swmss OF sscumcgg c_o_u RNA-DNA
HYBRID AND or METHYLMLON 0: RNA
IN 1-4 ammo CEuS

Thesis for the Degree of Ph. D.
MICHIGAN STATE 0141va

Robert Lee Armstrong
1966

THESIB

This is to certify that the

thesis entitled

STUDIES OF ESCHERICHIA COLI RNA-DNA HYBRID
AND OF METHYLATION 0F RNA IN
T-h INFECTED CELLS

presented by

Robert Lee Armstrong

has been accepted towards fulfillment
of the requirements for

Ph 0 D 0 degree in Biochemistry

W fl/Bae

/ Major professor]

Date _J_LLly__29_,_1966____

0-169

 

LIN? 4RY

Michigan Stan
[Inivcrghqr

 

ABSTRACT

STUDIES OF ESCHERICHIA COLI RNA-DNA
HYBRID AND OF METHYLAIION OF RNA IN
T-4 INFECTED CELLS

by Robert Lee Armstrong

The reaction between Escherichia gel; (3H) RNA and
denatured DNA to form ribonuclease stable hybrid was studied.
The reaction rate is optimal at 78° when 0.50 M KCl, 0.01 M
Tris (pH 7.3) is used as the solvent. Hybridization is
dependent on homologous, denatured DNA. Native or denatured
DNA from Bacillus subtilis or gseudomonas fluorescens will
not react with (3H) RNA from g. gall. RNA-DNA hybrid forma-
tion is dependent on both the RNA and DNA concentrations as
well as incubation at an elevated temperature (65-870).

The RNA-DNA hybrid melts 4-5° below the melting point of
native DNA (9u-95° and 99°, reSpectively) in 0.50 M KCl,

0.01 M Tris (pH 7.3). The stability of the RNA-DNA hybrid

in the presence of ribonuclease is dependent upon the salt
concentration. A sharp transition from ribonuclease resist-
ant to ribonuclease sensitive occurs when the salt concentra~
tion is decreasedo The midpoint of this transition occurs

at 0.135 M KCl.

Hybridization was used to show that there is less
messenger RNA present in glucose starved cells than is
present in cells grown on minimal media, enriched media, or
treated with chloramphenicol. Hybridization and nitro-

cellulose chromatography were used to isolate messenger RNA

Robert Lee Armstrong

from the rest of the cellular RNA. The Specific activity of
the isolated messenger RNA was higher than the pulse labeled
RNA from which it was isolated. The molecular weight of the
isolated messenger RNA was shown to be low by both methylated
albumin-kicselguhr chromatography and sucrose gradient
centrifugation. Hybridization does not occur with low molec-
ular weight RNA more readily than with high molecular weight
RNA. Most of the degradation of the isolated RNA apparently
occurred during the denaturation of the hybrid. Isolated
messenger RNA hybridized more readily with denatured DNA than
did pulse labeled RNA.

Methylation of RNA occurs in T-4 infected g. ggli'Klz W6.
About 60-70% of the RNA molecules which are methylated sedi-
ment with soluble RNA. The other 30-40% are a mixture of RNA
molecules of diverse size which sediment faster than u S and
which elute from a methylated albumin-kieselguhr column at
salt concentrations greater than that required to elute
soluble RNA. The methylation pattern of soluble RNA extracted
from infected cells is different from the pattern of RNA
from uninfected cells. Little or no methylation of ribosomal
RNA occurs in infected cells. -(3H) methyl labeled RNA from
infected cells hybridizes with1T-4 DNA but not with g, gel;
DNA. Some soluble RNA synthesis may occur in infected cells
since some of the (3H) uracil labeled RNA extracted from T-h
infected cells elutes from the methylated albumin-kieselguhr

column with soluble RNA.

STUDIES OF ESCHERICHIA COLI RNA-DNA
HYBRID AND OF METHYLNTION OF RNA IN
T-4 INFECTED CELLS

BY

Robert Lee Armstrong

A THESIS

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

DOCTOR OF PHILOSOPHY

Department of Biochemistry

1966

r” ,9,— /L’£ ‘/

I'l/V/

ACKNOWLEDGMENTS

I wish to express my sincere appreciation to Dr.
John A. Boezi for his encouragement, guidance, and construc-
tive criticism throughout the course of this work. Thanks are
also given to Lucy Lee, James Johnson, and Ken Payne for
helpful discussions. The financial support of the National

Institutes of Health is appreciated.

ii

TABLE OF CONTENTS
page
INTRODUCTION
Studies of E, 93;; RNA-DNA Hybrid. . . . . . . . 1

Studies of Methylation of RNA in T-h Infected
CGIISeeeeooeeeeeeoooeeeeee 7

MATERIALS AND METHODS

Studies of E. coli RNA-DNA Hybrid. . . . . . . . 8
GrOWth Of BaOteriao e e e e e e e e e e o o 8
Preparation Of DNA. 0 o e e e e e 8
Pulse Labeling and Purification of RNA. . . 9
RNA‘DNA Hybrid Formation. e e o e o e e o e 10
Nitrocellulose Chromatography . . . . . . . ll
Methylated Albumin-Kieselguhr

Chromatography. e e e e o e e e o o e e e 11

Sucrose Gradient Centrifugation . . . . . . 12

Studies of Methylation of RNA in T-4 Infected
Gel-18000000eoeeeeeeeeeeeoe 12

Growth of Bacteria and Labeling . . . . . . 12
Purification or RNA 0 e o e e e e o e e o o 13
HYbridization e e e e e e e e e e o e e e 0 1’4
Paper EleCtrOphoreSIS e e e e e e e e e e 0 11+

RESUDTS AND DISCUSSION
Studies Of E0 0011 RNA-DNA Hybrid. 0 e e e e o o 16

Kinetics of (3H) RNA-DNA Hybrid Formation . 16
Effect of RNA and DNA Concentration on

Hybrid. Formation. e e e e e e o 1.6
Effect of Temperature on (3H) RNA-DNA

HYbrid Formation. e e e e e e e 21
Thermal Denaturation of (3H) RNA-DNA

HYbrid. O O O O O O O O O O O O O O O O 21
Effect of Salt C ncentration on the

Stability of ( H) RNA-DNA Hybrid in the

Presence of Ribonuclease. . . . . . . . 2#
Comparison of RNA's from E. coli Cultured

under Different Conditions. . . . . . . 27
Behavior of DNA and RNA-DNA Hybrid on

NitrocelIUIOse (301111111180 0 e e e e o e e e 32

Isolation and Characterization of
MessengerRNA.............. 31+

iii

TABLE OF CONTENTS (continued)

page

Studies of Methylation of RNA in T-h Infected
C31130 0 e e e o e e e e e e e e e e e e e e o “9

Characterization of (3H) Methyl Labeled

RNA from T.“ InfeCted Cells 0 e e e e e o 49
Sucrose Gradient Sedimentation Analysis

of (3H) Methyl Labeled RNA from T-u

Infected Cells. 0 e e 30 e e e e e e e e o 52
MAK Chromatography of ( 3H) Methyl Labeled

RNA from T-# Infected Cells . . . . 52
MAK Chromatography of (3H) Methyl Labeled
RNA from Uninfected C 113 . . . 57

MAK Chromatography of ( H) Uracil Labeled
RNA from T‘u InfeCted Cells 0 e e e e e e 57
Hybridization of RNA from T-b Infected
Cells with Denatured T-4 DNA. . . . . . . 60
DISCUSSION. 0 O O O O O C O O O O O O O O O O C O O O 64

REFERENCES. 0 O O O O O O O O O O O O O O O O 0 O O O 67

iv

LIST OF TABLES

Table page
I. Hybridization of Messenger RNA . . . . . . . . . #8
II. Hybridization of Pulse Labeled RNA . . . . . . . 48
III. Hybridization of (3H) Methyl Labeled RNA . . . . 63
IV. Hybridization of (3H) Uracil Labeled RNA . . . . 63

LIST OF FIGURES

Figure page
10 Kinetics or Hybrid Formation e o e o e e o e e o 18

2. The Effect of Nucleic Acid Concentration on
HYbrid Formation e e e e e e e e e e e e e e o o 20

3. The Effect of Temperature on Hybrid Formation. . 23
4. Action of Heat or Low Salt on Hybrid . . . . . . 26

5. Comparison of RNA's from E, coli grown under
different Conditions 0 e e o e e e e e e e e e e 30

6. Behavior of Pulse Labeled and Messenger RNA
on a MAK Column. 0 e e e e e e e e e e e e e e e 37

7. Fractionation of Pulse Labeled RNA on a MAK
COlumn e o e e e e e e e o e e e e e e o e e e e 39

8. Sucrose Gradient Centrifugation of 2 different
Fractions frOm a MAK C01umne e e e e e e e e e e 41

9. Sucrose Gradient Centrifugation of Pulse
Labeled and Messenger RNA. 0 e e e e e e e e e e #u

10. Electrophoresis of Nucleotides . . . . . . . . . 51

ll. Sucrose Gradient Centrifugation of Labeled RNA
from T-u Infected Cells. . . . . . . . . . . . . 54

12. Behavior of (3H) Methyl Labeled RNA from T-u
Infected Cells on a MAK Column . . . . . . . . . 56

13. Behavior of (3H) Methyl Labeled RNA from
Uninfected Cells on a MAK Column . . . . . . . . 59

14. Behavior of (3H) Uracil Labeled RNA from T-4
Infected Cells on a MAK Column . . . . . . . . . 62

vii

INTRODUCTION

PART I. STUDIES OF E, coli RNA-DNA HYBRID.

