University of Southern California Dissertations and Theses

Changes In Leucine Transfer Ribonucleic Acid And Leucine Transfer Ribonucleic Acid Synthetase During Cotyledon Senescence

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Changes In Leucine Transfer Ribonucleic Acid And Leucine Transfer Ribonucleic Acid Synthetase During Cotyledon Senescence

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Content CHANGES IN LEUCINE TRANSFER RIBONUCLEIC ACID
AND LEUCINE TRANSFER RIBONUCLEIC ACID
SYNTHETASE DURING COTYLEDON SENESCENCE
by
Michael David Bick
A Dissertation Presented to the
FACULTY OF THE GRADUATE SCHOOL
UNIVERSITY OF SOUTHERN CALIFORNIA
In Partial Fulfillment of the
Requirements for the Degree
DOCTOR OF PHILOSOPHY
(Cellular and Molecular Biology)
June 1971
I
I
71-27,909
BICK, Michael David, 1945-
CHANGES IN LEUCINE TRANSFER RIBONUCLEIC
ACID AND LEUCINE TRANSFER RIBONUCLEIC ACID
SYNTHETASE DURING COTYLEDON SENESCENCE.
University of Southern California, Ph.D.,
1971
Bio logy-G en et i cs
j University Microfilms, A XEROX Company , Ann Arbor, M ichigan
THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED
UNIVERSITY OF SOUTHERN CALIFORNIA
THE GRADUATE SCH O O L
UNIVERSITY PARK
LOS ANGELES, CALIFO RNIA 9 0 0 0 7
This dissertation, 'written by
....................OT.Ctfm.mYm.BJCK
under the direction of h.±s Dissertation C om
mittee, and approved by all its members, has
been presented to and accepted by The G radu
ate School, in partial fulfillment of require
ments of the degree of
D O C T O R O F P F I I L O S O P I I Y
(j Dean
D ate ..........Jjuna—L9.7.L
IASSENTATION COMMITTEE
ACKNOWLEDGEMENTS
I wish to express my appreciation to Dr. Bernard
Strehler for stimulating my interest in molecular biology
and aging, and for demonstrating an early confidence in
my abilities. His comments and criticisms during the
course of this work have always served to stimulate my
thinking and his own breadth of interest has kept my
interests from becoming too narrow. His guidance and
friendship will always be remembered.
Gratitude is also due Dr. Joe Cherry who provided
an early impetus for the initiation of this ttfork and for
maintaining an interest in its progress, as well as for
providing me with a manuscript of his work prior to
publication. I would also like to thank Drs. Pete
Shugarman, John Petruska, Jacek Szafran for serving on
my dissertation committee and for their various comments
and discussions during the course of this work.
My friends and colleagues Gerald Mirsch, Richard
Nordgren, and Leo Andron have also contributed immeasurably
to this work through their many discussions and sugges
tions .
I would also like to acknowledge receipt of a NICHD
Predoctoral Traineeship through the Gerontology Center,
USC. My association with the Center has been stimulating
and rewarding.
ii
My wife especially deserves thanks for her support,
tolerance, and encouragement throughout these years of
study.
To Sharon,
Geri , and David
and to
Pauline
iv
TABLE OF CONTENTS
Page
ACKNOWLEDGEMENTS ii
LIST OF TABLES vii
LIST OF FIGURES v m
LIST OF ABBREVIATIONS x
Text
I. INTRODUCTION
1
General
Theories and Background on
Translational Control
Implications for Aging
The Cotyledon as a Model System
Growth Conditions
Isolation of Transfer RNA
Isolation of Synthetase
Aminoacylation Reaction
Chromatography of Transfer RNA
Fractionation of Synthetase
Phenoxyacetylsuccinimide Derivatization
Procedure
Carboxyanhydride Derivatization
Procedure
Changes in tRNA eu
Cytolcinin Effect on tRNA^eu
Changes in Leucyl-tRNA Synthetase
Chromatographic Comparison of Leucyl-
tRNA Synthetase Activities
Synthetase Derived from 21 Day Old
Cotyledons Cannot Fully Acylate
tRNALeu
Fractionation of Leucyl-tRNA Synthetase
II. METHODS AND MATERIALS
20
III. RESULTS
30
v
Text Page
IV. DISCUSSION.......................... 99
Changes in Transfer RNA
Changes in Leucyl-tRNA
Synthetase
Codon Response
Summary and Conclusions
LITERATURE CITED .................... 115
vi
LIST OF TABLES
Table Page
1. Relative Amounts of tRNA^eu Isoaccepting
Species Present in Different Aged
Cotyledons............................... 39
2. Relative Amount of tRNA^eu Species in
BAP Treated Cotyledons ................. 44
3. Relative Amount of tRNALeu Species
Charged by 5 and 21 Day Synthetase . . 57
4. Leucine Acceptor Activity of 5 and 21
Day tRNA and Synthetase................60
5. Effect of Pre-incubation on Charging . . 66
6. Codon Response of Some Aminoacyl-tRNA's
from Guinea Pig Liver.................. Ill
vii
LIST OF FIGURES
Figure Page
1. Aminoacylation of tRNA^eu ................ 31
2. Go-chromatography of tRNA^eu from 2 and
15 Day Old Cotyledons.................... 34
3. Co-chromatography of 5 and 21 Day tRNALeu 36
4. Chromatography of tRNA^eu from BAP
Treated Cotyledons .................... 42
13.
Chromatography of Permanganate Treated
tRNALeQ ............................. 46
6. Chromatography of Hypocotyl tRNA^eu Pre
incubated with Cotyledon Enzyme .... 49
7. Relative Activity of tRNALeu and Synthetase
from 5 and 21 Day Cotyledons............52
8. Co-chromatography of 5 Day tRNA^eu Acylated
with 5 and 21 Day Synthetase............55
9. Co-chromatography of tRNA^eu Acylated
with 5 and 21 Day Synthetase........... 58
10. Additive Effects of 5 and 21 Day Synthetase63
11. Labeling of Phenoxyacetylsuccinimide
Derivatized tRNALeu ...................... 69
12. Chromatography of Phenoxyacetylsuccinimide
Derivatized tRNA .......................72
Chromatography of Carboxyanhydride
Derivatized tRNALeu ........... 75
14. Hydroxylapatite Fractionation of 5 Day
Synthetase............................... 78
15. Hydroxylapatite Fractionation of 21 Day
Synthetase................................80
Figure Page
16. Chromatography of tRNALeu Acylated with
Fraction 1 Synthetase .................. 82
17. Chromatography of tRNA^eu Acylated with
Fraction 2 Synthetase .................. 85
18. Chromatography of tRNA^eU Acylated with
Fraction 3 Synthetase .................. 87
1 pi i
19. Aminoacylation of tRNA with Fractionated
5 Day Synthetase...................... 89
20. Aminoacylation of tRNALeu with Fractionated
21 Day Synthetase.........................91
21. Additive Effects of Fraction 2 Synthetase 94
22. Additive Effects of Fraction 3 Synthetase 96
ix
ABBREVIATIONS
A
adenine
Ac
acetate
AMP
adenos ine-5'-monophosphate
ATP
adenosine-5'-triphosphate
Asp
asparagine
C cytosine
°C
degrees centigrade
CMP
cytidine-51-monophosphate
CPM
counts per minute
CTP cytidine-51 - triphosphate
DNA
deoxyribonucleic acid
EDTA
ethylenediaminetetraacetate
g
grams
G
guanine
Glu
glutamic acid
Gly glysine
Leu
leucine
Lys lysine
M
molar
Met methionine
mg
milligram
ml milliliter
x
mji
pmoles
Pro
RNA
mRNA
tRNA
tRNA
Ser
Thr
Tris
Tyr
U
UCA
Leu
jamole
x 5
millimicron
picomoles
proline
ribonucleic acid
messenger ribonucleic acid
transfer ribonucleic acid
tRNA capable of accepting
leucine
serine
threonine
tris (hydroxymethyl) amino-
methane
tyrosine
uracil
an example of a trinucleotide
diphosphate with a 5'-
terminal hydroxyl attached
to U and 2', 3'-terminal
hydroxyls attached to A,
(UpCpA)
micromole
times gravity
CHAPTER I
INTRODUCTION
General
Higher animals and plants are organized into specific !
l
cell sets. These cell sets are in turn arranged as
I
tissues, tissues arranged as organs, and organs arranged
as organisms. Different cell sets perform specialized
functions and are therefore said to be differentiated. It j
is now clear that specialized cells arise during develop- j
ment due to cell-specific protein synthesis. Our current
understanding of molecular biology suggests that all cells
of a given organism (with certain notable exceptions)
contain the same genetic material [DNA) in both quanti
tative and qualitative terms. Cell-specific protein
synthesis then is equivalent to differential gene expres
sion, where all cells of a given organism have the genetic
capacity to produce all structural and functional proteins
for that organism, yet any particular cell type is
restricted to the production of only a few.
The extensive studies over the past several years
of Nirenberg (Nirenberg et al., 1966) , Ochoa (Smith et al.,
1966) , Khorana (Khorana et al., 1966) , and their col-
leagues have clearly established that the sequence of
amino acids in a polypeptide chain is determined by a
series of trinucleotide sequences in mRNA. The basic
information concerning the amino acid sequence of a
specific polypeptide is coded in the sequence of nucleotide
bases in DNA. The process of protein synthesis consists
of first transcribing RNA from a DNA template, producing
an mRNA with a complimentary base sequence. The mRNA
then becomes associated with ribosomes in a specific
manner which serves as the site for protein synthesis.
The attached mRNA determines what sequence of amino acids
will be incorporated into the polypeptide by an inter
action between triplet sequences in mRNA and specific
adaptor molecules (aminoacyl-tRNA). Thus, a particular
amino acid is introduced into a growing polypeptide chain.
The control of gene expression may exist at a number
of levels in the overall mechanisms leading to the final
protein product. The most notable, and most often
considered, possibilities exist at the level of RNA
transcription from a DNA template and at the level of
translating mRNA into protein. A number of potential
mechanisms exist in the former case including repressors
of gene activity (Bonner et al., 1968) , multiple RNA
polymerases with specificity of transcription (Roeder and
Rutter, 1969; Roeder and Rutter, 1970), and modifiers
of RNA polymerase activity (Burgess et al., 1969; Travers,
1970), This study is concerned with the second possibility
however, that of regulating protein synthesis at the
translational level.
Theories and Background on Translational Control
Degeneracy of the genetic code is now well estab
lished (Nirenberg et al. , 1963; Nirenberg et al., 1966;
Caskey et al., 1968) and this degeneracy is also reflected
in the multicomponent, heterogenous population of tRNA's
for each amino acid. The term isoaccepting tRNA's has
been suggested by Novelli (1967) to describe those
multiple tRNA species which accept the same amino acid.
Similar to the multicomponent system of tRNA's, at least
some amino acids have also been shown to have multiple
aminoacyl-tRNA synthetases (Ceccarini et al., 1966;
Strehler et al., 1967; Novelli, 1967; Sueoka and Kano-
Sueoka, 1970).
Itano (1963) was the first to implicate translational
mechanisms in regulating protein synthesis by suggesting
that the inability to produce sufficient amounts of the
beta-chain of hemoglobin in thallassemia may in fact be
due to the presence of a poorly translatable codon in the
mutant gene. The degenerate nature of the code and of at
least some components of translation has resulted in the
generation of a number of hypotheses during the last
decade implicating translational control mechanisms in
cell-specific protein synthesis. Most notable among these
hypotheses are the following:
1) The Modulation Hypotheses of Ames and Hartman
(1963) suggests that modulating triplets may cause ribo
somes to fall off of mRNA when such triplets are encoun
tered. Modulating triplets would in fact be due to their
correspondence to modulating tRNA's. The interruption
of translation would most likely occur due to rate
limiting concentrations of those tRNA's which correspond
to modulating triplets. This hypothesis was first
suggested to account for polarity in the histidine operon
but was later extended by Stent (1964) to include
regulation of other genes as well.
2) The Adaptor Hypothesis was suggested by Sueoka
and Kano-Sueoka (1964) subsequent to their work demon
strating the appearance of a new tRNA^eU species following
bacteriophage infection of E. coli. It was reasoned that
if the codon recognition of a particular adaptor (tRNA)
out of a set of degenerate adaptors for a given amino
acid is changed by modification, any mRNA which accomodates
the codon corresponding to the modified adaptor will not
be properly translated. Messenger RNA not containing
that particular codon will be translated normally. It
was considered that modification of the adaptor might take
place at the anticodon, the enzyme recognition site, or
at a ribosome binding site,
3) The Codon-Restriction Hypothesis of Strehler ;
(1966) was in large part an extension and bringing !
together of the two above hypotheses, A more direct role
for translational control in differentiation and
regulation in higher cells was implied, and differed from j
the other two hypotheses in implicating aminoacyl-tRNA ;
synthetases in control as well as tRNA's, The collection .
|
of codons (language set) that a particular cell uses
consists of the codons for which aminoacylated tRNA speciesj
are available in sufficient concentration to bring about j
translation. Therefore, regulation may occur either
through the production of usable tRNA species or their
cognate synthetases. Regulation by way of synthetases
was stressed by Strehler because the mRNA for synthetases
must themselves be translated if they are to be produced.
i
And if the synthetases were self-coding (i.e., correspond
to a codon contained in their own mRNA) it was possible
that new synthetases could be generated as the cells
language set changed during its developmental program.
