[CANCER RESEARCH 31, 688-693,
May 1971]
Transfer RNA Modifications and Synthesis in Animal Cells
M. W. Taylor, S. A. S. Volkers, B. K. Choe, and J. G. Zeikus
Department of Microbiology, Indiana University, Bloomington, Indiana 47401
SUMMARY
and even between species of animals. A minor difference was
observed between seryl-tRNA's from kidney and liver (23).
A review of alterations found in Chromatographie patterns
of specific species of tRNA's from both developing systems
However, when tRNA from different types of cells in
cultures were compared, or when tRNA from neoplastic cells
were compared with normal cells, varying Chromatographie
profiles were found (9, 23). As is summarized in Chart 1 for
tyrosyl-tRNA, these differences were demonstrated as shifts in
elution profile or new peaks. It could be demonstrated that
tyrosyl-tRNA from epithelial cells and fibroblastic cells
chromatographed
differently. Likewise, one could correlate
shifts in profile of tyrosine tRNA with the population of cell
types in culture. These were the first reported variations in
tRNA noted in mammalian systems.
The tumors (Ehrlich ascites tumor and Sarcoma-1), cell
lines, and viral transformed-cells used in this study have
undergone thousands of transfers, in which subpopulations of
cells may have been selected. It is also possible that in certain
cell lines, such as HeLa in which both peaks of tyrosine tRNA
were noted, there is population heterogeneity. We also realize
that a limitation of this system is the use of MAK
chromatography. The number of isoaccepting species detected
by this method is low in comparison to the number found by
reverse
phase
chromatography
or
BD-cellulose
chromatography. The best example of this discrepancy is in
serine tRNA, in which 1 or perhaps 2 species are observed by
MAK chromatography compared to BD-cellulose in which 4
to 5 species may be noted (Chart 2). We have noted
quantitative variations in tRNA species between cell types on
BD-cellulose, whereas the same tRNA's eluted as single peaks
and tumors is presented. Such differences have been observed
in the early stages of sea urchin development and between
tumors and their tissues of origin. In particular, the meaning of
differences found between tRNA from Morris hepatomas and
normal rat liver is discussed. Evidence is presented that tRNA
synthesis in mammaliam cells proceeds via a short-lived
precursor state and that the finished tRNA's are heterogeneous
in size. It is suggested that the Chromatographie variations
noted in these systems may reflect differential gene
transcription.
That cellular differentiation
may be controlled at the
translational level was first suggested by Ames and Martin (1)
and Stent (19). Both of these authors suggested a modulation
hypothesis to explain the polarity of gene products in the
histidine-biosynthetic system of Escherichia coli and variations
in hemoglobin synthesis in higher animals. This hypothesis
states that variations in the amount of different amino
acyl-tRNA's or modifications of tRNA molecules will limit the
synthesis of specific proteins by blocking the continuation of
protein synthesis, e.g., by the premature release of polysomes
from the mRNA-polysome complex.
This model can also be used to explain events subsequent to
virus infection. Host protein synthesis, which is shut off
shortly after virus infection, can be explained as resulting from
modification of a preexisting tRNA or formation of a new
tRNA that no longer recognizes a specific host codon. There
are other mechanisms now known that explain host shutoff,
such as a factors (4); however, this modulation hypothesis
prompted Sueoka and Kano-Sueoka (20) to investigate tRNA
synthesis after T2 infection of E. coli. This resulted in the
finding of a new species of leucyl-tRNA formed within 3 min
of T2 infection (review, Ref. 21).
During the last 4 years, we have been actively engaged in
examining tRNA variations in different developing systems.
This work was initiated in the laboratory of Dr. J. Holland. I
shall briefly review our techniques and original observations.
tRNA from 2 different sources, labeled with 14C- or
3H-labeled amino-acid was cochromatographed
on MAK1
columns. Fractions were collected and counted, and the
elution profiles of the labeled tRNA's were compared. Very
few differences were found between organs of the same animal
' The abbreviations used are: MAK, methylated albumin-Kieselguhr;
BD-cellulose, benzoylated diethylaminoethyl cellulose.
688
at different salt concentrations on MAK columns.
To overcome the objections of population heterogeneity,
we studied 2 other systems in this laboratory.
