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Mitochondrial RNA synthesis and ribonucleotide incorporation studies in Euglena gracilis

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Title:
Mitochondrial RNA synthesis and ribonucleotide incorporation studies in Euglena gracilis
Creator:
Brown, George Erwin, 1939-
Publication Date:
Language:
English
Physical Description:
xii, 109 leaves : ill. ; 28 cm.

Subjects

Subjects / Keywords:
Chloroplasts ( jstor )
DNA ( jstor )
Enzymes ( jstor )
Euglena ( jstor )
Genes ( jstor )
Mitochondria ( jstor )
Molecular weight ( jstor )
Ribonucleosides ( jstor )
Ribonucleotides ( jstor )
RNA ( jstor )
Dissertations, Academic -- Microbiology -- UF
Euglena gracilis ( lcsh )
Microbiology thesis Ph. D
Mitochondria ( lcsh )
RNA ( lcsh )
Genre:
bibliography ( marcgt )
non-fiction ( marcgt )

Notes

Thesis:
Thesis--University of Florida.
Bibliography:
Bibliography: leaves 98-107.
General Note:
Typescript.
General Note:
Vita.
Statement of Responsibility:
by George Erwin Brown.

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University of Florida
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University of Florida
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Copyright [name of dissertation author]. Permission granted to the University of Florida to digitize, archive and distribute this item for non-profit research and educational purposes. Any reuse of this item in excess of fair use or other copyright exemptions requires permission of the copyright holder.
Resource Identifier:
025798379 ( ALEPH )
03329329 ( OCLC )
AAV1741 ( NOTIS )
AA00004917_00001 ( sobekcm )

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Full Text













MITOCHONDRIAL RNA SYNTHESIS AND RIBONUCLEOTIDE INCORPORATION STUDIES
IN Euglena gracilis










by








GEORGE ERWIN BROWN


A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF THE UNIVERSITY
OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY





UNIVERSITY OF FLORIDA
1976




MITOCHONDRIAL RNA SYNTHESIS AND RIBONUCLEOTIDE INCORPORATION STUDIES
IN Euglena gracilis
by
GEORGE ERWIN BROWN
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF THE UNIVERSITY
OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1976


This dissertation is dedicated to my wife, Carolyn M. Brown, my parents,
Mr. and Mrs. Marshall V. Brown, and my children, George E. Brown and
Erika N. Brown, for their endless encouragement and personal sacrifices
during the time I spent in graduate school.


ACKNOWLEDGEMENTS
The author wishes to express his gratitude to the members of his
doctoral committee for their advice, guidance and assistance in the
preparation of this dissertation. Special acknowledgements of thanks
are given to the author's major professor and advisor, Dr. J. F. Preston,
for providing the laboratory facilities and supplies for conducting the
research reported here. The laboratory techniques learned in his labora
tory have already proved to be rewarding for the author in his pursuit
of other research projects. The use of Euglena gracilis as a test organism
for studying organelle development originated with Dr. Preston. He con
tributed much of his normal off duty time offering advice and suggestions
toward the completion of this study.
The author also thanks Dr. R. F. Mans for the personal interest he
took in the author's research and academic development. The advice and
guidance he provided proved to be valuable in pursuing the study of
nucleotide incorporations. The consultations held with Dr. Mans provided
the stimulation to complete this research rather than to terminate it.
In addition the knowledge obtained in the "Rusty Mans Molecular Journal
Club" was most rewarding in terms of the author's academic development.
Expressions of gratitude are also in line for Dr. L. 0. Engram for
his encouragements when things looked bad and for his valuable suggestions
that led to the completion of this research. Dr. Engram provided chemicals
and laboratory facilities for conducting several experiments.
iii


Dr. E. M. Hoffmann will be long remembered as the author's first
graduate instructor and for inspiring the author's interest in microbiology.
The author also thanks him for his assistance in the preparation of this
dissertation and for providing laboratory equipment for conducting this
research.
The author wishes to express his gratitude to Dr. T. W. O'Brien for
serving as the mitochondria expert and for providing advice and guidance
in the initial phase of this research. The consultations held with
Dr. O'Brien provided the author with the assistance and encouragements for
completing this dissertation.
iv


TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS iii
LIST OF TABLES vi
LIST OF FIGURES vii
KEY TO ABBREVIATIONS viii
ABSTRACT x
INTRODUCTION 1
LITERATURE REVIEW 8
MATERIALS AND METHODS 24
RESULTS 34
DISCUSSION 91
CONCLUSIONS 97
REFERENCES 98
BIOGRAPHICAL SKETCH 108
v


LIST OF TABLES
Page
1. DISTRIBUTION OF SUCCINIC DEHYDROGENASE ACTIVITY 41
2. NUCLEOTIDE INCORPORATION BY ISOLATED MITOCHONDRIA 42
3. INCORPORATION OF RIBONUCLEOSIDE TRIPHOSPHATES BY ISOLATED
MITOCHONDRIA 46
4. THE DEPENDENCY OF THE MITOCHONDRIAL ACTIVITY INCORPORATING LABEL
FROM 3H-CTP UPON ADDED RIBONUCLEOTIDES AND DNA 47
5. DISTRIBUTION OF 3H-CMP INCORPORATING ACTIVITY 48
6. INHIBITION OF 3H-CMP INCORPORATION 50
7. CHARACTERIZATION OF PRODUCT 51
8. PHOSPHOLIPID EXTRACTION OF THE H-CMP LABELED PRODUCT 60
9. A COMPARISON OF THE INCORPORATION OF 3H-UTP AND 3H-CTP BY
ISOLATED MITOCHONDRIA PURIFIED BY TREATMENT WITH DNASE 1 71
10. RNA POLYMERASE ACTIVITY SOLUBILIZED FROM MITOCHONDRIA 73
11. SUCCINIC DEHYDROGENASE ACTIVITY IN THE MITOCHONDRIAL SUPERNA
TANTS AND PELLETS AFTER SOLUBILIZATION TREATMENT 75
12. THE EFFECT OF AMMONIUM SULFATE PRECIPITATION ON MITOCHONDRIAL
RNA POLYMERASE ACTIVITY 77
3 3
13. A COMPARISON OF TRITIUM INCORPORATION FROM H-UTP AND H-CTP
BY THE ACTIVITY IN THE DEAE-SEPHADEX A-25 COLUMN FRACTION 3 82
14. THE DEPENDENCY OF THE DEAE COLUMN FRACTION 38 INCORPORATION OF
3H-UMP UPON ADDED RIBONUCLEOSIDE TRIPHOSPHATES AND DNA 83
15. INHIBITORS OF 3H-UMP INCORPORATION BY THE DEAE COLUMN FRACTION
38 84
16. TEMPLATE SPECIFICITY OF THE DEAE COLUMN FRACTION 38 90


LIST OF FIGURES
Page
1. SCHEMA FOR THE ISOLATION OF MITOCHONDRIA 35
2. DENSITY EQUILIBRIA ANALYSIS OF MITOCHONDRIAL DNA 38
3. RIBONUCLEOSIDE TRIPHOSPHATES INCORPORATION BY ISOLATED
MITOCHONDRIA 43
4. SUCROSE GRADIENT ZONAL CENTRIFUGATION 53
5. BIOGEL P-4 COLUMN CHROMATOGRAPHY 55
6. ULTRA VIOLET ABSORBANCY SPECTRA 58
7. THIN LAYER CHROMATOGRAPHY OF THE 3H-CMP LABELED PRODUCTS ELUTED
FROM THE BIOGEL P-4 COLUMN 61
8. THIN LAYER CHROMATOGRAPHY OF THE PRODUCTS OF THE ENZYMATIC
CLEAVAGE OF THE 3H-CMP LABELED MATERIAL ELUTED FROM THE
P-4 COLUMN 64
9. THE MIGRATION OF THE ENZYMATIC CLEAVAGE PRODUCTS CONTAINING
TRITIUM LABEL ON PEI CELLULOSE 66
10. DENSITY EQUILIBRIA ANALYSIS OF DNA FROM DNASED MITOCHONDRIA 69
11. PROCEDURE FOR THE PARTIAL PURIFICATION OF MITOCHONDRIA RNA
POLYMERASE 78
12. ION EXCHANGE CHROMATOGRAPHY ON DEAE-SEPHADEX A-25 80
13. DEPENDENCE OF THE ACTIVITY OF THE DEAE FRACTION 38 ON THE
CONCENTRATION OF Mn++ 86
14. DEPENDENCE OF THE ACTIVITY OF THE DEAE FRACTION 38 ON THE
CONCENTRATION OF Mg++ 88
vii


