THE 5’ -LENKED TERMINI
0F PLAN RIBOSDMAL RIBONUCLEIC ACIDS

Thesis for the Degree cf Ph. D.
MECHiGAN STATE UNIVERSETY
JOHN MCDGNELL HALLOEN
1%8

News“

This is to certify that the
thesis entitled

THE 5'—LINKED TERMINI
OF PLANT
RIBOSOMAL RI BOZV UCLE I C A CI 03

presented by

JOHN MCDONELL HALLOI IV

has been accepted towards fulfillment
of the requirements for

Ph.D. degree in Botany

P

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I 1 Maon‘ professor A

Date Feburary 2, 1968

 

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ABSTRACT

THE 5'—LINKED TERMINI OF PLANT
RIBOSOMAL RIBONUCLEIC ACIDS

by John McDonell Halloin

This thesis results from an investigation of the
5'-linked termini of plant ribosomal ribonucleic acids
(r—RNA). The objectives were to determine (a) the identity
of 5'-linked termini, (b) if plant r—RNA's are preferentially
terminated by a particular nucleoside or nucleosides and (c)
if the same termini occur on r—RNA from several plant species,
RNA was obtained from microsomal pellets of cauliflower,

cabbage and mushroom (Agaricus campestris) by extraction and

 

deproteinization with phenol—sodium dodecyl sulfate, It was
hydrolyzed with 0.3 N KOH, and the hydrolysate was neutralized
with a sulfonic acid resin, Dowex 50-H+. Nucleosides re—
leased from 5'-linked chain terminal positions by KOH
hydrolysis were separated and identified by paper chromato-
graph and spectrOphotometry,

Adenosine and uridine were the only nucleosides
obtained upon alkaline hydrolysis of unfractionated (18 S +
28 S) cauliflower r—RNA, and adenosine was approximately

twice as abundant as uridine, Similarily, only adenosine

 

3:6 uridi .e were
:-?\'A from cabba
cases uridine we

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r-FXA, after sej.
revealed that b
Phosphorylatior;
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meleosides, a
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John McDonell Halloin
and uridine were isolated from hydrolysates of unfractionated
r-RNA from cabbage and mushrooms, however, in both of these
cases uridine was approximately twice as abundant as
adenosine. Studies on the individual subunits of cauliflower
r-RNA, after separation on sucrose density gradients,
revealed that both adenosine and uridine terminate 28 S r—RNA.
Phosphorylation of the nucleoside fraction from 18 S r—RNA
with 32P-polypohosphate revealed that uridine is present as
a 5'—linked terminus on this RNA: cytidine was not detected,
This method is applicable only for the detection of pyrimidine
nucleosides, as purine nucleosides are not phosphorylated by
polyphosphate. Other studies in our laboratory showed that
adenosine and uridine are also the only nucleosides isolated
from alkaline hydrolysates of parsnip and spinach r—RNA.

The results presented above suggested that all plant
r-RNA's may terminate preferentially in adenosine and
uridine, however, other laboratories reported that all four
ribonucleosides were isolated from alkaline hydrolysates of
wheat germ r-RNA, A study was made, therefore, to determine
the 5'-1inked termini of wheat germ r—RNA, using the
techniques employed in our laboratory. RNA was isolated

from wheat germ by direct phenol extraction of raw wheat

 

 

 

95;; by direct P
are and ur ldlfu.
of the total nu.
from preparatic:
to be free of c
preparation of '
and s-Rh‘A, yie‘.
hydrolysis, A1
following hydr;
3N HCl (the p:
Still another
latter results
dePhOSphorylat
HCl was the so.
H‘Jcleosides in

The pr
uridine of all
termini sUbSEQ
LRNA by a met

f experiment 5

 

John McDonell Halloin
germ by direct phenol extraction of raw wheat germ, Adeno—
sine and uridine, plus a small amount of cytidine (5 percent
of the total nucleoside) were the only nucleosides isolated
from preparations of wheat germ r-RNA which had been shown
to be free of contamination by DNA and s—RNA. Another
preparation of wheat germ r-RNA, contaminated with both DNA
and s-RNA, yielded all four nucleosides upon alkaline
hydrolysis. All four nucleosides were also isolated,
following hydrolysis with l M NaOH and neutralization with
3 N HCl (the procedure used in other laboratories), from
still another wheat germ r-RNA preparation, Since the
latter results were similar to those reported by others,
dephosphorylation of nucleosides during neutralization with
HCl was the suspected cause of the appearance of all four
nucleosides in their studies,

The preferential termination with adenosine and
uridine of all r-RNA's studied suggested that addition of
termini subsequent to RNA chain synthesis might occur with
r-RNA by a method similar to that known for s—RNA. A series
of experiments was done using cauliflower r—RNA labeled with
2—14C-uridine in the presence and absence of actinomycin D.

which has been shown to inhibit DNA—dependent RNA synthesis,

Ll

 

 

 

to determine if
tents were base
are added after
to non-terminal
labeled under 1
dependent RNA
the absence of
because termin
hiSher rate up,
absence of int
l4
Cmucleotide
cauliflower r.
ited conditior
subsequwt to

for the Pre fe

John McDonell Halloin
to determine if end—group addition occurred, These experi—
ments were based on the assumption that if 5'-linked termini
are added after r—RNA chain synthesis, the ratio of terminal

. l4 . . . . . .
to non—terminal C-uridine Will be higher With r—RNA chains
labeled under conditions of actinomycin D-inhibited DNA—
dependent RNA synthesis than with r—RNA chains labeled in
the absence of such inhibition. This would be expected,
because terminal addition would occur at a proportionately
higher rate under conditions of inhibition than in the

. . . . . l4 .
absence of inhibition. The same ratio of C—nucleOSide:
1‘IVC-nucleotide was obtained upon alkaline hydrolysis of
cauliflower r-RNA labeled under both inhibited and uninhib-
ited conditions, suggesting that addition of termini

subsequent to ribosomal chain synthesis is not responsible

for the preferential termination of cauliflower r—RNA,

THE 5'-LINKED TERMINI OF PLANT

RIBOSOMAL RIBONUCLEIC ACIDS

by

John McDonell Halloin

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Botany and Plant Pathology

1968

 

slog Luann I'

 

I woulc
my major profes
assistance thr<
also extended 1
J. A. Boezi, A
assistance and

I also‘
Patience and w

manuscript .

 

 

ACKNOWLEDGMENTS

I would like to express my sincere appreciation to
my major professor, Dr. C. J. Pollard, for his advice and
assistance throughout this investigation. Appreciation is
also extended to the members of my guidance committee, Drs.
J. A. Boezi, A. H. Ellingboe and N. B. Good, for their
assistance and criticism of this manuscript.

I also wish to thank my wife Jeanne, for her
patience and willing assistance in the preparation of this

manuscript.

TABLE OF CONTENTS

ACKNOWLEDGMENTS . . . . . . . . . . . . . . . .
TABLE OF CONTENTS . . . . . . . . . . . . . . .
LIST OF TABLES . . . . . . . . . . . . . . . .
LIST OF FIGURES . . . . . . . . . . . . . . . .
INTRODUCTION . . . . . . . . . . . . . . . . .
LITERATURE REVIEW . . . . . . . . . . . . . . .

The Primary Structure of Ribosomal RNA . . .
Methods of Detecting RNA Termini . . . . . .

Enzymatic Methods . . . . . . . . . . . .

Escherichia coli alkaline phosphatase
Snake venom phosphodiesterase . . . .
Spleen phosphodiesterase . . . . . . .
Pancreatic ribonuclease . . . . . . .
Polynucleotide kinase . . . . . . . .
Nucleoside phosphotransferase . . . .

 

Chemical Methods . . . . . . . . . . . .

Alkaline hydrolysis . . . . . . . . .
Periodate oxidation . . . . . . . . .

Amine catalyzed cleavage of dialdehydes

Formation of bisphenylhydrazones .

Formation of trialcohols . . . . .

Formation of semicarbozones and
thiosemicarbazones . . . . . . .

ii

Page

ii
vi

viii

ll
11
12
12

l3

13
15

15
l6
l7

l7

Formation of isonicotinyl hydrazones . .

Acetylation . . . . . . . . .
Phosphorylation with polyphosphate . . . . .
Phosphorylation with cyanoethyl phosphate .
Formation of methyl phosphate derivatives

Formation of phosphoroanilidate derivatives

The Termini of Ribonucleic Acids . . . . . . . . .
Viral RNA . . . . . . . . . . . . . . . . . . .
Soluble RNA . . . . . . . . . . . . . . . . . .

Ribosomal RNA . . . . . . . . . . .

3'-Linked termini . . . . . . . . . . . . .
5'-Linked termini . . . . . . . . . . . . .
Enzymatic Addition of Termini . . . . . . . . .
MATERIALS AND METHODS . . . . . . . . . . . . . . . .
Experimental Materials . . . . . . . . . . .

Preparation and Hydrolysis RNA . . . . . . . . . .

Preparation of Ribosomal RNA by Direct
Extraction . . . . . . . . . . . . . . . . .

Extraction with phenol and sodium dodecyl
sulfate . . . . . . . . . . . . . . . .

Extraction with phenol and phosphate buffer

Preparation of CytOplasmic RNA by Differential
Centrifugation and Phenol Extraction . . . .

Ribosomal RNA . . . . . . . . . . . . . . .
Soluble RNA . . . . . . . . . . . . . . . .

Sucrose Density Gradient Centrifugation . . . .
Alkaline Hydrolysis . . . . . . . . . . . . . .

Chromatoqraphy and Electrophoresis . . . . . . . .

Anion-exchange Chromatography . . . . . . . . .
Paper Chromatography . . . . . . . . . . . . .

iii

Page
18
18
19
20
20
21
22
22
25
28

28
34

39
42
42
43

43

43
45
45

45
46

47
48

48

48
49

F
" "all-D 1L‘i01lfld

 

 

 

Paper

Radioisot
Phosphor}

Use 0:
Use 0;

Incorpor;
HYPOCI

RESULTS .
RNA 1801.

CytOp
Total

The 5 ' ‘L

Ribos

The Sl‘

HYPOC

EffeC
Time
The R

in

RNA I801
The 5 I ‘L

Paper Electrophoresis . . . . . . . . . . .
Radioisotope Counting . . . . . . . . .
Phosphorylation of Nucleosides . . . . . .

Use of Nucleoside Phosphotransferase
Use of P-polyphosphate . . . .

Incorporation of 2—14C-Uracil by Cauliflower
Hypocotyls . . . . . . . . . . .

RESULTS . . . . . . . . . . . . . .
RNA Isolation Procedures . . . . . . . . . . .

Cytoplasmic Ribosomal RNA . . . . . . . . .
Total Ribosomal RNA . . . .

The 5'-Linked Termini of Cytoplasmic RNA
Ribosomal RNA . . . . . .
Unfractionated r-RNA . . . . . . . .

28 S RNA . . . . . . . . . . . . . . . .
18 S RNA . . . . . . . . . . . . . . . .

Soluble RNA . . . . . . . . . . . . . . . . . .

The 5'-Linked Termini of Total Wheat Germ Ribosomal
RNA . . . . . . . . . . . . . . . . . . . . . .

Incorporation of 2—14C-Uracil by Cauliflower
Hypocotyls . . . . . . . . . . . . . . . . . .

Effect of Actinomycin D Concentration . . .

Time Course of 2-14C—uracil Incorporation . . .

The Ratio of Terminal to Non-terminal Uridine
in Cauliflower Hypocotyl r-RNA . . . . . . .

DISCUSSION . . . . . . . . . . . . . . . . . . . . .

RNA Isolation Procedures . . . . . . . .
The 5' —Linked Termini of Cytoplasmic r-RNA . . . .

iv

Page
52

52
53

53
54
54
57
57

57
58

6O
60
60
64
64

66

7O
82

83
86

87
95

95
98

Page

The 5'—Linked Termini of Total Wheat Germ r-RNA . .100
Incorporation of 2-14C-Uracil by Cauliflower

Hypocotyls . . . . . . . . . . . . . . . . . . 101
Future Application of the Results . . . . . . . , 101

LITERATURE CITED . . . . . . . . . . . . . 104

LIST OF TABLES
Table Page

1. Summary of data on the termini of viral
ribonucleic acid . . . . . . . . . . . . . . 24

. Summary of data on the 5'-linked termini of
soluble ribonucleic acids . . . . . . . . . 29

3. Summary of data on the 3'-linked termini of
soluble ribonucleic acids . . . . . . . . . 3O

4. Summary of data on the 3'-linked termini of
ribonucleic acids. I. The termini of
bacterial and yeast ribosomal RNA as a
reported by Sugiura and Takanami (86) . . . 32

5. Summary of data on the 3'-linked termini of
ribosomal ribonucleic acids. II. The
termini of ribosomal RNA from E, coli, L.
cells and wheat germ . . . . . . . . . . . . 33

6. Summary of data on the 5'—linked termini of
ribosomal ribonucleic acids . . . . . . . . 37

7. Summary of data on the effect of nutrients on
the nature of the 5'-1inked termini of E,
coli ribosomal RNA, as reported by Midgley
and McIlreavy (60) . . . . . . . . . . . . . 38

8. Nucleosides resulting from the hydrolysis of
unfractionated, cytoplasmic r-RNA in
typical experiments . . . . . . . . . . . . 63
9. Nucleosides resulting from the hydrolysis of

S—RNA...................69

vi

Table

10.

11.

12.

13.

14.

15.

Radioactivity ogznucleotides produced by the
reaction of P-p—nitrophenyl phosphate
with the nucleoside fraction from wheat
germ r-RNA . . . . . . . . . . . .

Summary of data on the 5'-linked termini of
wheat germ r-RNA . . . . . . . . . . . .
Yield (percent of expected) and identity of
nucleosides obtained from alkaline
hydrolysates of RNA preparations studied
in this thesis . . . . . . .
. . . . . l4 .
Inhibition of incorporation of 2— C-uraCil
into cauliflower hypocotyl r-RNA by 60
ug/ml of actinomycin D . . . . . . . . . . .
Radioactivity of nucleosides and nucleotides
isolated from alkaline hydrolysates of
r-RNA labeled in the presence and absence
of 60 ug/ml of actinomycin D . . . . . . .
Ratios of terminal to non—terminal uridine in
r—RNA isolated from cauliflower hypocotyls
incubated with 2-14C-uracil in the presence
and absence of 60 ug/ml of actinomycin D . .

vii

Page

73

80

81

86

92

93

Figure

1A.

1B.

7A.

