PROCEEDINGS OF THE BIOCHEMICAL SOCIETY
(Laird et al., 1969; Uhlenbeck et al., 1971; Wells
et al., 1970) of hybrids is only just becoming apparent.
A more direct approach to the problem of heterogeneity within repeated coding sequences is the
determination of nucleic acid sequences (Brownlee
& Sanger, 1967; Southern, 1970). These techniques
have successfully been employed to determine heterogeneity in the 5 S RNA coding sequences of
Escherichia coli (Brownlee et al., 1968; Jarry &
Rosset, 1971), but there is no unequivocal report of
sequence heterogeneity in an RNA species from a
eukaryote. On the contrary, the 5 S RNA species from
man (Forget & Weissman, 1969) and mouse
(Williamson & Brownlee, 1969) are known to be
identical, implying high evolutionary stability. E. M.
Southern and I have shown that in Xenopus there are
at least three and possibly five different 5S RNA
sequences. The differences are not due to polymorphism in the population, nor to variable end
group, which might occur on precursor molecules
such as in E. coli (Monier et al., 1969). There is
tissue-specific expression of the different sequences,
in that only one appears to be expressed in kidney
cells but all are found in total RNA from ovary. The
ovary unique 5S RNA sequences have been shown
to be incorporated into ribosomes; however, it is not
proven that they are functional.
These observations have clear implications for
ideas about the evolution and organization of repeated coding sequences, the control of genetic
activity and the tissue specificity of ribosomes in
eukaryotic organisms.
I am grateful for support from the Medical Research
Council.
Alloni, Y., Hatleu, L. E. & Attardi, G. (1971) J. Mol.
Biol. 56, 555
Birnstiel, M. L. & Grunstein, M. (1972) Proc. FEBS
Meet. 7th., Varna, in the press
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(1966) Nat. Cancer Inst. Monogr. 23, 431
Birnstiel, M. L., Chipchase, M. & Spiers, J. (1971a)
Progr. Nucleic Acid Res. Mol. Biol. 11, 351
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J. Mol. Biol. 63, 21
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Nat. Acad. Sci. U.S. 68, 3175
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Brownlee, G. G., Sanger, F. & Barrell, B. G. (1968)J. Mol.
Biol. 34, 329
Forget, B. G. & Weissman, S. M. (1969) J. Biol. Chem.
244, 3148
Jarry, B. & Rosset, R. (1970) Biochem. Biophys. Res.
Commun. 41, 789
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Laird, C. D., McConaughy, B. L. & McCarthy, B. J.
(1969) Nature (London) 224, 149
Moore, R. L. & McCarthy, B. J. (1968) Biochem. Genet.
2, 75
33P
Monier, R., Feunteun, J., Forget, B., Jordan, B., Reynier,
M. & Varricchio, F. (1969) Cold Spring Harbor Symp.
Quant. Biol. 34, 139
Smith, I., Dubnau, D., Morrell, P. & Marmur, J. (1968)
J. Mol. Biol. 33, 123
Southern, E. M. (1970) Nature (London) 227, 794
Southern, E. M. (1971) Nature (London) 232, 82
Sutton, W. D. & McCallum, M. (1971) Nature (London)
232, 83
Uhlenbeck, 0. C., Martin, F. H. & Doty, P. (1971)
J. Mol. Biol. 57, 217
Wells, R. D., Larson, J. B., Grant, R. C., Shantle, B. H. &
Cantor, C. R. (1970) J. Mol. Biol. 54, 465
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3, 306
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639
Ribosome Formation in Yeast
By THOMAS H. ROZIJN (Laboratory for Physiological
Chemistry, State University of Utrecht, Utrecht, The
Netherlands)
The synthesis and processing of rRNA both in
bacteria and in eukaryotic cells has been the subject
of considerable investigation (for reviews see Darnell,
1968; Attardi & Amaldi, 1970).
The formation of mammalian rRNA involves a
complex multi-stage maturation process. The two
major rRNA components derive from a common
large precursor molecule containing the sequences
of one molecule of each of both species of mature
rRNA and some excess of non-ribosomal RNA that
is discarded during the maturation process. In bacteria
each of the two species of rRNA is synthesized as
an independent unit that is probably somewhat
larger than the corresponding mature rRNA.