Jacob and Monod postulated the existence of messenger
RNA in 1961 (l). Gros, et. el., (2) and Brenner, 22. 31., (3)
verified the existence of messenger RNA in bacterial systems.
The existence of messenger RNA in plants and animal cells
has been well established (4, 5). Messenger RNA is syn-
thesized in the nucleus by RNA polymerase using DNA as the
template. The resulting RNA is complementary to the strand
of DNA which served as its template and is able, therefore,
to carry the biological information from the DNA to the sites
of protein synthesis. At these sites of protein synthesis,
information is translated from a sequence of nucleotides
into a sequence of amino acids in a protein. ~

In E. 221;, growing logarithmically, about 1% of the
total RNA is messenger RNA, 20% is soluble RNA, andrthe
remaining 79% is ribosomal or ribosomal precursor RNA (6).
If such cells are given a pulse of radioactive uracil for a
short time (5% of the generation time in the presence of (3H)
uracil) then much«of the radioactivity will be found in the
messenger RNA fraction (2). This relatively selective label—
ing of messenger RNA can be used to follow the course of
purification of messenger RNA.

Hall and Spiegelman (7) observed the formation~of com-
plex (hybrid) between T-2 denatured DNA and T-2 Specific RNA.
Hybrid formation is the reaction of RNA and denatured DNA to

-2-

form a RNAFDNA hybrid. The two complementary strands of
RNA + dDNA ——9' RNA-DNA
denatured DNA can also react to form the renatured DNA

dDNA + dDNA —-> DNA-DNA
duplex. In each of the above reactions there is a hydrogen
bonded, double stranded structure formed similar to the
Watson-Crick (8) structure of native DNA.

In order for hybrid formation to occur there must be
sufficient ionic strength in the reaction mixture to shield
the negatively charged phosphate groups and allow the two
separate strands of nucleic acids to approach each other
closely. The reaction mixture must be heated, apparently to
dissociate any non-Specific hydrogen-bonding which may occur
in a given nucleic acid molecule and to allow the complemen-
tary strandSAto form a hybrid. Denatured DNA is required
and native DNA will not form any hybrid with RNA. -Denatured
DNA.must be homologous to the RNA if hybridization is to
occur. E, 221i,denatured DNA.will not hybridize with pulse
labeled RNA from T-2 infected cells but this RNA does
hybridize with T-2 denatured DNA (7).

RNA present in the RNA-DNA hybrid is insensitive to
pancreatic ribonuclease in comparison to free RNA which is
rapidly degraded by ribonuclease. The RNA-DNA hybrid has a
density intermediate between the densities of free RNA and
denatured DNA (9). The RNA-DNA hybrid has a ratherrsharp
melting profile similar to that of double stranded,rhydrogen
bonded DNA (10). The RNA-DNA hybrid is adsorbed on nitro-

cellulose in the presence of high salt concentrations while

-3-
free RNA is not adsorbed.

Several techniques have been used to detect the presence
of RNAéDNA.hybrid. Hall and Spiegelman (7) used 0301 density
centrifugation (9). Free RNA has a density of 1.9 gm/ml and
DNA has a density of about 1.7 gm/ml. The RNA-DNA hybrid will
have a density intermediate between the density for DNA and
RNA. CsCl equilibrium centrifugation can, therefore, separate
the RNA from RNAPDNA hybrid from DNA but is expensive, time
consuming, and applicable only to small samples. Bautz and
Hall (11) developed a DNA cellulose column in which the
glucosylateerNA of certain phages was chemically bound to
cellulose. RNA was then heated with the column containing
DNA and the unreacted RNA was washed from the column while
RNAeDNA.hybrid was retained. The technique is limited to
those few DNA's containing glucosyl groups. Boltonwand
McCarthy (12)-used agar to immobilize denatured DNA. The
agar was out into pieces and packed into a column. «RNA was
then heated on the column and the unreacted RNA.was removed
by washing. The agar column presents the problem of manip-
ulating nucleic-acids in agar and also prevents the isolation
of RNAéDNA.hybrid. . .

Nygaard and Hall (13) introduced nitrocellulose membrane
filtration as a technique for the detection of RNA-DNA
hybrids. By'sometunknown mechanism, nitrocellulose is able
to selectively retain denatured DNA or RNA-DNA hybrid but
not free RNA if the nucleic acids are placed in contact
with the nitrocellulose in the presence of high-salt concentra-
tions. The method allows fast and simple analysis for the
presence of RNA-DNA hybrid. Gillespie and Spiegelman (14)

14-
reported that denatured DNA could be irreversibly bound to
the nitrocellulose filters by filtering the DNA onto the
filter, air drying the filter, and heating at 80° for a
short time. With denatured DNA bound to the membrane filters
DNA—DNA interactions cannot occur. Therefore, it is possible
to detect,quantitatively,the RNA able to hybridize with only
a limited portion of the genome (for example, soluble and
ribosomal RNA homologous to 0.025% (15, 16) and 0.2% (17, 18)
of the genome, respectively) because DNA-DNA interactions
are prevented. Nitrocellulose column chromatography (10,

19, 20) can be used for the isolation of larger amounts of
RNA-DNA hybrid. It works in the same manner as the membrane
filters but readily allows the isolation of larger amounts
of hybrid.

Since Hall and Spiegelman (7) observed the formation of
a specific T-Z RNA-DNA hybrid, the reaction has been widely
used to measure complementarity between RNA and DNA<nucleotide
sequences. Hybrid formation has been used to show that pulse
labeled RNA is complementary to homologous DNA (21,»22, 23)
and to demonstrate that DNA contains nucleotide sequences
complementary to ribosomal RNA (17, 18, 24, 25, 26) and
soluble RNA (15, 16). Hybrid formation has indicated the
portion of the bacterial genome complementary to soluble
(15, 16) and ribosomal (18) RNA. The hybridizationereaction
has been used to show that with most systems RNA synthesized
ig,zitgg is complementary to both strands of DNA whereas RNA
synthesized in'zizg appears to be complementary to only one
of the DNA strands (27-30). Hybrid formation has shown that

when intact phage DNA is transcribed ig,vitro, only one of

-5-
the two strands is transcribed but both strands are transcribed
if the DNA was previously sheared (31). RNArDNA hybrid forma-
tion has shown that pulse labeled RNA synthesized in ¢X 174
infected cells is not able to hybridize with vegetative ¢X 174
DNA. The RNA is able to hybridize with denatured, replicative
DX 174 DNA indicating that the RNA is complementary to the
replicating strand of the replicating form of ¢X 174 DNA
and not to the vegetative DNA.

More recently, RNA-DNA.hybrid formation has been used
to map the location of genes directing the synthesis of
ribosomal (32) and soluble (33) RNA in Bacillus subtilis.
The nucleolus organizer region of the sex chromosome of
Drosoghila melanogaster has been shown to contain loci cor-
responding to thecribosomal RNA by using RNArDNA,hybrid
formation (24, 25). The similarity of RNA has been compared
by measuring the ability of the RNA to compete withea known
RNA for hybridization sites on DNA (5, 10, 26,.34).a Hall,
22, 31., (34) showed by the competition technique that mes-
senger RNA for early phage enzymes was also transcribed during
the late portion of infection. RNA-DNA hybrid formation has
been used tOrShOW~that there is Specific messenger RNA.made
after induction of the galactose and lactose operons (34, 35).
Imamoto, 22..al., (37) used hybridization to show that, for
the tryptophan synthetase operon, repression occursdat the
level of transcription and derepression caused‘the synthesis
of messenger RNA for the tryptophan cperon. Bautz,2et, 31.,
(38) used hybridization and nitrocellulose columns to isolate

messenger RNA for the rII region of T-4 phage.

-6-

The objectives of the work to be reported in this thesis
were: 1. To study hybrid formation between E. 921i,nucleic
acids. 2. To study the properties of the hybrid. 3. To use
hybrid formation to isolate messenger RNA. 4. To char-
acterize the messenger RNA isolated.

Previous studies of hybrid formation (7) had used phage
nucleic acids and such a system is a great deal simpler than
the bacterial system studied here. In order to obtain the
most hybrid possible, the conditions required for hybridiza-
tion were studied. A careful study of the properties of the
hybrid may indicate new uses and/or means of detecting and
isolating the hybrid. The central role of messenger RNA in
information transfer makes the study of messenger RNA.desir-
able. However, messenger RNA comprises only about 1% of the
cellular RNA-and therefore messenger RNA.must be separated
from the other 99% of the RNA in the cell. Hybridization is
able to accomplish such a separation since 99% of the DNA
genome codes-for messenger RNA synthesis and 1% codes for the
synthesis of soluble and ribosomal RNA (the latter two com-
prise 99% of cellular RNA). Thus, most of the RNA which
hybridizes with denatured DNA will be messenger RNAJ Isola-
tion of the RNA-DNA hybrid from unhybridized RNA by nitro-
cellulose chromatography will give a mixture of denatured
DNA and RNA-DNA hybrid. Denaturation of the hybrid and
passage over a second nitrocellulose column will allow the
isolation of“messenger RNA. Such isolated messenger RNA
can be characterized by a number of techniques, some of

which will be described in this thesis.

-7-
PART II. STUDIES OF METHYLATION OF RNA IN T-4 INFECTED CELLS

The presence of methylated bases has been reported in
Escherichia ggli_DNA (39), soluble (40) and ribosomal RNA (41).
The existence of methylated bases in messenger RNA has not
been described. However, none have been reported in viral
RNA (42, 43) which serves as messenger RNA for viral Specific
proteins.

Methylation of nucleic acids occurs at the polynucleotide
level (44). .Methylases which catalyze the transfer of methyl
groups from S-adenosylmethionine to DNA (45), soluble (46)
and ribosomal (47) RNA have been described. Although the
mechanism of biosynthesis is understood, the biological
function of the methylated bases is still obscure.