LIST OF FIGURES

Page

Stylized representation of an RNA molecule,

adapted from Sugiyama and Fraenkel—Conrat

(88)................... 7
Products of alkaline hydrolysis of RNA:

nucleoside-2'(3'),5'—diphosphate from

3'-linked terminus, mixed 2'(3')—monophos-

phates from internal positions and

nucleoside from 5'—1inked terminus . . . . 7
Pattern of elution of cytidylic acid from

Rexyn 201. Cl‘ . . . . . . . . . . . . . . 50
Sedimentation profile of a typical preparation

of cauliflower cytoplasmic r-RNA . . . . . 61
Sedimentation of profiles of individual

cauliflower r—RNA subunits . . . . . . . . 65
Radioactivity of chromatogram, from system A,

of uridine monophosphates resulting from

phosphorylation of cauliflower 18 S r-RNA

nucleoside fraction with 32P-polyphosphate 67
Radioactivity of paper electrophoretogram of

uridine-2'(3')-monophosphate, resulting

from phosphorylation of cauliflg er 18 S

r-RNA nucleoside fraction with P-poly—

phosphate, and eluted from a paper

chromatogram developed in system A . . . . 68

Sedimentation profile of 48 OD units of wheat
germ r-RNA isolated by direct extraction
of wheat germ with phenol and solution A . 72

viii

Figure Page

73. Sedimentation profile of 42 OD units of
wheat germ r-RNA isolated by direct
extraction of wheat germ with phenol
and solution B . . . . . . . . . . . . . . 72

8. Radioactivity on chromatogram, developed in
system B, of nucleoside monophosphates
resulting from phosphorylation of wheat
germ r—RNA nucleoside fraction with
2P-p—nitrophenyl phosphate and nucleoside
phosphotransferase . . . . . . . . . . . . 74

9. Sedimentation profile of 48 OD units of wheat
germ r-RNA isolated by direct extraction
of wheat germ with phenol and solution A . 76

10. Sedimentation profile of wheat germ r-RNA
isolated by the methods of Singh and Lane
(80) 78

ll. Incorporation of 2-14C-uracil into alcohol-
insoluble material by cauliflower hypocotyls
with increasing concentrations of
actinomycin D . . . . . . . . . . . . . . . 84

12. Incorporation of 2-14C—uracil, into alcohol-
insoluble material, by cauliflower
hypocotyls with increasing time, in the
presence and absence of 60 ug/ml of
actinomycin D . . . . . . . . . . . . . . . 88

13. Sedimentation profiles of r—RNA isolated from
cauliflower hypocotyls incubated for 8 hours
in 2—14C—uracil, and of unlabeled carrier
r-RNA . . . . . . . . . . . . . . . . . . . 89

14. Sedimentation profiles of r—RNA isolated from
cauliflower hypocotyls incubated for 8 hours
in 2-14C-uracil and 60 ug/ml of actinomycin
D, and of unlabeled carrier r-RNA . . . . . 9O

ix

 

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Ind.

INTRODUCT I ON

The termination of soluble ribonucleic acid (s-RNA)
(5, 26, 27, 29, 32, 46, 78, 103, 104) and tobacco mosaic
virus RNA (TMV-RNA) (79, 88, 89, 96) with Specific nucleo—
tides is well established. Information on the termini of
ribosomal RNA (r-RNA), with the exception of wheat germ r—RNA
(45, 46, 49, 80), was not available prior to the start of
these investigations; and although the role of ribosomes in
protein synthesis is well established, little is know of
either the primary structure or function of r-RNA.

Evidence existed that the base ratios of r-RNA's
from different Species were different, as were those of the
r-RNA subunits from a single species (14, 66, 67). It has
been established that there is a multiplicity of DNA sites
for the synthesis of r—RNA (2, 101, 102), and that the sub-
units are synthesized on different sites (2, 102). Based on
these facts, an investigation was undertaken to determine
the identity of plant r-RNA termini. It was conceived that

such an investigation might lead to a more intimate knowledge

to

to

 

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an:

of the role of the RNA, if any, in protein synthesis, or
possibly contribute to knowledge concerning the synthesis

of r-RNA. For example, preferential termination of all
r-RNA with a single nucleoside could be indicative either
that this nucleoside participates somehow in binding of

the r-RNA to ribosomal protein, or that the nucleoside

could be necessary for biological activity. Finally, since
the 5'-1inked nucleoside is apparently the last one attached
to a growing chain of RNA, such information could contribute
to knowledge of the mode of biosynthesis of r-RNA.

An abstract of the first phase of this investigation,
dealing with the nature of 5'—1inked termini of plant r—RNA's,
has been published (69), and these results together with the
results of others (36, 43, 47, 53, 59, 62), showed that the
5'—linked end of r—RNA chains terminate preferentially in
adenosine and uridine. Because our results were quite
different from the results obtained with wheat germ r—RNA
by Lane and co¢workers (45, 46, 80) and by Lee and Gilham
(49), and because their isolation and alkaline hydrolysis
procedures differed from the methods usually employed in
this thesis, we reinvestigated the 5'-linked termini of
wheat germ r-RNA using several isolation procedures and two

different procedures of alkaline hydrolysis.

The existence of enzymes which preferentially add
the sequence p-cytidine—p—cytidine—p—adenosine (CCA) to the
5'-linked termini of s-RNA's (11, 12, 35, 26, 27, 55), and
enzymes from rat liver which add homopolymer chains to the
5'-linked termini of snythetic and natural polyribonucleo—
tides, including r—RNA (40, 41, 65); together with the
observation that growth conditions could influence the
proportion of 5'-linked terminal uridine in r-RNA from

Escherichia coli (60), suggested to us that such an enzyme

 

might be involved in the addition of uridine to the 5'—1inked
termini of plant r-RNA's. Accordingly, 14C-labeled uracil
was incorporated into RNA by cauliflower hypocotyls, in the
presence and absence of actinomycin D. It was hoped that

by inhibiting DNA—dependent r-RNA synthesis with actinomycin
D, it would be possible to detect any addition of uridine

to the 5'-linked terminal group.

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LITERATURE REVIEW

The Primary Structure of Ribosomal RNA

 

The structure of ribosomes and the ribonucleic acids
associated with them have been reviewed by Ebel (20),
Peterman (66), Spirin (81, 82) and Ts'o (92). Ribosomes are
subcellular particles which have sedimentation coefficients
between 70 and 80 S, and are composed of 40 to 60 percent
protein and 40 to 60 percent RNA. Lowering the magnesium
ion concentration in the surrounding medium causes a dissoci-
ation of the particles into two unequal subunits; one with
a sedimentation coefficient of 30 to 40 S, and the other
with a sedimentation coefficient of 50 to 60 S. The smaller
subunits contain RNA molecules with sedimentation coeffici—
ents varying from 13 to 19 S, which corresponds to molecular
weights of 5 to 7 x 105, or 1400 to 2000 nucleotides;
whereas RNA molecules isolated from the larger subunits
have sedimentation coefficients of 23 to 34 S and molecular
weights of 1.0 to 1.5 x 106, corresponding to 3000 to 4500

nucleotides.

End group studies on wheat germ r—RNA by Lane and
coeworkers (45, 46, 80) and by Lee and Gilham (49) showed
one 5'-linked end group for each 1200 1500 nucleotides.
McIlreavy and Midgley (53) found one 5'-1inked terminus
for each 1200 to 1500 nucleotides in the larger r-RNA sub-
unit from E, 99;}, Midgley (56, 58) found that two smaller
RNA molecules corresponding in size to the smaller r—RNA
subunit, were produced when the larger subunit of E, 52;;
r-RNA was incubated at pH 9, in the absence of magnesium
ion or in the presence of ethylenediamine tetraacetic acid
(EDTA). These results suggested that the larger molecules
may be discontinuous, being composed of two RNA chains
similar to the smaller subunit. Beck gE_§l, (3), have
demonstrated, however, that when yeast r—RNA is isolated
and incubated in the presence of polyvinyl sulfate, which
inhibits ribonuclease, no dissociation of the large subunit
occurs. This indicates that in the absence of enzymatic or
chemical hydrolysis, the larger subunit is a continuous
polynucleotide chain.

The existence of a difference in base ratios between
the large and small subunits (14, 66, 67) is evidence that
the larger subunit is not a dimer of smaller subunits.

Further evidence are the results of Yankofsky and Spiegelman

(102), who found through hybridization studies that the two

subunits of Bacillus megaterium r-RNA are non-competitive in

 

their binding to DNA. They concluded that the two subunits
are synthesized on different genetic loci.

A third r—DNA subunit associated with the larger
ribosomal subunit has recently been isolated from E, gel;

(9, 17, 22, 77), KB cells (22) and Blastocladiella emersonii

 

(17). This r-RNA is 100 to 105 nucleotides in length and
has a sedimentation coefficient of 5 S. It differs from
s—RNA in that it lacks methylated bases and amino acid
accepting activity.

The generalized structure of RNA may be represented
as a continuous polynucleotide chain, in which the ribonuc—
leosides adenosine (A), guanosine (G), cytidine (C) and
uridine (U) are joined together by phosphodiester linkages
between the 5' position of one nucleoside and the 3' position
of the nucleoside adjacent to it. The nucleoside in the
3'—linked terminal position contains a 5'-1ined phosphomono-
ester and a hydroxyl group in the 2' position, whereas the
,5'-linked terminal nucleoside contains hydroxyl groups in
both the 2' and 3' positions. Non-terminal nucleosides con—
tain hydroxyl groups only in the 2' position. This is

illustrated in Figure 1A. Alkaline hydrolysis of various

 

 

 

 

 

 

 

 

 

 

 

 

(A)
Base Base Base
2' -OH 2' -OH 2' -OH
3' 3' 3' —OH
P \P\ \K
\ 5. L_\ 5| 4n\ 50
3'-Linked 5'-L1nked
Terminus Terminus
08‘
(B)
Base Base Base Base
P ‘1
2' -OH 2' —OH 2' P-P 2' l—OH
3' —p 3' —r 31 3' —on 3' —ou
.r-d 5' Lao-4 5' Ho-l 5' __n 30‘ 5'
Nucleoside Mixed, 2'(3') Nucleoside
Diphosphato Monophosphstu
Figure l: (A) Stylized representation of an RNA molecule,

adapted from Sugiyama and Fraenkel-Conrat (88)f
(B) Products of alkaline hydrolysis of RNA:
nucleoside—2'(3'),5'-diphosphate from 3'-1inked
.terminus, mixed 2'(3')—monophosphates from
internal positions and nucleoside from
5'-linked terminus.

RNA's has confirmed this structure (see later discussion of
alkaline hydrolysis in Literature Review), as both nucleo-
sides and nucleoside—2'(3'),5'—diphosphates were obtained
upon alkaline hydrolysis of r—RNA from E, 32;; (9, 62, 77)
L. cells (47), and wheat germ (45, 80), as well as s-RNA
from E, gglé, yeast (5), rat liver (29), rabbit liver (78)
and wheat germ (46). The only exception to this structure,
observed to date, is TMV-RNA. Sugiyama and Fraenkel—Conrat
(88) observed that no phosphate was released when TMV-RNA

was incubated with E, coli alkaline phosphatase, indicating

 

that this RNA molecule is not phosphorylated at either
terminus. This was substantiated by the observation that
nucleosides are released both by alkaline hydrolysis (88),
and venom phosphodiesterase hydrolysis (89) of TMV-RNA.
These hydrolytic procedures release nucleosides from non-
terminally phosphorylated 5'-linked and 3'-linked termini
respectively.

The rest of this review is divided into three parts.
The first part deals with methods of detecting RNA termini,
the second with the nature of RNA termini and the third with

the enzymatic addition of terminal groups to RNA.

Methods of Detecting RNA Termini
Rathandary and Stuart (70) have recently reviewed
methods used to detect RNA termini. Most of these methods
depend upon the existence of polynucleotides of the type
illustrated in Figure 1A, and many of the methods are used

in conjunction with each other.

Enzymatic Methods

 

l. Escherichia coli alkaline phosphatase

 

Literature dealing with E, ggEE_alkaline phosphatase
has been reviewed by Hummel and Kalnitsky (33). The enzyme
is synthesized adaptively when E, gglé_cells are grown on
phosphate-deficient media. Heppel gg_§l, (28) found that
the purified enzyme acts nonspecifically, only on phosphate
monoesters and is therefore a useful tool for the removal of
terminal phosphates from nucleic acid chains, thereby render—
ing the termini accessible to certain reagents used for
detecting terminal groups. Sugiyama and Fraenkel—Conrat (88)
used this enzyme on 32P-labeled TMV-RNA, and determined that
neither terminus is phosphorylated in this RNA.

2. Snake venom phosphodiesterase
The action of phosphodiesterase from the venom of

Crolatus g2, (rattlesnake) and other snakes has been reviewed

10

by Laskowski (48). This enzyme acts primarily as an exonu-
clease, starting at the end of the chain bearing a free 3'
hydroxyl group, and sequentially liberating 5' nucleoside
monophosphates (73). If RNA is incubated first with E, EELA
alkaline phosphatase, and then with venom phosphodiesterase,
the 3'—1inked terminus will be released as a nucleoside,
while all other chain components will be released as monophos-
phates (89). Another use of the enzyme is for the sequential
degradation of RNA, with subsequent identification of released
nucleotides, to determine 5'—linked termini (23, 79). Singer
and Fraenkel-Conrat (79) recommended that this enzyme be used
at 00 C for sequential degradation. Disadvantages to this
procedure, however, are that values for number of cleavages
are only average values and do not represent identical degra-
dation of all chains (79, 84), and also, even purified
preparations of the enzyme exhibit limited amounts of
endonuclease activity (73, 79). This internal cleavage
appears to occur preferentially at sequences of the type
-pUpA;X-, which could lead to disproportionately high values
for terminal A and U (79). This enzyme was utilized by
Sugiyama and Fraenkel-Conrat (89) to identify the 3'-linked

terminus of TMV-RNA.

11

3. Spleen phosphodiesterase

The action of spleen phosphodiesterase has been
studies by Razzell and Khorana (74). This enzyme acts in
a manner opposite to that of venom phosphodiesterase, as
it hydrolyzes nucleic acids by sequential attack from the
end bearing a free 5' hydroxyl group and releases nucleoside
3' monophosphates. The 3'—linked terminus may be freed of
its 5'-linked phosphate by the action of E, gglg_alkaline
phosphatase. A 5'-1inked terminus with a free 3' hydroxyl
group is released as a nucleoside, which may be separated
from the nucleotides. No endonuclease activity is detected
in purified preparations of this enzyme.

4. Pancreatic ribonuclease

The mechanism of action of pancreatic ribonuclease
has been reviewed by Witzel (99) and by Hummel and Kalnitsky
(33). This enzyme cleaves phosphodiester linkages of the
type pyrimidine-p-X, giving as products pyrimidine—3'-mono-
phosphates and oligonucleotides of the type —X—p-pyrimidine-p.
Nucleosides are released from the 5'-linked terminus, if the
penultimate base is a pyrimidine. If the penultimate base
is a purine, the oligonucleotide will contain 2' and 3'
hydroxyls on the 5'-linked terminus. Hydrolysis by this

enzyme involves the formation of a cyclic

12

pyrimidine-2'(3')-phosphodiester, which then gives rise,
preferentially, to the pyrimidine 3'-phosphomonoester.
Hunt (36) has utilized this technique in conjunction with
isonicotinic acid hydrazide—labeling to determine the
5'-linked terminal and penultimate nucleosides in rabbit
reticulocyte r-RNA.
5. Polynucleotide kinase
Polynucleotide kinase has been isolated from cell-

free extracts of E, coli infected with bacteriophage T (63)

2
or T (75). This enzyme catalyzes the transfer of phosphate

4
from adenosine triphosphate (ATP) to the free 5' hydroxyl
position of a 3'—1inked terminal nucleoside of either RNA
or DNA. Selectivity of the enzyme is limited, however, as
it will also transfer phosphate, at a reduced rate, to the
5' position of adenosine—2(3')—monophosphate, but not to
adenosine or cytidine (75). This enzyme has been used to
transfer 32F from 6—32P—ATP to RNA in attempts to identify
3'-linked nucleosides (86, 90). RNA in these studies was
first incubated with E, gglE_alkaline phosphatase to remove
the 5'—1inked terminal phosphate moieties.