Yeast, a simple eukaryotic protist, resembles in
certain aspects both animal cells and bacteria. Like
all eukaryotes it contains a nucleus, surrounded by
a nuclear envelope. The envelope consists of two
membranes and contains typical nuclear pores
(Robinow & Marak, 1966; Matile et al., 1969;
Molenaar et al., 1970). On the other hand yeast
resembles prokaryotes in that it has a short generation time and no mitotic apparatus.
The DNA content of yeast (about 12 x 109 daltons/
haploid genome) is only about ten times that in
bacteria (Bicknell & Douglas, 1970).
Compared with other cells, yeasts are very rich in
RNA. The amount of RNA is 50-100 times greater
than the DNA content. Most of the total RNA of
the yeast cell is rRNA (Fukuhara, 1967).
The 80S ribosomes of yeast contain a 19S rRNA
species with a molecular weight ofabout 0.7 x 106 and
a 26 S rRNA species with a molecular weight of about
1.3 x106 (Retel & Planta, 1968; Loening, 1968). In
DNA-RNA hybridization experiments about 2 %
of the yeast genome was found to be complementary
34p
PROCEEDINGS OF THE BIOCHEMICAL SOCIETY
to yeast rRNA (Retel & Planta, 1968; Schweizer
et al., 1969). In most other organisms, prokaryotes
as well as eukaryotes, only 0.1-0.3% of the DNA is
complementary to rRNA. From the data it can be
calculated that about 140 cistrons for both 18 S and
26S rRNA are present on the (haploid) yeast genome,
a number comparable with that of higher organisms,
which have a DNA content/haploid genome some
50 times that in yeast. This large redundancy of
ribosomal genes may explain the high rate of rRNA
synthesis in yeast and its high content of cytoplasmic
ribosomes. The number of rRNA cistrons appears
to be constant in yeast cultures of very different
growth rates. The number of rRNA cistrons is thus
not a controlling factor in rRNA synthesis (Schweizer
& Halvorson, 1969).
Yeast resembles other eukaryotes in that the two
rRNA species are initially synthesized as a complex
precursor molecule with a sedimentation coefficient
of about 38S. This large precursor molecule breaks
down to give the 18S rRNA and an intermediate
precursor (28-30S) for the 26S rRNA (Taber &
Vincent, 1969a,b; Sillevis-Smitt et al., 1970, 1972;
Retel & Planta, 1967).
Simultaneously or immediately after their synthesis, the 38S RNA molecules undergo a chemical
modification through the action of methylating
enzymes (Retel et al., 1969; Taber & Vincent, 1969b;
Sillevis-Smitt et al., 1972). This methylation, as in
other eukaryotic organisms, was found to be mainly
ribose methylation (Retel et al., 1969; Isaksson &
Phillips, 1968). A secondary methylation, comprising
about 20% of the total methylation, occurs at a later
stage of the rRNA formation, presumably at the level
of mature rRNA. The secondary methylation is
mainly base methylation.
Polyacrylamide-gel electrophoresis showed the
large rRNA precursor (38S) to be heterogeneous,
comprising two species with molecular weights of
about 2.6 x 106 and 2.3 x 106 (Grierson et al., 1970),
or 2.9 x 106 and 2.4 x 106 (Retel & Planta, 1970),
corresponding to sedimentation coefficients of about
42S and 37S.
From the combined molecular weight of the rRNA
in yeast (about 2.0 x 106), it can be calculated that the
large precursor RNA contains an amount of nonribosomal RNA excess that is about 30% of the
weight of the precursor. This is in accordance with
the results of competition hybridization experiments,
which show that about 40 % ofthe precursor molecule
is of non-ribosomal type and is not conserved during
the processing into mature rRNA (Retel & Planta,
1970).
The development of a technique for fractionating
yeast cells into nuclear and cytoplasmic fractions
(Rozijn & Tonino, 1964) made it possible to study the
site of synthesis and processing of the rRNA precursor molecules (Sillevis-Smitt et al., 1970, 1972)
The heterogeneous 38 S precursor is located exclusively in the nucleus and is present in fairly large amounts.