KEY TO ABBREVIATIONS
A260
absorbancy at 260 nm
A280
absorbancy at 280 nm
AMP
adenosine monophosphate
ATP
adenosine triphosphate
ATPase
adenosine triphosphatase
BGP- 1
the first peak of tridium labeled material
eluted from Biogel P-4 column
BGP- 2
the second peak of tridium labeled material
eluted from Biogel P-4 column
BSA
bovine serum albumin
CAP
catabolite gene-activator protein
CDP-diglyceride
cytidine diphosphate diglyceride
CMP
cytidine monophosphate
CpC
cytidylyl (3' 5) cytidine
cpm
counts per minute
CTP
cytidine triphosphate
dCTP
deoxycytidine triphosphate
cyclic AMP
adenosine 3' : 5'-cyclic monophosphoric acid
DEAE
diethylaminoethane
DNA
deoxyribonucleic acid
DNase
deoxyribonuclease (EC 3.1.4.5)
dpm
disintegrations per minute
DTT
dithiothreitol
viii


EDTA
ethylenediamine tetraacetate
&
gravity
GMP
guanosine monophosphate
GTP
guanosine triphosphate
hr
hour
3h
radioactively labeled with tridium
I
Iodine
NTPs
ATP, CTP, GTP, UTP (minus the labeled substrate)
poly d(AT)
alternating copolymers of deoxyadenylic and
deoxythymidylic acid
poly (C)
poly cytidylic acid
POP
2, 5 diphenyloxazole
POPOP
1, 4 di- [2-(5-phenyloxazolyl)]-benzene
RNA
ribonucleic acid
Hn RNA
heterogeneous nuclear ribonucleic acid
in RNA
messenger ribonucleic acid
r RNA
ribosomal ribonucleic acid
t RNA
transfer ribonucleic acid
RNase
ribonuclease (EC 2.7.7.16)
S
sedimentation coefficient
SDS
sodium dodecyl sulfate
SSC
standard saline citrate
SVP
snake venom phosphodiesterase
TCA
trichloroacetic acid
tris
tris (hydroxymethyl amino) methane
UMP
uridine monophosphate
UTP
uridine triphosphate
UV
ultra violet
ix


Abstract of Dissertation Presented to the Graduate Council of the
University of Florida in Partial Fulfillment of the Requirements for
the Degree of Doctor of Philosophy
MITOCHONDRIAL RNA SYNTHESIS AND RIBONUCLEOTIDE
INCORPORATION STUDIES IN
Euglena gracilis
By
GEORGE ERWIN BROWN
August, 1976
Chairman: James F. Preston, Ph.D
Major Department: Microbiology
Two experimental approaches were utilized to study the in vitro
mitochondrial RNA synthesis in Euglena gracilis. First the RNA syn
thesis activity was examined in isolated and highly purified mitochondria
of Euglena gracilis strain z streptomycin bleached aplastic mutants. The
mitochondria obtained after isopynic centrifugation in sodium diatrizoate
3
gradients yield mitochondrial DNA (p=1.691 g/cm ) and little nuclear DNA
3 3
(p=l.707 g/cm ). The incorporation of label from the H-ribonucleoside
triphosphates into acid insoluble products showed no dependence upon the
presence of the other ribonucleotides or that of added DNA. The preferred
3
substrate was H-CTP from which label was incorporated into acid insoluble
products at a rate 100 times greater than the other labeled ribonucleotides.
This activity incorporates CMP from CTP into acid insoluble products
x