In addition, the opening,up of the synthesis of a single
protein (a specific synthetase) could lead to the synthesis
of a number of others.
6
All three of the above hypotheses have a number of
features in common and are not mutually exclusive. A
more generalized statement of translational control relies
on the degenerate nature of the code which results in
an average of three code words for each of the 20 amino
acids. The triplet nature of the code results in 64
different code words and theoretically there could be an
equal number of different tRNA species, each with a
unique anticodon. A minimum of 56 separable tRNA species
have been demonstrated in a mammalian system (Gallo, 1969;
Gallo and Pestka, 1970) and an equal number have been
shown in E. coli by Muench and Safille (1968) . If the
presence of isoaccepting tRNA species reflects different
anticodons (or at least different code reading abilities),
then on an average each tRNA species will respond to a
unique codon. By regulating the presence of a specific
aminoacyl-tRNA, or class of aminoacyl-tRNA's, the usage
of the corresponding codon(s) in coding for protein can
also be regulated. However, since there would be an
average of two additional codons for any given amino
acid, protein synthesis can continue which utilizes those
codons. Only those messages containing the 'restricted'
codon(s) will be untranslatable.
The same argument holds true for limiting concen
trations of specific aminoacyl-tRNA synthetases. There is
currently evidence that at least some amino acids have
more than a single synthetase. By limiting the concen
tration of specific aminoacyl-tRNA synthetases rather
than (or in addition to) species of tRNA the same result
is achieved, i.e., limiting concentrations of an
essential molecule in protein synthesis, aminoacyl-tRNA,
Methods are now available for examining the comple
ment of tRNA species in any given material. These
include methylated albumin kieselguhr (MAK) column
chromatography (Sueoka and Yainane, 1962) and the high
resolution Reverse Phase Chromatography system of Weiss
and Kelmers (1967). Upon fractionation, the number of
chromatographic peaks may reflect the total number of
tRNA species an organism, or particular cell type, is
capable of generating under any given set of conditions.
By comparing the chromatographic profile of charged tRNA
species one can obtain a measurement of the complement,
as well as the relative amounts, of isoaccepting tRNA's
from different sources and under different conditions.
There is considerable evidence consistent with
generalized models of translational control of protein
synthesis through modulation of specific species of tRNA
and/or synthetases. In B. subtilis Kaneko and Doi (1966)
have observed that the complement of tRNA^al changes
during sporulation. Since sporulation involves the
8
expression of a number of genes and represents a major
shift in the cells metabolic activity, these workers
suggest that a regulatory role is played by the changing
pattern of tRNA^al species. A similar study (Lazzarini,
1966) has demonstrated the presence of a tRNA^^s species
in B. subtilis spores which is not present in vegetative
cells. It was suggested that this unique tRNA^ys species
in spores may be related to spore-specific metabolic
processes. Kano-Sueoka and Sueoka (1966) and Waters and
Novelli (1968) have independently demonstrated the
appearance of a new tRNALeu species following infection
of E. coli with T-even bacteriophage. The time course of
alteration of tRNA^eu species revealed that this t^as one
of the earliest events taking place after phage infection
and it was suggested that this alteration may play some
role in the arrest of host-specific protein synthesis.
More recently, Kan et al. (1970) have shown a shift
in the relative amounts of two of the five tRNALeu species
in E. coli following infection with T2 bacteriophage,
accompanied by a possible change in the codon response
of at least one of these tRNALeu species. Arceneaux
and Sueoka (1969) have demonstrated a dramatic change
in the ratio of two tRNA^yr species during a period of
active growth in B. subtilis. However, these workers
were unahJLe to demonstrate any difference in codon
9
response between the two species and differential usage
of the two species in protein synthesis seemed unlikely.
In higher cells, Taylor et al. (1967) have used
the MAK column to examine the tRNA complement from
different organs as well as from cultured cells. Signif
icant differences were observed in tRNA^er species when
comparing rabbit liver to kidney as well as between
mouse liver and muscle. Tissue differences in the
complement of tRNA^ly were also demonstrated. In a later
report Holland et al. (1967) demonstrated a difference
in the complement of tRNA^^1* species between fibroblasts
and epithelial cells. And while there was a great
disparity in elution profiles between mammalian and
chicken organ tRNATyr, fibroblasts derived from these two
sources gave nearly identical profiles.
An organ specific deficiency in a specific alanyl-
tRNA synthetase has been demonstrated by Strehler et al.
(1967) in the rabbit, as well as a difference in the
charging capacity of leucyl-tRNA synthetases isolated
from rabbit liver and reticulocytes. In plants Anderson
and Cherry (1969) have demonstrated an organ specific
deficiency in the complement of tRNALeu as well as in the
cognate synthetase (Kanabus and Cherry, 1971). The
soybean cotyledon was shown to contain six isoaccepting
species of tRNALeu by fractionation on a Freon column
10
(Weiss and Kelmers, 1967), while the hypocotyl contains
only four of these species, In addition, hypocotyl
tissue is deficient in one of the three leucyl-tRNA
synthetases which are found in the cotyledon. As might
be expected the synthetase which is lacking in the
hypocotyl is specific for acylating those tRNA^eu species
which are also missing in hypocotyl tissue.
To look for changes in specific tRNA's during
developmental processes, Lee and Ingrain (1967) have
examined the complement of tRNA's present in 4 day old
chick red blood cells as compared to reticulocytes of
adult chickens. Their results revealed a major shift in
the proportion of the two tRNA^et species as well as a
possible difference in tRNA^eu species. Void and Sypherd
(1968a, 1968b) have compared the aminoacyl-tRNA1s of
wheat embryos and wheat seedlings and shown that quantita
tive changes occur in a number of aminoacyl-tRNA's
during development.
A number of reports have demonstrated aminoacyl-tRNA
differences between normal and neoplastic cells and
tissues (Axel et al., 1967; Taylor et al., 1968; Yang £t
al. , 1969; Taylor, 1970). Gallo and Pestka (1970) have
recently made an exhaustive study of the tRNA's present
in normal and leukemic lymphoblasts. A total of 56 tRNA
species for the 20 amino acids were demonstrated.
11
Pronounced differences were apparent for the complement of
tRNA^r and tRNA^lu species, while small but reproducable
differences were found in tRNA^eu, tRNA^er, tRNA^^r, and
tRNAPro species.
More direct evidence for isoaccepting tRNA involve
ment in cell-specific protein synthesis comes from the
study of Yang and Novelli (1968) which revealed different
tRNA^er chromatographic profiles between two mouse plasma
cell tumors that produce different kinds of myeloma
immunoglobulins. One cell line was an IgG producer while
the other synthesized only IgA. This is particularly
interesting since serine is one of the few amino acids
found to differ significantly in amount in different
antibody immunoglobulins (Koshland, 1966) . Also using
mouse plasma cell tumors Mushinski and Potter (1969) have
demonstrated a difference in the chromatographic profile
of tRNA^eu species. These particular tumors were all
kappa-light chain producers so these cells are producing
very similar, but not identical end products. It was
suggested that these tRNA^011 differences might be related
to immunoglobulin variability, particularly at the level
of translation.
Nearly all of the studies which have shown tRNA
changes during growth, differentiation, neoplasia, or
other metabolic alteration have revealed relative changes
in isoaccepting tRNA's rather than absolute gains or
losses in specific species. A reduction in a specific
species may, however, still reflect a loss in code reading
ability and therefore limit translational capacities. ;
Using an in vitro protein synthesizing system derived from i
E. coli Anderson (1969) has demonstrated that the rate of
i
protein synthesis can be regulated by the concentrations of
tRNA in the reaction mixture. The concentration range
over which tRNA was rate limiting coincides with the
calculated levels of tRNA species present in intact cells.
In a later report Anderson and Gilbert (1969) utilized a i
cell-free system derived from rabbit reticulocytes to !
synthesize hemoglobin. It was shown that the concentration
of one or more tRNA species can specifically change the
relative amounts of alpha and beta hemoglobin chains
produced. This is particularly interesting since both
chains contain all 20 amino acids. Their preliminary
results suggested that it may be a tRNA^Y species
responsible for limiting the production of alpha chains.
Yamane (1965) has reported that the relative
i
abundance of tRNA species in E. coli reflects the amino
acid composition of total protein from the organism.
More recently Lanks and Weinstein (1970) have studied
granulation tissue in an attempt to correlate specific
tRNA content with a cell's specialized function. Since j
collagen contains approximately 30 per cent proline plus
hydroxyproline it was reasoned that tRNA from granulation
tissue should be enriched in tRNAPro species if tRNA
content was related to a cell's specialized products.
When compared to liver, granulation tissue was signifi
cantly enriched in tRNA^ro, which increased even more
during extensive collagen production. There were no
differences observed for six other amino acids.
In the mealworm Tenebrio molitor Ilan and co-workers
(Ilan, 1969; Ilan et al., 1970) have studied the in vitro
translation of cuticular protein. At least part of the
message for adult cuticular protein was known to be
present at the first day of pupation although it was not
translated until the last days of adult development, 5 to
7 days later. Adult cuticular protein contains a high
level of tyrosine relative to leucine which can serve as
a marker for it's synthesis. These studies have demon
strated that there is a very low level of tyrosine
incorporation into protein utilizing microsomes from first
day pupae n/ith'first day tRNA and charging enzyme. When
microsomes from first day pupae are supplemented with
tRNA and enzyme from seventh day pupae however, there is
a significant increase in the incorporation of tyrosine
relative to leucine. The same results are seen when
microsomes from seven day pupae are used, except that ther
14
is an even greater stimulation. Tryptic digests of the
protein products produced in vitro are nearly identical to
those of cuticular protein. The salient feature is that
both tRNA and charging enzymes from the seventh day pupae
are needed for the synthesis of adult cuticular protein,
even though the message is present at the first day.
Neither of these components can be exchanged by either
first day tRNA or first day synthetase.
Additional experiments have shown that leucyl-tRNA
synthetase from animals after seven days of adult develop-
T p 11
ment can recognize more or different subspecies of tRNA
This new leucine accepting tRNA cannot be demonstrated in a
tRNA preparation from one day pupae. Similar results are
found for the cognate synthetase. These results suggest
that cuticular mRNA translation is limited in the 1-day
pupae due to limiting species of tRNA and synthetase and
demonstrates an apparent coordinate regulation of the avail
ability of a tRNA species and it's cognate synthetase.
Implications for Aging
The various models of cellular differentiation may
also have important implications for biological aging.
Weismann (1891) originally suggested that the senescence
of cells and organisms may be, at least in part, a long
term consequence of limitations in functional capacities
15
which occur as a result of cell specialization during
developmental processes. Medvedev [1967) similarly
concluded that it is necessary to understand the
principles underlying the molecular-genetic control of
morphogenesis and differentiation in order to understand
the mechanisms of aging.
Differentiation is generally associated with a
decrease or loss in the capacity for further growth and
cell division. It may be that concomitant with growth
cessation there is a loss or suppression of the synthetic
capacities characteristic of actively growing cells. The
Codon-Restriction Hypothesis was originally suggested by
Strehler [1966) as a model for molecular mechanisms of
aging. Clearly, if the cessation of cell [and organism)
growth is due to repressed synthetic capacities which
occur as a result of cell specialization, there may also
be a loss in the capacity to synthesize certain cellular
components essential to the long-term functional integrity
of the cell. The 'locking in' of certain specialized
syntheses, as suggested by most theories of cell differ
entiation, will almost certainly lead to the 'locking
out' of other syntheses. Deterioration of non-replenish
ing components through wear and tear will lead to
senescence and ultimate death in such cases. The exis
tence of long lived protein components in the mouse which
are synthesized early in the life cycle has been
established by Nordgren et al. (1969) and the study of
Gee et al. (1969) indicates the presence of many long
lived lipid components in mouse brain and skeletal
muscle. Such non-replenishing components might be
expected to provide natural loci for the accumulation of
damage, possibly leading to decreased functional capacities
If aging can be considered a post-mitotic event and
due primarily to a failure in non-dividing cells, then
any major control mechanisms of cellular metabolism may
have important implications for senescence. Jackson et al.
