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incorporation in anterior forebrain roof of exposed versus dark-box birds (Eso/Dso
= 1.25; P ~ 0 . 0 3 )The
.
same trend appeared in posterior forebrain roof hut was not
significant.
Thus in the forebrain roof of experimental birds, especially the anterior portion,
an elevation of pool radioactive lysine was accompanied by an increased incorporation
of this amino acid into protein as compared with dark-box birds. However, increases in
pool radioactivity of an even greater magnitude occurred in the forebrain base and
midbrain, but these were not accompanied by differential changes in incorporation.
The fact that pool radioactive lysine changes were accompanied by changes in incorporation only in the anterior forebrain roof in the present experiments, taken in
conjunction with the results of previous experiments, suggests that a population of
cells in that region responds to the imprinting stimulus with metabolic changes involving
protein synthesis. The degree to which this may or may not involve specific proteins must
await the results of more refined biochemical and cellular dissections.
This work was supported by an M.R.C. grant to S.P.R.R. which we gratefully acknowledge.
Bateson, P. P. G.,Horn, G . & Rose, S. P. R. (1972) Brain Res. 39,449-465
Hambley, J. & Rose, S. P. R. (1972) Biochem. J. 1 2 7 , 9 0 ~
Haywood, J., Rose, S. P. R. & Bateson, P. P. G . (1970) Nature (London)229,373-374
Haywood, J., Hambley, J. & Rose, S. P. R. (1973) Znt. SOC.Neurochem. 4th, Tokyo, 41 3
Horn, G., Bateson, P. P. G . & Rose, S. P. R. (1973) Science 181, 506-514
Developmental Changes in the Potassium Chloride-Induced Dissociation
of Rat Cerebral Free Polyribosomes
JOHN 0. WHITE, LOUIS LIM and ALAN N. DAVISON
Department of Neurochemistry, Institute of Neurology, National Hospital,
Queen Square, London WC 1N 3BG, U.K.
Mammalian free cytoplasmic polyribosomes can be dissociated into constituent 60s
and 40s subunits by treatment with O.~M-KCI.A proportion remains as 7 5 s particles
that appear to be single ribosomes stabilized by the presence of nascent peptides, since
preincubation with puromycin, followed by the KCl treatment, results in complete
dissociation of the polyribosomes. This procedure has been used to isolate haemoglobin
mRNA released from dissociated reticulocyte polyribosomes (Blobel, 1971).
We have used this method in an attempt to isolate subunits and mRNA from the
cerebral polyribosomes of the developing rat. The average size of cerebral free polyribosomes isolated from newborn rats is greater than that of corresponding polyribosomes
from the immature or adult rat (Zomzelly et al., 1971). When these polyribosomes are
subjected to the KCI treatment, preparations from the immature and adult rats show the
same general mammalian pattern in that a population of ribosomes refractory to the
KCI treatment can be separated together with the subunits. Although preincubation
with puromycin caused the complete dissociation of the polyribosomes with KCI, no
mRNA appeared to be released by this procedure. The possibility that mRNA was
released but co-sedimented with the 40s subunit on analysis on sucrose density gradients
cannot be discounted. Complete dissociation of polyribosomes from the newborn rat
could be achieved by the KCI treatment with or without the use of puromycin. The
‘refractory’ 7 5 s particle could be detected by the first week after birth. Similar studies
with hepatic polyribosomes showed that at all times after birth, preincubation with
puromycin was essential to ensure complete dissociation of polyribosomes with KCI.
The relative ease of dissociation with KCl alone appears to be a characteristic property
of the cerebral free polyribosomes isolated from newborn rats.
This research was supported by the Nuffield Foundation and the Medical Research Council.
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BIOCHEMICAL SOClETY TRANSACTlONS
Blobel, G . (1971) Proc. Nut. Acad. Sci. U.S.68,832-835
Zomzelly, C. E., Roberts, S., Peache, S. & Brown, D. M. (1971) J. Biol. Chem. 246,2097-2103
Nucleic Acid Metabolism in the Developing Rat Brain
WOLFGANG BERTHOLD, LOUIS LIM and ALAN N. DAMSON
Department of Neurology, National Hospital, Queen Square, London WC1N 3BG, U.K.
In the developing rat brain, the early period after birth is characterized by extensive
cellular proliferation and differentiation. This period is followed by one of growth and
maturation in which myelination is one of the most significant events taking place.
We have begun investigations into the changes in nucleic acid metabolism that arise
during the transition from the state of cellular proliferation to the process of myelination
in the rat brain.
[32P]Piwas injected intracisternally into rats of different ages. Nucleic acids were
extracted and purified from cerebral preparations at different time-intervals after the
injection. High-molecular-weight RNA was separated from tRNA and DNA by
precipitation with 2M-LicI. The nucleic acids were analysed by electrophoresis on
polyacrylamide gels of various concentrations, before and after the LiCl step, and uptake
of label into the various fractions was assayed. DNA synthesis was marked up to 10
days after birth and barely detectable in the actively myelinating rat (3-4 weeks old).
In the newborn, the rate of labelling of DNA was higher than that of ribosomal RNA for
several hours after the administration of [32P]Pi.At early time-periods there was extensive uptake into the high-molecular-weight RNA, including ribosomal RNA, with
most of the label appearing in the 28s and 185 RNA species 5 h after injection. In the
preparations from the myelinating rat, there was also extensive labelling of the highmolecular-weight RNA with a pronounced uptake of label into the ribosomal RNA by
5h. Even at this time, and up to 12h, a highly labelled heavy RNA fraction (>30S)
could still be detected, as distinct from the case of the labelled RNA isolated from the
newborn.
The LiCI-precipitated RNA, labelled for short time-periods, was analysed by oligo(dT)
cellulose column chromatography for mRNA content (Aviv & Leder, 1972). Since the
RNA analysed was the total cellular high-molecular-weight RNA, the ‘mRNA’ fractions
would include both ‘mRNA’ and the precursors to the ‘mRNA’. About 30% of the
labelled RNA in the myelinating rat was ‘mRNA’ up to 5 h after labelling, whereas the
corresponding values for the RNA from the newborn were 12 % in 1h, falling to 4-5 %
by 5h. These results suggest that in the newborn rat cerebral cortex, the synthesis of
ribosomal RNA represents a greater proportion of total RNA synthesis than that in the
myelinating rat, where a substantial portion of the RNA synthesized is ‘mRNA’, as
judged by the presence of poly(A) regions (Lim & Canellakis, 1970).
This research was supported by the Nuffield Foundation and the Medical Research Council.
Aviv, H. & Leder, P. (1972) Proc. Nut. Acad. Sci. U.S.69,1408-1412
Lim, L. & Canellakis, E. S . (1970) Nature (London)227, 710-712
1974