which contain phosphodiester bonds by a pyrophosphorolysis reaction. The
activity was inhibited by pyrophosphate and actinomycin D at 100 mg/ml but
not by inorganic phosphate, a-amanitin, rifampicin or low concentrations of
actinomycin D (50 mg/ml). The products were sensitive to snake venom phos
phodiesterase and alkaline hydrolysis but not pancreatic ribonuclease. The
products were isolated by phenol extraction followed by ethanol precipita
tion of the aqueous phase. Two classes of molecules with mean molecular
weights of 800 and 680 respectively were isolated. They were found to have
absorbancy spectra identical to that of C^C and were resistant to pancreatic
ribonuclease digestion. However, snake venom phosphodiesterase cleaved the
products into 5'-CMP, the expected cleavage product of C^C or poly C.
Mitochondria, purified by either isopycnic centrifugation or by treat
ment with pancreatic deoxyribonuclease, contained an associated activity
3
which incorporated label from H-UTP into acid insoluble products which
were sensitive to pancreatic ribonuclease digestion. This activity was
DNA dependent in the DNase mitochondria which was in contrast to that
observed for sodium diatrizoate purified mitochondria.
The second approach was to. study the activity of solubilized and
partially purified mitochondrial RNA polymerase. An enzyme purification
procedure is described utilizing a solution of Triton X-100 detergent and
KC1, centrifugation, ammonium sulfate precipitation, and DEAE-Sephadex A-25
chromotography that resulted in a partially purified enzyme with a specific
activity of 0.3 nmoles UTP/mg/10 min at 37. The incorporation of UMP into
acid insoluble material required DNA the four ribonucleoside triphosphates:
GTP, CTP, UTP, ATP, and metal, either Mn"*""1" or Mg"*-1". The product of the
reaction was sensitive to pancreatic ribonuclease digestion. The mito-
xi


chondrial enzyme is distinctly different from the nuclear RNA polymerase II
with respect to chromatographic behavior and antibiotic sensitivity. It
eluted from the DEAE-Sephadex column in a single peak between 0.32 M and
0.37 M ammonium sulfate in TGMED buffer, was stimulated by low concentration
| |
of the metals Mn and was insensitive to a-amanitin and rifampicin.
/
Chairman
xii


INTRODUCTION
Informational macromolecules are those through which genetic in
formation actually flows. Genetic information may be defined as that
primarily required to assemble a protein needed to perpetuate biological
order, but the definition applies also to the information for ribonucleic
acids (RNAs) which are not translated into protein. Deoxyribonucleic
acids (DNA) functions as the repository for information or coding se
quences ultimately destined to appear either in RNA or protein, and in
noncoding sequences which can act as signals for such things as initiation
and termination of the biosynthesis of a RNA chain. Within the cell the
informational sequences are transmitted on which specific proteins are
synthesized.
The DNA dependent RNA polymerases (E. C. 2.7.7.6.) are the key
enzymes implicated in the first step of genetic information flow from DNA
to protein. The term DNA dependent RNA polymerases is used to designate
enzymic activities which, using DNA templates, catalyze the sequential
assembly of the four ribonucleoside triphosphates into RNA molecules.
The synthetic reaction requires the presence of a DNA or a polydeosy-
ribonucleotide template and a divalent metal ion. The main features of
the transcription reaction consist of the enzyme locating and binding to
specific sites on the DNA, initiating a RNA chain de novo, extending the
chain by moving along the DNA using only one strand as a template to
direct the polymerization of the four ribonucleoside triphosphates into
1


2
a complementary RNA molecule, and finally, in some cases, termination of
the chain with the release of the enzyme from the DNA template. RNA
polymerases are responsible for the synthesis of ribosomal RNA's, transfer
RNA's and messenger RNA's, all of which are needed for translation of the
mRNA template into proteins.
Substantial data have been accumulated on the regulation of RNA
polymerases in prokaryotes, where a single enzyme is responsible for the
synthesis of all types of cellular RNA, but comparatively little is
known in the case of eukaryotes, where the situation is more complex.
Within the last few years, it has been demonstrated that eukaryotic cells
contain several types of RNA polymerases, which differ in their locali
zations, structures, and functions.
Nuclei contain several different RNA polymerases. Nuclear RNA
polymerase I is localized in the nucleolus (1). It is primarily con
cerned with the transcription of genes specifying the larger (45s)
ribosomal precursor RNA. Nuclear RNA polymerase II is localized in the
nucleoplasm and presumably transcribes messenger RNA. It is inhibited
by a-amanitin (2,3) an octapeptide produced by several species of the
genus Amanita (4). In several organisms a third (5,6) or even a fourth
(7,8) nuclear RNA polymerase activity was found. A separate RNA poly
merase III, which is distinct from forms I and II, has been shown to
transcribe (4s) t-RNA and 5s RNA (9,10). In mammals this enzyme is
slightly sensitive to a-amanitin, being 1,000 fold more resisistant than
RNA polymerase II.
RNA polymerase activity has also been found localized in mito
chondria and chloroplasts. The mitochondrial enzymes exhibit properties