Little is known of the methylation of nucleic acids in
T-even infected cells. Several reports deal with ig,!itgg
methylation. Gold, 23, e1., (48) reported that extracts from
T-2 infected cells are loo-fold more active in methylating
methyl deficient g, 921i,DNA than extracts prepared from
uninfected cells. These authors were unable to detect any
increase in RNA methylation activity of extracts following
infection. However, Wainfan, 22. g1., (49) did observe
altered RNA methylase activity (using methyl deficient RNA
as substrate and measuring the ratios of methylated bases
formed) in extracts prepared from T-2 infected cells. The
in,zizg,methylation of RNA in infected cells has not been
reported previously. Part II of this thesis deals with such
methylation in T-4 infected cells.

MATERIALS AND METHODS

PART I. STUDIES OF E. 221; RNA-DNA HYBRID.

ngwth of Bacteria. E. 921i BB was grown at 320 on a
gyrorotary Shaker or in a fermentor in C medium (50) with
0.4% glucose (doubling time = 60 minutes). In one experiment,
RNA extracted from cells growing exponentially in the above
medium was compared to RNA extracted from cells: (a) grow-
ing exponentially in C medium with 1.0% vitamin free casamino
acids and 0.5% yeast extract, (b) starved of an energy source
by limiting the glucose to 0.04% and incubating 150 minutes
after growthastcpped, (c) treated with 56 pg/ml of ehloramphen-
icol (Parke, Davis and Company) for 90 minutes.

Preparation of DNA. DNA was purified by the phenol
extraction method of Saito and Muira (51). After the iso-
pr0panol precipitation DNA was dissolved in 0.06 M KCl,

0.01 M Tris (pH 7.3) and dialyzed in the cold against the

same buffer for 14-16 hours. For the experiments on isolation
of messenger RNA, the ribonuclease digestion was omitted from
the above purification of DNA because traces of ribonuclease
were found in certain of the purified DNA preparations. In
the preparations of DNA not treated with ribonuclease, DNA
was denatured and separated from the RNA by nitrocellulose
column chromatography. DNA was denatured by heating to 1000
for 10 minutes in 0.06 M KCl, 0.01 M Tris (pH 7.3) and then
rapidly cooling to 0° by plunging into an ice bath.* Denatured
DNA could be stored in the frozen state without loss of
activity provided the solution was not more concentrated than

n
mum

-9-
200 pg/ml. If solutions more concentrated than 200 pg/ml
were frozen, it was necessary to denature the DNA after each

freezing and thawing in order to maintain 100% activity in

RNA-DNA hybrid formation.
gulse labeling and purification of RNA. (3H) RNA was
labeled by feeding (3H) uracil (Nuclear Research Chemicals,

Inc., Orlando, Florida, 4700 mc/mM) at 0.2 pg/ml to cells
growing exponentially in glucose-C medium. After 3 minutes
the culture was quickly cooled to 0°, centrifuged, and frozen
in a dry ice-acetone bath. The frozen cells were transferred
to 0.01 M acetate (pH 5.0) containing 1-2% sodium dodecyl
sulfate (SDS). After lysis the nucleic acids were purified
by the phenol method (52). DNA was removed by deoxy-
ribonuclease treatment or by heating to 100° for 10 minutes
and then quickly cooling. The KCl concentration was raised

to 0.5 M and the mixture was passed through nitrocellulose
membrane filters (13) or a nitrocellulose column (10 x 1 cm).
Denatured DNA is retained on the nitrocellulose but RNA

is not held on the nitrocellulose. The purified RNA was
dialyzed overnight in the cold (3°) against 0.50 M KCl, 0.01 M
Tris (pH 7.3). The denatured DNA content of certain (3H)
RNA's was determined by incubating an aliquot under condi»
tions known to favor RNA-DNA hybrid formation (0.50 M KCl

0.01 M Tris (pH 7.3), 78°, 240 minutes) and testing for the
presence of an (3H) RNA-DNA hybrid which would result from

the presence of any denatured DNA in the sample. If any
hybrid was formed, the RNA sample was passed through another
nitrocellulose column and retested. One passage over a nitro-
cellulose column was usually sufficient to remove all denatured

DNA. Using these procedures, (3H) RNA's were prepared having

-10-
specific activities of 3740-7125 counts/min/pg. The majority
of the pulse labeled RNA prepared by these methods sediments
between 8 and 148 but there is a portion which sediments
more rapidly than 148. The presence of any denatured DNA in
purified nonradioactive RNA samples was determined, after
incubation of the unlabeled RNA with (3H) RNA previously
Shown to contain no denatured DNA, by testing for (3H)
RNAPDNA hybrid.

The concentration of RNA and DNA was calculated from
the absorbancy at 260 mp, using an extinction coefficient
of 20 cmZ/mg. For analysis of radioactivity, 5-10% tri-
chloroacetic acid (TCA) insoluble material was collected on
a nitrocellulose membrane filter. Commercial salmon Sperm
DNA was added as "carrier”. The membrane filters were dried
and counted in a liquid scintillation spectrometer.

RNA-DNA hybrid formation. RNA-DNA hybrid was synthesized
by incubating RNAzand DNA in 0.50 M KCl, 0.01 M Tris (pH 7.3)
at 780 unless otherwise Specified. To assay for hybrid
formation, 0.1 ml samples were removed at various times and
added to 0.5 ml of 0.06 M KCl, 0.01 M Tris (pH 7.3) contain-
ing lo‘pg of ribonuclease. After incubation at 37° for 10
minutes, thexsample was diluted with 15 ml of 0.50 M KCl,
0.01 M Tris (pH 7.3). The presence of ribonuclease resistant
(3H) RNA hybridized with DNA was determined by the nitro-
cellulose membrane filtration technique of Nygaard and Hall
(13). The ribonuclease digestion lowered the amount of
unreacted RNA trapped on the filters to less than 0.1% of the
total and gave less scatter in the data. For the later

experiments the salt concentration during the ribonuclease

-11-
treatment was raised to 0.50 M with only a slight increase
in background and a doubling of the amount of hybrid detected.

Nitrocellulose Chromatography. Nitrocellulose (type RS),
a gift from Hercules Powder Co., Wilmington, De1., was
thoroughly washed with water, homogenized in a Waring blender,
and incubated overnight in 0.06 M KCl, 0.01 M Tris (pH 7.3)
at 65°. Used nitrocellulose was washed with water and then
0.06 M K01, 0.01 M Tris (pH 7.3), and incubated overnight
at 65°. After this treatment, it was indistinguishable from
unused material. The material was Slurried, poured into a
column, and packed to about one third the unpacked volume by
tamping with a glass rod. The packed column was washed with
several cycles of water followed by 0.50 M KCl, 0.01 M Tris
(pH 7.3). Finally, the column was washed with several column
volmmes of the salt solution. The washing of the column is
necessary to remove fragments of nitrocellulose from the
column. This process can readily be followed by observing
the absorbancy-of the eluant at 230 mp. The sample was
applied to the washed column in 0.50 M KCl, 0.01 M Tris
(pH 7.3). However, lower or higher salt concentrations may
be used. Denatured DNA or RNAPDNA hybrid was eluted from
the column by 0.01 M Tris (pH 7.3) or by water. Two or
three column'volumes were usually sufficient to remove most
of the adsorbed material.

Methylated albumin-kicsel r chromato ra h .
Methylated albumin-kieselguhr (MAK) column chromatography was
similar to that used by Sueoka and Cheng (53) except that both
linear gradients and stepwise elution procedures were used.

It was found that bits of methylated albumin eluted from the

-12-
column at higher salt concentrations and interferred with
hybrid formation. Washing the column with about 5 column
volumes of high salt (1.3 M NaCl in 0.05 M phOSphate (pH 6.8))
was usually sufficient to remove all fragments of methylated
albumin. Before applying the sample the column was washed
with 5 volumes of 0.3 M NaCl, 0.05 M phosphate (pH 6.8).

Sucrose gradient centrifugation. A 0.2 ml sample of
RNA was layered on 4.8 ml of 20%-5% sucrose dissolved in
0.1 M NaCl, 0.01 M Tris (pH 7.3). This sucrose gradient was
centrifuged in the cold (3°) for 5% hours at 39,000 rpm.
Three drop fractions were collected from the bottom of the
tube. If cptical densities were desired, 0.5 ml of-water
was added to each tube and the absorbancy at 260 up was
determined. Radioactivity was determined by collecting the
TCA precipitable material on a membrane filter and counting

in a scintillation counter.

PART II. STUDIES OF METHYLATION OF RNA IN T-4 INFECTED CELLS.

The following description of materials and methods
applies to the experiments involved in the studies of methyla-
tion of RNA. Except for the changes listed below procedures
were the same as those given above.

growth of Bacteria and Labeling. E. 231;,K12 W6, kindly
supplied by Dr. E. Borek, was grown at 32° on a gyrorotary
Shaker in C medium (50) supplemented with 12.5‘ug/l'of biotin,
30 mg/l of Lemethionine, and 0.4% glucose. The doubling time
was about 90 minutes. When the cell density of a exponential

culture reached 5 x 108 cells/ml, 20 mg/l of L-tryptophan and

-13-
wild type T-4 phage, at a multiplicity of 5-10, were added.
Approximately 10 minutes after infection the cells were
collected by centrifugation. The pellet of infected cells
was resuSpended in 0.4 volumes of C medium and repelleted.
This pellet was suspended in 1 volume of C medium containing
12.5 ug/l of biotin and 0.4% glucose. The cells were
"starved" for methionine for 4-5 minutes. The culture was
split into 2 parts and to the one part 1 pg/ml of (3H methyl)
L-methionine (Nuclear Chicago, 150 mc/mM) was added. To

the other part of the culture 1 pg/ml of unlabeled

3

Ldmethionine and 4Juc/m1 of H uracil (Nuclear Research
Chemicals, Orlando, Florida, 4700 mc/mM) was added. Label-
ing was allowed to proceed for 7 minutes (27-34 minutes after
infection) and then the culture was rapidly cooled on ice and
centrifuged in the cold. The pellets were quickly frozen by
dipping into a bath at -25°. Control uninfected cultures
were starved of methionine and labeled in a similar manner.
In one experiment chloramphenicol (10 pg/ml) was added with
the (3H methyl) methionine. In this case, labeling was
allowed to proceed for 60 minutes.