6. Nucleoside phosphotransferase

Brawerman and Chargaff (7) isolated an enzyme from

malt diastase which catalyzes the transfer of phOSphate from

13

phenyl phosphate to nucleosides. Tunis and Chargaff (93)
have isolated and partially purified a similar enzyme from
carrot. The enzyme from carrot leaf tissue has been further
purified by Becker (4), who studied its ability to transfer
phosphate from 32P-p—nitrOphenyl phosphate to the 5' position
of nucleosides. The enzyme can be used to study nucleosides
released from the termini of RNA by the nucleolytic enzymes
Which have been discussed, or by alkaline hydrolysis. Puri—
fied enzyme preparations posess phosphomonoesterase activity,
which remains associated with the enzyme through all purifi-
cation steps which have been studied, making precise
quantitative estimates of chain terminal groups difficult.
Becker (4) used this technique to phosphorylate nucleosides

isolated from alkaline hydrolysates of cauliflower r-RNA.

Chemical Methods

 

1. Alkaline hydrolysis
The mechanism of alkaline hydrolysis of RNA has been
reviewed by Witzel (99). As is shown in Figure 1B, hydrol—
ysis releases mixed 2'(3')-mon0phosphates from non-terminal
positions on the RNA chain, nucleosides—2'(3'),5—diphosphates
from the 3'—linked terminus and nucleosides from the
5'-linked terminus. As in the case of pancreatic ribonuclease,

hydrolysis proceeds via the formation of a cyclic 2',3'—-

l4

nucleoside phosphodiester intermediate (51, 99), however,
in alkali this diester is cleaved non-preferentially, to
give mixed nucleoside—2'(3')-monophosphates. Alkaline

hydrolysis is thus dependent upon the presence of a free

hydroxyl in the 2' position, and is therefore ineffective

34,

on DNA or on dinucleotides in which the 3'—1inked member
is substituted in the 2' position. Hudson §E_§E, (32) have
shown that alkali-stable dinucleotides in wheat germ s-RNA
have the general structure prNp, where Nx is the 2'-0-methy1
derivative of a nucleoside.

Whitfeld (96) and Lee and Gilham (49) have discussed
a side reaction which can occur in alkaline hydrolysis, and
which can give misleading results in analyses of 5'-1inked
termini. This reaction involves the transfer of a phosphate
from the 2' or 3' position of a nucleotide, to the 2' or 3'
position of a nucleoside, and thereby converting the original
nucleotide to a nucleoside. The reaction, which apparently
proceeds by the formation of a phosphodiester linkage between
the two nucleoside moieties, is slow, due to the low concen-
tration of nucleosides in alkaline hydrolysates, and is
apparently of importance only in long—term hydrolyses. Cohn
(16) has reported that use of excess alkali may cause further

hydrolysis of nucleotides.

15

2. Periodiate oxidation

Whitfeld and Markham (98) discovered that ribonucleo—
sides react with sodium meta periodate, giving a dialdehyde
oxidation product. The reaction involves cleavage of the C—C
bond between the 2' and 3'-hydroxy1ated positions of the
ribosyl moiety, with concomitant formation of aldehyde groups
at these positions. This reaction is the first step in each
of the next five methods.

A. Amine catalyzed cleavage of dialdehydes

The dialdehydes produced by periodate oxidation of
dinucleoside phosphates were shown to react with glycine, to
yield free bases and nucleoside-3'-monophosphates (8, 98).
Brown, gg_§l, (8) suggested that this method might be used
for the sequential degradation of RNA. Ogur and Small (64)
found that lysine was more effective than glycine, and it
now appears that any primary amine is effective in catalyzing
this reaction. Among the other amines which have been used
are cyclohexylamine (103), phenylhydrazine (38) and aniline
(84). This method may be used on intact RNA chains and on
nucleosides and 5' nucleotides produced by hydrolysis. Neu
and Heppel (61) have described a modification of the amine
catalyzed cleavage reported by Whitfeld and Markham (98),

which involves the use of higher temperature (450 C, rather

16

than room temperature) and excess periodate, thereby facili-
tating recovery of the purine of pyrimidine base in a free
state.

Amine catalyzed cleavage of the dialdehydes involves
the formation of Schiff's bases (38, 39, 61, 103), which are
stable at pH 9, but which break down at pH 7-8 to yield free
base plus an aglycone (38, 39).

Periodate oxidation and amine catalyzed cleavage
have been used to characterize the 5'-linked termini of a
variety of nucleic acids (69, 84, 97, 103). Whitfeld (97)
has demonstrated the use of this technique, in conjunction
with E, gglg_alkaline phosphatase, to determine the 5'—linked
terminal sequence of TMV-RNA. The terminal nucleoside was
determined by subjecting the TMV-RNA to periodate oxidation
followed by amine catalyzed cleavage. The terminal base
released was subsequently identified, and the TMV-RNA was
dephosphorylated with E, gglg_alkaline phosphatase, and the
procedure repeated, to identify the penultimate base.

B. Formation of bisphenylhydrazones

Khym and Cohn (38) have described conditions for the
formation of bisphenylhydrazones by the reaction of dialde-
hydes, formed by periodate oxidation, with phenylhydrazine.

A serious disadvantage to this procedure, however, is the

17

observation that excess phenylhydrazine causes a dissociation
of the derivatives to bases and two- and three-carbon frag—
ments. This disadvantage probably accounts for the fact that
this method has not been used in end group assays.

C. Formation of trialcohols

Another reaction described by Khym and Cohn (38) is
the formation of trialcohols by the reduction of the dialde-
hyde oxidation products with sodium borohydride. This
method, employing tritium—labeled sodium borohydride. has
been used to characterize nucleosides produced by alkaline
hydrolysis of cauliflower r-RNA (69).

D. Formation of semicarbozones and thiosemicarbazones

Methods analagous to the previous two, in that they
involve formation of derivatives of dialdehydes, have been
described by Steinschneider and Fraenkel-Conrat (83) and by
Dulbecco and Smith (19) for the formation of semicarbazones
and thiosemicarbazones, respectively. These methods involve
the reaction of either l4C—semicarbazide or 35S-thiosemi—
carbazide with dialdehydes, with subsequent formation of
either semicarbazones. Like the previous method, these allow
the detection of small quantities of nucleoside by means of
radioactivity. Steinschneider and Fraenkel-Conrat (84) have

used 14C—semicarbozones to determine the rate of release of

18

terminal bases, by amine catalyzed cleavage following
periodate oxidation of TMV-RNA.

E. Formation of isonicotinyl hydrazones

Hunt (34) has described the synthesis of isonicotinyl
hydrazone derivatives of the dialdehyde oxidation products
described previously. These derivatives are formed by the
reaction of the dialdehydes with isonicotinic acid hydrazide,
and, in contrast with the amine Schiff bases which are stable
at pH 9 but not at pH 7-8, these derivatives are stable at
low pH, but are destroyed at pH 7 or above.

Periodate oxidation and formation of isonicotinyl
hydrazones, followed by hydrolysis of RNA with pancreatic
ribonuclease, has been used by Hunt (36) to determine the
5'—linked terminal sequences in rabbit reticulocyte r-RNA.

In these studies he used 3H-labeled isonicotinic acid
hydrazide. Midgley (57, 58) has used this technique in
attempts to determine chain length in E, EQEE_r-RNA, and
McIlreavy and Midgley (53, 59) have used it to determine
the 5'-linked chain termini in E, Egll r-RNA.

3. Acetylation

A method which has not been used on RNA, which should
be applicable for detecting and quantifying small amounts of

nucleoside isolated from RNA hydrolysates, has been developed

19

by Stuart and Khorana (85). They found that 3H-acetic anhy—
dride reacts specifically with terminal hydroxyls of DNA.
Jackson §E_§l, (37) used this method to determine the 5'—linked
chain termini of DNA associated with crystalline yeast

cytochrome b This method would not be applicable for use

2.
with intact RNA chains, as acetic anhydride would react with
free 2' hydroxyls of the ribonucleotides.
4. Phosphorylation with polyphosphate

Weiss gE_§l, (95) have described a method for the
synthesis of cytidine monophosphate from cytidine and poly-
phosphate. This method, when used with 32P-polyphosphate,
allows the detection of small quantities of nucleoside. A
similar synthesis has been described by Hall and Khorana (24),
for the synthesis of pyrimidine nucleoside—2'(3'),5'—diphos—
phates, and appreciable quantities of monophosphates are
formed as by-products of the reaction. This method is only
suitable for the phosphorylation of pyrimidine phosphates, as
purines are not phosphorylated. Waehneldt and Fox (94) used
the polyphosphate method to synthesize both 2'(3') and 5'
monophosphates of all four ribonucleosides, but yields in
their experiments were low, being in the range of 10 to 15

percent.

20
5. Phosphorylation with cyanoethyl phosphate

The use of cyanoethyl phosphate and dicyclohexylcar—
bodiimide (DCC) for the synthesis of mono— and diphosphates
from nucleosides has been described by Tener (91). He found
that cyanoethyl phosphate reacts with alcohols, such as free
hydroxyl groups on the ribosyl moiety of nucleosides, yield-
ing a phosphodiester of the cyanoethyl group and the alcohol.
DCC acts as a coupling agent in the reaction. The cyanoethyl
moiety is then removed by mild alkaline hydrolysis, yielding
nucleotides. Tener (91) used nucleosides which had 2' and 3'
hydroxyls blocked with isopropylidine, to synthesize 5'
nucleotides. If this blocking is not done, phosphorylation
of nucleosides by this method gives mixed products of all
possible monophosphates and diphosphates. This method has
been used in the synthesis of many of the ribo— and deoxyribo—
nucleosides (21, 91) and in the characterization of
nucleosides released upon alkaline hydrolysis of cauliflower
r—RNA subunits (68).

6. Formation of methyl phosphate derivatives

Another method, related to the previous one, employs

condensation reactions to add a 14C label to the 3'-linked

terminus of nucleic acids. This reaction, described by

Rathandary gE_§l, (71), involves the reaction of the

21

5'—terminal phosphate with l4C—methyl phosphomorpholidate.
Methanol-14C is reacted with cyanoethyl phosphate in the
presence of DCC. The methyl phosphate produced by alkaline
hydrolysis of this product is then reacted with morpholine,
in the presence of DCC, to give methyl phosphormorpholidate.
This product reacts Specifically with a phosphomonoester to
form a perphosphate linked methyl phosphate derivative.

This method has been used to determine the 3'-linked
termini of yeast s-RNA (71) and of the DNA associated with

yeast cytochrome b (37). This method Should also be appli-

2
cable for the introduction of a 32F label to the 3'—linked
chain terminus, as well as a 14C label, Since both a phosphate
and a methyl group are added in the reaction.
7. Formation of phosphoroanilidate derivatives

Ralph EE.2ln (72) have described the synthesis of
14C-phosphoroanilidate derivatives of 5' nucleotides. The
reaction involves the condensation of phosphomonoesters with
aniline, in the presence of diisopropylcarbodiimide to form
a phosphodiester. This method has been used to attach a 14C
label to the 5' phosphate of the 3'—linked terminal nucleotide

in yeast s—RNA, and could have general application for the

detection of this terminus in other nucleic acids.

22

The Termini of Ribonucleic Acids

 

Viral RNA

 

Lee and Gilham (49) found all four ribonucleosides
(A, G, C and U) in alkaline hydrolysates of both f2 and MS 2
bacteriophage RNA, but concluded that adenosine was the
5'-linked terminus of both, Since this nucleoside composed
more than 75 percent of the product in both cases. These
results, for MS 2 bacteriophage, were confirmed by Sugiyama
(87), using dyrolysis with both alkali and pancreatic ribo—
nuclease.

The termini of TMV—RNA have been investigated by
Fraenkel-Conrat and co-workers (79, 84, 88, 89) and by
Whitfeld (96, 97). Their results, obtained by alkaline
hydrolysis (88, 89, 96), hydrolysis with venom phosphodi—
esterase (79) and pancreatic ribonuclease (96) and by
periodate oxidation followed by amine cleavage (84, 97)
showed adenosine to be the major nucleoside component of
the 5'-1inked terminus. Steinschneider and Fraenkel—Conrat
(84) observed that elimination of the terminal nucleoside
by periodate oxidation and amine cleavage resulted in a 95
percent loss of infectivity of the RNA. Activity was not

restored by removal of the 3' terminal phosphate by E, coli

alkaline phosphatase. These findings were in conflict with

23

results obtained by Singer and Fraenkel-Conrat (79), which
Showed only a 10 percent loss of infectivity with an average
of one cleavage per RNA chain with venom phosphodiesterase.
Steinschneider and Fraenkel—Conrat (84) concluded that values
for cleavage of RNA chains with phosphodiesterase actually
represent average numbers of cleavages per chain, rather

than actual cleavage of every chain.

Sugiyama and Fraenkel-Conrat (88, 89) studied the
3'-linked terminus of TMV-RNA by the use of E, ggii_alkaline
phosphatase and extensive digestion with venom phosphodi-
esterase. Using E, 99;} alkaline phosphatase (88) they
determined that, unlike other RNA'S, TMV—RNA contains no
5'-linked phosphate at its 3'-linked terminus. Studies with
venom phosphodiesterase (89) revealed that about 50 percent
of the nucleoside released was adenosine. They concluded
that adenosine probably represented the 3'-linked terminus,
thereby indicating that TMV—RNA has adenosine at both termini.

Thus, in summary, it may be said that the 5'-linked
end group of all the viruses studied is adenosine. Results
discussed for the termini of viral RNA'S are summarized in

Table l.

24

 

 

 

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.H magma

25

Soluble RNA

 

All s-RNA'S studied to date have been found to
terminate preferentially in either cytidine or adenosine at
the 5'-linked end. Zillig, EE.E$3 (104) found that adenosine
was the only nucleoside produced by alkaline hydrolysis of
E, ggli_s—RNA, whereas McIlreavy and Midgley (53) found both
adenosine and cytidine as termini when samples of E, 921$
s-RNA were labeled with 3H-isonicotinic acid hydrazide.
Eighty percent adenosine and 20 percent cytidine were found
in alkaline hydrolysates of yeast s-RNA by Bell, g3_§l, (5),
whereas Yu and Zamecnik (103) found about 30 percent adenosine,
60 percent cytidine and 10 percent uridine, using both
alkaline hydrolysis and periodate oxidation followed by amine
cleavage. Hecht and Zamecnik (26) and Herbert and Canellakis
(29) found mostly adenosine and cytidine in alkaline hydroly—
sates of rat liver s-RNA; Similarily, Singer and Cantoni (78)
found mostly adenosine and cytidine (at least 50 percent
adenosine) upon alkaline hydrolysis of rabbit liver S-RNA.
Studies on wheat germ s—RNA by Lane and Allen (46) and by
Hudson, 33.31: (32) revealed that adenosine and cytidine
comprised most of the nucleoside function released by

alkaline hydrolysis.

26

Heidelberger, gE_§l, (27) found that an enzyme system
from rat liver would incorporate adenosine at the 5'-linked
terminus of s—RNA, and that the penultimate nucleoside was
cytidine. Canellakis (11) found that this enzyme system
would only add adenosine to a 5'-linked terminal cytidine
residue, and Hecht, 33 3E. (26) reported that the enzyme was
specific for s—RNA, and that it sequentially added cytidine
and adenosine. The enzyme had a very low activity for the
incorporation of uridine (18, 26). Herbert and Canellakis
(30) observed that when s—RNA is degraded by approximately 10
percent with venom diesterase, its ability to accept cytidine
or uridine in the rat liver enzyme system is increased,
whereas its ability to accept adenosine is decreased. The
presence of all four ribonucleoside triphosphates in the re—
action mixture increases incorporation of adenosine and
cytidine, and decreases incorporation of uridine. Among the
uridine-containing termini added sequentially in a similar
system studied by Klemperer and Canellakis (42) were: -pU,
-pUpA: —pUpC and -pCpU.

