochondria were able to incorporate thymidine and could also be
stained by the Feulgen reaction, although there did not appear to be
a loss of D N A from the nucleus. These puzzling results pose several
problems: could the treatment result in a slight loss of nuclear D N A not
large enough to be detectable by the measurements, but sufficient to
act subsequently as a "primer" in the cytoplasm? Or could the mitochondria contain a small amount of D N A which is not normally in the
DNA-priming state but which could be activated by the DNAase?
Under certain conditions, DNAase has been shown to increase the
priming ability of D N A in Kornberg's in vitro system for D N A synthesis
(Lehman, 1959; Mantsavinos and Canellakis, 1959). O n the other
hand, in the absence of a D N A primer, could an " A T " co-polymer be
produced in the mitochondria in the presence of cytoplasmic polymerase? The polymerase enzyme has been found in cytoplasmic extracts in
some systems (Bollum and Potter, 1958; Smellie, Keir and Davidson,
1959; Prescott, Bollum and Kluss, 1962). Light could perhaps be
thrown on these matters by analysis of the nucleotide-base composition
of the D N A synthesized in the cytoplasm in the experiments of Chevremont et al.
Firket, Chevremont-Comhaire and Chevremont (1955) have also
observed an effect of RNAase on chick fibroblasts in culture. In a
325
In spite of the last results, the use of thymidine uptake alone to
assess D N A synthesis does need caution, particularly where no synthesis
is indicated. Thymidine is not a normal D N A metabolite and some
cells may be incapable of utilizing it; this may be so for certain leukaemic
cells in culture (Gavosto, Maraini and Pileri, 1960a, b ) , as was pointed
out by Bianchi (1962) who has, in fact, demonstrated a very low level of
thymidine kinase in leukaemic cells.
With the aid of rat-fibroblast cultures partially synchronized with
respect to mitosis, Harris (1959a) has attempted to follow the relative
timing of thymidine incorporation into the nucleolus and chromatin
separately. He has concluded that nucleolar incorporation precedes
chromatin incorporation during interphase in his material. However,
this does not appear to be the case in randomly-growing cultures of the
five types of cells I have investigated (see above), where the nucleolar
incorporation of thymidine during interphase closely parallels the
timing of incorporation into the chromatin (Seed, unpublished experiments) .
An interesting exception to the nuclear localization of thymidine is
given by the autoradiographic experiments of Chevremont and co-workers (Chevremont, Chevremont-Comhaire and Baeckeland, 1959a, b ) .
In these experiments, chick-heart cells in culture were treated with an
acid DNAase. After the treatment, it was observed that the mitochondria were able to incorporate thymidine and could also be
stained by the Feulgen reaction, although there did not appear to be
a loss of D N A from the nucleus. These puzzling results pose several
problems: could the treatment result in a slight loss of nuclear D N A not
large enough to be detectable by the measurements, but sufficient to
act subsequently as a "primer" in the cytoplasm? Or could the mitochondria contain a small amount of D N A which is not normally in the
DNA-priming state but which could be activated by the DNAase?
Under certain conditions, DNAase has been shown to increase the
priming ability of D N A in Kornberg's in vitro system for D N A synthesis
(Lehman, 1959; Mantsavinos and Canellakis, 1959). O n the other
hand, in the absence of a D N A primer, could an " A T " co-polymer be
produced in the mitochondria in the presence of cytoplasmic polymerase? The polymerase enzyme has been found in cytoplasmic extracts in
some systems (Bollum and Potter, 1958; Smellie, Keir and Davidson,
1959; Prescott, Bollum and Kluss, 1962). Light could perhaps be
thrown on these matters by analysis of the nucleotide-base composition
of the D N A synthesized in the cytoplasm in the experiments of Chevremont et al.
Firket, Chevremont-Comhaire and Chevremont (1955) have also
observed an effect of RNAase on chick fibroblasts in culture. In a