Pagification of RNA} The frozen cells were lysed by
stirring with 1% SDS in 0.01 M acetate buffer (pH 5.0).
An equal volume of phenol was added and the mixture stirred
10 minutes. The aqueous layer was removed and then the
phenol and interphase material were extracted with 1/2 volume
of SDS. The aqueous layers were combined and precipitated
with 2 volumes of cold ethanol. After 1 hour in ice the
precipitate was collected and dissolved in 0.1 M Trizma

(pH 8.0) 0.01 M MgClZ, 0.1 mM EDTA. 25 pg/ml of pancreatic

-14-
deoxyribonuclease (Sigma) was added and the solution was
incubated at 37° for 10 minutes. Two more phenol purifica-
tions were performed. The aqueous phase was dialyzed exten-
sively against 0.50 M KCl, 0.01 M Tris (pH 7.3) to remove
traces of phenol. The Specific activity of RNA obtained
3

from H methionine fed cells was 15-50 counts/minépg. The
Specific activity of uracil labeled RNA was 1000 counts/minéug.

Hybridization. The assay for RNA-DNA hybrid using DNA
fixed to nitrocellulose filters was according to the method
of GilleSpie and Spiegelman (14). The DNA was fixed to the
filters by heating in an oven at 90° for 30 minutes. Hybrid
formation was carried out at 79° for 2 hours in 0.50 M KCl,
0.01 M Tris (pH 7.3). After hybrid formation the filter was
washed on both sides with 15 ml of 0.5011KC1, 0.01 M Tris
(pH 7.3) andvthen incubated at 370 for 10 minutes in 3.3 ml
of 0.50 M KCl, 0.01 M Tris (pH 7.3) containing 10 pg/ml of
pancreatic ribonuclease. Finally, each side of thezfilter was
washed with 15 ml of 0.50 M KCl, 0.01 M Tris (pH 7.3), dried,
and counted in a scintillation counter.

Paper electrophoresis. To test for the presence of
methylated bases in (3H) methionine labeled RNA, the RNA
was hydrolyzed by incubating 18 hours at 37° in 0.3 M KOH.
The solution was neutralized by adding HClOu and then con—
centrated to a small volume by lyophilization. The tube
was cooled in ice and centrifuged to remove K010” and then
a portion of the clear supernatant was Spotted-on Whatman
3 MM paper. After the paper was wetted with 0.05 Miammonium
formate (pH 2.8),.electr0phoresis was conducted for 2 hours

at 2500 volts in a Gilson model D using Varsol as a coolant.

-15-

The major nucleotides were located by ultraviolet light and
marked. Squares 1.9 x 1.9 cm were cut from the paper and
placed in scintillation bottles and counted. The pattern of
radioactivity obtained was compared with the published
electrophoretic mobilities for the methylated bases (49).

RESUDTS AND DISCUSSION

PART I. STUDIES or E, coli RNA-DNA HYBRID.

Kinetics of (3H) RNA-DNA hybrid formation. Figure 1

illustrates the time course for the reaction of pulse labeled
(3H) RNA and denatured DNA to form ribonuclease stable RNA-DNA
hybrid. No reaction is observed if denatured DNA is omitted
or if native DNA is substituted for denatured DNA (Fig. l).
Incubation of denatured DNA from either Bacillus subtilis

or Pseudomonas fluorescens with pulse labeled (33) RNA from

E. 921; results in no measurable hybrid formation. .

ngect of RNA and DNA concentration on hybrid formation.
The formation of RNA-DNA hybrid is dependent upon the

 

concentration of both denatured DNA and RNA (Fig. 2).
Figure 2a illustrates that the amount of hybrid formed increases
linearly with increasing RNA concentrations for both DNA
concentrations tested (5 and 50 pg/ml.) Figure 2b Shows that
the amount of hybrid formed depends on the amount of added
denatured DNA. The lack of linearity between hybrid syn-
thesized and denatured DNA added at the higher concentrations
is presumably due to the removal of denatured DNA from the
reaction by DNA-DNA interactions which result in the formation
‘of material not reactive with (3H) RNA. For example, denatured
DNA preincubated at 60 pg/ml for 60 minutes at 650 then assayed
at 48‘ug/ml, shows a 40% decrease in the initial rate of
reaction with (3H) RNA. If the DNA was preincubated as above
but assayed at 5 pg/ml there was no loss in reactivity with
-16-

-17-

Figure 1. Kinetics of hybrid formation. Each reaction mix-
ture contained 48 pg/ml of (3H) RNA (3740 counts/minéug).
DNA, if present, was present in a concentration of 50 pg/ml.

The upper line, 0 0, indicates hybrid formed when

 

 

 

denatured DNA is used. The lower line, 0 O and A A ,
indicates the lack of hybrid formation when no DNA is added

or if native DNA is added.

 

COUNTS/MIN. x I0"2

l6?-

l4-

l2-

5

 

~18-

 

 

 

 

: A
8
’I‘ I 8 1 ‘i ‘1)
0 3O 60 90 '20 I50 '80
TIME (MIN)

Figure l

-19-
Figure 2. The effect of nucleic acid concentration on

hybrid formation.

Figure 2a. The effect of RNA concentration. The concentra-

 

tion of denatured DNA in reaction mixtures was 50, O O,

and 5, 0

 

0,1pg/ml. Each point represents the average of

3 samples assayed after 210 min incubation.

Figure 2b. The effect of DNA concentration. All reaction
mixtures contained 48 pg/ml of (3H) RNA (3740 counts/min/pg).
Each point is the average of 3 samples assayed after 210

min incubation.

5000

4000

COUNTS/MIN.
3

N
C)
(D
C)

I000

2000

I500

I000

COUNTS/MIN.

500

 

-20-

 

 

O
O
O
I 1 I 1
50 I00 ISO 200
RNA , ing/ml —.

 

 

l l l
50 l00 I50

DENATURED DNA. uq/ml

Figure 2

«21-“

(BR) RNA. The loss in reactivity observed thus appears to
be due to some type of aggregation and not renaturation
because renatured DNA would be stable to dilution. The lack
of linearity between denatured DNA added and RNA-DNA hybrid
formed could also be due to self absorption of the radio-
activity by increasing amounts of denatured DNA on each
filter. It was shown, however, that no correction for self
absorption is necessary for the DNA concentrations used in
this experiment.

gffect of temperature on (332 RNA-DNA hybrid formation.
Figure 3 gives the kinetics of hybrid formation at 3 dif-
ferent temperatures. Reaction mixtures for experiments shown
in Figure 3a contained 48‘pg/ml of denatured DNA and #O‘pg/ml
(33) RNA (7125 counts/minfipg). Reaction mixtures for experw
iments shown-in Figure 3b contained S‘pg/ml of denatured
DNA and 212‘ug/ml of (3H) RNA (#100 counts/minfiug). In
both cases, 780 is the optimum temperature. At 65° hybrid
formation ceases after 90 minutes in the reaction mixture
containing uBng/ml denatured DNA and ho‘pg/ml (3H) RNA.
Under these conditions, DNA-DNA interaction rather than
RNA-DNA reaction is favored. At higher temperatures or

lower concentrations of denatured DNA these DNA interactions
don't remove DNA from reaction with (3H) RNA and hybrid

formation occurs for longer times.

ghggmal denaturation of 13H) RNArDNA hybrid. (3H) RNA~
DNA hybrid, isolated free of unreacted RNA by nitrocellulose
column chromatography, was heated at various temperatures in
0.50 M KCl, 0.01 M Tris (pH 7.3) for 10 minutes. After heat«

ing the samples were assayed for ribonuclease resistant (3H)

-22-

igure 3. The effect of temperature on hybrid formation.

Figure 3a. Effect of temperature on hybrid formation at
high DNA concentration. The concentration of denatured
DNA and (3H) RNA (7125 counts/minéng) was #8 and holpg/ml,

0, represents 78°; the

 

respectively. The upper line, 0

 

 

middle one,;3 ti, 87°; and the bottom one, 0 0,

65° incubation.

Figure 3b. Effect of temperature on complex formation at
low DNA concentration. The concentration of denatured

DNA and (3H) RNA (4100 counts/minéug) was 5 and 212‘pg/ml,

 

respectively. The upper line, C 0, represents 78°;

the middle one, 0 0, 65°; and the lower one,A-—-—A,

 

87° incubation.

4000

3200

N
A
C)
<3

COUNTS/MIN.
8
C)

800

I200

I000

800

600

COUNTS/ MIN.

400

200

 

-23-

 

 

 

 

 

_ 78°
V
L O
87
o 5 Ir
A
_ A
A
A
_ 65"
A T i 4 °
1 1 1 1 1 1
30 60 90 I20 I50 I80
._ TIME (MIN)
0
z"
' I I l I I 1 _
60 I20 I80 240 300 360
TIME (MIN)

Figure 3

-24-
RNA-DNA hybrid. The results are shown in Figure ha. The
observed tm is 9h-95o. The tIn is defined as the temperature
at which one half of the complex remains after-treatment at
that temperature for 10 minutes.