The requirement for sequential addition of a -pCpCpA
terminus for acylation of s-RNA with amino acids, was dis—
covered by Hecht, gE_§l, (25), through a study on the kinetics

of nucleotide incorporation with the rat liver enzyme system.

27

They found that two cytidine residues were incorporated for
each adenosine, and that adenosine was not added until after
cytidine had been incorporated. Acylation would not occur
until a terminal adenosine was present. The CCA terminus

was lost when s-RNA was incubated in the presence of pyrophos—

phate. They concluded that the CCA terminus is necessary for

“fin“ :1 ~ 1

the incorporation of nearly all amino acids, because all of

the 14 amino acids studied showed increased acylation with

 

addition of the CCA terminus, and were non—competitive with *"
each other. Merits (55), studying incorporation of 32P-labeled
nucleosides into s-RNA, found that in the presence of
actinomycin D, most of the radioactivity was in the CCA
terminus, indicating that incorporation was due to end group
addition, and that this process was independent of DNA—
dependent RNA synthesis. Rosset and Monier (76) found that
s-RNA from exponentially growing yeast cells had mostly
adenosine as its 5'—linked terminus, whereas cells in a
stationary growth state had both adenosine and cytidine at

this terminus. They concluded that there was a slow turn—

over of terminal adenosine in exponentially growing yeast
cells, but not in those in a stationary state of growth,
resulting in the observation of cytidine in the latter cells,

due to enzymatic hydrolysis of terminal adenosine.

28

Guanosine has been shown to be the 3'-linked terminus
of s-RNA. Zillig, §E_§l, (104) found pGp as the only diphos—
phate produced upon alkaline hydrolysis of E, EQEE_s—RNA,
whereas Bell, §§_§l, (5) and McLaughlin and Ingram (54) found
that it comprised approximately 80 percent of the diphosphate
produced upon alkaline hydrolysis of yeast S—RNA. Studies on
rat liver s—RNA and rabbit liver S-RNA by Herbert and
Canellakis (29) and Singer and Cantoni (78), respectively,
showed that pGp was the main diphosphate produced by alkaline
hydrolysis of these s—RNA'S. Similar results were obtained
by Hudson, gg_gl, (32) in their study on S-RNA from wheat germ.
Ralph, §E_gl, (72) formed l4C-phosphoroanilidate derivatives
of yeast s-RNA, and found that most of the label was bound
to quanosine; and Rathandary, 3E.il- (71) found that when
l4C-methl phosphate derivatives of yeast s—RNA were formed,

75 percent of the label was associated with guanosine.

Tables 2 and 3 summarize data on the termini of s-RNA.

Ribosomal

 

Studies on the termini of r-RNA'S have been reviewed
by Ebel (20).
1. 3'—1inked termini
Studies done by Sugiura and Takanami (86), on the

3-‘1inked termini of r-RNA's of yeast (Saccharomyces cerevisiae)

 

29

Table 2. Summary of data on the 5'-linked termini of soluble
ribonucleic acids.

 

Mole Percent

 

Source of RNA

 

 

A G C U Ref.

E, coli 100 n.d. n.d. n.d. 104
51 - 38 n.d. 53

Yeast 80 n.d. 20 n.d. 5
28.6 n.d. 61.4 10.0 103

Rat liver + n.d. + n.d. 26
98.5 0.5 0.5 0.5 27

67 14 7 12 29

Rabbit liver >50 tr. + tr. 78
Wheat germ 77.3 1.8 17.0 3.9 32
75.5 6.8 12.0 5.7 46
+ detected, but quantitative data not available

- no information given
tr. trace

n.d. not detected

30

Table 3. Summary of data on the 3'—linked termini of soluble
ribonucleic acids

 

Mole Percent

 

Source of RNA

 

 

 

A G C U Ref.
E, coli n.d. 100 n.d. n.d. 104
Yeast 7 78 5 10 5
4.1 75.5 5.1 10.1 71
16.6 83.4 n.d. tr. 54
+ + n.d. + 72
Rat liver n.d. 100 n d. n.d. 29
Rabbit liver - >80 — — 78
Wheat germ 3.8 89.5 1.0 5.7 32
+ Detected, but quantitative data not available

— no information given
tr. trace

n.d. not detected

31

and four Species of bacteria: Bacillus cereus, E, subtilis,
E, stearothermophilus and Saracina lutea, using K—BzP—ATP

and polynucleotide kinase, revealed that all r—RNA'S studied
terminated preferentially in uridine except the heavy sub—
unit of E, E3332, which had mostly adenosine at the 3'-1inked
terminus. These results are summarized in Table 4.

Nichols and Lane (62), using alkaline hydrolysis, and
Takanami (90), using plynucleotide kinase, -32P-ATP and
alkaline hydrolysis, determined that E, gglE_r-RNA has mostly
adenosine on the 3'linked terminus of 16 S r-RNA, and mostly
guanosine on 23 S r-RNA. The 5 S r-RNA from E, Egll (9, 77)
has been shown, through alkaline hydrolysis, to have uridine
at the 3'—linked terminus.

Alkaline hydrolysis of r-RNA from L cells revealed
that this r-RNA has mostly uridine at the 3'—linked terminus
of the 16 S subunit, and cytidine at the 3'—linked terminus
of the 28 S subunit. Unfractionated wheat germ r—RNA has been
shown by alkaline hydrolysis to have no preferential termina—
tion at the 3—linked end, as all four nucleoside diphosphates
were present in hydrolysates (45, 80).

Data on the 3'—linked termini of r—RNA from E, SELE;
L cells and wheat germ are summarized in Table 5.

The observations that TMV-RNA, s—RNA and E, coli r-RNA

32

 

 

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33

 

 

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.Euom umo£3
new maaoo .A .flaoo am Eoum 42m HmEomOQAH mo ASHEHop one .HH .moflom
oeoaoocnnflu HmEomonflu mo flcflfiuop omxcflan.m on“ no mumo mo >HmEEdm .m manna

 

34

all have purines at their 3-linked termini may have some
relationship to the observation of Maitra and Hurwitz (50),
that products of DNA—dependent RNA polymerase from E, gglE_
are preferentially initiated by purines. The results of
Sugiura and Takanami (86), however, suggest that this is
not a universal phenomenon.
2. 5'-1inked termini

Lane (43), using alkaline hydrolysis, found that the
16 S + 23 S r-RNA of E, gglé_had mostly adenosine and uridine
at the 5'-linked terminus. These results were confirmed by
Midgley and McIlreavy (59) and by McIlreavy and Midgley (53),
both my means of alkaline hydrolysis, and by experiments
involving isonicotinyl hydrazones. These latter experiments
involved periodate oxidation of 5'-linked r—RNA chain termini
and labeling with 3H—isonicotinic acid hydrazide, followed by
hydrolysis of the isonicotinyl-RNA with either pancreatic
ribonuclease or ribonuclease Tl. Studies on the 5'-1inked
termini of individual subunits of E, gglE_r—RNA by Nichols
and Lane (62), using alkaline hydrolysis, and by McIlreavy
and Midgley (53) using 3H-isonicotinic acid hydrazide deriva—
tives, revealed that the 16 S subunit terminates primarily
as adenosine, and the 23 S subunit primarily as uridine.

Rabbit reticulocyte r—RNA was studied by Hunt (35.

35

36) via formation of isonicotinyl hydrazones and enzymatic
hydrolysis. He found that there were essentially two types
of 5'-linked termini in 30 S r-RNA, one being -pyrimidine-p-U
and the other —G—p-U. The 17 S r-RNA terminates preferen-
tially as —pyrimidine-p—A. Lane and Tamaoki (47) used
alkaline hydrolysis to determine that r—RNA from L cells has
mostly adenosine at the 5'-linked terminus of the 16 S subunit,
and uridine at that terminus of the 28 S subunit.

Hadjiolov, gg_gl. (23) have reported that upon
sequential degradation of rat liver r-RNA with venom phos—
phodiesterase, the contents of adenosine and uridine in early
hydrolysates (30 percent hydrolysis) are higher than are
those obtained upon complete hydrolysis. Nucleotides
isolated following 30 percent hydrolysis of 18 S r—RNA were
composed of 20.8 percent adenosine monophosphate and 24.5
percent uridine monophosphate, and those from 28 S r—RNA were
composed of 19.4 percent adenosine monophOSphate and 20.9
percent uridine monophosphate. Complete hydrolysis with
alkali, however, gave products from 18 S r-RNA containing
12.1 percent adenosine monophosphate and 22.3 percent uridine
monophosphate; and from 28 S r—RNA containing 14.9 percent
adenosine monophosphate and 20.9 percent uridine monophos—

phate. It was suggested that the segments of the chains

36

near the 5'-1inked termini may be higher in adenosine and
uridine than in quanosine and cytidine. These results are
subject to question, however, because of the observations
that venom phosphodiesterase has some endonuclease activity
(73, 79), and that this gives preferential cleavage at
sequences of the type —pUprX- (79).

Alkaline hydrolysis of unfractionated wheat germ
r—RNA has revealed that there is no preferential termination
at the 5'-linked end of this r—RNA. Because of this finding,
and that reported previously for the 3'-linked terminus,
wheat germ r-RNA provides the only exception observed to the
preferential termination of RNA's. The possible Significance
of this will be discussed in greater detail later.

Table 6 summarizes data on the 5'—linked termini of
r-RNA'S.

Midgley and McIlreavy (60) also found that when E,
ggll_cells were grown on "casamino acids" or broth, rather
than on glucose or succinate, the percent of terminal uridine
increased on both subunits. A summary of their results is
presented in Table 7.

Alkaline hydrolysis of the 5 S r-RNA from E, ggll_

(9, 77) revealed that this subunit has uridine at its

5'-linked terminus.

37

 

co>flm coHumEHOMCH o: I

manmaflm>m no: mumo m>HpmuHucwso Don .Umuomuoo +

 

 

om a.vm v.vm m.om m.om

mo ¢.mm H.Hm o.mm m.mH

ma o.mm o.mm m.om m.na

we m.om a.n~ o.Hm a.Hm m mm + m on Show omens
he mm 6 am NH m mm

be ma a 6 mu m 6H mHHoo .q
0m + I I I m Om

um I I I + m SH mesooHsoHpmu panama
me On a 6 ma

mm we I I mm m mm

mm ma 0H 0H om

mm ma I I an m 0H

mm mm m Ha mo

mm mm I I me _I
ma m.om m.ma New o.>v m mm + m 0H Aaoo .m
.mmm o o o a

42m mo oNHm «2% mo ouudom

 

unmouom oaoz

 

.moflom oaoaosconfiu HmEOmOQHH mo HSHEHou poxcHHI.m onu so mumo mo >umEEom .o oHQmB

38

 

 

0 mm ma m mm

OH mm hm m ma

0 on om m mm + m 0H muamm ocm moflom osHEmmmo
Ha mm 0H m mm

Ha mm om m 0H

m mm mm m mm + m 0H Spoum
em mm mm m mm

NH om mm m ma

0H 0v on m mm + m 0H ounceoosm
em we mm m mm

Ha ma oh w ma

am on mo m mm + m 0H muamm pom omOUSHU
Honpo D d

 

mscwauoe mo unmoumm maoz

:ofluomum 42m

Eoflpmz np3ouw

 

pom >oamoflz an oouuomou mm

 

.azm HmEomoaHu Haou

.Aoov >>mmuHHuz
.m no Hananmu omxcusu.m

mnu mo ououmc map so mucmfluusc mo poowwo on» so none mo wumfifidm .h manna

39

Enzymatic Addition of Termini

 

The action and importance of enzymes which add the
terminus -CCA to the 5'-linked ends of s—RNA have been dis—
cussed in the section on s-RNA termini.

An enzyme which incorporates uridine derived from
uridine triphosphate into the 5'-linked termini positions of
RNA of an unspecified type, has been isolated from Ehrlich

Ascites carcinoma cells by Burdon and Smellie (10). Action

 

of this enzyme was unaffected by the addition of triphosphates
of adenosine, guanosine and cytidine, but was surprisingly
stimulated by the addition of a mixture of amino acids.
Another enzyme isolated in the same study gave incorporation
of uridine into both 5'—linked terminal and non—terminal
positions, and was stimulated by the addition of a mixture of
nucleoside triphosphates.

Klemperer (40, 41) isolated an enzyme from the pH 5,
105,000 g supernatant fraction of rat liver homogenate which
incorporated adenosine monophosphate or uridine monophos—
phate, derived from the corresponding triphosphates, as
homopolymer chains on the 5'-1inked end of natural or synthe—
tic polyribonucleotides. Although this enzyme would add to
the termini of S-RNA, it differed from previously studied

enzymes in that it was not specific for s—RNA, and would also

40

work on rat liver r—RNA and synthetic polyribonucleotides.
Klemperer found that incorporation of uridine monophosphate
was dependent upon the presence of magnesium ions, and
incorporation of adenosine monophosphate was dependent upon
the presence of manganese ions.

Recently, Page, 93.2ln (65) reported that a similar
enzyme from rat liver microsomes incorporates all four

ribonucleotides into homopolymer chains on the 5'—linked

 

termini of r—RNA, and that such incorporation is dependent
upon the presence of manganese ions. That all four nucleo—
tide triphosphates Show competitive inhibition indicates
involvement of the same enzymatic sites for incorporation of
all of them. When manganese ions are replaced by magnesium
ions in the system, only uridine monophosphate is incorporated
into the terminal chains.

Enzymes which add terminal groups may be involved in
the findings of Midgley and McIlreavy (60) summarized in
Table 6, which Showed an increased ratio of 5'-linked terminal
uridine to terminal adenosine in cells grown on broth or
casamino acids, as compared to those grown on glucose or
succinate. These authors concluded, however, that the in-
crease in terminal uridine was due to a different response

of genetic elements controlling the synthesis of r—RNA, in

41

different environments. They reported that the synthesis of
"A-terminal" r—RNA was constant in all systems, whereas the
synthesis of ”U-terminal” r-RNA was stimulated in broth or
casamino acids. Taken on the basis of molecules per cell,
the quantity of “A-terminal“ r—RNA was constant under all
conditions, whereas the quantity of ”U-terminal" r—RNA was

increased by broth or casamino acids.

 

MATERIALS AND METHODS

Experimental Materials

 

 

Plant tissues used in these studies, cauliflower
(Brassica oleracea, L. var. botrytis, L.), cabbage (E,

oleracea, L. var. capitata), mushroom (Agaricus campestris,

 

L. ex Fr.) and wheat germ (Triticum aestivum, L.), were

 

 

purchased from local commercial sources; cauliflower seeds
(Snowball) were purchased from Joseph Harris Company,
Rochester, New York. Gills and the outer surface of mush—
room caps were removed prior to use, as were leaves and heavy
stem sections of cauliflower, while cabbage heads were
shredded.