(1970) have studied the effect of growth hormone on the
livers of hypophysectomized rats. Fractionation of tRNA's
revealed the appearance of two new species of tRNA^sP
which were not present in control hypohysectomized
animals. Similar changes in tRNA^sP were found to occur
in the regenerating liver following partial hepatectomy.
These results suggest that the appearance of new tRNA^sP
species is directly related to the initiation of growth.
Vanderhoef and Key (1970) have compared six aminoacyl-
tRNA’s from dividing and non-dividing cells of a plant
tissue. Transfer RNA was taken from the rapidly dividing
meristem of roots from pea seedlings and compared to the
tRNA taken from non-dividing, fully elongated cells of
maturing root tissue. This study revealed that a larger
percentage of the tRNA from dividing cells (70 per cent)
can be aminoacylated than the tRNA from non-dividing
cells (54 per cent). In addition, fractionation of the
various aminoacyl-tRNA's demonstrated a significant
decrease in the relative amounts of two of the three
tRNATyr species in non-dividing cells when compared to
dividing cells.
The Cotyledon as a Model System
The present study was initiated to test certain
aspects of the Codon-Restriction Hypothesis, particularly
as it relates to aging. The soybean cotyledon was chosen
as the material for examining possible age-related
alterations in certain components of the organ's trans
lational machinery.
The cotyledon was chosen as a model system for the
following reasons. 1) It is generally considered to be a
senescing organ throughout its lifespan as its function
is to provide metabolic nutrients to the growing embryonic
axis from the hydrolytic products of its own storage
materials. Upon germination of the seed there is an
ordered series of events leading to the degradation of
storage materials in cotyledonary cells (Cherry, 1967),
These metabolic events are not accompanied by cell
division. 2) The cotyledon has a relatively short life
18
span (about 3 weeks under the growth conditions employed
in this study) and is available in large quantities.
3) Anderson and Cherry (1969) have reported a difference
in the complement of tRNA^eu between soybean cotyledon
and hypocotyl tissue. This suggested that there may be
different translational capacities between these two
tissues and therefore represented a potential model to
study losses in translational capacities as well. 4) A
class of naturally occurring plant hormones, the
cytokinins, are known to retard or delay senescence in
detached leaves and has prompted Osborne (1965) to suggest
that the hormone's action may be through its maintenance
of nucleic acid and protein synthesis. Interestingly,
cytokinin analogs (which include a variety of ^-substi
tuted purines) are also found to be components of certain
tRNA molecules in bacteria, yeast, plants, and animals
(Hall, 1970). Additionally, when -substituted purines
are found as components of tRNA, their location is always
adjacent to the anticodon. This suggested that there
was a possible relationship between the function of
specific tRNAs, hormone action, and plant senescence.
Using the cotyledon as an aging system then, tRNA
and aminoacyl-tRNA synthetases were extracted from dif
ferent aged cotyledons, tRNA was aminoacylated in vito
using either homologous or heterologous synthetase, and
19
subsequently fractionated on a Freon column for detection
of isoaccepting species. Total amino acid acceptor
activity was calculated and the relative amounts of each
isoaccepting species determined. In this way the tRNA
acceptor activity and aminoacyl-tRNA synthetase charging
capacities could be determined from different aged
cotyledons.
Specifically, tRNALeu and leucyl-tRNA synthetase
(L-leucine:tRNA ligase (AMP)t EC 6.1.1.4) were chosen
for study. This was because of the previous work of
Anderson and Cherry (1969) showing tissue differences in
these components, and the fact that there are six
isoaccepting species of tRNA^eu and six leucine codons
(two of which begin with U) made it a potential candidate
for modulation of isoaccepting species.
CHAPTER II
METHODS AND MATERIALS
Growth Conditions
Soybean seeds (Glycine max var. Hawkeye 64) were
purchased from Fred Gutwein and Son, Francesville, Indiana,
and kept at 4°C until use. Seeds were allowed to imbibe
water overnight, sown in 9 inch x 15 inch Pyrex trays
containing moist Vermiculite, and placed in a dark
humid growth chamber maintained at 29-30°C. Water was
added as needed during the growth period to keep the
Vermiculite moist. After various times of growth the
cotyledons were excised, the seed coats removed if still
present, and the cotyledons washed three times in ice-
cold deionized water and tRNA and synthetase extracted
as described below.
Isolation of Transfer RNA
50-100 g of cotyledons were routinely used for
extraction of tRNA by a modification of the method of
Anderson and Cherry (1969) . Cotyledons were homogenized
in a Sorvall Omni-Mixer at high speed for 2 minutes
in Buffer A (1 ml/g tissue) which consisted of: 0.03M
21
MgAc, 0.003M Tris-Cl (pH 7.4), 0.003M EDTA, and 0.005M
2-mercaptoethanol, along with 0.75 ml/g tissue of 88
per cent phenol. Homogenization and all other steps
were carried out at 4°C unless otherwise stated. The
homogenate was shaken for two hours and spun at 20,000 x g
for 20 minutes. The upper aqueous phase was removed by
syringe and the phenol phase re-extracted with an equal
volume of Buffer A. The two aqueous phases were combined
and nucleic acids precipitated by the addition of 2 volumes
of 9 5 per cent ethanol and 0.1 volume of 2M potassium
acetate (pH 5.5) at -20°C, The precipitate was collected
at 20,000 x g, dissolved in 1M NaCl (approximately 1 ml/g
starting tissue) and magnetically stirred for 1 hour in
the cold. This solution was centrifuged at 20,000 x g
for 20 minutes, the supernatant decanted, and the precip
itate re-extracted with 3M potassium acetate. After
centrifugation the supernatants from both salt extractions
were combined and tRNA precipitated by the addition of
2 volumes of 9 5 per cent ethanol and 0.1 volume of 2M
potassium acetate. The precipitate was collected and
dissolved in 1M Tris buffer (pH 9.2) and incubated at
37°C for 30 minutes to deacylate the tRNA as described by
Mushinski and Potter (1969) . Transfer RNA was again
precipitated in ethanol and potassium acetate, collected
by centrifugation, dissolved in a small amount of
22
deionized water containing 0.005M 2-mercaptoethanol.
Light absorbance was determined at 280 nip and 260 mp.
The sample was routinely diluted to approximately 40 A2^q
units/ml and stored at -20°C until use. An A__ unit is
260
defined as that amount of sample per ml of solution which
produces an absorbance of 1 in a 1-cm light path at
260 mp.
Isolation of Synthetase
50-100 g of cotyledons were homogenized at high
speed in a Sorvall Omni-Mixer for 2-3 minutes in Buffer B
fl ml/g tissue) which consisted of: 0.04M KC1, 1M
Sucrose, 0.01M Tris-Cl (pH 7.9), 0.04 MgC^, and 0.01M
2-mercaptoethanol, along with an equal weight of insoluble
polyvinylpyrrolidone (Polyclar AT from General Aniline
and Film Corp., South San Francisco, California) saturated
with one-half strength Buffer B. The homogenate was
filtered through 4 layers of cheesecloth followed by a
single layer of Miracloth, and spun at 20,000 x g for
15 minutes. The supernatant was spun at 80,000 x g for
1 hour in a Beckman Ultracentrifuge, Model L2-65B, using
an SW-27 rotor. The supernatant was filtered through a
layer of Miracloth and protein precipitated by the
addition of solid ammonium sulfate to 70 per cent
saturation with stirring in the cold for 1 hour. The
23
precipitate was collected at 20,000 x g for 30 minutes
and dissolved in a small amount of 0.01M sodium phosphate
buffer (pH 6.0) and dialysed for 4 hours against one-half
strength Buffer B with sucrose omitted. Light absorbance
was determined at 260 mp and 280 mp and protein content
was estimated by the method of Layne (1957). The
synthetase preparation was stored in 40 per cent glycerol
at -20° without detectable loss in activity for up to
4 months.
Aminoacylation Reaction
Transfer RNA was aminoacylated at 29-30°C in a 1 ml
reaction mixture containing 10 pmoles Tris-Cl (pH 7.8),
5 pmoles MgC^, 0.2 per cent soluble polyvinylpyrrolodone,
5 pmoles ATP, approximately 1 mg synthetase protein and
10 A20Q units of tRNA, and 10 pmoles of leucine (either
or labeled). Radioactive leucine was a product of
Schwarz BioResearch, Orangeburg, New York, with Specific
Activities of 56.0 C/mmole for and 316 mc/mmole for
■^C. The charging reaction was initiated by the addition
of synthetase and at appropriate time intervals 0.1 ml
samples were pipetted onto glass fiber filters (Whatman
GF/A, 2.4 cm). The tRNA was precipitated by allowing
the filters to stand in ice-cold 10 per cent trichloro
acetic acid (TCA) for 30 minutes, followed by two washes
24
each of 10 per cent TCA and ethanol-ether (1:1, volume/
volume). The filters were dried under infrared lamps
and radioactivity determined in a Nuclear Chicago Unilux
II liquid scintillation counter. The scintillation
solvent consisted of a liter of toluene containing
4 g 2,5-Diphenylaxazole and 0.1 g [l ,4-bis-2-(5-Phenyl-
oxazolyl)-BenzeneJ , both products of New England Nuclear,
Boston, Massachusetts.
Chromatography of Transfer RNA
IVhen aminoacylated tRNA was to be used for fraction
ation by reverse phase chromatography it was routinely
treated as follows: the aminoacylation reaction was
allowed to proceed to a predetermined plateau level, which
was assumed to be the maximum level of charging. The
reaction was stopped by the addition of 2 ml of ice-cold
Buffer C which consisted of: 0.01M NaAc (pH 4.5), 0.01M
MgAc, 0.001M EDTA, and 0.00 5M 2-mercaptoethanol. The
sample was subsequently loaded onto a 1 cm x 6 cm
diethylaminoethyl cellulose column (Whatman DE-52) which
was equilibrated with Buffer C. The sample was pumped
onto the column, free amino acids washed clear with 10 ml
of Buffer C, and the aminoacyl-tRNA subsequently eluted
with 10 ml of Buffer C containing 1M NaCl. The sample
was diluted to a final concentration of 0.4M NaCl with
25 (
Buffer C and then loaded onto a Freon column as described
below.
A Freon column was prepared essentially by the
method of Weiss and Kelmers (1967). The organic phase was
prepared by mixing 475 ml of 1,1,1, 3-Tetrachlorotetra-
fluoropropane (Freon 214 from E. I. duPont de Nemours,
and Company, Los Angeles, California) with 25 ml of
methyl tricaprylyl ammonium chloride (Aliquat 336 from I
General Mills, Kankakee, Illinois), which was then washed !
successively with 2 volumes each of IN NaOIl, IN HC1, and
0.5M NaCl. The washed organic phase was then dried over
CaCl2 crystals overnight. Chromasorb W (purchased from ;
Johns-Manville Products Corporation, New York, New York)
was coated with organic phase (600 g Chrom W and 336 ml
organic phase) by tumbling the mixture in an air tight
container for 7 days. The coated Chromasorb was then
packed into a column (90 cm x 2.5 cm) at room temperature
and washed with 2 liters of Buffer C containing 0.4M
NaCl.
Aminoacyl-tRNA was loaded onto a Freon column in a
volume of approximately 20 ml (following DEAE chromatog
raphy) and eluted with a 2 liter linear gradient of 0.4M
to 0.8M NaCl in Buffer C. Fractions of 8 ml were collected:
at room temperature at a flow rate of 1.3 ml/minute.
Fractions were placed in a cold room at 4°C and tRNA
precipitated by the addition of 1.5 ml of 50 per cent TCA.
After at least 30 minutes in the cold the fractions were
collected on GF/A glass fiber filters (2.4 cm) by
filtration on a Millipore stand. The filters were washed
twice with 10 per cent cold TCA and twice with ethanol-
ether (1:1, volume/volume), dried under infrared lamps,
and radioactivity determined as described above.
Fractionation of Synthetase
Leucyl-tRNA synthetase was fractionated on a hydroxyl-
apatite column essentially by the method of Kanabus and
Cherry (1971). A 2.5 cm x 15 cm column was prepared by
suspending 10 g of hydroxylapatite (Bio-Gel IITP, Bio-Rad
Laboratories, Richmond, California) in 0.05M potassium
phosphate (pH 6.5) containing 0.01M 2-mercaptoethanol
(Buffer D), along with 1 g of cellulose powder (Whatman
CF-11). This slurry was poured into the column with
small pads of cellulose powder both above and below the
HTP mixture. The column was washed with 500 ml of Buffer
D prior to use.