3
which clearly distinguish them from the nuclear RNA polymerases. The
mitochondrial enzyme is insensitive to a-amanitin and although some
functional differences have been described in mitochondrial RNA poly
merase, there is general agreement that the molecular weight of the
enzyme, consisting of a single polypeptide chain, is about 60,000
daltons (11-15).
Control of RNA synthesis for regulating cell
growth, development, and differentiation
If considerations were based purely on energy requirements, it
would be reasonable to exclude DNA replication and mRNA translation as
primary levels where gene expression is controlled. Several reasons may
be given for excluding changes in the kinds or numbers of genes and post-
transcriptional modification as steps at which regulation generally takes
place. Nuclei of cells in different stages of development in the same
individual appear to be genetically identical and changes in the number of
gene copies from which mRNA is transcribed have not been observed. Like
wise, there is no good evidence that changes in the kinds of proteins
synthesized in development can be attributed to controls at the trans
lational level and in addition eukaryotic cells have no mechanism for

excluding the translation of messages characteristic of other cell
types (16). It seems that translational controls are used to make quanti
tative adjustments to a pattern of protein synthesis determined primarily
by the synthesis of new messages.
It can be concluded by elimination that transcription must be the
level at which gene expression is primarily controlled. In bacterial
systems, there are two well-characterized examples of programmed trans-


scription: bacteriophage development and bacterial sporulation. These
processes are similar in some ways to differentiation. Therefore, under
standing the control of transcription in these processes may provide some
clues to the regulation of transcription in eukaryotic systems. Studies
of transcriptive regulation of phage A show how the expression of specific
genes is regulated by other gene products (17). In both T^ bacteriophage
development and Bacillus subtilis sporulation, significant changes that
are coordinated with development occur in the RNA polymerase molecule
and associated proteins (18,19,20). In _B. subtilis there is evidence
suggesting that these changes may be related to the onset of sporulation.
The primary need for regulating transcription is that, by turning
it on and off, protein synthesis is controlled at the origin. In both
prokaryotic and eukaryotic organisms regulation must enable cells to
cope with fluctuation in their immediate environment. In the prokaryote
most of the genes come into operation sooner or later during each cell
cycle, but this is not true of many genes in the higher eukaryote where
differentiation has resulted in a strategy of regulation unlike that in
the prokaryote. By contrast the lower unicellular eukaryote has a re
gulatory resemblance to the prokaryote. Evidence that gene transcription
is controlled in eukaryotic development is provided by the Balbiani rings
of polytene chromosomes. The ring is characteristically expanded and
active in RNA synthesis or unpuffed and inactive in transcription at
different stages of development (19). Also, agents such as actinomycin
which inhibit RNA synthesis but which have little immediate effect on
translation arrest eukaryotic development within a short time. These
effects suggest an essential role for the regulation of transcription in
development.


5
Regulation of transcription by alteration
in molecular properties of RNA polymerase
The regulation of transcription may be affected by control of any
of the components involved in the RNA synthesis: the template, the poly
merase, associated factors and other environmental conditions. However,
the multiplicity and different intranuclear localization of DNA-dependent
RNA polymerases have suggested that gene expression in eukaryotic cells
is regulated, in part, by distinct RNA polymerases with different template
specificities. The quantity and quality of RNA synthesis can depend
either on the concentration of RNA polymerase or on the level of activity
of the enzyme. In growing _E. coli cells, there is a pool of excess RNA
polymerase (21) although the polymerase subunits seem to be synthesized
at the same rate as other cellular proteins (22). Therefore, the regula
tion of growth depends upon modulation of the RNA polymerase activity
rather than upon the concentration of the enzyme. Although the rRNA
genes account for only about 0.4% of the genome (23), rRNA accounts for
40% of the total RNA synthesized in growing cells and is therefore
preferentially synthesized (24) .
On the other hand, bacteriophage infection results in the inhibition
of host cell transcription presumably by the destruction or alteration of
the host RNA polymerase (25,26). Similar effects on host RNA synthesis
have been found in viral infections in mammalian cells (27,28). This
may be analogous to terminal differentiation where the loss of nuclear
function is involved (29)
Regulation of transcription and control of
organelle development
An understanding of the mechanisms by which a eukaryotic cell
controls organelle development should give valuable insight into many