Rexyn 201 (Cl--quartenary amine resin) was purchased
from Fisher Scientific Company, Detroit, Michigan; Dowex 50
(H+—sulfonic acid resin) was purchased from Sigma Chemical
Company, St. Louis, Missouri. Both resins were washed
successively with portions of acetone, 50 percent ethanol and
20 percent concentrated hydrochloric acid, followed by exten-

sive washing with distilled water, prior to use. Carrier—free

42

43

2 . .
3 P-phosphoric aCid was purchased from New England Nuclear

Boston, Massachusetts, and 2-14C—uraci1 was purchased from
Schwarz BioResearch, Inc., Orangeburg, New York. Nucleoside
phosphotransferase from carrot leaf tissue and 32P—p—nitro-
phenyl phosphate were gifts of Veryl E. Becker; Actinomycin D
was a gift of Merck Sharp and Dohme, Research Laboratories,
Rahway, New Jersey. Other chemicals were reagent grade and

are available at most chemical supply houses.

 

Preparation and Hydrolysis of RNA

 

Preparation of Ribosomal RNA by Direct Extraction

 

l. Extraction with phenol and sodium dodecyl sulfate

Plant tissues were homogenized for two to three
minutes at 80 percent line voltage in a Waring Blendor, with
two volumes each (w/v) water—saturated phenol and a solution
consisting of 0.001 M magnesium chloride, five percent (w/v)
sodium dodecyl sulfate, 0.28 M lithium sulfate and 0.1 M
sodium acetate, pH 5.0. The latter solution, which will
subsequently be referred to as solution A, was adapted from
McCarthy and Hoyer (52).

The homogenate was then centrifuged for 10 minutes,
at 10,000 g_and the upper aqueous phase was removed. It was
added to an equal volume of phenol and deproteinization was

repeated. Following centrifugation, the RNA was precipitated

 

1‘... .F. I. 1;. I
A

44

from the aqueous phase with an equal volume of cold absolute

ethanol and the resulting RNA precipitate was collected by

centrifugation. The pellet was then homogenized in cold 3 M

sodium acetate, pH 6.0, and the insoluble material was

collected by centrifugation. Unless otherwise noted, all

r-RNA was washed two times with 3 M sodium acetate to remove l
S-RNA. This procedure was modified by increasing the solution

A: plant tissue ratio to 4:1 for the extraction of r—RNA from

wheat germ.

 

Phenol used in the deproteinization steps was Baker
Analyzed Reagent, since use of other brands often resulted in
a discolored, pink or gray, product. Click and Hackett (15)
reported that use of phenol other than Baker Analyzed Reagent
resulted in low yields and poor resoltuion of r-RNA.

Other solutions used for extraction were modified
from those described by Click and Hackett (15). These con-
sisted of equal volumes of water-saturated phenol prepared
by dissolving phenol in distilled water containing 0.001 M
ethylenediamine tetraacetic acid (EDTA), and a solution
containing one percent (w/v) sodium dodecyl sulfate, 0.01 M
EDTA, pH 9.5. This latter solution will be referred to as
solution B. The extraction procedure was the same as the

one described previously.

45

2. Extraction with phenol and phosphate buffer
The methods of Singh and Lane (80) were used to prepare
one batch of wheat germ r-RNA. Wheat germ was placed in 6.5
volumes (w/v) 0.05 M phosphate buffer, pH 7.0, containing 6.5

volumes of water—saturated phenol and shaken vigorously at

room temperature for 20 minutes. The suspension was then I
centrifuged at 10,000 g for 10 minutes and the aqueous phase

was siphoned off and made 1 M in sodium chloride. This

 

solution was allowed to stand for 24 hours, at O0 C. The
insoluble RNA was collected by centrifugation, and subse—
quently washed by homogenization successively in 100 m1
portions of absolute ethanol and diethyl ether. The RNA was
then resuspended in phosphate buffer and again collected by
making the solution 1 M in sodium chloride.

Preparation of Cytoplasmic RNA by Differential Centrifugation
and Phenol Extraction

 

The following methods of preparing cytoplasmic RNA
were develOped as a part of this research.
1. Ribosomal RNA
Equal quantities (w/v) of chilled plant tissue and a
solution of 0.01 M magnesium chloride, 0.01 M calcium chloride,
0.25 M sucrose and 0.05 M Tris, pH 7.8; were homogenized in

a Waring Blendor at 50 percent line voltage for two minutes.

46

The resulting slurry was filtered through two layers of

cheesecloth, and the filtrate reused as homogenizing medium

for three to four successive batches of plant tissue. The

homogenizing medium was chilled in ice water before each

homogenization, so that the temperature never exceeded 80 C.

The last filtrate from the cheesecloth was filtered through I

four layers of facial tissue, and the resulting filtrate was

.nk.“ .—_ =_. 2.1--

centrifuged at 15,000 g for 15 minutes, to remove nuclei,

 

Ike—

mitochondria and cell wall material. The 15,000 g_supernatant
solution was spun for 30 minutes at 105,000 g, with a number
40 rotor in a Spinco Model L. ultracentrifuge. Microsomal
pellets were then suspended in solution A, combined with an
equal volume of water-saturated phenol, and homogenized in
a Tenbroeck all-glass homogenizer. RNA was extracted by the
procedure described previously for preparation of RNA by
direct extraction with phenol and solution A.
2. Soluble RNA

An equal volume of absolute ethanol was added to the
105,000 g supernatant solution (postmicrosomal fraction), and
the precipitate was collected by centrifugation. Soluble RNA
was prepared from this pellet by the same procedure described

for the preparation of r-RNA by direct extraction with phenol

and solution A, except that the phenol deproteinization step

47

was done four times, and the material soluble in 3 M sodium
acetate was collected, as S-RNA, by ethanol precipitation.
Because of suspected contamination with DNA, wheat
germ r-RNA preparations were checked for DNA content with
the diphenylamine test described by Ashwell (1). This test
involved the addition of 1 m1 of sample (RNA solution) to EN-

2 ml of a solution formed by the addition of l g of diphenyl—

amine and 2.75 ml of concentrated sulfuric acid, to 100 m1

 

of glacial acetic acid. The mixture was heated for 10

am

minutes in a boiling water bath, and following cooling, the
absorbance was read at 595 and 650 mu.

All RNA used in these studies was of high Optical
purity (0D 260/280 = 1.95 - 2.15), and quantitative estima-
tions of RNA were based on the assumption that 1 mg of RNA

in 1 ml would have an CD of 24 at 260 mu.

Sucrose Density Gradient Centrifugation

Linear sucrose gradients (4—20 percent, w/v) were
prepared in either 0.15 M sodium chloride, 0.015 M sodium
citrate, pH 7 (saline citrate), or 0.05 M sodium chloride,
0.05 M sodium acetate, 0.0001 M magnesium chloride, pH 5.3
(saline acetate). Ordinarily, from 12 to 50 OD units
(260 mg) of RNA per bucket were centrifuged for routine

examination of sedimentation profiles, whereas 80 to 100 0D

48

units per bucket were centrifuged in runs for preparation of
r-RNA subunits. Gradients were centrifuged at 00 C for 15 -
19 hours at 23,000 rpm with a Spinco 25.1 rotor. RNA subunits
were recycled without precipitation, after dialysis at 20 C
overnight against the buffer used to prepare the gradients.
Centrifuge tubes were pierced through the bottom and the
gradients collected in 5—drop fractions which were then
diluted with 3 ml of the gradient buffer. unless otherwise

noted. Sedimentation profiles were obtained routinely for

 

all r—RNA preparations.

Alkaline Hydrolysis

 

Unless otherwise noted, RNA was hydrolyzed for 18—24
hours, at 370 C, in 0.3 M potassium hydroxide. Hydrolysis
was stopped by adjusting the solution to pH 7, in the cold,

+
with Dowex 50 H resin.

Chromatography and Electrophoresis
Anion-exchange Chromatography.

Nucleosides were collected from neutralized alkaline
hydrolysates as described by Cohn (16), by passing them
through a column of Rexyn 201-C1-, quartenary amine resin.
Material which came off the column during loading, during a

water wash and before 0.003 N hydrochloric acid began to elute

49

cytidylic acid, was taken as the nucleoside fraction. AS is
illustrated in Figure 2, cytidylic acid is eluted as a sharp
peak with 0.003 N hydrochloric acid, and fractions preceeding
this peak may be considered devoid of nucleotides.

The nucleoside fraction was then neutralized with
dilute sodium hydroxide, evaporated to dryness under reduced :
pressure, and the residue extracted with boiling pyridine.

Following filtration, to remove undissolved material, the

 

pyridine was evaporated under reduced pressure. Successive la—
small quantities of concentrated ammonium hydroxide were

added to the residue and evaporated to remove last traces of

pyridine. The nucleoside residue was then dissolved in 0.05

N hydrochloric acid and applied to paper for chromatography.

The nucleotide fraction from cauliflower hypocotyl

r—RNA was eluted with 1 N hydrochloric acid.

Paper Chromatography

All paper chromatography was descending, on Whatman
No. 1 paper, with chromatograms develOped in the following
solvent systems: (A) 2-propanol, one percent ammonium sul-
fate, 2:1, paper impregnated with one percent ammonium
sulfate; (B) 75 percent ethanol, paper impregnated with 10
percent-saturated ammonium sulfate; (C) 2-propanol, concen-

trated hydrochloric acid, water, 65:15:20; (D) water—saturated

Figure 2:

50

3.0- f

 

260 mu
I

d
n
I

 

 

Absorbance,

 

 

“b‘fizo 2'4 2'3 3'2 3'6

Fraction number

Pattern of elution of cytidylic acid from Rexyn
201, Cl‘. A neutralized alkaline hydrolysate
of 450 mg of RNA was loaded on to a 2 X 10 cm
column of Rexyn 201, Cl‘, rinsed with distilled

water and eluted with 0.003 N HCl. Seventy ml
fractions were collected, and all material which

was passed through the column during loading,
the distilled water rinse and to the point
indicated by the arrow, was saved as the nucleo-
side fraction. Cytidylic acid was identified by

its absorption spectrum and by chromatography in
system B.

 

51

butanol, in an ammonia—saturated atmosphere; (E) isopropanol,
concentrated ammonium hydroxide, 0.1 M boric acid, 7:1:2;

(F) t—butanol, 88 percent formic acid, water, 70:15:15; and
(G) 0.1 M sodium phosphate, pH 6.8 (1000 m1), ammonium sul-
fate (600 g), n-propoanol (20 ml). Systems A, B, C and D
have been described by Hall and Khorana (24), Lane (44),

Wyatt (100), and Hotchkiss (31), respectively, whereas systems
E, F and G are listed in the 1967 catalog from Schwartz Bio-
Research Inc., Orangeburg, New York.

Following paper chromatography, spots were detected
with ultraviolet light and were eluted from the paper with
0.1 N hydrochloric acid. Often areas adjacent to standards
were eluted, whether ultraviolet light absorption was
detected or not. Identification of nucleosides was based on
chromatographic mobilities and on absorption Spectra at both
acidic and alkaline pH. Quantitative determinations were
based on AOD (260 mu — 280 mu) at acidic pH (pH 1).

Samples containing undesired inorganic salt, such as
those eluted from papers developed in systems A and B, were
absorbed on Norit A charcoal (0.02 - 0.1 g), in the presence
of 0.05 N hydrochloric acid, prior to further chromatographic
purification. After washing the Norit A 24 2O times with

distilled water, the samples were eluted with two 10 m1

52

portions of 80 percent ethanol containing one percent con-
centrated ammonium hydroxide, followed by one 10 ml portion
of 50 percent ethanal containing one percent concentrated
ammonium hydroxide. Norit A was removed from the solutions,
at each step, by centrifugation, and the combined alcoholic

solutions were evaporated to dryness under reduced pressure.

Paper Electrophoresis

Paper electrophoretic separation of nucleotides was

 

achieved in a Beckman RD—2 electrophoresis unit, using two
different systems. The first System, as described by Bell,
EE.§$: (5) employed 0.05 M citrate buffer, pH 5, with a
potential of 300 volts. The second system, described by
Chandra and Varner (13), and modified by Becker (4), con—
tained 0.01 M EDTA (tetrasodium salt) and 0.34 ml of pyridine
in 600 ml of water. The pH was adjusted to 3.5 with acetic
acid. A potential of 400 volts was applied across the paper

to achieve separation.

Radioisotope Counting
Radioactive samples were counted in a Packard Tri—
Carb liquid scintillation Spectrometer, using a solution
consisting of 0.3 g of l,4—biS—2—(4-methyl-5-phenyloxazoly1)—

benzene and 5 J of 2,5—diphenyloxazole per liter of toluene.

 

53

Chromatograms and electrophoretograms were cut in strips
which were counted directly in the toluene solution, whereas
14 . . . . .

C-labeled plant tissues were dried in counting bials at

o . . . .
95 C, prior to addition of the solution. Aqueous samples

1 . . .

of 4C-labeled nucleOSide and nucleotides, from cauliflower
hypocotyl r—RNA, were pipetted into counting vials and l

evaporated to dryness at 950 C, prior to counting. Cauli_ E

flower hypocotyl r-RNA from sucrose density gradients was ,

 

prepared for counting according to a method modified from
Bollum (6). Fractions from sucrose gradients were spotted

on filter paper strips, air dried and then rinsed successively,
twice in five percent tricholoracetic acid, once in 75 per-
cent ethanol, twice in absolute ethanol and once in diethyl
ether. Air dried strips were then placed in vials for count-
ing.

Phosphorylation of Nucleosides

 

Use of Nucleoside Phosphotransferase

 

Nucleosides, purified from wheat germ r—RNA hydroly-
sates, were incubated for six hours, at 370 C, with 0.1 ml
of 0.1 M sodium acetate, pH 5.1, 50 ul of 32P—p-nitrophenyl—
phosphate (15.8 mg/ml, activity = 10,000 cpm/ug) and 0.1 m1
of a nucleoside phosphotransferase preparation. The

reaction was stopped by spotting the material on

54

chromatography paper and evaporating to dryness.

2
Use of 3 P-polyphosphate

 

The techniques of Weiss, §£_§E, (95) were used for the
phosphorylation of nucleosides derived from 18 S cauliflower
r—RNA. Nucleosides were evaporated to dryness and were Ir—
reacted with 40 ul of 0.1 M 32P-phosphoric acid (Specific
activity = 0.75 mc/u mole) and approximately 5 mg of phos—

phorus pentoxide for two hours in an 800 C incubator. The

 

reaction was stopped by the addition of 4 ml of water, and the
mixture was placed in a boiling water bath for 45 minutes.

The sample was then diluted 10 ml, centrifuged and the residue
discarded, after which the dissolved products were adsorbed
onto Norit A and washed 20 times to remove traces of radio—
activity due to unreacted 32F. Following elution from Norit
A, the products were co—chromatographed with nucleotide
standards.

Incorporation of 2—14C-Uracil by
Cauliflower Hypocotyls

 

 

Cauliflower seeds were germinated three to four days
at room temperature, prior to excision of hypocotyls. The
hypocotyls were stored in cold, one percent sucrose until
ready for use. Experiments on incorporation into the ethanol-

insoluble fraction were done with seven to ten hypocotyls per

55

tube, whereas experiments involving RNA extraction were done
with 100-200 hypocotyls per tube.
The incubation medium consisted of one percent sucrose,
. -2 .

50 ug/ml of streptomyCin sulfate and 10 M potaSSium phos-
phate, pH 6.8. This medium was also used to make up solutions
. . l4 . .
of actinomyCin D and 2- C-uraCil. Hypocotyls were preincu-

bated for 2 hours, in either the incubation medium or

incubation medium and actinomycin D, prior to the addition of

 

2-14C-uraci1.