Sea Urchin
The sea urchin has been an ideal system for the study of
early development. One has the opportunity to compare the
events that lead from the unicellular, unfertilized egg to the
multicellular blástula stage within a period of 24 hr. At about
this time, the blástulabegins to form at its vegetative pole, the
primary mesenchyme. Mesenchyme formation continues at the
vegetative pole by invagination to form the primitive gut (26).
The part of the developmental process that we have
examined so far is the time from fertilization to the formation
of the late mesenchyme blástula. During this period, no
growth takes place, metabolic activity is low, and the shape of
the embryo does not change. In the mature, unfertilized egg,
RNA synthesis and protein synthesis are halted. The absence
of protein synthesis appears to be limited by RNA activity and
not by the absence of any species of RNA. rRNA, tRNA, and
CANCER RESEARCH VOL. 31
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tRNA Modification and Synthesis in Animals
mRNA are all present in the unfertilized egg and are functional
in in vitro systems (5, 18, 28). The reason for the lack of
activity of mRNA in the unfertilized egg is unknown.
On fertilization of the sea urchin egg, there is a rapid
increase in protein synthesis. This protein synthesis is due to
the utilization of previously existing mRNA and not due to
synthesis of new mRNA (8). Thus the initial regulation of
protein synthesis in the fertilized egg is at the translational
level. The proteins synthesized at this early stage are primarily
associated with mitotic apparatus and histones. During this
period, there is a rapid turnover of the CCA end group of
tRNA molecules (7,13, 27).
The 2nd period of translation of mRNA occurs at the
mesenchyme blástula stage. The rate of protein synthesis rises
quite steeply, leveling off after gastrulation. This 2nd period of
TYHOSINl
FROM
[PITHEUAL
WHITE
AND
TYROSINE
IHNA
FROM
MAMMALIAN
BOTH
CELLS.
BLOOD
(BOM
AND
CELLS
TYROSINE
tRNA
FIBÜOBI ASÕS
FROM
MAMMALIAN
CHICKEN
tRNA
ORGANS
CHICKEN
DIFFERENTIATED
~"~
TYROSINE
FROM
tRNA
CERTAIN
MAMMALIAN
TUMOR
new RNA is synthesized prior to late gastrulation. Others,
however, have found that a low level of mRNA is synthesized
from the 4-cell stage and some rRNA is synthesized at the
blástula stage. No de novo synthesis of tRNA has been
reported prior to gastrulation.
The question that we were asking is whether any new
species of tRNA (not necessarily newly synthesized) occur in
response to the presence of new mRNA. If such new tRNA
were found, it would give credence to the role of tRNA in
modulating or initiating gene transcription during differentia
tion.
MAK
column
chromatography
of
8
different
aminoacyl-tRNA's from unfertilized eggs and mesenchyme
blástulawere compared. No elution differences could be found
for arginyl-, tyrosyl-, valyl-, phenylalanyl-, or aspartyl-tRNA.
However,
repeatable
Chromatographie
differences
were
detected for leucyl-, seryl-, and lysyl-tRNA. In the case of
leucyl-tRNA, we have a distinct quantitative difference in
serine, an overall shift in profile, and, in lysyl-tRNA, both a
shift and a new species (Chart 3). That these are tRNA
differences and not changes in acylating enzymes was shown
by charging with heterologous enzymes (30).
This type of data conclusively shows that, even in
"controlled"
systems, new tRNA species are formed or
DIFFERENTIATED
MAMMALIAN
ORGANS
protein synthesis arises in response to gene transcription
during cleavage (8).
Prior to gastrulation, very little new RNA is synthesized.
Comb et al. (7) has shown by pulse labeling with 32P that no
CELLS
modified during differentiation.
These data suggest that
certain species of blástulatRNA's differ from egg tRNA. Since
reports in the literature agree that no or very little new tRNA
is formed during this stage of development, these differences
in profile must result from modifications of existing tRNA's.
Chart 1. Schematic presentation of the MAK column elution
positions of tyrosyl-tRNA from a number of sources (9).
However, these modifications, whether they be due to
methylation or thiolation of other enzymatic modification
must be selective in nature, since not all tRNA's are altered.
1000
9OO
8OO
g
7OO
<3H> EHRLICH
ASCITES
TUMOR
Chart 2. A comparison of the elution
profile of seryl-tRNA from a MAK
column (insert) and a BD-cellulose
column.
8"eoo
50O
4OO
3OO
2OO
1OO
-L.