Thermal denaturation of DNA was determined by measuring
the reactivity of. the resulting denatured DNA with (33) RNA.
Samples of DNA at a concentration of 60 pg/ml in 0.50 M KCl,
0.01 M Tris (pH 7.3) were heated for 10 minutes at various
temperatures. (33) RNA was added and the reaction mixture
was incubated at 780 for 150 minutes then assayed for ribo-
nuclease resistant hybrid. The results are plotted on
Figure 4a. The tm for DNA denaturation is 99°. In-this
case, the tm refers to the temperature at which the DNA is
50% denatured and thus one half as reactive invhybridization
as fully denatured DNA. The DNA duplex is more stable than
the RNA-DNA hybrid as shown by the difference in the tmfs
of ”-50. This greater stability of the DNA duplex relative
to the RNArDNA hybrid was also observed by Chamberlin and
Berg (54). They noted the 25x 1711 RNA-DNA hybrid melts u-5°
below the double strand derivative of ¢X 17h DNA. Also,
Bolton and McCarthy (55) observed that RNA elutes from a
DNA agar column at a temperature 4° below thatnecessary to
elute the corresponding DNA.

Effect of salt concentration on the stability of g3g}
RNAADNA hybrid in the presence of ribonuclea§2.* In the

presence of sufficient ionic strength, RNA in the RNA-DNA

 

 

*This experiment was first performed by James C. Johnson.

-25-
Figure 4. Action of heat or low salt on hybrid.

Figure 11a. Thermal denaturation of (3H) RNA-DNA hybrid.
The hybrid was formed by incubating (BR) RNA (37h0 counts/
minéug) and denatured DNA at 212 and S‘pg/ml, respectively,

for 4 hours. 0 O, ribonuclease stable hybrid remaining

 

after 10 min. incubation. O O, ribonuclease resistant

 

(3H) RNA-DNA hybrid formed after 150 min incubation using
native DNA treated for 10 min at each temperature before

incubating with (33) RNA at 78°.

Figure 4b. Sensitivity of hybrid to low salt concentration
in the presence of ribonuclease. (3H) RNArDNA hybrid was
treated with 16.7Jpg/ml of ribonuclease for 10 min at 37°
in a series of different salt concentrations. Samples were
diluted with 15 m1 of 0.50 M KCl, 0.01 M Tris (pH 7.3) and

filtered through nitrocellulose membrane filters.

COUNTS/ MIN.

COUNTS/ MIN.

I200

800

400

2200

I800

I400

I 000

600

200

-26-

 

 

 

 

 

 

 

 

 

 

.. o O ‘K
o
Tm = 94° —" \ O

a nf=99

i 1 1 I I i 1 I 1° - 1

20 40 60 80 I00
TEMPERATURE, °C

" o
L .

I I I I I I I I I L
0 0.2 0.4 0.6 0.8 |.0

MOLARITY 0F KCI

Figure u

-27-
hybrid is not degraded by pancreatic ribonuclease. If, how-
ever, the salt concentration is too low, the RNA in the
hybrid is degraded by ribonuclease. Figure #b shows the
transition of the RNA in the hybrid from ribonuclease sen-
sitive at low salt concentrations to ribonuclease stable
material at higher salt concentrations. The am is defined
as the salt concentration at which one half of the RNA in
the RNA-DNA hybrid is stable to ribonuclease treatment at 37°.

(3H) RNA-DNA hybrid was formed by incubating uo‘pg/ml of
(3H) RNA and 50 Pg/ml of denatured DNA in 0.50 M K01, 0.01 M
Tris (pH 7.3) for # hours. The hybrid was isolated from
unreacted RNA by nitrocellulose column chromatography and
treated for 10 minutes at 37° with 16.7‘pg/ml of pancreatic
ribonuclease at several different salt concentrations.
Samples were diluted with 15 ml of 0.50 M K01, 0.01 M Tris
(pH 7.3) and filtered through a nitrocellulose membrane. The
Sm obtained was 0.135 M KCl. All experiments previously
described were conducted with salt concengrations during the
ribonuclease treatment of 0.135NIand therefore only one half
of the hybrid formed was actually detected.

Comparison of RNA's from E. coli cultured-under different

conditions. RNA's extracted from cells cultured under different

 

conditions may be compared by a measuring the degree to which
they compete for reaction with denatured DNA (3“). One of

the RNA's used for comparison is labeled. The other is used
without label. If the two RNA's are identical, the‘addition
of increasing concentrations of unlabeled RNA to a reaction
mixture containing labeled RNA and denatured DNA will result

in a decrease in the amount of label found in the hybrid. With

-28-
a large excess of unlabeled RNA, no labeled hybrid will be
formed. If the two RNA's are totally different, no competi-
tion between the two will occur. Consequently, equal amounts
of labeled hybrid will be synthesized in the absence and in
the presence of a large excess of unlabeled RNA.

Using this technique, unlabeled RNA's extracted from
cells, (a) grown on glucose-C medium, (b) grown on C medium
plus casamino acids and yeast extract, (c) treated with
chloramphenicol, (d) starved of an energy source, were used
to compete in hybrid formation with pulse labeled (3H) RNA
extracted from cells grown on glucose-C medium. The results
are shown in Figure 5. The lower line shows the competition
between identical RNA's, i.e., unlabeled RNA extracted from
cells grown on glucose-C medium and pulse labeled (3H) RNA
from cells grown on the same medium. The data for the RNA's
from cells grown on enriched medium and from those treated
with chloramphenicol fit the same line. Therefore, within
the sensitivity of the technique, these cells contain RNA
homologous to pulse labeled (3H) RNA obtained from.cells
grown on glucose-C medium.

The upper line describes the competition between the
pulse labeled (3H) RNA and the RNA extracted from glucose
starved cells. Little or no competition is observed. Thus,
glucose starved cells contain little or no RNA homologous
to pulse labeled RNA from cells grown on a glucose-C medium.

Cells grown on C medium supplemented with casamino acids
and yeast extract and those treated with chloramphenicol
contain RNA homologous to and in the same proportion as the

RNA which is pulse labeled in cells growing on glucose-C

-29-

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o “condone

 

Hooasozaamnoaso .‘ ‘34..“de cosoaham 80.5 q Q

 

”adapts ouomoosaw so sconw mHHoo Bonn «2m wsapoaaoo .0

 

o
.Amoaasmm opmoaaadv no owmnobmv aoameSOQH mas oam poems
season manna: Ammv pacemamon ommoaossopaa mo pdfioss on» msosm
message one .«zm Ema do ass: was and 42a ecascssme .8

H8\w1.m dosaspaoo onspxaa codpomcn scam .mnoapadnoo pamaomeb

 

sodas macaw Haoo an Bong m.<zm mo somanmaaou .m ondwam

I2

 

 

 

I

-30-

I

J

—

 

 

I0-

m

CD

0'

9-01 x 'NIW/SiNflOO

Figure 5

750 l000 I250 I500 I750 2000
UNLABELED RNA, pg/ml

500

250

-31-
medium. If cellular regulation occurs at transcription
(DNA«-€> RNA), it may be expected that cells grown on enriched
medium, where the synthesis of many enzyme systems is repressed,
would not contain all of the messenger RNA molecules found in
cells grown on glucose-C medium where these enzymes would be
present. However, the messenger RNA's which direct the syn-
thesis of these repressed enzymes probably constitute a small
proportion of the total messenger RNA present in the cell.

The competition technique might not detect such small dif-
ferences. Alternatively, cellular regulation may occur at
translation (RNA ——) protein).

Following treatment of a culture with chloramphenicol
for 90 minutes, a condition stopping growth by inhibiting
protein synthesis, the messenger RNA content of the cells
remains the same as before treatment. However, if growth
is stOpped by depletion of the energy source, the messenger
RNA content of the cells is greatly decreased. This is under-
standable in view of the fact that chloramphenicol blocks
protein synthesis but allows RNA synthesis (56). However,
depletion of the energy source would result in the end of
synthesis of all macromolecules followed by degradation of
the unstable messenger RNA.

The lack of messenger RNA in glucose starved cells may
explain the unusual sedimentation pattern observed for ribo»
somes extracted from such cells. McCarthy (57) observed that
these ribosomes, when examined at 10"2 M Mg++, sediment as
100-8 particles. .At this Mg++ concentration, however, ribo~
somes from exponentially growing cells sediment at 70 S and

85 8. These ribosomes would be expected to contain fragments

-32-

of messenger RNA as a result of degradation of polysomes
during isolation. The attached fragments of messenger RNA
may hinder aggregation of the particles in 10"2 M Mg++.
Ribosomes extracted from glucose starved cells, a condition
which depletes their messenger RNA content, should not have
these fragments attached. Accordingly, they aggregate to

2 M Mg++.

Behavior of DNA and RNA-DNA hybrid on nitrocellulose

columns. A study of native, denatured, and renatured DNA

lOO—S particles at 10'

 

and of RNAPDNA hybrid on nitrocellulose columns was conducted.
In 0.50 M KCl, 0.01 M Tris (pH 7.3) denatured DNA and RNA-DNA
hybrid is retained by the column but RNA, native, and fully
renatured DNA are not retained.

A firmly packed column of nitrocellulose 1 cm in diameter
by 10 cm in height was able to retain 500 pg of denatured DNA
in the presence of 0.50 M KCl, 0.01 M Tris (pH 7.3). 80-100%
of this DNA could be washed from the nitrocellulose column by
4-5 column volumes of water. Capacity was determined by
pouring excess denatured DNA over the column and measuring
the amount of denatured DNA which was retained on the column.