Incorporation was stOpped, in studies on ethanol-
soluble incorporation, by the addition of five volumes of
ethanol to each tube, followed by five minutes of boiling.
Hypocotyls were then washed by boiling five successive times
in 5 m1 portions of 75 percent ethanol, containing 0.2 mg/ml
of unlabeled uracil. Following two successive washings in
boiling absolute ethanol, the hypocotyls were dried at 950 C
and their radioactivity determined.

Synthesis of 14C-labeled RNA was stopped by direct
extraction of RNA from the hypocotyls with phenol and solution
B. Hypocotyls were removed from the incubation medium and
placed in a mixture of 15 ml of water-saturated phenol, 12 m1
of solution B, 1 ml of a solution containing 0.2 mg/ml of

unlabeled uracil and 5 mg of unlabeled cauliflower cytoplasmic

56

r-RNA. The mixture was homogenized in a Tenbroeck all—glass
homogenizer, and the r—RNA was extracted and purified as

described previously.

 

RESULTS

RNA Isolation Procedures

 

Cytoplasmic Ribosomal RNA

 

Cytoplasmic r—RNA was isolated from the microsomal
fraction obtained by differential centrifugation of homo—

genates of cabbage, cauliflower and mushrooms. The microsomal

 

fraction was extracted with phenol and solution A. Because

it was felt that contamination with r-RNA from cellular
organelles might give misleading results, and because large
quantities of r-RNA (300-500 mg) were needed for detection

of terminal residues by means of chromatography and Spectro-
photometric analysis, a method was needed which would provide
relatively high yields of cytoplasmic r-RNA. This was achieved
through repeated reuse of the homogenizing medium (tris:sucrose,
pH 7.8) for the homogenization of plant tissue. That no
noticeable degradation of the r—RNA or preferential loss of

the large subunit resulted may be accounted for by the
relatively high concentration of magnesium ion (0.01 M) in

the homogenizing medium. This method was found unsuitable

57

58

for the preparation of wheat germ r—RNA, because the sucrose
homogenizing medium became so viscous that the appropriate
subcellular fractions failed to sediment out at either the
15,000 g_or 105,000 g_steps. This high viscosity was appar—
ently due to the presence of dissolved polysaccharide and DNA.
Attempts to isolate r-RNA from giant ragweed (Ambrosia
trifida, L), goldenrod (Solidago gp,) and burdock (Arctium I
EEEEEJ [Hill], Bernh.) were abandoned, because a black

material, associated with the first ethanol precipitate,

 

could not be separated from the RNA.

In a typical preparation, three to four kg of cauli—
flower yielded 250 mg of r—RNA. However yields varied greatly
between batches of cauliflower. Yields of r-RNA were lower
from cabbage and higher from mushrooms, than those obtained
from cauliflower. Good resolution of r-RNA subunits on
sucrose density gradients was achieved with these preparations,
as is illustrated in Figure 3. Yields of s—RNA were found to

be 20 mg per Six to eight kg of plant tissue.

Total Ribosomal RNA

 

Subunits of wheat germ r—RNA isolated by direct extrac-
tion with phenol and solution A were not resolved on sucrose
density gradient as well as the r—RNA's extracted from the

microsomal fractions, as described above. In preparation of

59

this RNA, difficulty was experienced in sedimenting the
insoluble material, following each of the first two homo—
genizations in 3 M sodium acetate, due to the high viscosity
of the homogenate. Wheat germ RNA preparations were found

to be contaminated with DNA, which was eliminated only after

"'I

repeated homogenization in 3 M sodium acetate. Pollard (68),
however, experienced no such difficulties, and achieved good

resolution of subunits upon sucrose density gradient centri-

 

fugation, with r-RNA prepared from cauliflower by this method.

None of the problems of viscosity or DNA contamina-
tion encountered with the previous system were found when
wheat germ r-RNA was isolated by direct extraction with phenol
and solution B. This material gave less satisfactory
resolution with sucrose density gradient centrifugation
(Figure 7B), however, and the profile was skewed toward the
heavy side of the 28 S subunit.

Isolation of wheat germ r-RNA by direct extraction
with phenol and phosphate buffer, pH 7.0, gave a product
which was free of DNA and whose subunits could be resolved
well upon sucrose density gradient centrifugation, as is

illustrated in Figure 10.

60

The 5'-Linked Termini of Cytoplasmic RNA

 

Ribosomal RNA

 

l. Unfractionated r-RNA
Cytoplasmic r—RNA was prepared from cauliflower,
cabbage and mushrooms, by means of differential centrifuga-
tion, followed by extraction of microsomal pellets with
phenol and solution A. A sedimentation profile of caulflower

r—RNA is shown in Figure 3. This figure shows the usual

 

r-RNA subunits, having a distribution of 67 percent 28 S ei_
and 33 percent 18 S, as calculated from areas under the
peaks; as well as an absence of material in the S—RNA area
(4-5 S). Further evidence of the absence of S—RNA will be
presented in the section dealing with 5'—linked termini of
s—RNA. Similar profiles were obtained with r-RNA'S from
cabbage and mushrooms.

The nucleoside fraction isolated after hydrolysis of
550 mg of unfractionated cauliflower r-RNA was chromato—
graphed in system, B, and was found to contain 0.33 n moles
of adenosine and 0.19 u moles of uridine. In four repetitions
of this experiment, adenosine was consistently found to
predominate over uridine. No cytidine or guanosine was
detected in any of these preparations, although in some

experiments spots were detected adjacent to the quanosine

Absorbance, 260 mu

Figure 3:

61

28 8

F
'T’

18 8

s:
‘f’

p
'7‘

 

 

 

 

 

2'0 3'0 4'0 5'0
Fraction number

Sedimentation profile of a typical preparation
of cauliflower cytoplasmic r—RNA. Hatched
areas represent the portions of the gradient
saved for studies on the termini of the
individual subunits. RNA (24 OD units) was
placed on a linear sucrose gradient (4—20
Percent, in saline citrate buffer) and was
centrifuged for 16.5 hours.

 

62

standard on Chromatograms developed in system B. Absorption
spectra of these spots, however, differed from those of
guanosine, and upon rechromatography in acidic solvents,

they were resolved into several spots, with mobilities differ-
ing from quanosine.

When mixtures of 50 to 100 ug each of authentic
nucleosides were subjected to these procedures, recoveries
of from 50 to 90 percent were realized, no preferential
losses being noted.

In three experiments on cabbage r-RNA only adenosine
and uridine were detected in the nucleoside fractions. How-
ever, these results differed from the results obtained with
cauliflower r—RNA, in that uridine was more abundant than
adenosine. In a typical experiment, hydrolysis of 455 mg
of 4-RNA gave 0.091 n moles of adenosine and 0.318 n moles
of uridine. Similarily, 464 mg of mushroom r-RNA yielded
0.308 u moles of adenosine and 0.691 n moles of uridine.
Again, no cytidine or quanosine was detected.

The yield of nucleoside did not exceed the theoret-
ical yield, based on equal molar ratios of 18 S and 28 S
RNA, in any of the experiments on unfractionated cauliflower
or cabbage cytoplasmic r—RNA, but was twice the expected

yield with mushroom cytoplasmic r—RNA. The reason for

 

63

higher than expected yields of nucleosides from mushroom
r-RNA is not known, but may have been due to ribonuclease
activity, although no such enzymatic hydrolysis was evident
in sedimentation profiles of this RNA. Data comparing
expected and actual yields of nucleosides are presented in

Table 12.

lll‘t .

Results of typical experiments with unfractionated

 

cytoplasmic r-RNA are summarized in Table 8. Values are

 

expressed as percent of total nucleoside detected.

Table 8. Nucleosides resulting from the hydrolysis of
unfractionated, cytoplasmic r—RNA in typical
experiments.

 

Mole Percent of Nucleosides
Source of

 

 

RNA A G C U
Cauliflower 63 n.d. n.d. 37
Cabbage 22 n.d. n.d. 78
Mushroom 31 n.d. n.d. 69

 

n.d. not detected

Cauliflower r—RNA subunits, represented by the
hatched areas of Figure 3, were collected in attempts to
determine if any preferential termination with either

adenosine or uridine could be found on individual subunits.

64

Sedimentation profiles obtained by recycling of the individ—
ual subunits are presented in Figure 4.
2. 28 S RNA
Eighty-nine mg of 28 S cauliflower RNA were collected
from sucrose gradients by ethanol precipitation, over a
period of one month. The nucleoside fraction obtained from

the alkaline hydrolysate of this RNA was chromatographed in

system B, and the individual nucleoside areas located by

’ | ‘- .V.UM'..'Q“I'. ‘

 

means of an ultraviolet light. Spectrophotometric analysis
showed that the sample contained 0.037 n moles of adenosine
and 0.064 n moles of uridine. No other nucleosides were
detected.
3. 18 S RNA

The nucleosides isolated from an alkaline hydrolsate
of 29 mg of 18 S cauliflower RNA were phosphorylated with
32P-polyphosphate, in order to facilitate detection of
pyrimidine nucleosides. As previously mentioned, this method
is unsatisfactory for phosphorylation of purine nucleosides.
Preliminary experiments indicated that 0.5 ug samples of
pyrimidine nucleosides could be phosphorylated by this tech—
nique and could be easily detected. Products included 2'(3')—
and 5'-monophosphates and 2'(3'), 5'-diphOSphateS. Half of

this sample was chromatographed successively in systems B and

Figure 4:

65'

 

 

 

288
2 0.3-
c:
a:
on
o“ 0.2-
e:
c:
co
4:
h
s; 0.1-
4:
I:
I I I r l
10 20 30 40 50
Fraction number
Sedimentation profiles of individual cauliflower

r—RNA subunits. Subunits from hatched areas of
Figure 3 were centrifuged simultaneously for

15 hours in separate linear sucrose gradients,
using saline acetate buffer. Four 0D units of
18 S r-RNA and Six OD units of 28 S r-RNA were

used. Fractions were diluted with one m1 of
saline acetate buffer.

‘ r’J-fll

 

66

E, and was found to contain 2693 cpm in uridine—5'—monophos-
phate and 6175 cpm in uridine -2'(3')-monophosphate. The
distribution of these products upon rechromatography in
system A is shown in Figure 5, and the distribution of the
2'(3')-monophosphate upon electrophoresis in 0.05 M citrate
buffer, pH 5, is illustrated in Figure 6. The other half

of the phosphorylated sample, following successive chromato-
graphy with cytidine monophosphate standards in systems B,

C and A gave only 15 cpm above background.

 

Ir

Pollard (68) reacted the periodate-oxidized nucleo—
side fraction of cauliflower 18 S RNA with tritiated sodium
borohydride, and was able to isolate both adenine and
uridine trialcohols.

These results constitute good evidence that nolecules
of both the 18 S and 28 S subunits of cauliflower r—RNA may

terminate as either adenosine or uridine.

Soluble RNA

 

The nucleoside fraction derived from alkaline
hydrolysis of 20 mg of cauliflower S—RNA was found following
chromatography with system B, to contain 0.021 n moles of
adenosine, 0.034 u moles of guanosine, 0.076 n moles of
cytidine and 0.045 u moles of uridine. These results

demonstrate that all four nucleosides may be produced by

counts x 10'2

Figure 5:

67

 

CD 33 J

U-S'-P U-2'(3')-P

 

 

7'1
'I
II
Jl
IL

I

1F

 

 

7 r
I .I
I _I
|
I

.I-IJ
lad
H

 

I
l I l I I

10 20 30 40 50

Centimeters from origin

 

Radioactivity of chromatogram, from system A,
of uridine monophosphates resulting from
phOSphorylation of cauliflower 18 S r—RNA
nucleoside fraction with 32P—polyphosphate.
The isomers were chromatographed successively
on Whatman No. 1 paper in systems B and E,
and then chromatographed in adjacent lanes in
system A. The drawing at the top represents
the ultraviolet-absorbing areas of unlabeled
standards with which the samples were
co-chromatographed. Counts per 10 minutes
are given for the 5' isomer (broken line) and
counts per minute for the mixed 2'(3') isomers
(solid line).

Figure 6:

cpm X 10'2

68

 

 

 

 

 

'"l r1 I. I
i 15 25

Centimeters from origin

 

Radioactivity of paper electrophoretogram of
uridine—2'(3')-monophosphate, resulting from
phosphorylation of cauliflower 18 S r-RNA
nucleoside fraction with 32P-polyphosphate, and
eluted from a paper chromatogram developed in
system A. Electrophoresis was done for 3 hours
at 300 V, in a Beckman RD—2 electrophoresis
unit, using 0.05 M citrate buffer, pH 5.0, and
Whatman 3 mM electrophoresis paper. The hatched
area at the tOp represents the ultraviolet-
absorbing area of unlabeled standard.

 

69

alkaline hydrolysis, thereby indicating that guanosine and
cytidine were not preferentially lost in experiments on
r-RNA. Although the finding of all four nucleosides in
alkaline hydrolysates of s—RNA is not unique, typical re-
sults of others usually show that adenosine and cytidine

are the only nucleoside products of alkaline hydrolysis of
s-RNA (see Table 2). The results we obtained upon hydrolysis
of 20 mg of cabbage s—RNA, showed that 0.049 u moles of

adenosine and 0.055 u moles of cytidine were released. No

 

quanosine or uridine were detected. These results, expressed
as percent of total nucleoside fraction, are summarized in
Table 9.

Table 9. Nucleosides resulting from the hydrolysis of
S-RNA.

 

Mole Percent of Nucleosides
Source of

 

 

5 'RNA A G c U
Cauliflower 12 19 43 26
Cabbage 47 n.d. 53 n.d.

 

n.d. not detected

1

Yields of nucleosides from s—RNA were only 13 to 22
percent of those which would be expected from molecules

With molecular weights of 2.5 X 104, indicating that s—RNA

7O

preparations may have been contaminated with larger RNA
species. Purity of s-RNA preparations was not varified by
sucrose density gradient centrifugation. Data comparing
expected and actual yields of nucleosides are presented in
Table 12.

The 5'-Linked Termini of Total
Wheat Germ Ribosomal RNA

 

 

The results of Lane and co—workers (45, 46, 80) and
of Lee and Gilham (49), who found all four nucleosides in
alkaline hydrolysates of wheat germ r—RNA is different from
our finding of only adenosine and uridine in hydrolysates of
cauliflower, cabbage and mushroom r-RNA. Attempts were made
to explain these differences. The experimental approach
followed was to identify the nucleosides resulting from
alkaline hydrolysis of r—RNA (a) isolated and hydrolyzed
under the conditions used in our laboratory and (b) under
the conditions used by Lane and co—workers (80). In
addition, Special attention was given to the sedimentation
profiles of the RNA since large quantities of RNA had to be
employed. The contribution of nucleoside from 1 mg of
S-RNA is approximately equivalent to that from 20 mg of
18 S RNA or 40 mg of 28 S RNA. Contaminating s—RNA could
very easily escape notice unless sufficient amounts of RNA

were applied on the gradients.

 

3'51..-
I

71

A sample of wheat germ r-RNA, which will be referred
to as preparation I, was obtained by direct extraction of
wheat germ with phenol and solution A, and was washed six
times by homogenization in 3 M sodium acetate. A sedimenta-
tion profile of this r—RNA is presented in Figure 7A. No
DNA was detected in this preparation. Only adenosine
(0.240 u moles) and uridine (0.159 u moles) were detected

in the hydrolysate of 350 mg of this preparation, following

 

n.—

chromatography in system B.