10
20
JL
30
_U
40
FRACTION
50
J_
60
_L
7O
8O
9O
1OO
NUMBER
MAY 1971
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689
Taylor, Volkers, Choe, and Zeikus
Chart 3. The elution profile of leucyl-, seryl-, and lysyl-tRNA's from unfertilized sea urchin eggs and 24-hr mesenchyme blástula.Methods are
described in Ref. 30.
Morris Hepatomas
The Morris hepatomas (12) were developed as a system of
tumors that were highly differentiated and similar in many
respects to the normal liver. A review by Wu (29) has recently
shown, however, that these tumors do differ from each other
and from normal liver in many enzyme activities. Chromosome
numbers may vary from the diploid number, 42 to 96 for
5123C. There appears to be some correlation between rate of
growth, chromosome number, and enzyme activity, but no
definite conclusions can be drawn.
However, if tRNA modifications correlate with degree of
differentiation or neoplasia, it should be possible to correlate
these changes with differing hepatomas. We have, therefore,
been studying tRNA from 3 hepatomas of varying degrees of
differentiation, namely 9618A (highly differentiated), 5123D
(well-differentiated),
and 3924A (poorly differentiated). We
have compared the isoaccepting species of leucyl-, lysyl-,
tyrosyl-, seryl-, histidyl-, and phenylalanyl-tRNA's from these
3 hepatomas, normal rat liver, and regenerating rat liver, by
reverse phase partition cochromatography.
Regenerating rat liver and normal rat liver demonstrate no
690
detectable differences, suggesting that growth rate (or rate of
synthesis) is not a factor in modification. No differences were
detectable between hepatoma 3924A and tRNA isolated from
rat liver. However, with hepatoma 5123D and 9618A, distinct
repeatable differences were found. Seryl-tRNA from hepatoma
5123D shows 2 distinct peaks over and above those found in
normal liver, one large and the other small. An extra species of
phenylalanyl-tRNA 5123D is found when compared to rat
liver and differences in histidine tRNA 5123D (Chart 4).
Hepatoma 9618A exhibited two more lysyl-tRNA species and
one more phenylalanyl-tRNA than did normal rat liver. All of
these differences are repeatable with heterologous enzymes.
The fact that these differences are not detectable in
hepatoma 3924A but present in 5123D and 9618A would
suggest that these changes are not the result of humoral
processes, effect of location, or even rate of growth, since
hepatoma 5123D is an intermediate-growing
tumor. Also,
regenerating rat liver did not differ from normal rat liver. One
therefore must conclude that these differences are intrinsic to
the cells of hepatoma 5123D and specific for this tumor.
However, whether they are related to the neoplastic process
and involved in modulation we cannot tell. There may be
CANCER RESEARCH VOL. 31
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tRNA Modification and Synthesis in Animals
modifications
in aminoacyl-tRNA's
of hepatoma 3924A which
These modifications may not be unique to tumor cells but
may be present in other tissues. Turkington and Riddle (25),
for instance, have found that 7-methylguanosine found in
mammary carcinoma tRNA, is also present in other tissues.
regulation. However, thus far there is no evidence for the Enzymatic modifications may occur in other cell types and
biological function of these differences. We have examined the may reflect physiological conditions (e.g., hormonal effects)
codon response of an altered phenylalanyl-tRNA from Ehrlich on the tissues.
Differential Gene Transcription. Genes for tRNA are highly
ascites tumor (22) without detecting any differences. It is
possible that these altered tRNA's have a very subtle biological redundant in cells of higher organisms (14, 16). This is
role not related to codon response and not detectable with probably true of most genes. A similar situation exists in
heterologous systems.
plants. E. Williams (personal communication) has reported
Following are some models that might explain these that there may be as many as 50 copies of each leucyl-tRNA
different profiles although their biological significance is not DNA in higher plants. This redundancy probably arose by gene
discussed.
duplication during continuous recombination and mitosis
Modification of tRNA. Sharma and Borek (17), Tsutsui et through evolutionary
periods.
Evidence from proteins
al. (24), and Turkington and Riddle (25) have shown that the demonstrates that many changes at the single nucleotide level
specific activity of tRNA-methylating enzymes is higher in may take place in DNA with a concomitant change in amino
tumor cells then in nontumor cells. Turkington has shown that acids in protein. Such changes may have neither a positive nor
mammary carcinomas contain methylases not normally found a deleterious effect on the protein and may not have a
in the mammary gland. One must assume that these enzymes selective advantage (10). One can easily imagine that, during
are using tumor tRNA as template with the addition of extra the course of gene duplication, speciation, etc., redundant
methyl groups to the tRNA.
tRNA genes with slightly different nucleotide sequences may
we have not examined.