For fractionation of leucyl-tRNA synthetase a sample
containing approximately 20 mg of protein (prepared as
previously described) was pumped onto the column in 50 ml
of Buffer D, and the protein was then eluted with a
linear gradient of potassium phosphate (pH 6,5) from 0.05M
27 i
to 0.4M in Buffer D. The total volume of the gradient
was 500 ml, using a flow rate of 1.5 ml/minute at 4°C,
collecting approximately 6 ml per fraction.
Fractions were assayed for leucyl-tRNA synthetase
activity in a 0.1 ml reaction mixture containing 0.05 ml
of fractionated protein and other components as previously
i
I
described. The reaction was allowed to proceed for 20
minutes at 29-30°C, collected on GF/A glass fiber filters, !
precipitated in 10 per cent TCA, washed, dried, and ,
counted as previously described.
Phenoxyacetylsuccinimide Derivatization Procedure '
i
Transfer RNA was aminoacylated in a 2 ml reaction as
described above containing 45 A2^q units of tRNA and
approximately 4 mg of synthetase protein. The reaction
was allowed to proceed to a predetermined plateau level
and tRNA was precipitated by the addition of 2 volumes of
95 per cent ethanol and 0.1 volume of 2M potassium acetate,,
and collected by centrifugation. Derivatization of
aminoacyl-tRNA was carried out by Peterkofsky and
Jesensky's (1969) modification of the method of Gillam
et al. (1968). Aminoacyl-tRNA was dissolved in 4 ml of
0.1M triethanolamine buffer (pH 4.0) containing 0.01M
Mg2S04 at 4°C, and 0.4 ml of tetrahydrofuran containing
10 mg of phenoxyacetic acid N-hydroxysuccinimide ester
28 i
(Schwarz BioResearch, Orangeburg, New York). The pH was
immediately raised to 8.0 by the addition of IN NaOII, the
reaction stirred for 10 minutes while maintained at 4°C,
and the pH lowered to 4.5 by the addition of IN acetic
acid. Derivatized aminoacyl-tRNA was precipitated with
ethanol and potassium acetate, collected by centrifugation, .
i
and dissolved in a small amount of deionized water. j
i
i
I
N-Carboxyanhydride Derivatization Procedure
i
This procedure utilized Peterkofsky and Jesensky's
(1969) modification of the method of Simon et al. (1964) j
to produce water insoluble tRNA-polyamino acid derivatives. I
Transfer RNA was aminoacylated in a 5 ml reaction mixture
containing 40 A250 uni‘ ts of tRNA and approximately 12 mg
of synthetase protein, and I I labeled leucine. The
reaction proceeded to a plateau level and was terminated
by the addition of ethanol and potassium acetate. The i
precipitate was collected by centrifugation and washed
twice with 0.5M NaCl in 6 7 per cent ethanol.
For derivatization, the washed precipitate was
dissolved in 10 ml of deionized water and 0.4 ml of
NaIiC03 was added. Then, 500 mg of /9-benzylaspartate
N-carboxyanhydride (Miles Laboratories, Elkart, Indiana)
was added in 6 ml of dioxane with stirring. A precipitate ;
forms immediately. The mixture was then left at 4°C
29
without stirring. After 2 hours, 10 ml of dioxane-water
(6:10, volume/volume) was added. The precipitate was
collected and washed three times in the dioxane-water
mixture. An aliquot was subsequently used to determine
the amount of charging with ^11-leucine. The supernatant
containing underivatized, and therefore, uncharged, tRNA
was concentrated and later used for charging with -^C-
leucine and other enzyme preparations.
CHAPTER III
RESULTS
Changes in tRNA^0U
In order to examine for possible changes in iso
accepting tRNA's and the corresponding synthetases as a
function of age, these components were isolated from
soybean cotyledons after various times of plant growth.
Under the growth conditions employed in this study the
maximum lifespan of the cotyledon was just over 21 days.
Following extraction, tRNA was aminoacylated in vitro
with radioactive leucine. The aminoacylation of tRNALeu
follows standard acylation kinetics; i.e., there is a
linear increase in the incorporation of radioactive amino
acid for the first 10 minutes, followed by a stationary
level after 15 to 20 minutes. A typical curve showing
the time course of acylation is demonstrated in Figure 1.
The plateau level is taken to be the maximum level of
charging which can be achieved under any given set of
conditions, utilizing specific tRNA and synthetase
preparations. The conclusion that charging is essentially
complete is reinforced by the fact that the addition of
inorganic pyrophosphatase (8.5 units/ml; Sigma Chemical
30
FIGURE 1
Aminoacylation of tRNA^eu ;
Time course of aminoacylation of tRNALeu. Both
tRNA and synthetase were derived from 5 day old cotyledons.
The reaction mixture contained 11 A ^ q units of tRNA and ;
approximately 1 mg of synthetase protein. The complete
acylation mixture and sampling techniques are described
under Methods and Materials.
The curve represents an average of duplicate
samples (O and □ ).
31
LEUCINE CPM X10
AMINOACYLATION OF tRNALeu
40
M
30
20
s c
10
60 50 40 30 20 10
MINUTES
33
Company, St. Louis, Missouri) did not enhance maximum
levels of charging (Kull et al., 1969). The stationary
level of charging is generally maintained for at least
30 to 40 minutes indicating that the synthetase preparation
is relatively free of ribonuclease activity.
To determine differences in the complement of iso
accepting species of tRNA^eu present in different aged
cotyledons, tRNA was extracted from different aged tissue
and aminoacylated in two separate in vitro reaction
mixtures. One reaction contained 3H-leucine while the
other had ^C-leucine. After the incorporation of radio
active leucine reached a stationary level the samples
were processed as described under Methods and Materials
and loaded onto a Freon column. The tRNALeu from soybean
cotyledons can be fractionated into six distinct peaks of
activity on a Freon column. These peaks are numbered
1 through 6 according to their elution pattern. Figure 2
shows a comparison of the relative amount of tRNA^eu
isoaccepting species present in 2 and IS day old cotyledons
by co-chromatography on a Freon column. Figure 3 is a
similar comparison between tRNA^eu from 5 and 21 day old
cotyledons. A synthetase preparation derived from 5 day
old cotyledons was used in the aminoacylation of each of
these tRNA samples. Reversing the radioactive labels had
no effect on the profiles, indicating that these dif-
FIGURE 2
Co-chromatography of tRNA^eu from
2 and 15 Day Old Cotyledons
Samples of tRNALeu from 2 and 15 day old cotyledons
were aminoacylated in two separate reaction mixtures.
Each reaction contained approximately 10 A25q units of
tRNA and 1 mg of synthetase protein from 5 day old
cotyledons. The tRNALeu from 2 day cotyledons is charged
with 14C-leucine C -3 while 15 day tRNALeu is charged
with ^H-leucine (--------).
The two aminoacylated tRNA samples were combined
and co-chromatographed on a Freon column as described
under Methods and Materials. The radioactive peaks are
numbered according to their elution pattern.
34
I
o
r— I
X
O i
u
w
s
H
u
D
w
J
I
sc
m
CO-CHROMATOGRAPHY OF tRNALeU FROM 2 AND 15 DAY COTYLEDONS
16
40
12
30
20
10
100 20 40 60 80 120
m
i
0
i — i
X
2
0 1
U
w
s
H
0
S3
w
1
o
w
in
FRACTION NUMBER
FIGURE 3
Co-chromatography of 5 and 21 Day tRNA^eu
Freon column co-chromatography of tRNALeu from
5 day old cotyledons (-------) and 21 day old cotyledons
(------ ). Both samples contained approximately 10 A26O
units of tRNA and 1 mg of S day synthetase protein.
^11-leucine was used with 5 day tRNA and ^C-leucine was
used with 21 day tRNA.
36
Leu
CO-CHROMATOGRAPHY OF 5 AND 15 DAY tRNA
32
12
24
E
ft
U
a
s
M
U
0
w
a
1
rF
16
100 120 20 40 60 80
( N
I
O
rH
x
E
U
W
s
H
0
£ >
a
> 4
1
u
' j *
r— i
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FRACTION NUMBER
38
ferences are in fact due to quantitative differences in the
T 0 11
tRNA isoaccepting species present in different aged
cotyledons.
Table 1 summarizes these data. The key changes
observed are a decrease in the relative amount of Peak 2,
with sharp increases in the relative amounts of Peaks 5
and 6. Peak 1 does not show any consistent pattern of
change with age. This peak may partially represent de
graded tRNA (Anderson and Cherry, 1969) which is present in
different amounts in different tRNA preparations. Peaks 3
and 4 do not significantly change with age of the tissue.
Cytokinin Effect on tRNALeu
A specific class of compounds, N^-substituted purines,
are known to occur in at least certain tRNA's of yeast,
bacteria, plants, and animals (Hall, 1970). Specifically,
the modified adenine residue is known to be adjacent to
the anticodon and has been demonstrated only in those
tRNAs which respond to codons beginning with U. N^-substi-
tuted purines also comprise a class of plant hormones, the
cytokinins, which have been shown to retard or delay
senescence in excised leaves. Osborne (1965) suggested
that the mode of cytokinin action in delaying senescence
may be through maintenance of nucleic acid and protein
synthesis. It was therefore of interest to determine
TABLE 1
Relative Amounts of tRNA^611 Isoaccepting Species
Present in Different Aged Cotyledons
Approximately 11 A2^q units of tRNA from cotyledons
of various ages was acylated in a 1 ml reaction mixture
and fractionated on a Freon column. A synthetase
preparation from 5 day old cotyledons was used in every
case. The amount of radioactivity in each peak was
summed and expressed as per cent of the total radioactivity
in the six peaks.
39
]
TABLE 1
Relative Amounts of tRNA^eu Isoaccepting Species
Present in Different Aged Cotyledons
AGE (Days) PEAK
(tRNA) 1 2 3 4 5 6
2 10.3 66.0 8.3 6.8 3.9 4.5
5 13. 2 57.6 9.2 7.3 5.2 6.7
15 7.2 53.6 6,6 5.8 12.4 14.4
21 11. 3 37.5 10,2 7.9 14.6 18. 5
40
41
what possible effect cytokinin application might have on
the complement of tRNA^eu species. This seemed particu
larly pertinent since two of the six codons assigned to
leucine (Nirenberg et al., 1966) begin with U. It might
therefore be expected to find the modified adenine residue
in at least some of the tRNALeu species.
Figure 4 shows a comparison of elution profiles of
tRNA^eu from control plants and plants sprayed with the
synthetic cytokinin, 6-benzylaminopurine (BAP; purchased
from Cal Biochem, Los Angeles, California). The concen
tration used and details of treatment are given in the
legend to Figure 4. The relative amount of each of the
tRNALeu isoaccepting species is given in Table 2. Peaks
5 and 6 are seen to become a larger percentage of the
total leucine accepting tRNA species. These results are
consistent with the preliminary observations of Anderson
and Cherry (1969) who showed that treatment of soybeans
with 6-benzylaminopurine causes an increase in tRNALeU
species 5 and 6 from hypocotyl tissue. These tRNA species
are normally present in only barely detectable quantities
in this tissue. It is not yet known whether the changes
determined in the present study reflect an absolute
increase in Peaks 5 and 6 or a decrease in Peaks 1 through
4 relative to 5 and 6.
The effect of cytokinin application on the relative
FIGURE 4
Chromatography of tRNA^eu from
BAP Treated Cotyledons
Freon column elution profile of tRNA^eu from
6-benzylaminopurine treated (------) and control (-------)
cotyledons, 4 days of age. For BAP treatment seeds were
imbibed in 2 x 10 "SM BAP for 6 hours and then planted
in Vermiculite moistened with the same concentration of
BAP. Cotyledons were excised after 4 days of growth and
tRNA isolated as described under Methods and Materials.
Control plants were imbibed and planted with water in
place of BAP.
Control tRNA was acylated with ^C-leucine and
BAP tRNA with 3H-leucine. Approximately 8 A260 units
tRNA and 0.5 mg of 5 day synthetase protein was used in
each reaction mixture.
42
ro
i
0
i — I
X
S
c u
u
H
s
H
u
3
H
1
U
" 3 *
i- 4
CHROMATOGRAPHY OF tRNALeU FROM BAP TREATED COTYLEDONS
12
6
4
2
120 100 80 60 40 20
fi
J
0
i — i
X
s
£ u
U
[ 4
a
H
u
3
M
i 4
1
a
n
o - j
FRACTION NUMBER
TABLE 2 j
Relative Amount of tRNA^eu Species
I
in BAP Treated Cotyledons*
I
PEAK
Treatment
1 2 3 4 S 6
Control 16.0 55.1 10.3 7.6 4.7 6.3
BAP 10.8 56.0 9.2 7.2 7.6 9.1
*Data presented as in Table 1
44
45
amount of isoaccepting tRNA^eu species suggested that the
changes might be due to modification of tRNA species.