RNA-DNA hybrid is retained by the nitrocellulose column
in 0.50 M KCl, 0.01 M Tris (pH 7.3) and is eluted from the
column with “-5 column volumes of water. The yield‘from the
column is 90-100%. The first 1—2 column volumes will elute
70% of the material. Free RNA is not retained by the nitro-
cellulose column in either water or salt solution.

All of the native DNA from gs. fluorescens and from
fig. fluorescens phage gh-l (58) but only 85% of the E. 221;

DNA passes through a nitrocellulose column. The 15% of the

-33...

E. coli which is retained by the nitrocellulose column in

 

salt can be washed from the column by water. The nature of
this 15% of E. 93;; DNA which behaves abnormally on nitro-
cellulose is unknown but it is thought to contain denatured
regions. E. ggEE’DNA fractioned in the 2 phase system of
Albertsson (59) as if it contained native and denatured DNA.
The 2 phase system is able to separate native from denatured
DNA (59). The phage gh-l DNA fractionated as expected for
native DNA. This evidence also indicates that the E. 22;;
DNA as prepared by the method of Saito, 22. gE., (51) may
contain some denatured regions.

In order to study the behavior of ”renatured" DNA on a
nitrocellulose column, denatured DNA was "renatured” by
heating at 78° for u hours in 0.50 M KCl, 0.01 M Tris (pH 7.3).
When the "renatured" DNA was applied to a nitrocellulose
column, 60-80% of the DNA was retained by the column. This
probably represents denatured DNA and "renatured” DNA which
still has large regions of DNA retaining the denatured
character. It could be eluted from the nitrocellulose column
by water. Rechromatography of the 20-40% of the ”renatured"
DNA which passed through the first column results in only
a small portion being retained by the second column.

Double stranded nucleic acid molecules will exhibit an
increase in absorbancy at 260 mp after being heated to 1000
and quickly cooled. The amount of hyperchromicity is prOpor~
tional to the degree of double stranded structure. The por-
tion of the "renatured" DNA which was retained by the nitrom
cellulose column was heated to 1000 for 3 minutes and cooled

by dipping in ice water. The increase in absorbancy at 260 mp

-3h-
was 5 8% after heating. The "renatured" DNA which passed
through the nitrocellulose column gave a hyperchromicity of
.15%. Native E. 22E;_DNA heated and quickly cooled gave an
increase in absorbancy of .22%. Therefore, the nitrocellulose
column is able to distinguish the degree of secondary struc-
ture present in a DNA preparation and to separate highly
structured molecules from less structured molecules.

Isolation and characterization of messenger RNA. In
order to obtain enough isolated messenger RNA for characterizam
tion, 20 mg of RNA and 5 mg of denatured DNA were dissolved
in 100 ml of 0.50 M KCl, 0.01 M Tris (pH 7.3) and incubated
for # hours at 78°. Nitrocellulose columns (4.5 x 20 cm) were
used to separate the resulting hybrid from the unreacted RNA.
After eluting the RNA-DNA hybrid from the nitrocellulose
column, the hybrid was denatured by heating at'100o for 3
minutes. The salt concentration was raised to 0.50 M KCl
and a second nitrocellulose column was used to separate the
denatured DNA from the messenger RNA. The messenger RNA was
extensively dialyzed against distilled water in the cold and
concentrated by lyophilization. Approximately 300 pg of
messenger RNA was isolated by this technique. Isolated mes-
senger RNA was then characterized by several methods which
are described below.

Isolated messenger RNA was chromatographed on a MAK
column. The messenger RNA was eluted from the MAK column by
low salt concentrations. Experiments to be described below
will show that material eluted from a MAK column by low salt
concentrations is low molecular weight material. Therefore,

isolated messenger RNA is low molecular weight material.

-35-
Figure 6 shows the comparison of the MAK column elution pro—
file for pulse labeled RNA and purified messenger RNA. The
isolated messenger RNA could be low molecular weight material
either because of degradation during isolation or because the
hybridization reaction is selective against high molecular
weight RNA and selective for low molecular weight RNA. To
test which of these two alternatives is correct, (3H) pulse
labeled RNA was fractionated on a MAK column. An aliquot
of each fraction was assayed for TCA precipitable radio-
activity. Denatured DNA was added to selected fractions
and the solution was incubated at 780 for 7 hours to allow
hybridization to occur. The percentage of radioactivity
found in the hybrid was determined by filtering on a nitro-
cellulose membrane filter and counting in a scintillation
counter. The results are plotted in Figure 7. The 16 and
23 S ribosomal RNA probably peaks in tubes 55 and 60 (Fig. 7)
and hybridization of radioactive RNA is inhibited by the
competition with unlabeled ribosomal RNA. Those fractions
which eluted from the MAK column with a low salt concentra-
tion hybridized to a lesser extent than did fractions
eluted from the column by higher salt concentrations. Hence,
the hybridization reaction is not selecting only the lower
molecular weight RNA.

Sucrose'gradient centrifugation of fractions 18 and 60
(Fig. 7) was performed to check the correlation between the
Isalt concentration necessary to elute the RNA from the MAK
column and the molecular weight. Figure 8 illustrates the
result of this centrifugation. RNA which was eluted from

the MAK column by low salt (fraction 18) is low molecular

-36-
Figure 6. Behavior of pulse labeled and messenger RNA on

a MAK column.

Figure 6a. MAK chromatography of pulse labeled RNA. The
cpen circles represent the NaCl concentrations as obtained
from the refractive index of selected fractions. The
smooth curve without points represents the radioactivity

of the eluted material. Each fraction contained 5 ml.

Figure 6b. MAK chromatography of messenger RNA. The smooth
curve indicates the radioactivity of the messenger RNA
eluting from the column. The dashed line represents the
behavior of (14C) labeled ribosomal RNA used as a marker.

The eluting gradient was similiar to that in Fig. 6a.

COUNTS! MIN.

COUNTS! MIN.

I000

800

600

400

200

800

600

400

200

 

-37...

I
O
b

I
.0
a

I
.0
a

 

 

 

FRACTION NUMBER

0.2
70

 

 

20 30 40 50 60
FRACTION NUMBER

Figure 6

MOLARITY 0F NaCl

-38-

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Bonn Homz mo psoaumnw hmosaa m an conmaamaooom was soapzam
.omm pm 429 condudsoo spas soaumpdosa mo undo: m use m nouns
soxmp moaascm mo awesobc on» mpsomonaon usaoa comm .42Q
confinescc nods cudoannas on maps ma scans msoapomam oopooaom
ad moabupocoacmn mo cwcpsoonca can mopmoausa mafia common

one .szo coasessoo nods meanaoaanar «zm beacons chasm one no
case or» .osaa echoes the .sssaoo as: or» sons ooosao soars
anaedsosoaoss co soassnasomao one ensconced osaa eaaom one

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-39..

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9 8 8 9
T I r j

 

   
  

 

 

 

  
 
    

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3.0: x 'NlIN/SiNnOD

Fi :zjure 7

20 30 40 60 70 BO
FRACTION NUMBER

I0

-40-
Figure 8. Sucrose gradient centrifugation of 2 different

fractions from a MAK column.

Figure 8a. Sucrose gradient centrifugation profile of frac-
tion 60 in Fig. 7. Material was centrifuged at 39,000 rpm
for 5% hours. The markers were present as (14C) labeled

ribosomal RNA mixed with the sample before centrifugation.

Figure 8b. Sucrose gradient centrifugation profile of frac-

tion 18 in Fig. 7. The sample was treated the same as Fig. 8a.

COUNTS/ MIN.

COUNTS/ MIN.

I200

IOOO

800

600

400

200

 

 

 

 

 

 

o 5 I0 15 20 25 so 35
FRACTION NUMBER
800 b 233 l6 s 43
I I 7"
600 -
400 ~—
200 -
I I I I I I
o 5 IO 15 20 25 30 35

FRACTION NUMBER

Figure 8

-42-
weight RNA while RNA requiring higher salt for elution (frac~
tion 60) is of higher molecular weight. Thus the MAK column
can be used to obtain an estimate of the molecular weight of
RNA.

The profiles of isolated messenger RNA and of pulse
labeled RNA after sucrose gradient centrifugation are given
in Figure 9. The results again indicate that isolated mes-
senger RNA is low molecular weight material. Most of the
radioactivity in the pulse labeled RNA sediments faster than
8 8 material while all of the messenger RNA sediments slower
than 8 3 material.

Sucrose gradient centrifugation and MAK chromatography
indicated that the incubation at 780 was causing some degrada-
tion of the labeled RNA. MAK chromatography of RNA incubated
at 65° showed little, if any, degradation of pulse labeled
RNA. Therefore, hybridization at 650 was tried. The yield
of RNA in the hybrid decreased by 20-30% without any increase
in the molecular weight of the isolated messenger RNA.

Pulse labeled RNA was not degraded during nitrocellulose
chromatography. To check if degradation of RNA occurred dur_
ing the denaturation of the hybrid, pulse labeled RNA
dissolved in 0.01 M Tris (pH 7.3) was heated to 1000 for

3 minutes and quickly cooled. After the heat treatment both
MAK chromatography and sucrose gradient centrifugation
indicated degradation of RNA. Therefore, another method of
denaturing the RNA-DNA hybrid was sought. An attempt to
denature the hybrid by lowering the pH of the solution was
made. The RNA-DNA hybrid was placed in 0.05 M phosphate
buffer (pH 6.8) and when one equivalent of HCl was added the

-43-
Figure 9. Sucrose gradient centrifugation of pulse labeled

and messenger RNA.