The results with both sea urchins and hepatomas are good
circumstantial
evidence for the "modulation"
theory of
UFFALO
RAT
LIVER
PATOMA
BUFFALO
HEPATOMA
RAT
51230
LIVER
51
: 2
0.
u
22
126
FRACTION
13O
134
138
142
146
9O
15O
NUMBER
FRACTION
FRACTION
94
98
1O2
106
NUMBER
NUMBER
Chart 4. A comparison of rat liver and hepatoma 5123D servi-, histidyl-, and phenylalanyl-tRNA by reverse phase chromatography
cpmx 100; 3H, X 1000).
MAY
1971
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(I4C
691
Taylor, Volkers, Choe, and Zeikus
by ultracentrifugation
that it is a unique molecular species
with a sedimentation value of 4.7 S. By calculation, it is about
20 nucleotides longer than the bulk of the tRNA molecules. In
in vitro charging experiments, this longer species accepts all
amino acids tested. Therefore, it appears to be neither a
precursor molecule nor a unique species of tRNA. It does not
appear to be a dimer of tRNA.
We would like to speculate that this molecule may reflect
the "master" gene that perhaps contains the signal for the
so
o
SLICE NUMBER
Chart 5. Acrylamide gel (5%) agarose electrophoresis of tRNA
purified by BD-cellulose chromatography. Note similarity of gel
patterns of both salt-eluted and alcohol-eluted material.
have arisen, which would give rise to tRNA molecules of
slightly different primary structure and conformation but with
identical biological activity.
If we accept that gene redundancy exists, we must ask what
the effect of differential gene transcription is. It has been
shown in a number of systems that gene transcription occurs
on different discrete areas of the chromosome at differing
times (15). Therefore, if there is a population of DNA
molecules on different chromosomes (or even on the same
chromosome) that code for the same tRNA and if these DNA
molecules differ one from the other, one might expect to see,
as a result of differential gene expression, the synthesis of
different tRNA molecules.
The differences between tRNA molecules in tumor cells and
in normal cells may either reflect a breakdown in the regular
transcription
process
or
result
from
chromosomal
abnormalities. The inability to detect a species of tRNA in
liver, present in a hepatoma, may reflect the processes of gene
transcription.
In the course of examining tRNA synthesis in Ehrlich
ascites tumor cells and Chinese hamster ovary cells, we made a
number of surprising observations that might explain some of
the heterogeneity and number of isoaccepting species of
tRNA. A number of groups (2, 3, 11) have reported the
presence of a precursor to tRNA in HeLa cells, human
lymphocytes, and KB cells. This RNA was defined as precursor
on the basis of actinomycin D-chase experiments. Utilizing
very short pulses of 5 min or less, we can demonstrate on
agarose acrylamide gels a transient precursor molecule to 4 S
RNA in both Chinese hamster ovary cells and Ehrlich ascites
tumor cells. This species may be only 1 of a number of
precursor states in the formation of tRNA. That this precursor
has a very short half-life, about 1/100 of a generation time, has
been confirmed by detailed kinetic analysis.
In the course of this work, we discovered the occurrence of
a 2nd group of tRNA molecules, larger than 4 S, which do not
fit the kinetic requirements of precursor tRNA. These
molecules are methylated, contain both dihydrouridine and
pseudouridine, and are synthesized in parallel with bulk tRNA.
We have purified this species of tRNA and can demonstrate
692
initiation of transcription of a specific species of tRNA. If this
is the original gene for tRNA and genetic redundancy has
arisen by duplication, then we might expect that segments of
this tRNA gene would be lost during evolution. If this is
correct, we would then expect heterogeneity in size of tRNA
species.
Using acrylamide gel electrophoresis and partially purified
tRNA, we have noted, as have other (6), a wide distribution of
tRNA molecules on gels. There are at least 3 to 4 subgroups
(Chart 5). Since this is unacylated tRNA labeled with '4C-or
3H-orotic acid, the ionic charge on these tRNA's should be
uniform, so that separation should be on the basis of size
distribution.
Furthermore,
when this tRNA was run on
BD-cellulose and the fraction eluting with ethanol which
contains different species of tRNA was run separately on gels,
a similar distribution
was observed. This would imply
heterogeneity in sizes of tRNA molecules.