Specifically, such modification might take place through
the isopentenyl adenosine residue (IPA) which is the most
common form of N^-substituted purine found adjacent to
the anticodon. Gefter and Russel (1969) have reported
the separation of three tRNATyr species from E. coli
which differed only in the extent to which an adenosine
adjacent to the anticodon was modified. And in a later
report Rosenberg and Gefter (1969) showed that the
elution profile of a number of tRNA’s was altered when
modification of this adenosine residue was not complete.
This suggested that there may be an interconversion of at
least some of the tRNA^eu isoaccepting species through
modification of this cytokinin-active residue. To test
this possibility, tRNA preparations were incubated with
potassium permanganate which has been shown to cleave the
isopentenyl side chain of IPA containing tRNA’s (Robbins
et al., 1967). These results are presented in Figure 5
which shows that there is no difference in the relative
amount of isoaccepting tRNALeu species between control
and permanganate treated tRNA,
It appeared possible that since cytokinin treatment
apparently affects tRNA^e^ ^ these two species might be
modified forms of any of the other four species. The
FIGURE 5
Chromatography of Permanganate
Treated tRNA^eu
Approximately 40 ^260 units of tRNA from 5 day
old cotyledons was dissolved in 6 ml of water, to which
0.04 ml of 0.1 per cent potassium permanganate was added.
This mixture was incubated for 15 minutes at room temper
ature. Transfer RNA was precipitated with the addition
of ethanol and potassium acetate and subsequently used in
an aminoacylation reaction. Control tRNA was the stock
preparation without pre-incubation.
Permanganate treated tRNA was acylated with ^C-
leucine (-------) and control with 3H-leucine (-------),
Both reactions contained approximately 8 A26Q units of
tRNA and 0.5 mg of synthetase protein.
Although there appears to be a slight change in
peak ratios, duplicate samples have shown that this is
not significant.
46
20
10
CHROMATOGRAPHY OF PERMANGANATE
TREATED tRNALeU
16
m
O
rH
12
W
2
H
u
D
W
a
i
a
120 100 80 60 20 40
CM
I
0
rH
X
s
p j
u
a
s
H
u
a
J
1
U
T
rH
- p= >
FRACTION NUMBER
48
report of Anderson and Cherry (1969) demonstrated that
Peaks 5 and 6 are normally not detectable in hypocotyl
tissue, but are detected in very small quantities if
hypocotyl tRNA is acylated with cotyledon synthetase. If ;
Peaks 5 and 6 are modified forms of any of the other j
four peaks, hypocotyl tissue must lack the enzymes
necessary for this modification. In contrast, the !
cotyledon would have such enzymes since it contains
Peaks 5 and 6. Therefore, the presence of small quantities,
of Peaks 5 and 6 when hypocotyl tRNA is acylated by
cotyledon synthetase might be due to modification of
I
hypocotyl tRNA during the acylation reaction. To test i
this possibility, hypocotyl tRNA was incubated with i
cotyledon synthetase in the absence of amino acids,
isolated, and subsequently acylated with cotyledon syn
thetase. Figure 6 shows that incubation of hypocotyl
tRNA with cotyledon enzyme has no apparent effect on the
relative amounts of tRNA^eu species present. Although
these experiments cannot rule out the possibility, the
interconversion of isoaccepting tRNALeu species seems
unlikely, particularly through modification of the IPA
moiety.
Changes in leucyl-tRNA Synthetase
The possibility of specific synthetase changes as a
FIGURE 6
Chromatography of Hypocotyl tRNA^eu
Pre-incubated with Cotyledon Enzyme
Approximately 130 units of hypocotyl tRNA were
incubated for 40 minutes at 29°C with 12 mg of synthetase
protein from 5 day old cotyledons. The incubation
mixture contained 0.01M Tris-Cl (pH 7.8) and 0.01M ATP.
The tRNA sample was then phenol extracted and used for
aminoacylation, The control tRNA is hypocotyl tRNA which
has not been incubated.
The control tRNA^eu sample was charged with 14c-
leucine (------) and the pre-incubated sample with ^H-
leucine ( ). Both samples were aminoacylated with
synthetase from 5 day old cotyledons.
49
Leu
CHROMATOGRAPHY OF HYPOCOTYL tRNA PRE-INCUBATED
25
WITH COTYLEDON ENZYME
30
20
25
o
20
k
Z
H
u
D
h
a
15
x
10
ro
100 60 80 120 20 40
C M
I
O
H
X
s
a .
u
w
s
H
u
D
w
t
u
Ln
O
FRACTION NUMBER
function of cotyledon age was also of interest in this
study. Therefore, synthetase was extracted from cotyledons
of 5 and 21 days of age and used to aminoacylate both
homologous and heterologous tRNA^eu. Figure 7 shows the
capacity of synthetase preparations from young (5day) and
old (21 day) cotyledons to acylate tRNA^eu from 5 and 21
day old cotyledons. Eleven A25Q units of tRNA was used
in each reaction and the tRNA concentration was shown to
be limiting. The addition of inorganic pyrophosphatase
(8.S units/ml) did not enhance the maximum level of
charging in any case, indicating total aminoacylation of
tRNA species chargeable by a given synthetase preparation.
It can be seen from Figure 7 that there is not only a
reduction in the capacity of tRNALeu derived from 21 day
old cotyledons to be charged, but there is also a corre
sponding reduction in the capacity of synthetase from
21 day old cotyledons to aminoacylate tRNA^eu. Thus,
leucyl-tRNA synthetase derived from 21 day old cotyledons
is only 1/3 as effective in charging 5 day tRNA as is the
5 day synthetase, and similar results are seen in
charging tRNA derived from 21 day old cotyledons.
Chromatographic Comparison of leucyl-tRNA
Synthetase Activities
In order to determine the nature of the changes which
FIGURE 7
Relative Activity of tRNA^eu and Synthetase
from 5 and 21 Day Cotyledons
Time course of aminoacylation of tRNA^eu and
synthetase extracted from cotyledons after 5 and 21 days
of plant growth, using homologous and heterologous
components. Each reaction mixture contained 11 &2(>0 units
of the appropriate tRNA sample and approximately 1 mg of
synthetase protein.
A) 5 day synthetase, 5 day tRNA
B) 5 day synthetase, 21 day tRNA
C) 21 day synthetase, 5 day tRNA
D) 21 day synthetase, 21 day tRNA
LEUCINE CPM xlO
53
RELATIVE ACTIVITY OF tRNALeu AND SYNTHETASE
40
FROM 5 AND 21 DAY COTYLEDONS
30
20
10
40
30
20
10
50 30 50 10 10 30
MINUTES
54
occur in synthetase activity with advancing cotyledon age,
a 5 day tRNA preparation was aminoacylated in vitro
utilizing 5 and 21 day synthetase preparations in two
separate reactions. Figure 8 shows the co-chromatographic
profiles of these two synthetase activities in charging
the various tRNALeu isoaccepting species. The relative
amount of leucine acceptor activity in each peak is
shown in Table 3. The elution profiles have been normal
ized relative to Peaks 5 and 6 where the total amount of
leucine charged is nearly the same (data in Table 4). The
most notable difference in these profiles is the reduced
capacity of the synthetase derived from 21 day old
cotyledons to fully acylate Peaks 1 through 4.
Similar changes are observed when tRNA from 21 day
old cotyledons is aminoacylated with 5 and 21 day synthe
tase preparations. These data are shown in Figure 9 and
Table 3. The leucine acceptor activity in Peaks 1 through
4 is even further reduced and Peaks 5 and 6 are becoming
a major portion of the total leucine acceptor activity in
the 21 day old cotyledon.
From the data in Table 3 a comparison can be made of
the relative per cent of each acylated tRNA^eu species
present in the two homologous systems (S day tRNA-5 day
synthetase, and 21 day tRNA-21 day synthetase). Table 4
summarizes the total pmole acceptor activity in the six
FIGURE 8
Co-chromatography of 5 Day tRNALeu Acylated
with 5 and 21 Day Synthetase
Freon column elution profile of tRNA^eu derived
from 5 day old cotyledons acylated with either 5 day
synthetase (-------) or 21 day synthetase (-------} . H-
leucine was used in the former case, -^C-leucine in the
latter. Both samples were acylated to a predetermined
plateau level prior to being combined and fractionated.
Each sample contained approximately 11 A ^ q units
of tRNA and 1 mg of synthetase protein.
55
Leu
ACYLATED WITH
5 AND 21 DAY SYNTHETASE
CO-CHROMATOGRAPHY OF 5 DAY tRNA
12
m
o
i — i
X
120 100 80 20 40 60
32
CM
I
24 X
E
& 4
u
w
z
16
u
a
w
a
i
u
Ln
C T 4
FRACTION NUMBER
TABLE 3
Relative Amount of tRNA^eu Species Charged
by 5 and 21 Day
?EAK
Synthetase*
tRNA
(Age
Enzyme
in Days)
1 2 3 4 5 6
5 5 13.5 57.7 9.4 8.3 5.9 5.5
5 21 11. 3 37.5 10.2 7.9 14.6 18 . 5
21 5 12.9 49.9 9.9 8.6 9.1 9.4
21 21 9.9 31. 5 7.6 6.3 18.5 26.2
*Data presented as in Table 1
57
FIGURE 9
Co-chromatography of tRNA^eu Acylated
with 5 and 21 Day Synthetase
Freon column co-chromatography of tRNALeu derived
from 21 day old cotyledons acylated with 5 day synthetase
(-------) and 21 day synthetase (-------}, using ^H-leucine
and ^C-leucine respectively.
Each sample contained approximately 11 A2gp units
of tRNA and 1 mg of synthetase protein.
58
Leu
CO-CHROMATOGRAPHY OF tRNA ACYLATED WITH 5 AND 21
DAY SYNTHETASE
32
24
o
rH
* 18
£
04
u
24
w
s
H
u
D 12
w
► 4
1
16
m
120 100 F0
60 40 20
FRACTION NUMBER
TABLE 4
Leucine Acceptor Activity of 5 and 21 Day
tRNA and Synthetase
The total pmole acceptor activity was determined
from the maximum level of charging achieved in an in
vitro assay. The acceptor activity in each peak was
calculated from the total pmole incorporation by using
the per cent of total radioactivity in each peak. Values
are the average of two separate column runs which vary
- 10 per cent. Calculation of total pmole acceptor
activity in each peak by summation of the radioactivity
in each peak is in close agreement with the values given
in this table.
TABLE 4
Leucine Acceptor Activity in 5 and 21 Day tRNA and Synthetase
Enzyme tRNA Total pmole/^gg pmole in each Peak
1 2 3 4 5 6
A S S 36.6 4.4 19.2 3.0 2.4 1.9 2.3
B 5 21 18.2 2.1 6.8 1.9 1.5 2.6 3.3
C 21 5 11.0 1.5 5.8 1.1 1.0 1.1 1.2
D 21 21 6.0 0.6 1.9 0.4 0.4 1.1 1.5
Ratio A/D 6.1 7.3 10.1 7.5 6.0 1.7 1.5
62
tRNA^eu species in both young and old cotyledons with
respect to both tRNA acceptor activity and synthetase
charging capacity. It is particularly notable that the
pinoles of leucine charged in Peaks 5 and 6 is only
slightly reduced (on the basis of &26Q un^ts tRNA) in
the homologous system from old cotyledons when compared
to 5 day old tissue, while the acceptor activity in
Peaks 1 through 4 is reduced between 6 and 10 fold.
Synthetase Derived from 21 Day Old Cotyledons Cannot
Fully Acylate tRNALeu
The reduced capacity of synthetase derived from 21
day old cotyledons to acylate tRNA^eu was of particular
interest since there was no evidence that any tRNALeu
isoaccepting species i\rere being lost, as detected by Freon
column chromatography (Figures 8 and 9). Rather, only
maximal levels of label incorporation were affected. To
determine the nature of the loss in synthetase activity,
5 day tRNA was acylated to a plateau level with 21 day
synthetase, following which 5 day synthetase was added.
Figure 10 shows that under these conditions the addition
of 5 day synthetase promotes further charging. The
addition of more 21 day synthetase does not produce such
an effect however. Similarly, when 5 day tRNA is acylated
to a plateau level with 5 day synthetase, the addition
FIGURE 10
Additive Effects of S and 21
Day Synthetase
A) Transfer RNA from 5 day old cotyledons is acylated
with approximately 1 mg of 21 day synthetase protein
(O o) . After 32 minutes of charging 0.4 mg of addition
al 21-day synthetase is added to the reaction .
Incubation of 21 day synthetase in a reaction mixture
minus tRNA (□---□) shows no charging.