Although the 18 S rRNA arising from cleavage of the
38 S precursor is rapidly transported to the cytoplasm,
the intermediate precursor (28-30S, mol.wt. about
1.5 x 106) is retained in the nucleus and slowly processed to 26S rRNA, which then enters the cytoplasm.
Electron-microscopic radioautography of nuclei isolated from pulse-labelled yeast cells provided evidence
that the 38 S precursor RNA is confined to an
electron-dense region of the yeast nucleus ('dense
crescent') that has the chemical and morphological
characteristics of a nucleolus (Sillevis-Smitt et
al., 1972; Molenaar et al., 1970). The results of
preliminary experiments (W. W. Sillevis Smitt,
unpublished results) in which a deoxyribonuclease
treatment of isolated nuclei has been used to free
the nucleolar structures from chromatin, suggest that
the 28-30S intermediate precursor is located in the
'dense crescent'. It thus appears that in yeast, as in
animal cells, a nucleolus is the site of ribosome
synthesis.
At some time during the maturation of rRNA
the ribosomal proteins become associated with
the RNA. Warner (1971) has presented evidence
that before to their appearance on ribosomes a large
portion of the ribosomal proteins is found in the
nucleus, where it may be associated with maturing
rRNA. The newly formed ribosomes appear in the
cytoplasm as 40S and 60S ribosomal subunits
(Warner, 1971; Taber et al., 1969).
Attardi, G. & Amaldi, F. (1970) Annu. Rev. Biochem. 39,
183-226
Bicknell, J. N. & Douglas, H. C. (1970) J. Bacteriol. 101,
505-512
Damell, J. E. (1968) Bacteriol. Rev. 32, 262-290
Fukuhara, H. (1967) Biochim. Biophys. Acta 134, 143-164
Grierson, D., Rogers, M. E., Sartirana, M. L. & Loening,
U. E. (1970) Cold Spring Harbor Symp. Quant. Biol.
35,589-598
Isaksson, L. A. & Phillips, J. H. (1968) Biochim. Biophys.
Acta 155, 63-71
Loening, U. E. (1968) J. Mol. Biol. 38, 355-365
Matile, Ph., Moor, H. & Robinow, C. F. (1969) in The
Yeasts (Rose, A. H. & Harrison, J. S., eds.), vol. 1,
pp. 220-302, Academic Press, London and New York
Molenaar, I., Sillevis-Smitt, W. W., Rozijn, T. H. &
Tonino, G. J. M. (1970) Exp. Cell Res. 60, 148-156
RetWl, J. & Planta, R. J. (1967) Eur. J. Biochem. 3, 248258
RetWI, J. & Planta, R. J. (1968) Biochim. Biophys. Acta
169,416-429
Ret6l, J. & Planta, R. J. (1970) Biochim. Biophys. Acta
224,458-469
Ret6l, J., van den Bos, R. C. & Planta, R. J. (1969)
Biochim. Biophys. Acta 195, 370-380
Robinow, C. F. & Marak, J. (1966) J. Cell Biol. 29,
129-151
Rozijn, Th. H. & Tonino, G. J. M. (1964) Biochim.
Biophys. Acta 91, 105-112
PROCEEDINGS OF THE BIOCHEMICAL SOCIETY
Schweizer, E. & Halvorson, H. 0. (1969) Exp. Cell Res.
56, 239-244
Schweizer, E., MacKechnie, C. & Halvorson, H. 0. (1969)
J. Mol. Biol. 40, 261-277
Sillevis-Smitt, W. W., Nanni, G., Rozijn, Th. H. &
Tonino, G. J. M. (1970) Exp. Cell Res. 59, 440-446
Sillevis-Smitt, W. W., Vermeulen, C. A., Vlak, J. M.,
Rozijn, Th. H. & Molenaar, I. (1972) Exp. Cell Res.
70, 140-144
Taber, R. L. & Vincent, W. S. (1969a) Biochem. Biophys.
Res. Commun. 34,488-494
Taber, R. L. & Vincent, W. S. (1969b) Biochim. Biophys.