6
similar cellular control processes such as those involved in differentia
tion, morphogenesis and tumor production. Since RNA polymerase has been
implicated in controlling development in prokaryotic systems (18,19,25,
30,31) it could be a major controlling factor in Euglena gracilis organelle
development. Therefore, the research in this laboratory focuses upon the
RNA polymerases found in Euglena, and upon their role in organelle de
velopment. Euglena is an excellent organism to study transcription in
organelles since it contains both chloroplasts and mitochondria. The
organism is a unicellular flagellate with 10 to 12 chloroplast per cell,
numerous mitochondria, and a polyploid nucleus. Euglena can be grown
phototrophically, heterotrophically or mixotrophically. These growth
conditions will alter the development of the organelles and therefore
offer a useful system for studying organelle development. Phototrophic
conditions are restrictive for photosynthetic function, but the other
two growth conditions are permissive. In cells grown phototrophically,
mitochondrial development is repressed, while the chloroplasts function
to provide the bulk of the cell's energy. Cells grown mixotrophically
derive energy from functional chloroplasts and mitochondria. However,
wild type cells can be grown in the dark as etiolated heterotrophs
where the proplastids, which have stopped forming chlorophyll and
chloroplast membrane, exist as undeveloped proplastids which are
capable of differentiating into functional mature chloroplasts .upon
exposure to light. The heterotrophic dark grown cells primarily use
mitochondrial oxidative phosphorylation as a source of energy. It has
been shown (32) that cells grown heterotrophically have large active
mitochondria, and that exposure to light, which starts plastid develop-


7
ment, results in a decrease in mitochondrial density and function (33).
Mutants that completely lack plastids (aplastidic) have been produced by
treatment with streptomycin, heat, or ultraviolet light (34,35). These
permanently bleached cells are perfectly viable as long as they are
grown on a carbon source which can be respired. This makes it feasible
to distinguish processes controlling mitochondrial development from those
controlling chloroplast development.
This dissertation concerns itself with studies of mitochondrial RNA
synthesis in Euglena gracilis and is directed towards elucidating the
components of the transcriptional process, in particular, characterizing
the mitochondrial DNA dependent RNA polymerase with a view toward under
standing the control of transcription. The RNA synthesizing activity of
isolated mitochondria has been studied and the DNA dependent RNA polymerase
has been solubilized and partially purified. The experimental approach
in this study was to develop a method for preparing highly purified mito
chondria, to study the incorporation of labeled ribonucleoside triphosphate
precursors into RNA by isolated mitochondria and to identify the labeled
products. The mitochondrial DNA-directed RNA polymerase was partially
purified by ion exchange chromatography. The enzyme activity was charac
terized to determine its requirements for product synthesis and to compare
it to nuclear activities with respect to these requirements.


LITERATURE REVEIW
Guides to the Literature
In recent years, several review articles or symposia on RNA poly
merases from prokaryotes (20,36-46) and eukaryotes (47-52), on the
regulatory elements (53,54) and on transcription (55-59), have appeared.
There are several introductory texts (60-62) and collected papers (63-66)
which may be consulted for a more historical background discussion.
General Mechanisms of RNA Synthesis
The synthesis of RNA from DNA is mediated by a DNA dependent RNA
polymerase that uses ribonucleoside triphosphates as precursors. In
general the reaction involves binding of enzyme to DNA template and
the asymmetric transcription of the DNA. RNA synthesis appears to proceed
in the following steps: template binding, chain initiation, chain elon
gation and termination. The RNA polymerase binds to specific initiation
sites (promoter sites) on the DNA in a specific reaction in which the
strands of the DNA are opened over a short local region. Chain initiation
involves the binding of two ribonucleotide triphosphates to the RNA
polymerase followed by elimination of inorganic pyrophosphate to form a
dinucleotide tetraphosphate. The initial nucleotide retains its triphos
phate while each added ribonucleotide triphosphate has its two terminal
phosphates cleaved as pyrophosphate. The direction of elongation is
from 3' hydroxy to 5' phosphate in the DNA, the RNA being made 5' to 3'
8