Figure 9a. Sucrose gradient centrifugation profile of pulse
labeled RNA. Sample was layered on 5-20% sucrose dissolved
in 0.1 M NaCl and centrifuged at 39,000 rpm for 5% hours.

The three markers were obtained from (lac) labeled RNA mixed

with the sample before centrifugation.

Figure 9b. Sucrose gradient centrifugation profile of

messenger RNA. Treated as pulse labeled RNA above (Fig. 9a).

 

AA -—_——-—— -__‘——- — .————— —— -—

COUNTS/ MIN.

COUNTS/ MIN.

l200

800

400

I200

800

400

~4u-

 

 

 

 

 

- 23 8 I6 S 4 S
I I I
I 1 I 1 I 1 I 1
IO 20 30 40
— FRACTION NUMBER
L.
a 23 8 I6 3 4 S
I I I
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A I I 1 I 1 1 1
IO 20 3O 4O

FRACTION NUMBER

Figure 9

-u5-

resulting pH was 2. After a 5 minute incubation in an ice
bath, one equivalent of NaOH was added to raise the pH to 6.8.
All of the (36) RNA formerly in the RNA-DNA hybrid became sen-
sitive to ribonuclease indicating the low pH denatured the
hybrid. However, both MAK chromatography and sucrose gradient
centrifugation indicated that the isolated messenger RNA had
a low molecular weight. Pulse labeled RNA was degraded by
the low pH so the method was not used.

One preparation of isolated messenger RNA exhibited
4.1 times as great a Specific activity as the starting
material. The optimum increase in specific activity that
could be expected is 30-100 fold. This low increase in
specific activity obtained may have been caused by several
different factors. During the dialysis step after isolation
of messenger RNA from denatured DNA,about one half of the
TCA precipitable radioactivity was lost. This loss in radio-
activity is apparently caused by some of the degraded messenger
RNA passing through the pores of the dialysis membrane and
into the dialysis medium. Secondly, the ultraviolet Spectrum
of the isolated messenger RNA indicated there were bits of
nitrocellulose present since the relative value of the absorb~
ancy at 230 mp was high as compared to the absorbancy at 260 mp.
Thirdly, the preparation of messenger RNA may have contained
bits of denatured DNA which were too small to be retained by
the nitrocellulose column and these bits of DNA would decrease
the Specific activity of the product. Since the molecular
weight of the isolated messenger RNA was very low, no attempt

was made to achieve a greater fold purification.

-46-

Purified messenger RNA Should be able to hybridize more
readily than can pulse labeled RNA because the latter con-
tains some labeled ribosomal RNA precursors (to 60% of the
radioactivity, reference 12) which must compete with a large
amount of unlabeled ribosomal RNA for 1 limited hybridization
sites on denatured DNA. However, it was discovered that the
isolated messenger RNA formed very little hybrid. The ultra-
violet spectrum of the isolated messenger RNA indicated the
presence of nitrocellulose fragments. It was thought that
the nitrocellulose might inhibit hybrid formation, and there-
fore, a method to remove these fragments was sought. When
the isolated messenger RNA was placed on a MAK column and
eluted in a stepwise manner the ultraviolet Spectrum indica-
ted the removal of the nitrocellulose fragments. This RNA
was also able to readily hybridize with denatured DNA. Since
the MAK column was able to remove the nitrocellulose fragments,
all messenger RNA was placed on a MAK column and eluted in a
stepwise manner before characterization.

The ability of isolated messenger RNA to hybridize with
denatured DNA was tested as follows: Denatured DNA (final
concentration, 50 yg/ml) was added to isolated messenger RNA
and the solution was incubated at 780 for 4 hours to allow
hybrid formation. An aliquot was removed and tested for
ribonuclease stable RNA-DNA hybrid and for TCA precipitable
radioactivity. RNA-DNA hybrid and denatured DNA were removed
from unreacted RNA by filtration through a nitrocellulose
membrane filter. DNA was added to the filtrate to give a
final concentration of 50 pg/ml. The process of incubation,

sampling, filtration, and addition of denatured DNA was

-47-
repeated 5 times. The results are summarized in Table I.

AS a comparison, the ability of pulse labeled RNA to
form a hybrid was determined. The pulse labeled RNA was
placed on a MAK column and eluted in a stepwise manner as
used for the messenger RNA purification above. Denatured
DNA (final concentration, 50 pg/ml) was added and the solu-
tion was incubated at 78° for 4 hours. An aliquot was removed
and tested for ribonuclease stable RNA-DNA hybrid and for
TCA precipitable radioactivity. The RNA-DNA hybrid and
denatured DNA were separated from the unreacted RNA by filter-
ing through a nitrocellulose membrane filter. Denatured DNA
was added to the filtrate to give a final concentration of
50 pg/ml. The process of incubation, sampling, filtration,
and the addition of denatured DNA was repeated 4 times. The
results are summarized in Table II.

When the data of Table I is compared with that in Table II,
it can be seen that the isolated messenger RNA hybridizes with
denatured DNA more readily than the unfractionated pulse
labeled RNA. After five incubations, a total of 83% of mes-
senger RNA has hybridized with denatured DNA while only 48.8%
of the pulse labeled RNA has hybridized. The greater ease
with which messenger RNA hybridizes with denatured DNA is
probably due to presence labeled ribosomal RNA which is present
in the pulse labeled material (12) and which competes with

the cellular pool of ribosomal RNA for hybridization with DNA.

-hg-

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

TABLE I. HYBRIDIZATION OF MESSENGER RNA
Incubation (3H)RNA in Reaction (3HIRNA-DNA Hybrid 5 Radio-
Mixture Counts/min/ml activity
in ,
Hybrid
TCA Insoluble Counts/ 0 hr 4 hr
min/ml
1 20,820 0 5,190 20.1
2 11,240 180 2,670 17.8
3 7,000 80 1,435 12.7
4 3.850 85 935 12-7
5 2,410 125 505 15.9
TABLE II. HYBRIDIZATION 0F PULSE LABELED RNA
Incubation (3H)RNA in Reaction (3H)RNA-DNA Hybrid % Radio-
Mixture Counts/min/ml activity
in *
Hybrid
TCA Insoluble Counts/ 0 hr 4 hr
_# min/ml
1 16,760 200 2.790 12.5
2 9,030 120 1,070 8.5
3 6,110 290 585 7.7
4 4,100 95 590 9.8
5 2,620 50 383 10.3

 

*Zero time values were substracted from the 4 hour values
before calculating the percentage of radioactivity in the

hybrid.

The net count/min in the hybrid was multiplied

by 0.803 (To correct for greater efficiency in counting
( H) on nitrocellulose membranes as compared to TCA
precipitable counts) before dividing by the input TCA
precipitable counts.

-49-
PART II. STUDIES OF METHYLATION OF RNA IN T-4 INFECTED CELLS.

Characterization of (éE) methyl labeled RNA from T-4
infected cells. The specific activity of (3H) methyl labeled
RNA from T-4 infected cells was 15,000 cpm/mg. The Specific
activity of (3H) methyl labeled RNA from T-4 infected cells
treated with chloramphenicol was 53,000 cpm/mg. 93-95% of the
radioactive TCA insoluble material was alkali labile (0.3 M
KOH, 37°C, 18 hrs) and was also degraded by ribonuclease.

The 5-7% of the material which is alkali stable is not degraded
by a mixture of deoxyribonuclease and venom phOSphodiesterase.
This radioactive material is assumed to be a protein contam-
inant.

To demonstrate that the radioactivity is present in the
methyl groups of RNA and not due to a labeling of the purine
and pyrimidine bases themselves, a sample of RNA from the
T-4 infected, chloramphenicol treated cells was hydrolyzed
by alkali and analyzed by paper electrophoresis at pH 2.8.

The RNA from the chloramphenicol treated cells was used in
this experiment because its specific activity is greater than
that of RNA prepared from infected cells not treated with
chloramphenicol. The two RNA's are considered to be equiv~
alent since their MAK elution profiles are identical (see
below).

The results are presented in Figure 10. Some radioactive
material is observed at the origin. No appreciable radio»
activity is found in the cytidylic acid and adenylic acid
region. A second radioactive compound moves somewhat faster

than adenylic acid. Additional radioactive compounds are

-50-

 

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Soapmaaapaaom m S« oopSSoo use Honda scans one Bonn pdo macs
So m.a N m.H monmddm .m.m mm us was N 90% mpaob comm pm
cmSHomHoa was mamonosaonpocam .Aoaca as m seamen: so coppoam

mes oaascm .mouapocaods mo mamonosaonpocam .oa oasmam

-51-

on

mm

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cu

n.

O.

 

 

 

a:

 

 

 

wooz< II

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Figure 10

-52-
observed in the guanylic acid and uridylic acid regions.
ElectrOphoresis followed by solvent chromatography in the
second dimension (49) suggests that the radioactive compounds
in the Gp and Up regions are the methylated derivatives of
guanylic acid, and thymidylic acid.

Sucrose gradient sedimentation analysis of (3H) methyl
labeled RNA from.T-4 infected celEAAI The sucrose gradient
sedimentation profile of (3H) methyl labeled RNA from T-4
infected cells is given in Figure 11a. Approximately 70%
of the labeled RNA sediments at 48. The remaining labeled
RNA sediments faster--exhibiting a wide range of sedimenta-
tion velocities. For comparison, the sedimentation profile
of T-4 specific RNA labeled with (BB) uracil is presented in
Figure 11b.