B) Transfer RNA from 5 day old cotyledons is acylated
with approximately 1 mg of synthetase protein from 21 day
old cotyledons (O O) . After 32 minutes 0.4 mg of
5 day synthetase is added to the reaction •) .
Incubation of S day synthetase in a reaction mixture
minus tRNA (□— □) shows no charging.
Data are presented as CPM/A26Q to allow for the
concentration difference of samples taken before and after
the addition of new enzyme.
63
LEUCINE CPM/A260 xlO
64
ADDITIVE EFFECTS OF 5 AND 21 DAY SYNTHETASE
12
“O
8
4
20
16
/
8
4
10 20 30 40 50 60
MINUTES
65
of either 5 day or 21 day synthetase does not promote
further charging. When either synthetase preparation
was incubated in a charging reaction lacking added tRNA,
no detectable aminoacylation took place (Figure 10). This
rules out the possibility that significant amounts of
tRNA were present in either of the synthetase preparations.
In order to determine if the two synthetase prep
arations might differ in ribonuclease activity, or in
some other factor(s) which might modify tRNA so as to
alter its amino acid acceptor activity, the following
experiment was performed. Aliquots of 5 day tRNA were
separately incubated with either 5 day or 21 day synthetase
preparations in the absence of amino acids. CTP was
added to this otherwise complete charging mixture.
Incubation was carried out for 30 minutes. Following
phenol extraction and ethanol precipitation the tRNA
samples were aminoacylated with radioactive leucine and
the converse synthetase preparation. The results of
these pre-incubation experiments are shown in Table 5.
In neither case does pre-incubation of tRNA with one
synthetase preparation alter the relative distribution
of label incorporation into the various tRNALeu species
upon subsequent acylation with the converse synthetase.
These experiments rule out differential ribonuclease
activity in the two different synthetase preparations as a
TABLE 5
Effect of Pre-incubation on Charging
Transfer RNA from 5 day old cotyledons (25 A^^^ units)
was incubated with 2 mg of synthetase protein (either 5
or 21 day as indicated) in 20 jjmoles Tris-Cl (pH 7.8),
10 mmoles MgCl2, 0.2 per cent polyvinylpyrrolidone,
10 jumoles ATP, and 10 jjmoles CTP in a total volume of
2 ml. Incubation was carried out for 30 minutes at 30°C.
Transfer RNA was phenol extracted, precipitated in ethanol,
and collected by centrifugation. The converse synthetase
preparation was then used to acylate tRNA^eu which was
subsequently fractionated on a Freon column. Data are
presented as in Table 1.
66
TABLE 5
Effect of Pre-incubation on Charging
Charging Enzyme tRNA Pre-incubation
1 2
Peak
3 4 5 6
5 day 5 None 13.7 59.2 8.7 7.3 5.5 6.1
5 day 5 21 Enzyme 12.9 58.3 9.1 8.0 5.3 6.3
21 day 5 None 12.5 48.7 9.6 9.0 9,6 10.1
21 day 5 5 Enzyme 13.0 49.6 9.4 8.5 9.8 9.4
68
source of differences in peak charging. Additionally,
differences in the capacity for the removal or addition of
the -CpCpA terminus of tRNA is ruled out as a cause of
differences of relative charging of tRNA^eu isoaccepting
species, as well as other modifiers of tRNA amino acid
acceptor activity.
In order to determine which tRNA^eU species were
being charged when 5 day synthetase was added to the
completed S day tRNA-21 day synthetase reaction (Figure
10) , two experimental schemes were utilized.
Firstly, 5 day tRNA was aminoacylated with 21 day
synthetase and leucine to a predetermined plateau
level. The tRNA was then precipitated and derivatized
with phenoxyacetic acid N-hydroxysuccinimide ester to
stabilize the aminoacyl bond as described under Methods
and Materials. Following derivatization and isolation,
the tRNA was acylated in a reaction mixture containing
■^C-leucine and either 5 day or 21 day synthetase. These
data are shown in Figure 11. Using the 5 day synthetase
preparation there is a substantial amount of -^C-leucine
incorporation into the tRNA which was previously charged
with 21 day synthetase. There is only a low level of
-^C-leucine incorporation with 21 day synthetase, and
this incorporation appears to correlate with the loss in
label. A similar sample of derivatized tRNA (following
FIGURE 11
Labeling of Phenoxyacetylsuccinimide ;
|
Derivatized tRNA^eu
i
i
Transfer RNA from 5 day old cotyledons was acylated
with 21 day synthetase and ^H-leucine, following which ,
I
it was isolated and derivatized by the phenoxyacetyl-
j
succinimide procedure described under Methods and
Materials. Samples were then acylated with either 5 day
synthetase (A A) or 21 day synthetase (O O) , using
■^C-leucine. The loss of ^H-leucine from derivatized
tRNA^eu was also measured during subsequent charging
(□ D) .
69
70
LABELING OF PHENOXYACETYLSUCCINIMIDE
DERIVATIZED tRNALeu
16
12
16
8
12
4
10 20 30
(N
I
O
rH
X
E
P u
O
w
E S
H
U
D
W
vA
I
E C
n
MINUTES
71
acylation with 21 day synthetase) was acylated with -^C-
leucine and 5 day synthetase to a stationary level and
then loaded onto a Freon column for fractionation. These
results are presented in Figure 12 which shows that the ,
31I-leucine is incorporated into all six peaks of tRNALeu, 1
whereas significant -^C counts are found only in Peaks 1
through 4. This demonstrates that the synthetase derived !
from 21 day old cotyledons can fully acylate Peaks 5
and 6 but is apparently deficient in its capacity to fully
acylate isoaccepting Species 1 through 4. When tRNA^eu is i
i
aminoacylated with synthetase from 5 day old cotyledons,
derivatized, and subsequently acylated with additional j
5 day synthetase and a different radioactive label, there i
is no further incorporation of labeled amino acid.
The second experimental approach utilized a deriv-
atization procedure which renders aminoacyl-tRNA water
insoluble. The method depends on the capacity of the
free *t-amino group of an amino acid acylated to a specific ;
tRNA to act as a chain initiator for the polymerization
of an N-carboxyanhydride. The N-carboxyanhydride of
/3-benzylaspartate forms a water insoluble polymer. |
Therefore, 5 day tRNA was acylated with 21 day synthetase
and isolated and derivatized according to the N-carboxy
anhydride procedure as described under Methods and Materi
als, The precipitate which contains derivatized amino-
FIGURE 12
Chromatography of Phenoxyacetylsuccinimide
Derivatized tRNALeu
Freon column chromatography of tRNA^eu acylated
with both 21 and 5 day synthetase. Transfer RNA from
5 day old cotyledons was acylated in a 2 ml reaction
with 21 day synthetase and ^H-leucine (-------) for 35
minutes. After isolation and derivatization with phenoxy-
acetic acid N-hydroxysuccinimide ester as described under
Methods and Materials, the tRNALeu sample was acylated
with 5 day synthetase and l^c_ieucine ( ) for
10 minutes. The sample was subsequently fractionated on
a Freon column.
The radioactive profiles of the two separate labels
should represent the charging capacities of the two
synthetase preparations.
72
C M
I
o
E
Oj
0
w
s
H
u
p
w
p
1
K
n
CHROMATOGRAPHY OF PHENOXYACETYLSUCCINIMIDE DERIVATIZED tRNALeU
8
6
4
2
s » * ■
120 100 80 60 20 40
8
C M
I
0
rH
x
E
u
w
s
t - l
u
p
P
1
u
I —i
' - J
Lrl
FRACTION NUMBER
74
acyl-tRNA was washed and the initial supernatant and
washes combined. This solution contains all of those
tRNA species which were not acylated by the 21 day
synthetase. Transfer RNA was concentrated by ethanol
precipitation and subsequently used for aminoacylation by
either 5 day or 21 day synthetase preparations and
■^C-leucine. Figure 13 shows that the supernatant result- j
ing from this derivatization procedure contains only !
tRNA^e^, Attempts to acylate the supernatant tRNA with
21 day synthetase resulted in no incorporation of radio
active label. Similarly, if tRNA was first acylated with
5 day synthetase and subsequently derivatized by the j
N-carboxyanhydride procedure, no further label incorpora
tion can be demonstrated when the supernatant tRNA is
used for aminoacylation by either 5 day or 21 day
synthetase.
These results are consistent with those using the
phenoxyacetylsuccinimide derivatization procedure and
suggest that while the leucyl-tRNA synthetase derived from
21 day old cotyledons can fully acylate tRNA^|^, it is
deficient in its capacity to fully acylate isoaccepting
Species 1 through 4.
FIGURE 13
Chromatography of Carboxyanhydride
Derivatized Supernatant tRNALeu
Approximately 40 &26Q units of 5 day tRNA was
acylated in a 5 ml reaction containing 12 mg of 21 day j
1
synthetase protein. The reaction was carried out for 60 ,
minutes in the presence of ^M-leucine. Transfer RNA was
precipitated in ethanol and potassium acetate, washed with
0.5M NaCl in 67 per cent ethanol, and derivatized according-
to the carboxyanhydride procedure described under Methods ■
i
and Materials. The derivatized aminoacyl-tRNA precipitates !
in this reaction. The supernatant therefore contains
those tRNA species which were not charged in the initial
reaction with 21 day synthetase.
The supernatant was concentrated and aliquots used
for charging by either 5 day or 21 day synthetase, using
|
■^C-leucine. The samples were subsequently fractionated
on a Freon column. The data for subsequent charge with,
21 day synthetase is not shown as no significant counts
were detectable. The profile is ^^C-leucine incorporation
utilizing 5 day synthetase.
75
CHROMATOGRAPHY OF CARBOXYANHYDRIDE
DERIVATIZED SUPERNATANT tRNALeu
2
-i----------------1 1 1 1 ----------- -— “i --------------- r-
40 60 80 100 120
fraction NUMBER
77
Fractionation of leucyl-tRNA Synthetase
Kanabus and Cherry (1971) have reported the fraction
ation of leucyl-tRNA synthetase from cotyledons into
three peaks of activity on hydroxylapatite columns. The
peak which elutes first is specific for acylating tRNA^f^
5q 6
i
while the other two fractions are specific for acylating
i
isoaccepting Species 1~4. A possible reason for the loss |
in the capacity of 21 day synthetase to aminoacylate
Species 1-4 is that there is a specific loss in one of the
i
two leucyl-tRNA synthetase fractions specific for acylating ,
these tRNA^eu species. I
This possibility was examined by fractionating leucyl-
tRNA synthetase from 5 and 21 day old cotyledons on
hydroxylapatite columns. These results are shown in
Figures 14 and 15. It is apparent that leucyl-tRNA
synthetase is separable into three peaks of activity, and
that all three peaks are present in both the 5 day old
and 21 day old cotyledons. The relative heights of each
peak reflects the enzymatic activity in a 20 minute
charging assay, but not necessarily the absolute amount
of enzyme in each fraction. The peak tube from each of
the three fractions were used to aminoacylate a tRNA
sample which was subsequently fractionated on a Freon
column. Figure 16 shows the Freon column profile of those
s
FIGURE 14
Hydroxylapatite Fractionation of
5 Day Synthetase
Approximately 20 mg of synthetase protein prepared
from 5 day old cotyledons was loaded onto a column of
hydroxylapatite and eluted with a linear gradient of
potassium phosphate (0,05 to 0.4M). Fractions were
assayed for leucyl-tRNA synthetase activity in a 20 minute
reaction as described under Methods and Materials. Every
other tube was assayed for activity.
78
LEUCINE INCORPORATION CPM xlO
50
HYDROXYLAPATITE FRACTIONATION OF 5 DAY SYNTHETASE
40
30
20
10
V
20 40 60 80 100
FRACTION NUMBER
FIGURE 15
Hydroxylapatite Fractionation of
21 Day Synthetase
Approximately 20 mg of synthetase protein from 21
day old cotyledons was fractionated on a column of
hydroxylapatite as described under Methods and Materials.
Every other tube was assayed for leucyl-tRNA synthetase
activity. Once the peaks were located, additional tubes
were assayed in order to get a significant number of
points in each peak.
80
LEUCINE INCORPORATION CPM xIO
HYDROXYLAPATITE FRACTIONATION OF 21 DAY SYNTHETASE
8
6
4
2
20 40 60 80 100
FRACTION NUMBER
FIGURE 16
Chromatography of tRNA^eu Acylated
with Fraction 1 Synthetase
The peak tube from Fraction 1 of the hydroxylapatite
fractionated 5 day synthetase (Figure 14) was used to
acylate tRNA^0U. The reaction contained 10 ^2^0 units of
5 day tRNA, 3H-leucine, 0.4 ml of Fraction 1 synthetase,
and other components as previously described to produce
a complete reaction mixture. Aminoacylation was carried
out to a predetermined plateau level, following which
tRNA was fractionated on a Freon column.