We have tried to explain the basis for these differences and
for the large number of isoaccepting species as being a result of
gene duplication, without really touching on their biological
significance. Obviously, all of these factors (methylation,
differential gene transcription, etc.) might be functioning at
once. However, if we knew the origins of these differences, we
should perhaps be ready to say something of their biological
significance.
It should be remembered that these ideas are speculative.
Only nucleotide sequencing will give definitive answers.
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Precursor to t-RNA and the Effect of Methionine Starvation on
t-RNA Synthesis. J. Mol. Biol., 42: 43-56, 1969.
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4. Burgess, R. R., Travers, A. A., Dunn, J. J., and Bautz. E. K. F.
Factor Stimulating Transcription by RNA Polymerase. Nature,
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5. Comb, D. G., Katz, S., Brand, R., and Pinzino, C. J.
Characterization of RNA Species Synthesized during Early
Development of the Sea Urchin. J. Mol. Biol., 14: 195-213, 1965.
6. Friedlander, A., and Buonassiss, V. Kinetics of Synthesis of
Cytoplasmic t-RNA with Transfer Properties in Cultures of Adrenal
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7. Clisen, V. R., and Glisen, M. V. Ribonucleic Acid Metabolism
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tRNA Modification and Synthesis in Animals
8. Gross, P. The Immediacy of Genomic Control during Early
Development. J. Exptl. Zool., 757: 21-38, 1964.
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Differences between Tyrosyl Transfer RNA from Different
Mammalian Cells. Proc. Nati. Acad. Sei. U. S., 2437-2444, 1967.
10. Jukes, T. H., and Cantor, C. R. Evolution of Protein Molecules. In:
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Characteristics of Transplantable Rat Hepatoma No. 5123 Induced
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13. Nemer, M. Old and New RNA in the Embryogenesis of the Purple
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14. Quincy, R. V., and Wilson, S. H. The Utilization of Genes for
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Drosophila melanogaster. Genetics, 54: 663-676, 1966.
17. Sharma, O. K., and Borek, E. Hormonal Effect on Transfer
Ribonucleic Acid Methylases and on Serine Transfer RNA.
Biochemistry, 9: 2507-2513, 1970.
18. Slater, D. W., and Spiegelman, S. An Estimation of Genetic
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19. Stent, G. S. The Operon: On Its Third Anniversary. Science, 144:
816-820, 1964.
20. Sueoka, N., and Kano-Sueoka, T. A Specific Modification of
Leucyl-sRNA of Escherichia coli after Phage T2 Infection. Proc.
Nati. Acad. Sei. U. S., 52: 1535-1540,1964.
21. Sueoka, N., and Kano-Sueoka, T. Transfer RNA and Cell
Differentiation. Progr. Nucleic Acid Res. Mol. Biol., 10: 23-55,
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22. Taylor, M. W. Coding Response of an Altered Phenylalanine
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Chromatographie Alterations in Transfer RNAs Accompanying
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Discussion
Dr. Borek: With respect to your alterations in the
developing sea urchin embryo, it was reported some 4 or 5
years ago that there are no changes in methylation in the
developing sea urchin. This has been a source of distress to me
all along because this would be one system in which
differentiation
goes on, and yet there is no change in
methylation.
We have reexamined this problem recently with the
collaboration of Dr. Larry Loeb, of Philadelphia, who is an
expert in sea urchin development. We can say the enzymes are
present in the unfertilized egg and there is a burst of
methylation right after fertilization, and we have this both
with enzymes in vitro and with in vivo labeling of tRNA's.
So here is another case of concomitant or sequential
changes, methylation and then change of tRNA.
Dr. Stulberg: I was wondering whether in the hepatomas
where you see differences, i.e., new species of various tRNA's,
have you ever attempted to associate these new species with
abnormalities in the biosynthesis of their cognate amino acids,
either lack of or appearance of a particular enzyme activity?
Dr. Taylor: No. That would be a very big project, of course.
Dr. Stulberg: Have you attempted, on paper, to correlate
tRNA changes with enzymatic deficiencies in the hepatoma?
Dr. Taylor: No, we haven't.
MAY 1971
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693
Transfer RNA Modifications and Synthesis in Animal Cells
M. W. Taylor, S. A. S. Volkers, B. K. Choe, et al.
Cancer Res 1971;31:688-693.
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