The sedimentation profile of a second RNA preparation
(preparation II) is illustrated in Figure 7B. This RNA was
isolated by direct extraction with phenol and solution B,
was homogenized two times with 3 M sodium acetate, and like
preparation I, was found to be free of DNA. The nucleoside
fraction, following alkaline hydrolysis of 600 mg of RNA,
was chromatographed in system B, and was found to contain
0.425 n moles of adenosine, 0.039 u moles of cytidine and
0.400 u moles of uridine. No guanosine was detected.
Rechromatography of the adenosine and uridine products in
system G showed that they migrated with authentic standards
in that system as well.

Ten percent of the nucleoside fraction from prepa-

ration II was incubated with purified carrot leaf—nucleoside

72

(A)
1D-
05-

024

 

260 mu

(8)

Absorbance,

 

 

 

1'0 2'0 30 4'0 5'0
Fraction number

Figure 7: (A) Sedimentation profile of 48 OD units of ’
wheat germ r—RNA isolated by direct extraction
of wheat germ with phenol and solution A. The
preparation was washed six times with 3 M
sodium acetate, pH 6.0. Centrifugation was done
for 18 hours, using saline citrate buffer.

(B) Sedimentation profile of 42 OD units of
wheat germ r-RNA isolated by direct extraction
of wheat germ with phenol and solution B. The
preparation was washed two times with 3 M
sodium acetate, pH 6.0. Centrifugation was done
for 16.5 hours, using saline citrate buffer.

m1

 

 

73

phosphotransferase and 32P-p-nitrophenyl phosphate, Spotted
on chromatography paper, and the chromatogram developed with
system F for 37 hours. The nucleotides remained near the
origin, while inorganic phosphate and p-nitrophenyl phosphate
migrated more rapidly and were thus eliminated. Elution of
the nucleotide area, followed by chromatography in system B,
gave the distribution of radioactivity shown in Figure 8.
Subsequent electrophoresis of each of the nucleotide samples
individually in the pH 3.5, acetic acid:pyridine system,
showed the distribution of radioactivity presented in Table 10.
Table 10. Radioactivityzgfpnucleotides produced by the

reaction of - -nitropheny1 phosphate with
the nucleoside fraction from wheat germ r-RNA.

 

 

 

I‘
Nucleotide
Activity
AMP GMP CMP UMP
cpm 74 7 161 554
Percent 9 1 20 70

 

Radioactivity in the guanosine monOphosphate area of the
chromatogram shown in Figure 8 migrated with cytidine
monophOSphate in the electrOphoretic separation, and was
probably due to overlap of the two spots on the chromato—

gram. These results provide qualitative support for the

 

74

 

 

Figure 8:

@IDGDCD J

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

4 3 '12 16

Inches from origin

Radioactivity on chromatogram, developed in
system B, of nucleoside monophOSphates
resulting from phosphorylation of wheat germ
r—RNA nucleoside fraction with 32P-p—nitrophenyl
phosphate and nucleoside phosphotransferase.
The nucleotides were 3chromatographed first in
system F, to remove 2P and P—p—nitrOphenyl
phosphate. The drawing at the top represents
the ultraviolet—absorbing areas of unlabeled
standards with which the sample was
co-chromatographed.

 

75

identity of nucleosides reported for this RNA preparation,

as the monophosphates of adenosine, cytidine and uridine
formed the major portion of the product. The relative
amounts of radioactivity, however, are probably misleading,
as this criterion showed cytidine to be twice as abundant

as adenosine, whereas spectral evidence showed that adenosine
was more than ten times as abundant as cytidine.

Preparation III of wheat germ r-RNA (Figure 9) was

 

isolated by direct extraction with phenol and solution A,
followed by two homogenizations in 3 M sodium acetate, in-
stead of six homogenizations as done with preparation I.
The sample was shown to contain four percent DNA, as indi-
cated by the diphenylamine test. The hump on the light side
of the 18 S RNA peak, between fractions 31 and 40, was
believed to be enzymatically degraded r—RNA. Thus it is
clear from a comparison of Figure 9 with Figure 7A that
extensive treatment with 3 M sodium acetate is required for
obtaining a good preparation of wheat germ r—RNA, when
Solution A has been employed for extraction of RNA.

Nucleosides produced by hydrolyzing 675 mg of RNA
from preparation III were found, following chromatography
in system B, to consist of 0.381 n moles of adenosine,

0.220 n moles of guanosine, 0.336 n moles of cytidine and

260 mu

Abscrbanca

Figure 9:

O
05
l

O
b
l

P
N
1

76

 

 

 

l l I I

10 20 30 40 5'0
Fraction Iunnber

Sedimentation profile of 48 0D units of wheat
germ r—RNA isolated by direct extraction of
wheat germ with phenol and solution A. The
preparation was washed two times with 3 M
sodium acetate, pH 6.0. The gradient was
made up in saline citrate buffer, and was
centrifuged for 19 hours.

77

0.407 n moles of uridine.

The occurrence of all four nucleosides in the hydroly—
sate of preparation III, which was shown to be contaminated
with DNA and low molecular weight RNA, was in agreement with
the results of Lane and co-workers (45, 46, 80) and Lee and
Gilham (49), (see Table 7). This fact indicated the possi—
bility that wheat germ r-RNA preparations used by these
workers may have been contaminated in much the same manner
as preparation III. Another wheat germ r—RNA preparation
(preparation IV), was isolated, therefore, according to the
procedures of Singh and Lane (80). This involved direct
extraction of RNA with phenol and phosphate buffer pH 7.0,
and precipitation of the r—RNA from 1.0 M sodium chloride.
No DNA was detected in this preparation, and good resolution
of the r—RNA subunits was achieved with sucrose density
gradient centrifugation (Figure 10). The RNA was also
hydrolyzed according to Singh and Lane (80), using 1.0 M
sodium hydroxide at room temperature, rather than 0.3 M
potassium hydroxide at 370 C, as was done for our other
preparations. Another difference in this procedure was
that the hydrolysate was neutralized with 3 N hydrochloric
acid, rather than with Dowex 50-H+. Purification and chro-

matography of nucleosides were the same as described for

 

260 mu

Absorbance,

Figure 10:

78

 

 

10 20 30 40 50
Fraction number

Sedimentation profile of wheat germ r—RNA
isolated by the methods of Singh and Lane
(80). The gradient was made up in saline

citrate buffer. Forty—two 0D units of r-RNA
were centrifuged for 16 hours.

 

79

other preparations. Nucleosides from 300 mg of RNA were
shown to consist of 0.054 u moles of adenosine, 0.093 u
moles of guanosine, 0.165 n moles of cytidine and 0.216 n
moles of uridine, thereby supporting reports of all four
nucleosides in alkaline hydrolysates of wheat germ r-RNA
when these methods were used.

Results on all four wheat germ r—RNA preparations

 

are summarized and compared with those of Lane and co—workers

 

“IL

(45, 46, 80), and Lee and Gilham (49), in Table 11. ''''''

Yields of nucleosides varied between different wheat
germ r-RNA preparations, and may have influenced the nature
of the product which was obtained. The yield of nucleosides
from preparation I was approximately that which would be
expected from nolecules with an average weight of 106, and
only adenosine and uridine were detected. Preparation II
yeidled 44 percent more nucleosides than expected, and in
this case, in addition to adenosine and uridine, cytidine
was detected. Yields of nucleosides from preparations III
and IV were respectively 99 and 76 percent greater than

expected, and in both cases, all four nucleosides were

detected. These data are summarized in Table 12.

 

.4.‘;...

ill I l

80

Table 11. Summary of data on the 5'-linked termini of
wheat germ r—RNA.

 

Mole Percent of Nucleoside

 

 

Preparation

A G C U

I 60 n.d. n.d 40

II 49 n.d. 5 46

III 28 17 25 30

IV 10 18 31 41

a
A 20 31 26 23
Bb 20 24 31 25

 

 

n.d. not detected

average values from data of Lane and co—
workers (45, 46, 80) presented in Table 7.

data of Lee and Gilham (49).

81

 

 

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o .o .o .4 mm 4zmIm unsoHMHHsmo
o .o .S .4 men m mm + m ma >H :oHumumamnm
w .0 .D .4 mma m mm + m ma HHH Goapmummmum
o .o .4 444 m mm + m ma HH coaumummoum
o .4 444 m mm + m 44 H coflumumdmum

Enom among

2 .4 464 m mm HoBOHMHHSmo

S .4 64m m mm + m ma eoourmaz

D .4 om m mm + m ma ommnnmo

D .4 mm m mm + m ma HoBOAMAHSmO

oouoouon Aoopoomxm mo unmouomv

mooflmooaodz mooflmooaooz mo oaoflw 42% mo oNHm 42m mo mousom

 

ou one on mmE can

Hmommm mzmIm Eoum oocflmuno moaoflm 30A
maumoum ouo3 moaoflm CoSB nouoouoo Oman ouo3 oCHmocmDm pom mafioflpho
mmmHoSS .oouoomxm mm3 QUHEB umnu maoumEonummm mmB oaoflw onu

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.ooumHOmH mooflmooaooc osu mo ucoocomoocfl on CD
.oouooumxo omonu mo mmooxo Ca

.mnmmnu

was» SA moflodpm mcofiumnmmoum «2% mo moummhaouomn ocflamxam Eoum
oosHmqu mooflmooaosc mo xuflucmofl ocm Aoouoomxo mo unmouomv oaoflw .ma manna

82

Incorporation of 2-14C-Uracil by
Cauliflower hypocotyls

 

 

The preferential termination of plant r-RNA'S with
adenosine and uridine, and the existence of enzymes which
add 5'—linked terminal nucleotides to RNA'S other than s-RNA
(10, 40, 41, 65), suggest that terminal addition, subsequent
to DNA—dependent RNA synthesis, might be involved in the
preferential termination of plant r—RNA. Because all of

the enzymes mentioned above were found to add uridine mono-

 

phosphate, and because the proportion of terminal uridine on
E, gglé_r-RNA has been shown to vary with environmental
conditions, experiments were done to determine if uridine

is added to plant r—RNA chains subsequent to DNA-dependent
RNA synthesis. The experimental approach followed was to
isolate the nucleoside and nucleotide fractions from
alkaline hydrolysates of caulfilower hypocotyl r—RNA labeled
with 2-14C—uraci1, in both the presence and absence of
actinomycin D. It was conceived that if 5'-1inked—terminal
uridine is added to the r-RNA chains after DNA-dependent
chain synthesis, a higher ratio of 14C-nucleoside:l4C-nucleo—
tide should be obtained from RNA labeled in the presence of

actinomycin D than from that labeled in its absence.

83

Effect of Actinomycin D Concentration

 

Prior to studies on the ratio of terminal to non-
terminal uridine in cauliflower hypocotyl r-RNA, in the
presence and absence of actinomycin D; it was necessary to
determine suitable concentrations of actinomycin D and
optimum incorporation times. A concentration of actinomycin
D which would give approximately 90 percent inhibition of
incorporation was desired, as it was reasoned that at this

level of inhibition it would be possible to detect prefer—

 

ential addition of 5'-1inked terminal uridine to existing
RNA chains.

Ten hypocotyls were weighed and placed in each tube
where they were preincubated for two hours in incubation
media with actinomycin D concentrations of 0 to 60 ug per ml.
Each concentration was run in triplicate. Incorporation was
started by the addition of 0.2 uc of ZuéUT-uracil, was
allowed to proceed for Six hours and was stopped by boiling
the reaction mixture in ethanol. Following extraction of
alcohol—soluble material, the radioactivity was measured.
The results of this experiment, which was the third in a
series of experiments, are presented in Figure 11. An
actinomycin D concentration of 60 ug per m1 gave 90 percent

inhibition of 2-14C-uracil incorporation. In the first of

Figure 11:

84

NOW

160-

lncorporation, cpm/mg fresh weight

 

 

l I

20 40 0b
Actinomycin D, Jig/ml

Incorporation of 2—14C-uraci1 into alcohol-
insoluble material by cauliflower hypocotyls
with increasing concentrations of actinomycin
D. Ten hypocotyls were incubated in each
tube for 6 hours at room temperature following ‘
the addition of 2—14C—uracil. Each point
represents the mean of three replications.

 

85

these experiments no preincubation period was used, and much
less inhibition resulted at the same concentrations, indi—
cating that penetration of actinomycin D into the hypocotyl
cells occurs more slowly than penetration of uracil. The
second of these experiments involved the use of actinomycin

D concentrations ranging from 0 to 50 ug per ml, and

although results were similar to those reported for the third
experiment, the desired level of inhibition (90 percent) was
not achieved.

An interesting aside to these experiments, which was
not pursued in greater detail but which was consistently
observed, was a stimulation of uracil incorporation by acti-
nomycin D at concentrations of 0.2 to S'ug per m1 (Figure
11) .

The validity of the assumption that inhibition of
incorporation of uracil into ethanol—insoluble material is
equivalent to inhibition of RNA synthesis was established
in another set of experiments. Preincubated hypocotyls were
incubated for eight hours in the presence and absence of 60
ug per ml of actinomycin D, which had been Shown (Figure 11)
to give approximately 90 percent inhibition of alcohol—
insoluble uracil incorporation. RNA was isolated by

extraction with phenol and solution B. Results of these

 

86

experiments are presented in Table 13. Thus it is estab-
lished that incorporation into the ethanol—insoluble fraction
corresponds to incorporation into RNA.

Table 13. Inhibition of incorporation of 2—14C—uracil into
cauliflower hypocotyl r-RNA by 60 ug/ml of

 

 

 

 

actinomycin D. I-wm
Experiment CPM per 100 Hypocotyls Percent
Number Control + Actinomycin D Inhlbltlon
II 174,000 22,300 87

 

Time Course of 2-14C-uracil Incorporation

 

An experiment was done on the time course of uracil

incorporation to determine an appropriate incubation time

. . . . . l4 . .
for experiments involVing incorporation of 2- C—uraCil into
r—RNA. An incubation time was wanted which would insure

. . 14 . . .
adequate incorporation of 2— C—uraCil into r-RNA of acti—
nomycin D—inhibited hypocotyls to enable detection of
terminal nucleosides, but which would not be so long as to
lead to a breakdown of inhibition. For these experiments,
hypocotyls were treated as in the previous experiments,
except that only seven hypocotyls per tube were used, and

only one concentration of actinomycin D (60 ug/ml) was

87

compared with the control. Results are presented in Figure
12. Based on these results, an incubation time of eight
hours was selected for experiments involving incorporation
of 2-14C—uraci1 into RNA, as it was felt that adequate
incorporation could be obtained in that length of time, and
inhibition approximated the 90 percent level which was

desired.

The Ratio of Terminal to Non—terminal Uridine in Cauliflower
Hypocotyl r-RNA

 

 

 

Incorporation of uracil into RNA was accomplished
using an eight hour incubation period, with 100 hypocotyls
in 3 ml of incubation medium for controls, and 200 hypocotyls
in 6 ml of incubation medium for actinomycin D treatments.
Control hypocotyls were supplied with 50 uc of 2-14C—uracil,
whereas actinomycin D treated ones were supplied with 100 uc.