LEUCINE CPM xlO
Leu
ACYLATED WITH CHROMATOGRAPHY OP tRNA
FRACTION I SYNTHETASE
16
12
i
K
m
120 100 60- 80 20 40
FRACTION NUMBER
84
tRNA^eu species which are acylated by Fraction 1
synthetase from 5 day old cotyledons. Similarly, Figures
17 and 18 demonstrate the tRNA^eu species which are
acylated with Fractions 2 and 3 synthetase, respectively.
It is apparent that Fraction 1 synthetase is specific for
acylating tRNA^^ while the other two synthetase Fractions
are both specific for acylating Species 1-4. Additionally,
this specificity was seen to be the same in the fraction
ated synthetase from both 5 and 21 day old cotyledons.
T 611 •
The time course of aminoacylation of tRNA using
each of the three synthetase fractions from 5 day old
cotyledons is shown in Figure 19. Interestingly, both
Fractions 2 and 3 are seen to charge tRNA^eu to a higher
plateau level than the unfractionated synthetase. This
is true even tlyiugh the concentration of synthetase in
any of these three fractions must be more dilute than in
the unfractionated synthetase, at least by a factor of
three.
A similar study was made of the fractionated synthe
tase from 21 day old cotyledons. These data are shown in
Figure 20. In contrast to the activity of fractionated
5 day synthetase, Fraction 1 of the 21 day synthetase
charges the same tRNA sample to a higher plateau level
than the unfractionated 21 day synthetase. It is
particularly noteworthy that Fraction 1 synthetase from
FIGURE 17
Chromatography of tRNA^eu Acylated
with Fraction 2 Synthetase
The peak tube from Fraction 2 of the hydroxylapatite
fractionated 5 day synthetase (Figure 14) was used to
acylate tRNA^eu from 5 day cotyledons. Aminoacylation
reaction conditions are as described in the legend to
Figure 16 except that ^C-leucine was used. The sample
was then loaded onto a Freon column.
85
Leu
CHROMATOGRAPHY OF tRNA ACYLATED WITH
FRACTION 2 SYNTHETASE
120 100 80 60 40 20
FRACTION NUMBER
FIGURE 18
Chromatography of tUNA^011 Acylated
with Fraction 3 Synthetase
The peak tube from Fraction 3 of the hydroxylapatite
fractionated 5 day synthetase was used to acylate 5 day
tRNA^eu. The tRNA sample was then fractionated on a
Freon column. Aminoacylation conditions are as described
in the legend to Figure 16 except that l^C-leucine was
us ed.
87
Leu
ACYLATED WITH CHROMATOGRAPHY OF tRNA
FRACTION 3 SYNTHETASE
40 60 80 100 120 20
FRACTION NUMBER
FIGURE 19
Aminoacylation of tRNA^eu with
Fractionated 5 Day Synthetase
Approximately 10 ^260 units ^ ^ay t^NA was
acylated with l^c-leucine in a 1 ml reaction containing:
Co-----#) 1 mg of unfractionated 5 day synthetase,
(A----A) 0.4 ml of Fraction 1, hyroxylapatite
fractionated 5 day synthetase,
(O-----o) 0.4 ml of Fraction 2, hydroxylapatite
fractionated 5 day synthetase,
(□ □) 0.4 ml of Fraction 3, hydroxylapatite
fractionated 5 day synthetase.
89
LEUCINE CPM xlO
Leu
WITH FRACTIONATED 5 DAY SYNTHETASE AMINOACYLATION OF tRNA
« 12
20 40 60 100 80 120
MINUTES
91
FIGURE 20
Aminoacylation of tRNALeu with
Fractionated 21 Day Synthetase
Approximately 10 A26O units of 5 day tRNA was
icylated with ^C-leucine in a 1 ml reaction containing:
(•— — •) 1 mg of unfractionated 21 day synthetase,
(A A) 0.4 ml of Fraction 1, hydroxylapatite
fractionated 21 day synthetase,
( O— — o) 0.4 ml of Fraction 2, hydroxylapatite
fractionated 21 day synthetase,
(□---- □) 0.4 ml of Fraction 3, hydroxylapatite
fractionated 21 day synthetase.
AMINOACYLATION OF tRNAL8U WITH FRACTIONATED 21 DAY SYNTHETASE
100 120 80 20 40 60
MINUTES
93
both 5 and 21 day old cotyledons deomonstrate nearly the
same activity in acylating tRNALeu, while the activity of
Fractions 2 and 3 is greatly reduced in the 21 day old
cotyledon.
The earlier data which demonstrated a reduction in !
the charging capacity of 21 day synthetase suggested that
this reduction was primarily due to a loss in the capacity j
to fully acylate tRNA^e^. The data from the fractionated j
j
synthetases is also consistent with this suggestion. Thus, i
j
the charging capacity of Fraction 1 synthetase is nearly !
the same in both young and old synthetase preparations, ;
while the activity of Fractions 2 and 3 is reduced in j
the older cotyledon. In an attempt to determine whether
or not Fractions 2 and 3 were acylating the same tRNA^eu
species, as was suggested by the Freon column profiles,
two tRNA samples were aminoacylated with Fraction 3
synthetase in two separate reactions. Once a predetermined
plateau level was reached, Fraction 2 synthetase was
added to one reaction while more Fraction 3 was added to
the other. These data are shown in Figure 21. When i
tRNA^eu is aminoacylated to a plateau level with Fraction
3 synthetase, the addition of Fraction 2 synthetase does
not promote any further charging. The same results are
seen when tRNAkeu is first acylated with Fraction 2
synthetase (Figure 22), followed by the addition of
FIGURE 21
Additive Effects of Fraction 2 Synthetase
Two samples of 5 day tRNA were acylated with
"^C-leucine and Fraction 3 synthetase from 5 day old
cotyledons in a 1 ml reaction. After 78 minutes (arrow)
0.2S ml of additional Fraction 3 synthetase was added
to one sample (A A) , while 0,25 ml of Fraction 2
synthetase was added to the other ( o ©) .
Data are presented as CPM/A2^q to allow for dilution
of samples by additional synthetase.
94 i
LEUCINE CPM/A260 xlO
ADDITIVE EFFECTS OF FRACTION 2 SYNTHETASE
MINUTES
FIGURE 22
Additive Effects of Fraction 3 Synthetase
Two samples of 5 day tRNA were acylated with
■^C-leucine and Fraction 2 synthetase from 5 day old
cotyledons in a 1 ml reaction. After 78 minutes (arrow)
0.25 ml of additional Fraction 2 synthetase was added to
one sample (O— -O) while 0.25 ml of Fraction 3
synthetase was added to the other (A A) .
96
n q 7
OTX U3CV/WdD a N I O Q a i
ADDITIVE EFFECTS OF FRACTION 3 SYNTHETASE
MINUTES
98
Fraction 3. Control experiments in both cases show that
the addition of the same synthetase fraction is also
ineffective in promoting further charging.
These results, along with the earlier Freon column
profiles clearly demonstrate that Fractions 2 and 3 of
leucyl-tRNA synthetase are specific for acylating the
same isoaccepting species of tRNALeu. The loss in the
capacity of leucyl-tRNA synthetase from 21 day old
cotyledons to charge tRNA^0^ appears to be due to a
decrease in the concentrations of both Fractions 2 and
3 of the synthetase. The activities of Fractions 2 and 3
relative to Fraction 1 in both the 5 and 21 day old
fractionated synthetase preparations confirm this
conclusion (Figures 19 and 20).
CHAPTER IV
DISCUSSION
Changes in Transfer RNA
The results of this study are consistent with a
number of other reports which have demonstrated changes in
specific tRNA's during development, growth, neoplasia,
between different cell types, and under different growth
conditions.
Some care must be taken in interpreting results
similar to those reported here. Artifactual alterations
in tRNA profiles might come about by aggregation of tRNA
species, conformational changes in tRNA, incomplete
charging, nuclease attack, artifacts of isolation, or
artifacts of labeling. Most of these possible explana
tions do not seem applicable to the present study however.
Extensive studies have been carried out with tRNA^eu from
bacteria, plants, and animals, and the reverse phase
column profiles presented in the current study are in
close agreement with the profiles observed by others.
A notable difference is the presence of an additional
peak in the soybean cotyledon. Both bacterial and
mammalian systems have been reported to contain only five
100
isoaccepting species of tRNA^eu. The presence of six
species in the cotyledon has also been confirmed by
Cherry and co-workers (Anderson and Cherry, 1969; Cherry
and Osborne, 1970; Kanabus and Cherry, 1971) and may
reflect the presence of an additional organelle, the
chloroplast, in this organ.
The occurrence of factors which selectively modify
tRNA^eu acceptor activity in either young (5 day) or old
(21 day) synthetase preparations has been ruled out by
the experiment described in Table 5. This is applicable
only to in vitro modification, however, and cannot exclude
the possible presence of factors which modify tRNA in
vivo which are not present (or n^t detectable) in the
crude synthetase. Changes in isoaccepting species of
tRNA due to selective modification of it’s amino acid
acceptor function would be an equally interesting finding,
however. It is equivalent to quantitative changes in
isoaccepting species in that the end product is a
reduction in the amount of specific aminoacyl-tRNA.
Yegian and Stent (1969) have observed that during amino
acid starvation in E. coli there is a reduction in the
capacity of one tRNA^eu species to be charged. Further
work revealed that this reduction in amino acid acceptor
ability is due to the presence of a factor, bound to the
tRNA species, which is not leucine or any other amino
101
acid. The factor prevented this tRNA species from being
aminoacylated. Thus, this is a method to functionally
remove a specific species of tRNA from participation in
protein synthesis.
Transfer RNA which is lacking it's -CpCpA terminus
at the 3'-end cannot be charged. Different tRNA^eu
profiles, showing quantitative differences in the labeling
of isoaccepting species, could result if specific species
L 01 j
of tRNA (1-4 in this case) were partially lacking their
-CpCpA terminus and could not be charged in the in vitro
reaction. There is no evidence, however, to show
specificity in the addition of these bases to different
aminoacyl-tRNA species, much less specificity among iso
accepting species. In addition, the pyrophosphorylase
enzyme which catalyses the addition of CMP and AMP to
the 3*-end of tRNA is usually found in crude enzyme
extracts (Makman and Cantoni, 1966). If this were true
in the case of cotyledon synthetase preparations, the
incubation of tRNA with synthetase, ATP, and CTP, as
described in Table 5, should have regenerated the missing
-CpCpA termini. Since there was no change in chroma
tographic profiles this does not seem to be a particularly
1 a l l
tenable explanation for different tRNA profiles
between young and old cotyledons.
Artifacts of radioactive labeling can also be ruled
102
out as giving rise to different chromatographic profiles
as reversing the labels had no effect on the relative
amount of charging of isoaccepting species in any tRNA
preparation. Although specific artifacts generated
during the isolation of tRNA cannot entirely be ruled
out, the reproducibility of the different chromatographic
profiles between young and old tRNA make this an unlikely
possibility.
The changes in relative amounts of isoaccepting
tRNA^eu species reported here therefore seem to represent
actual quantitative differences between young and old
cotyledons. The reduced amino acid acceptor activity in
21 day tRNA^eu is apparent regardless of which synthe
tase preparation is used. That these changes are due to
a loss in isoaccepting Species 1-4 is indicated by the
total amino acid acceptance presented in Table 4. The
capacity of these four species to accept leucine is
decreased between 6 and 10 fold (on the basis of
units of tRNA), while there is only a slight decrease in
the leucine charged to Species 5 and 6. As mentioned
earlier, however, this reduced capacity to accept leucine
need not necessarily be due to an absolute loss in iso
accepting species. Modification which eliminates their
ability to accept an amino acid also effectively removes
these species from participation in protein synthesis (if
103
they become rate limiting in concentration). This is not
unlike the observations of Vanderhoef and Key (1970) that
a smaller percentage of the total tRNA is chargeable from
non-dividing root tissue when compared to dividing root
tissue.
Although there is no direct evidence from the present
study bearing on this point, the loss in tRNA^eu species
may be interpreted in light of Yamane's (1965) observa
tion that in E. coli the relative abundance of amino acid-
specific tRNA's reflects the amino acid composition of
total protein from that organism. A similar correlation
Pro
is seen in granulation tissue with respect to tRNA
(Lanks and Weinstein, 1970).