3 meth 1 labeled R o -

chromato ra h of

infected cells. The MAK elution profile of (3H) methyl

 

labeled RNA is presented in Figure 12. About 60% of (3H)
methyl labeled RNA elutes in the soluble RNA region. The
remaining 40% of the radioactive RNA elutes at higher salt
concentrations. In the soluble RNA region, the radioactive
profile, a measure of soluble RNA methylated during the
labeling period, and absorbancy profile, a measure of total
soluble RNA, are different-swith the specific activities in
this region differing by greater than lOO-fold. The elution
profile given in Figure 12 has been reproduced more than 6
times on two different RNA preparations. The MAK elution
profile for (3H) methyl labeled RNA from.T-4 infected cells
treated with chloramphenicol (not Shown) is identical to

that presented in Figure 12.

-53-
Figure 11. Sucrose gradient centrifugation of labeled RNA
from T-4 infected cells.

Figure 11a. Sucrose gradient centrifugation pattern of
(3H) methyl labeled RNA from T-4 infected cells. The
dashed line indicated absorbancy at 260 my and the solid
line, radioactivity. The three peaks in the absorbancy
profile correSpond to 23 S and 16 S ribosomal RNA and to
soluble RNA (left to right). Centrifugation was for 5%

hours at 39,000 rpm.

Figure llb. Sucrose gradient centrifugation pattern of
(3H) uracil labeled RNA from T-4 infected cells. Condi-

tions were as in Fig. 11a.

 

-54-

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.57-
Any (3R methyl) methionyl-soluble-RNA or (3H methyl)
methionine containing peptidyl-soluble-RNA present in the
RNA preparations would contribute to the radioactive profile
in the soluble RNA region. However, these compounds have not
been detected in the RNA preparations. Following treatment
of the RNA under conditions which would cleave either acyl
bond to soluble RNA (pH 8.8, 05 minutes, 35° and pH 10.0,
60 minutes, 37°), the TCA insoluble radioactive content is
unchanged and the elution pattern in the Soluble region is
essentially unaltered.

AAA chromatography of (3H) methyl labeled RNA from
Epinrected cells. The MAK elution profile of (3H) methyl

 

 

labeled RNA from uninfected cells is presented in Figure 13.
Radioactivity is observed in both the soluble and ribosomal
RNA region. The radioactive profile and the absorbancy
profile in the soluble RNA region are different. A comparison
with the elution profile of (3H) methyl labeled RNA from
infected cells (Fig. 12) reveals that the methylation pattern
of the two RNA's in the soluble RNA region are distinctly
different. Further, little or no methylation of ribosomal
RNA occurs in T-4 infected cells.

EAEEQhromatography of_(?E) uracil labeled RNA from T-4
infected cells. Methylation of soluble RNA in infected cells
could occur on pre-existing host soluble RNA'or on soluble RNA
synthesized Eg,ggyg. Although soluble RNA synthesis in T-even
infected cells supposedly does not occur, (60) an experiment
was designed to determine if any soluble RNA synthesis could
be detected under conditions where methylation of soluble RNA

was observed.

-58-

 

 

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FRACTION NUMBER

-60 -

(3R) uracil labeled RNA was prepared from infected cells
and analyzed by MAK chromatography. The elution profile is
shown in Figure 14. About 6% of the radioactive material
elutes in the soluble RNA region suggesting that soluble RNA
synthesis may occur in infected cells. A second fraction,
amounting to about 6% of the radioactive material elutes
immediately after the soluble RNA region. This material has
not been identified. The radioactive RNA's eluting in the
ribosomal RNA region probably correspond to T-4 specific
messenger RNA II, III, and IV described by Ishihama, 33. 21-
(61).

Hybridization of RNA from T-4 infected cells with
denatured T-4 DNA. 4-5.5% of the (3R) methyl labeled RNA

 

purified from infected cells reacts with denatured T-4 DNA
immobilized on a membrane filter to form ribonuclease resist-
ant product (Table 3). About one half of the product is T-4
Specific RNA-DNA hybrid. The remainder results from non-
specific adsorption (noise) of radioactivity to the filter.
The noise level was evaluated by treating a sample of RNA
with ribonuclease before incubation in the hybridization
test. About 2.2% of the radioactivity of the ribonuclease
degraded RNA sample is still adsorbed on the filter in the
hybridization test. This material is assumed to be radio-
active protein contaminating the (3H) methyl labeled RNA
preparation. (3H) methyl labeled RNA does not react with
denatured E. 22A;_DNA.

(3H) uracil labeled RNA from infected cells reacts with
denatured T-4 DNA in the hybridization test (Table 4).

Pretreatment of the RNA with ribonuclease stops completely

-6l-

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-62-

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Figure 14

III III Ill-II.|II|I llIla-II‘I I ’III. I.
II: I 1 III-ll

-53-

TABLE III. HYBRIDIZATION 0F (3R) METHYL LABELED RNA

 

(3R)RNAaDNA %(3H)RNA

 

 

 

(3H) Methyl Denatured
Labeled RNA DNA Hybrid in
Hybrid
Counts/min/ 125 Pg/Filter Counts/min
Reaction Tube 0 ar 2 hr
Exp. 1* 3618 T-4 29 172 3.95
EXp. 2 3246 T-4 7 182 5.48
Ribonuclease Pretreated T-4 6 73 2.06
Exp. 3 6492 T-4 24 302 4.56
Ribonuclease Pretreated T-4 10 153 2.20
Exp. 4 3246 E. coli 8 47 1.20
Exp. 5 6&92 E0 0011 0 100 1056‘

 

 

 

 

 

 

*Experiment 1 is the average
preparation of (3H) labeled

RNA.

of 5 trials with a single

Egperiments 2-5 were performed on a second preparation of
( H) methyl labeled RNA.

 

 

 

TABLE IV. HYBRIDIZATION 0F (3R) URACIL LABELED RNA
(3R) Uracil Denatured (3R)RNA-DNA %(3R)RNA
Labeled RNA DNA Hybrid in
Hybrid
Counts/min/ 125 pg/Filter Counts/min
Reaction Tube hr 2 hr
Exp. 1 28,780 T-4 23 7,357 25.48
Ribonuclease Pretreated T-4 29 34 0
Exp. 2 28,780 E. coli 6 73 0.23

 

 

 

 

 

 

-624...
subsequent hybrid formation. No Significant reaction is
observed between (3H) uracil labeled RNA and denatured
E. ggAA.DNA.

W

Methylation of RNA occurs in T-4 infected cells. About
60-70% of the RNA methylated is soluble RNA. The other 30-40%
is RNA's of diverse size which sediment faster than 48 and
which elute from a MAK column at salt concentrations greater
than that required to elute soluble RNA. Little or no methyla-
tion of ribosomal RNA occurs in T-4 infected cells.

The methylation pattern of soluble RNA in infected cells
is distinguishable from the pattern in uninfected cells. The
MAK elution profile of soluble RNA.methylated in infected
cells is different from the elution profile of total soluble
RNA (measured as the absorbancy profile) and is different
from the elution pattern of soluble RNA methylated in uninfec-
ted cells.

Methylation of soluble RNA in infected cells may occur
on soluble RNA made Eg,ggyg and/or may occur on pre-existing
cellular soluble RNA. Soluble RNA synthesis has not previously
been observed in T-even infected cells (60), however, in an
eXperiment described above, radioactive RNA which elutes from
a MAK column in the soluble RNA region was extracted from
T-4 infected cells labeled with (BB) uracil. This radio-
active RNA was not synthesized by uninfected cells present
in the culture. Infection of the culture was complete as
evidenced by the fact that none of the (3H) uracil labeled
RNA reacts with denatured E. EQAA,DNA in the hybridization

-65...
test (Table IV). The radioactive RNA may represent soluble
RNA synthesized gg.ggyg_in the infected cell or, may not be
soluble RNA at all, but rather fragments of T-4 Specific
messenger RNA produced by degradation of messenger RNA
during the purification procedure. However, the presence of
T-4 Specific messenger RNA in the (3H) uracil labeled RNA
preparation whose sedimentation profile appears normal (with
some RNA sedimenting faster than 23 S) and whose MAK elution
profile at the higher salt concentration is similar to mes-
senger RNA II, III, and IV fractions obtained by Ishihama,
23. 3A. (61), argues against degradation of messenger RNA
as the source of radioactive RNA eluting in the soluble RNA
region. Experiments are in progress to characterize more
completely this radioactive RNA. In the connection that
this RNA.may represent soluble RNA synthesized gg_ggy2_in
T-4 infected cells, it is interesting to note that Earhart
and Neidhardt (62) recently observed the appearance of a
valyl soluble RNA synthetase in infected cells.

Another possibility is that all or part of the methyla-
tion of soluble RNA in infected cells may occur on cellular
soluble RNA.= Modification of host soluble RNA by methyla-
tion may play some role in the control of protein synthesis
at the translation level. However, modification of host
soluble RNA might not effect its capacity to be charged by
host amino acyl synthetases. Sueoka and Kano-Sueoka (63)
observed that the amino acyl charging patterns of 16 soluble
RNA's (leucyl soluble RNA was an exception) prepared from
uninfected cells and T-2 infected cells were identical when

charged with amino acyl synthetases from uninfected cells.

-66-

Hybridization studies have Shown that RNA methylated in
T-4 infected cells reacts with denatured T-4 DNA but not
with denatured E. ggAA,DNA. Preliminary experiments, using
RNA fractionated by sucrose gradient centrifugation, indicates
that the methyl labeled RNA which hybridizes with T-4 DNA
sediments faster than 4 S. This hybridizable RNA may be
methylated T-4 Specific messenger RNA. Hybridization between
any soluble RNA synthesized gg_ggygland host soluble RNA
modified by methylation with their reSpective template DNA'S
would not be observed because of the low Specific activity
of the labeled RNA's and the small number of the complementary

sites on the DNA genome.

7.

8.

9.

10.

11.

12.

13.

14.

15.
16.

17.

18.

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