RNA extracted from the hypocotyls was applied to
sucrose density gradients; the resulting sedimentation pro-
files are Shown in Figures 13 and 14. Although the added
carrier RNA appears to have undergone a partial dissociation
of the 28 S subunit during the extraction procedure, the
l4C-labeled RNA remained intact and showed good resolution

into subunits on the sucrose gradient. In subsequent exper—

iments carrier RNA was added after homogenization with

ZOOw

Incorporation, cpm/mg fresh weight

Figure 12:

160-

88

120- E
80- g.
E.

40-

 

 

 

 

. I 0
4 8.12 10

0 I I

Hours of incubation

Incorporation of 2-14C-uracil, into alcohol-
insoluble material, by cauliflower hypocotyls
with increasing time, in the presence and
absence of 60 ug/ml of actinomycin D. Seven
hypocotyls were incubated in each tube at
room temperature. Each point represents the
mean of three replications.

*——————. Incorporation, cpm/mg fresh weight
in control hypocotyls

+
F

. Incorporation, cpm/mg fresh weight
in actinomycin D treated hypocotyls

@p.—-—-49 Percent inhibition

 

Figure 13:

89

opm X 10'2

 

Absorbance, 260 mu

 

 

 

 

1'0 2'0 3'0
Fraction number

Sedimentation profiles of r—RNA isolated from
cauliflower hypocotyls incubated for 8 hours

in 2—14C-uracil, and of unlabeled carrier r—RNA.
The gradient, prepared in saline citrate buffer,
was centrifuged for 16 hours. The fractions
between the arrows at the top were collected

for alkaline hydrolysis of the RNA.

u——————o Radioactivity of cauliflower
hypocotyl r-RNA

—-—-—-—o Absorbance at 260 mu of 15 OD units
of added carrier, cauliflower r—RNA

 

9O

 

=

E 03- ‘—
O

'0

N

w. 0.2-

O

=

G

a :L-w
:3 0.l~

(n .

a

a:

 

 

 

 

1’0 2‘0 3'0 40
Fraction number

Figure 14: Sedimentation profiles of r—RNA isolated from
cauliflower hypocotyls incubated for 8 hours in
2-14C-uraci1 and 60 ug/ml of actinomycin D, and
of unlabeled carrier r-RNA. The gradient, pre—
pared in saline citrate buffer, was centrifuged
for 16 hours. The fractions between the arrows
at the top were collected for alkaline hydrolysis
of the RNA. '

~——————o Radioactivity of cauliflower
hypocotyl r—RNA

-—-—-—o Absorbance at 260 mu of 15 0D units
of added carrier, cauliflower r—RNA

91

solution B and phenol, and good resolution was achieved on
gradients, as measured by absorbance at 260 mu. The pres—
ence of radioactive material in the s-RNA region of Figure

14, not eliminated by homogenization in 3 M sodium acetate,
made it necessary that all labeled RNA used in the remainder
of these studies be subjected first to sucrose density
gradient centrifugation, so that only the r—RNA was collected.
Inclusion of the low molecular weight fraction could have led
to disproportionately high values for terminal nucleoside in
alkaline hydrolysates.

RNA was collected from sucrose gradients, hydrolyzed,
and the nucleoside fraction collected as previously des—
cribed; after which the nucleotides were eluted from the
column with 0.1 N hydrochloric acid. These fractions were
evaporated to dryness under reduced pressure and redissolved
in 5 m1 of 0.05 N hydrochloric acid. One ml aliquots of
each fraction were pipetted into each of three counting vials,
evaporated to dryness, and their radioactivity determined.
Results of three repetitions of this experiment are presented
in Tables 14 and 15.

The results, with a 50 percent overlap of standard
deviations, indicate that no difference exists in the ratio

of terminal to non-terminal uridine in cauliflower hypocotyl

 

92

r . m .r. u 4.7.8.— D..1.II

 

 

 

 

 

hmvm ma humam mm m
moHo NH Hoomm hm N
Hmoao om mmmao mHH H
oofluooaosz ooflmooaosz oofluooaooz moflmooaooz
HoQEDZ
ucofifluomxm

o cfio>socfluo4 zmo

 

Homoeoo Smo

 

.Q aflomfioaflpom mo

HE\md ow mo oocomnm 0cm mocomonm on“ SH ooaonma «zqu mo moummmaouohn
oaflamxam Eoum ooumaomfl moofluooaooc pom mmofimooaosc mo >ue>apomoflomm .va manna

93

 

 

 

Hm H_mmo\H mm + mNm\H coflumfi>on Unmocmpm
pom ommuo>4
mmm\H mom\a m
mom\a mma\n m
oma\H omm\H H
oopmoue Q CAUAEOCHDod quEsz
SH oflumm Houucoo CH oaumm ucoefluomxm

 

 

 

.oumnmmozmocoEIA.mv.mIocHUHHD Ho

oceoflud MC Show map SH ma ooDMHOmH >DH>HuomoHomn Dam pmnp COHDQESmmm

ofip co comma mum muaomom

.Q aflohfiocfluom mo HE\md 00 mo monomnm pom

oocomoum onu CA HHomuoluoHIN LDHB ooumndocfl mawpooomhn HoSOHMAHDmo
Eonm oopmHOmH «zmIH SH ocfloflus HmcHEuoDrsoc ou Hmcflfiuop mo moflumm .mH manna

94

r-RNA labeled in the presence or absence of actinomycin D.
Surprisingly, the ratio of approximately 1/515 was much
higher than the ratio which was anticipated (1/1500 to

1/1600). A suitable explanation of this observation is not

known.

 

DISCUSSION

RNA Isolation Procedures

A potentially important contribution of these studies
is the development of techniques for obtaining large quanti-
‘ties of cytoplasmic r—RNA from plants. This may be
especially Significant if studies on the base sequence of
r-RNA are undertaken. Since it has become apparent that
organelles possess r—RNA, and since it has been possible to
demonstrate that chloroplast ribosomes have distinctly dif-
ferent S-values from cytoplasmic ribosomes, it might be
desirable to utilize cytoplasmic r-RNA as a basis for studies
on base sequences, in order to minimize the pssibility of

obtaining heterogeneous r-RNA, for example, r—RNA from

different organelles.

By improving techniques for the separation of RNA's.
it is possible that systems may be developed which will
separate r-RNA'S varying by very small differences in S
values, or differing in some other manner. If so, then it

may be possible to investigate the differences of r-RNA in

95

—— 3.1“}11

 

96

organelles, or even the structural heterogeneity of cytoplas—
mic r—RNA subunits.

In view of such possible future needs for large
quantities of r-RNA, it might be well to briefly asses the
methods used in this thesis.

Extraction of RNA from microsomal pellets from cauli-
flower, cabbage and mushrooms with phenol and solution A
yielded a product which showed good resolution on sucrose
density gradients. This method is particularily applicable
to tissues such as fleshy or leafy plant parts, where low
yields of RNA can be expected, and large volumes of tissue
must be used. Inadequacy of the method for tissues high in
nucleic acids and protein is demonstrated by the difficulties
encountered in attempts to isolate r-RNA from wheat germ.

Since DNA and s—RNA were also extracted in attempts
to isolate r—RNA from wheat germ with phenol and solution A,
it is probably the least satisfactory of the homogenizing
media used for the extraction of r-RNA from this tissue.

Use of either solution B or pH 7.0 phosphate buffer gave
products free of DNA or s-RNA contamination. The method of
Singh and Lane (80), using phosphate buffer and phenol, gave
the best results, based upon resolution of the subunits by

sucrose density gradient centrifugation.

 

97

A difficulty which may be encountered in the isola-
tion of RNA with phenol and solution B is illustrated by the
apparent decrease in the amount of 28 S r—RNA and concomi—
tant appearance of a larger, bimodal peak in the 18 S region
of carrier r-RNA added during the isolation of cauliflower
hypocotyl r—RNA (Figures 13 and 14). Beck, et_al, (3)
demonstrated that dissociation of the larger r-RNA subunit
into two smaller molecules, at alkaline pH (pH 9) and in
the presence of EDTA, as described by Midgley (56, 58), was
due to the action of ribonuclease. Since solution B has a
pH of 9.5, and contains EDTA, a dissociation of the type
described by Midgley (56, 58) appears to be involved; this
resulted in the sedimentation profiles illustrated in
Figures 13 and 14. Apparently sufficient ribonuclease was
present in the cauliflower to cause a partial hydrolysis of
the carrier r-RNA added prior to homogenization with phenol.
The reason that the hypocotyl r—RNA did not undergo this
same dissociation may have been that this r-RNA was shielded
by ribosomal proteins. Solution B was apparently not
directly responsible for the dissociation, because when
carrier r-RNA was added immediately following homogenization,
rather than prior to it, no dissociation was apparent.

These observations on the use of different isolation

ml

 

98

techniques demonstrate that in any attempts to isolate RNA,
the methods used must be checked to determine the quality
of the product obtained, and that one method is not neces—

sarily applicable to all tissues.

 

The 5'-Linked Termini of Cytoplasmic r—RNA -..,
Adenosine and uridine were the only nucleosides
isolated from alkaline hydrolysates of unfractionated cyto—

plasmic r-RNA from cauliflower, cabbage and mushrooms, and

 

from 28 S r—RNA from cauliflower. Uridine was identified as 4
a 5'—linked terminal groups of the 18 S subunit. The
presence of both adenosine and uridine as 5'-linked termini
of both subunits of cauliflower r-RNA has been confirmed by
Pollard gt El- (60) using tritiated trialcohols, formed by
reduction of the dialdehyde oxidation products of nucleosides.
Cauliflower r—RNA isolated by direct extraction with phenol
and solution A was found by Pollard (68) to also contain only
adenosine and uridine as 5'—linked termini, indicating that
this phenomenon is not confined to cytoplasmic r-RNA. The
difference in ratios obtained with r—RNA from cabbage and
cauliflower indicates that the ratios of the termini within
a species is not necessarily constant.

Similar reports, of a predominance of adenosine and

uridine as 5'-linked termini, have been made by others in

99

studies on r-RNA from E, ggli_(43, 53, 59, 60, 62), rabbit
reticulocytes (36) and L cells (47), (see Table 7). Pollard,
‘ggigl. (69) have demonstrated that adenosine and uridine are
the only nucleosides isolated from alkaline hydrolysates of
spinach and parsnip r-RNA. Although these results do not
constitute proof of the universality of this phenomenon,

they do suggest that it is wide-spread. Worthwhile extension
of these findings could be achieved through the use of dif-
ferent techniques to confirm them, and studies on a greater
diversity of organisms.

The results of Midgley and McIlreavy (60) demonstrate
that the ratios of termini may be changed by altering the
nutrient status of E, coli, Another line of investigation,
which might beproductive, would be to determine if organisms
other than E, ggli_exhibit this phenomenon.

The significance of these findings, a predominance
of 5'-1inked terminal adenosine and uridine, is unclear at
this time due to a deficiency of information on the structure
and function of r-RNA. It is possible that the nature of
the termini of r—RNA may be related to the activity of
ribosomes in protein synthesis. This possibility will be

discussed in greater detail later.

 

100

The 5'—Linked Termini of Total Wheat
Germ r-RNA

 

Results of experiments to determine the 5'—linked
termini of wheat germ r—RNA indicate that this RNA terminates
predominantly as adenosine or uridine. Hydrolysis of the
r-RNA with 0.3 M alkali gave adenosine and uridine primarily,
small amounts of cytidine and no quanosine. Qualitative

confirmation of these results was achieved by phosphorylation

 

of the nucleosides with nucleoside phosphotransferase and

32

Et?‘

—P—p-nitrophenyl phosphate. Hydrolysis with 1.0 M alkali,
and neutralization with 3 N hydrochloric acid, rather than
with Dowex 50-H+, resulted in the production of all four
nucleosides, confirming the results of Lane and co—workers
(45, 46, 80) and of Lee and Gilham (49). This conflict of
results may be due to artifacts introduced by the hydrolysis
or neutralization procedures. Sugiyama and Fraenkel—Conrat
(88) have reported that neutralization of alkaline hydroly—
sates with acid can result in dephosphorylation of
nucleotides. DephOSphorylation of this type was probably
responsible for the appearance of all four nucleosides in
the hydrolysate of preparation IV. A reexamination of the
procedures employed and use of other techniques, such as

labeling with 3H-isonicotinic acid hydrazide as described

101
by Hunt (36) are desirable. RNA isolated by the methods of
Singh and Lane (80) would probably be the best to use in such
studies, because of its good resolution on sucrose density
gradients.

Incorporation of 2-14C-Uracil by
Cauliflower Hypocotyls rr——

 

 

Experiments to determine the ratio of terminal to
non-terminal uridine in r—RNA synthesized by control and

actinomycin D—inhibited cauliflower hypocotyls, revealed

 

that there was no difference in this ratio between the two
treatments. These results suggest that termination of these
r-RNA molecules, unlike that of s-RNA, is not due to end
group addition or turn-over. Termination with Specific
nucleosides is probably due directly to genetic activity
(transcription), as suggested by Midgley and McIlreavy (60),
although the possibility also exists that termination is con—
trolled by ribonuclease activity, subsequent to RNA synthesis.
A worthwhile extension of this work would be a
determination of the ratios of terminal to non—terminal

nucleosides, using highly labeled 14C r-RNA.

Future Application of the Results

 

The consistent observation of adenosine and uridine

as 5'-linked termini on all r—RNA's studied to date suggests

102

that some mechanism, common to all of the organisms studied,
may control the termination of these RNA's. One possible
explanation of this phenomenon, discussed by Midgley and
McIlreavy (70), is that these termini occur at natural stop-
ping points in the DNA which codes for r—RNA: that is to say,
as”
at adenosine and thymidine moieties on DNA. Other possible

explanations would be (1) the existence of enzymes which

give preferential cleavage, at adenosine or uridine, of

 

larger RNA molecules, thereby giving rise to r-RNA, or (2) hr
the addition of adenosine and uridine to the 5'—linked

termini of r-RNA. Although the results of experiments with

labeled r-RNA presented in this thesis suggest that the

latter mechanism is not involved, they do not completely

preclude the possibility of its existence.

The possibility that specific r—RNA termini are
related to the activity of ribosomes was mentioned earlier.
Although conclusive proof of such a proposal is not possible
at this time, due to inadequacy both of techniques and of
knowledge concerning the structure and function of r-RNA,
circumstantial evidence for it could be obtained by experi-
ments based on the experiments of Midgley and McIlreavy (60),
which have already been discussed. These experiments would

involve the use of ribosomes from E, coli grown both on

103

glucose and on ”casamino acids," which are known to have
different proportions of 5'—linked terminal adenosine and
uridine. These ribosomes could be used in a cell-free amino
acid—incorporating system to determine their relative rates
of amino acid incorporation. Higher rates of amino acid
incorporation for either the “high adenosine—" or "high
uridine-terminal" r—RNA would provide circumstantial evidence
of the essentiality of that terminus. Findings of equal

rates of incorporation with both types of ribosomes would

 

provide no evidence either for or against the proposal.
Conclusive proof of the essentiality of particular
termini, such as exists for s-RNA and TMV-RNA (see Litera-
ture Review), will probably not be obtained until techniques
are developed by which biologically active ribosomes can be
disassembled and reassembled. In this way, the RNA mole—
cules could be removed from ribosomes, their termini removed,
such as by periodate oxidation and amine catalyzed cleavage,
followed by hydrolysis with E. coli alkaline hydrolysis, and

the RNA then put back into ribosomes.

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