Mitochondria and chloroplasts also contain tRNA and
synthetases (Barnett and Brown, 1967 ; Buck and Nass, 1968 ;
Epler, 1969; Kull and Jacobson, 1969). These may be
similar to their cytoplasmic counterparts in some cases,
but organelle specific in other cases. In cotyledons, the
only information on organelle tRNA's is Anderson and
Cherry's (1969) observation that tRNA^|u appear to be
major mitochondrial species. Their results cannot rule
out the possibility that other tRNA^eu species exist in
mitochondria, nor the possibility that Species 3 and 4
coexist in the cytoplasm. Therefore, evidence that is
presently available does not allow interpretation of the
104
present data on the basis of changes in organelle-specific
tRNA's and synthetases.
The nature of the effect of cytokinin application on
Lou
tRNA species is still obscure and does not allow
considerable speculation. A number of mechanisms could be
operative in changing the relative amounts of isoaccepting
species. These include a stimulation in the production
of Species S and 6, an increased degradation of Species
1-4, a specific protection of Species 5 and 6 against
degradation, or an interconversion of isoaccepting species
through modification of cytokinin-active residues in tRNA.
This last possibility seems unlikely, however, in light
of the attempts to modify tRNA^eU with permanganete
treatment.
Changes in leucyl-tRNA Synthetase
The changes in leucyl-tRNA synthetase activity are
somewhat difficult to assess. Only a limited number of
cases of multiple synthetases have previously been
reported (Kull and Jacobson, 1969; Strehler et al. , 1967;
Ceccerini et al. , 1967 ; Vescia, 1967 ; Kanabus and Cherry,
1971). And some of these cases may represent organelle
differences in the complement of amino acid-specific
synthetases rather than multiple cytoplasmic synthetases.
There is a measurable reduction in the capacity of
105
I oil
synthetase from 21 day old cotyledons to acylate tRNA
A number of experiments in the present study suggest that
this loss in activity is due exclusively to a loss in
synthetase Fractions 2 and 3 from hydroxylapatite
fractionation. Fraction 1, which is specific for acylating
tRNA^^, shows the same level of charging activity in
both young and old cotyledons (Figures 19 and 20). In
contrast, the c'.pacities of Fractions 2 and 3 to acylate
tRNA^eu is considerably reduced in the older cotyledon.
This is of particular interest as it is tRNA^e^ which
appear to decrease with greater cotyledon age. There
thus appears to be a co-ordinate regulation in maintaining
the levels of specific tRNA species and their cognate
synthetases. This is similar to the findings of Ilan and
co-workers (Ilan, 1969; Ilan et al., 1970) in Tenebrio.
It is interesting that those studies also concerned tRNA^eu
and leucyl-tRNA synthetase.
Even though there is no absolute loss in synthetase
activity, the in vitro charging reaction suggests that
synthetase Fractions 2 and 3 may be present in rate
limiting amounts in the 21 day old cotyledon. Therefore,
L eu
even though tRNA^ ^ are not fully charged in the in vitro
reaction using 21 day synthetase, the addition of more
21 synthetase cannot promote further charging. Similar
results have been seen by other workers, primarily in the
106
case of heterologous reactions. Roy and Soil (1970) have
shown that E. coli tRNA^er can be fractionated into five
species. When charged in a heterologous reaction using
Neurospora crassa synthetase, all 5 species are charged
but to a lesser degree than with homologous E. coli
synthetase. The reasons for only partial charging in
those studies, as well as in the present study, are
incompletely understood. There is, however, an interest
ing report concerning an 'enhancing factor' in the
heterologous charging reaction. Makman and Cantoni (1966)
observed that yeast synthetase was only about 40 per cent
effective in charging E. coli tRNASer as compared to
E. coli synthetase. They were able to obtain a fraction
from yeast which when added to the heterologous reaction
resulted in total charging of the tRNA^er species. This
’enhancing factor’ did not effect the level of charging
with homologous synthetase, did not contain detectable
amounts of CCA-pyrophosphorylase, and was heat labile.
Preliminary results also suggested the presence of a
similar factor for acylation of tRNA^eu by heterologous
yeast synthetase.
It is not yet possible to interpret the gain in
activity of fractionated synthetase over the unfraction
ated form (Figures 19 and 20), although the general
phenomenon of 'enhancing factors' may have some relevance
107
here also. Interestingly, it is Fractions 2 and 3 which
demonstrate greater activity in the synthetase preparation
from 5 day old cotyledons, but Fraction 1 is enhanced in
the 21 day synthetase preparation. These results correlate
with the pattern of change observed simply by looking at
the charging capacities of unfractionated 5 and 21 day
synthetase. This increase in activity might be due to
different optimal reaction conditions for the three
separate synthetases, which is apparent only in the
fractionated form. There is also the possibility of
inhibitors of synthetase activity, either specific or non
specific, which are fractionated out of the synthetase
preparation on hydroxylapatite, as well as other trivial
artifacts which cannot yet be assessed.
Codon Response
This study was initiated in large part to determine
if losses might occur in certain components of the trans
lational machinery with fige. If the results of this
* L n\i
study accurately reflect in vivo changes in tRNA and
synthetase, rather than artifacts of preparation and
analysis, there is still the question of whether or not
these changes are reflecting some regulatory processes.
Although codon response of isoaccepting species was not
determined in this study, some speculation is warranted
108
on the basis of other studies.
A number of studies have examined tRNA^eu species in
E. coli for a variety of experimental purposes (Kano-
Sueoka and Sueoka, 1966; Kano-Sueoka et al., 1968; Kan
et al. , 1970) . There are clearly five species of tRNA^eu
in E, coli as determined by reverse phase chromatography.
Results from these studies are in agreement in assigning
the folloiving codons to isoaccepting species:
tRNALeul: CUG
tRNAL6U2: cuu, cue
tRNALeU3: CUA, CUG
tRNALeU4: UUG
(?)
tRNALeu5: UUG
An additional codon has been assigned to leucine (Nirenberg
et al. , 1966) , UUA. A corresponding isoaccepting species
has not yet been determined. The major difference between
E. coli tRNA^eu and the soybean cotyledon is the presence
of an additional species in the latter case. A comparison
of tRNA^eu profiles on reverse phase columns shows the
E. coli Species 1 to be the major peak which suggests that
E. coli Species 1 through 5 may correspond to cotyledon
Species 2 through 6. The cotyledon contains Species 1
which is lacking in E. coli. This peak may account for
the usage of the additional leucine codon.
Assuming that the codon assignment for E. coli is
109
correct and that the correspondence between E. coli and
cotyledon tRNALeu is at least close to that suggested
above* then one can envision possible alterations in trans
lational capacities accompanying the changes in tRNA^eu and ;
synthetase determined in the present study. Thus, if
Species 1-4 decrease to become rate limiting it can be 1
seen that there would be a loss in the ability to read j
codons CUA, CUC, CUG, CUU, and possibly UUA. Since tRNAL®u i
w
is significantly larger than the other three species, ‘ it ;
is possible that even with this decrease it may not become
rate limiting. Hence, if only Species 1, 3 and 4 become ,
i
rate limiting, reading of the codons CUU, CUA, CUC, and j
possibly UUA is restricted. Therefore, even with some I
degree of redundancy (a unique species of tRNA responding
to more than a single codon) it is possible to have
absolute losses in code reading abilities if certain of
these tRNA species are present in rate limiting amounts.
There are cases of redundant codon responses where
it is difficult to see a loss in code reading abilities
due to losses in isoaccepting tRNA’s, Nishimura and
Weinstein (1969) have fractionated tRNA^r from rat liver
and determined the codon response for each of the two
major species. They were unable to show any differences
in the response to the codons UAU and UAC. In this same
study however, the two major tRNA^er species displayed
different codon responses. Peak 1 responds to UCU, UCC,
UCA, and UCG, while Peak 2 responds only to AGU and AGC.
In addition, it was suggested that Peak 1 probably
consists of at least two species. Therefore, even with
redundancy in the codon response of both major species,
the loss of either one will limit codon usage.
Caskey et al. (1968) have made an extensive study
of the codon response of seven aminoacyl-tRNA's in guinea
pig liver. These results are summarized in Table 6. In
the case of arginine, the loss of Species 1 eliminates
the usage of CGU and CGC; the loss of Species 4 eliminates
AGG; Species 2 and 3 must each be lost in conjunction with
another species to eliminate their respective codons. The
single isoleucine species responds to all three codons.
The loss of any one of the three tRNA®er species will
eliminate the usage of their respective codons as there
is no overlap in response. Cysteine tRNA species respond
equally well to both codons. If Species 1 of tRNA^et is
lost, so is the codon GUG. In the case of threonine, the
response of each species is unique, so that the loss of
either species will eliminate usage of their respective
codons.
The lysine response is particularly interesting as an
example of potential translational control through
modulation of specific tRNA species. There is redundancy
TABLE 6
Codon Response of Some Aminoacyl-tRNA's
from Guinea Pig Liver*
Amino Acid Peak
1 2 3 4
Arginine Codons CGU
CGC
CGA
CGG CGA
CGG
AGG
Isoleucine Codons AUU
AUC
AUA
Serine Codons AGU
AGC
UCU
UCC
UCA
UCG
Cysteine Codons UGU
UGC
UGU
UGC
Methionine Codons AUG
GUG
AUG
Threonine Codons ACU
ACC
ACA
ACG
Lysine Codons AAA
AAG
AAG
*Taken from Caskey et al
111
. (1968).
. -
in the codon response of one peak but not in the other.
Thus, there is no effect on code reading abilities if
Species 2 is lost. In contrast however, the loss of
Species 1 eliminates the use of the codon AAA without
affecting AAG. Thus, if the triplet AAA is of particular
importance in regulation, the modulation of tRNA^s can
limit translation of that codon, while still allowing
lysine to be used in protein synthesis (with the codon AAG)
Sekiya et al. (1969) have examined the tRNAGlu species
present in yeast. Two major species are present, one of
which responds only to the codon GAG, the other responding
only to GAA. Further, in a cell-free system synthesizing
rabbit hemoglobin it was determined that each of these two
p 1
tRNA u species incorporate glutamic acid at different
points in the hemoglobin chain.
Summary and Conclusions
The current study has demonstrated an apparent
quantitative change in the complement of isoaccepting
tRNA^eu species during the senescence of cotyledons.
Specifically, there is a 2 fold decrease in the leucine
acceptor activity of tRNA between cotyledons of 5 and 21
days of age. The cognate leucyl-tRNA synthetase is also
shown to decrease in charging capacity with greater
cotyledon age. These two changes taken together result in
113
a 6-10 fold decrease in the amount of aminoacyl-tRNA^eu
for 4 of the 6 isoaccepting species. The present results
suggest that a loss in both specific tRNA^eu isoaccepting
species and their cognate synthetase may result in rate
limiting quantities of at least three aminoacyl-tRNA^eu
isoaccepting species. Extrapolation of codon response
studies in E. coli and mammalian systems indicate that
rate limiting quantities of these three tRNA^eu species
could restrict the utilization of at least three leucine
codons in protein synthesis.
The fact that these changes in tRNA and synthetase
occur as a function of cotyledon age is consistent with
the suggestion (Strehler, 1966) that aging may be due to
a loss in translational capacities, leading to an inability
to replace worn or damaged components and hence, result
in the ultimate demise of the organism. These data do not,
however, allow for speculation on the cause or effect
relationship of these changes. Therefore, rather than
playing a regulatory role these changes may simply reflect
a changing metabolism of the cotyledon which includes a
reduced need for certain code reading abilities. For
example, mRNA in the 21 day old cotyledon might contain
little of the codons corresponding to tRNA^e^. The
measured changes would then reflect compensatory shifts to
eliminate those components of translation which are no
114
longer required.
In order to further examine this question attempts
should be made to determine the codon response of each of
the isoaccepting tRNA^eu species. The Wobble Hypothesis
(1966) predicts a minimum of three unique anticodons for
leucine codons, while there may in fact be more as is
suggested by the coding studies in h. coli. In addition,
the relative capacities of tRNA from young and old
cotyledons to maintain protein synthesis in a cell-free
system should be determined. Such studies should utilize
polysomes from both young and old cotyledons. These
studies might reveal whether or not the aminoacyl-tRNA^eu
complement in old cotyledons is rate limiting in trans
lating polysomes from young cotyledons.
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115
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Creator Bick, Michael David (author)

Core Title Changes In Leucine Transfer Ribonucleic Acid And Leucine Transfer Ribonucleic Acid Synthetase During Cotyledon Senescence

Degree Doctor of Philosophy

Degree Program Cellular and Molecular Biology

Publisher University of Southern California (original), University of Southern California. Libraries (digital)

Tag biology, genetics,OAI-PMH Harvest

Language English

Contributor Digitized by ProQuest (provenance)

Advisor Strehler, Bernard L. (committee chair), Petruska, John A. (committee member), Shugaram, Peter M (committee member), Szafran, Jacek (committee member)

Permanent Link (DOI) https://doi.org/10.25549/usctheses-c18-517246

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