Acta 186, 317-325
Taber, R. L., Vincent, W. S. & Coetzee, M. L. (1969)
Biochim. Biophys. Acta 195, 99-108
Warner, J. R. (1971) J. Biol. Chem. 246, 447-454
Ribosomal Ribonucleic Acid in Evolution
By U. E. LOENING (Department ofZoology, University
ofEdinburgh, West Mains Road, Edinburgh EH9 3JT,
U.K.)
Aspects of the structure of rRNA in eukaryotes
that illustrate the extent to which different regions of
the molecules have diverged in evolution will be
reviewed; the independent evolution of parts of the
precursors to rRNA will be discussed.
Lava-Sanchez et al. (1972) have shown that the
base composition of rRNA, and especially the relationship of the compositions of the '18 S' and '28 S'
components, can be correlated with the major plant
and animal Phyla. Thus the changes do not seem to
have been entirely random.
M. Grunstein & M. Birnstiel (unpublished work)
have shown, by studying the kinetics of hybridization
of heterologous rRNA to DNA, that regions of the
rRNA molecules have evolved to an extent that
varies with evolutionary separation of the species;
however, a small region of the molecule has been
conserved, even between widely different species such
as Drosophila and mouse and Xenopus.
Despite the large changes in composition and in
sequence, the overall structure of the ribosomal
subunits seems to have been conserved: P. Cammarano, A. Felsani, M. Gentile, C. Gualerzi, A.
Romeon & G. Wolf (unpublished work) have shown
that hybrid 80S ribosomes, prepared from pea and
mouse or rat subunits in either combination, are
active in the synthesis of polyphenylalanine stimulated by poly(U).
The above results may be correlated with a
previous finding (Loening, 1968) that the molecular
weight of the '18S' rRNA has been conserved in all
eukaryotes at 0.7 x 106, whereas that of the '28S'
rRNA increases above 1.3 x 106 only in the animals.
We have since found a number of small deviations
from this rule. The most striking exception, however,
is that of the amoeba Acanthamoeba (Loening, 1968),
35P
in which the molecular weights of the rRNA are
1.6 x 106 and 0.9 x 106. We have now shown that the
component of molecular weight 1.6 x 106 is cleaved
less than 2h after synthesis into two components
plus '7S' RNA, which can be detected only after
unfolding the molecule in 50 % formamide. The two
components have molecular weights of about 0.9 x
106 and 0.7 x 106; thus one of them has the same
molecular weight as the amoeba '18S' RNA and the
other the same molecular weight as the '18 S' RNA
from other eukaryotes.
This may be a coincidence, but it does again raise
the question whether the '28 S' rRNA has evolved
by duplication of the '18 S' rRNA gene followed by
some divergence. There are many species in which the
'28 S' RNA is cleaved in or near the middle. In most
plants and in yeast, but not in Xenopus, there is considerable homology between the '18S' and '28S'
rRNA species, as measured by competitive hybridization.
There is no such systematic evolution of the precursor to rRNA. The amount of excess of nonribosomal RNA in the precursor is much greater in
warm-blooded animals than in any other species, and
among the latter certain parts of the precursor seem
to have changed very rapidly in size. In the amoeba
mentioned above the amount of excess of RNA in
the precursor is also exceptionally large, so that the
molecular weight of the precursor is about 3.5 x 106,
compared with about 2.5 x 106 in other plants and
protozoa.
It is clear from all these results that different parts
of the ribosomal genes have changed at very different
rates in evolution.
Lava-Sanchez, P. A., Amaldi, F. & La Posta, A. (1972)
J. Mol. Biol. in the press
Loening, U. E. (1968) J. Mol. Biol. 38, 355-365
Regulation of Ribosome Synthesis
By ROBERT P. PERRY (Institute for Cancer Research,
Philadelphia, Pa. 19111, U.S.A.)
Knowledge of the mechanisms that regulate ribosome production in eukaryotic cells is still very
fragmentary and incomplete. However, a considerable number of phenomena concerned with the
regulation of ribosome synthesis have been described.
Although these phenomena are usually not well
understood in terms of mechanism, they nevertheless
provide some clues and insights into the general types
of regulatory mechanisms that are operative. Moreover, it is becoming evident that some of the general
principles involved in the control of ribosome biosynthesis are also applicable to the regulation of
other macromolecular constituents of the cell. A
brief review and analysis of some of the following
phenomena will be presented.