in the antiparallel direction. The initial product is complementary to
the region of the DNA employed as template and the RNA may be modified
9
post transcriptionally.
Prokaryotic Transcriptions
Background
There are several reviews of prokaryotic transcription available
(35,36,56,57) which may be consulted for discussions of aspects that
will not be considered here. The basic transcriptional studies have
been carried out in bacterial systems with and without phage infection
and these studies have served as a model system for understanding the
transcriptional process and its control. Only the properties of the
bacterial RNA polymerase and the regulation of bacterial RNA synthesis
will be reviewed here.
Prokaryotic RNA polymerase
The bacterial RNA polymerases are large molecules (molecular
weights between 400,000 and 500,000) and have complex subunit structures.
Two enzymatically active forms of the RNA polymerase are currently known:
the holoenzyme and the core enzyme. The holoenzyme contains the following
polypeptide chain subunits: one beta prime (3') subunit; one beta (3)
subunit; two alpha (a) subunits; and either one sigma (o) subunit or one
sigma prime (o') subunit. The subunits have the molecular weights of
approximately 160,000; 155,000; 90,000; and 40,000 respectively. Two
forms of the holoenzyme with the structures ct233'ci(67) and a233'o'(68)
have been found in Escherichia coli. The core enzyme lacks a sigma
subunit. Therefore, the holoenzyme can be separated into two functional
parts: a core enzyme, which is able to synthesize RNA but lacks the


10
ability to initiate such synthesis specifically; and a sigma subunit,
which acts catalytically to allow the efficient initiation of RNA at
specific promoter sites. Bacterial RNA polymerases from different
species appear to be closely related in subunit structure but show dif
ferences in the sizes of the subunits (42). Some preparations of the
.E. coli enzyme contain a minor component a) (69,70) which has a molecular
weight of about 10,000 and is present in the stoichiometry of two u per
holoenzyme. The subunits of the holoenzyme are functional subunits
since it has been demonstrated that these subunits are essential for the
reconstitution of enzymic activity when the enzyme is reformed from
separated subunits (71,72). The m subunit is not required for recon
stitution of RNA polymerase holoenzyme activity.
All transcription in E. coli is sensitive to inhibitors of the
purified enzyme, such as rifampicin and streptolydigin (73,74), which
block RNA synthesis through interaction with the 3 subunit of the
enzyme (75). Thus, all RNA synthesis appears to depend on an enzyme
complex in which the 3 subunit is functional. The beta subunit is also
altered in enzymes from mutants resistant to rifampicin (71) or
streptolydigin (76-78).
General mechanism of prokaryotic transcription
Transcription is a process involving a series of different bio
chemical events. The holoenzyme is able to recognize and to bind to
specific regions on the DNA template (the promoters) and to undergo a
conformational change, catalyzing the addition of substrate ribonucleo-
side triphosphates to the binding site (79), with the subsequent for
mation of the first internucleotide linkage of ATP or GTP with a second


11
ribonucleoside triphosphate. Inorganic pyrophosphate is eliminated,
resulting in the formation of a dinucleotide tetraphosphate of the general
structure pppPupX. The sigma subunit appears to be released (80,81) and
the core enzyme moves along the DNA template synthesizing the RNA chain
by adding ribonucleoside monophosphates to the 3' hydroxy terminus of the
nascent RNA chain from ribonucleoside triphosphate substrates. When the
RNA polymerase encounters a specific termination site on the DNA template,
RNA chain growth is terminated and the nascent RNA chain and the RNA
polymerase are released from the template. A termination protein factor,
rho, has been implicated in this last step in Eh coli (82). However, the
RNA polymerase can in some instances terminate accurately without rho.
Prokaryotic polymerases and regulation of transcription
Two basic mechanisms, negative control and positive control, appear
to be equally effective in controlling prokaryotic transcription. In
negative control, the regulator gene product (a repressor molecule) in
its active form binds to the operator gene to prevent the transcription
of the operon's structural genes. When the repressor is inactivated by
the specific inducer, transcription of the structural genes may proceed.
Thus the repressor controls RNA synthesis by negatively affecting RNA
polymerase activity with respect to specific sites on DNA. The classic
example of negative control is the lac operon of Eh coli (83). The lactose
operon is under the negative control of the lactose repressor which binds
specifically to the lactose operator (84,85) region of the DNA, thus pre
venting transcription of the DNA past the operator. The expression of
this operon requires induction by a galactoside. The inducers bind to
the repressor and decrease the repressor's affinity for the lactose operator.


12
The histidine operon is another example of regulation by negative
repression, with histidyl-tRNA as the co-repressor necessary for activa
tion of the histidine repressor which binds to the histidine operator
region of the DNA and prevents transcription of the DNA past the operator (86).
In positive control the RNA polymerase has low affinity for the
promoter site, and the controlling molecule in its active form helps the
RNA polymerase to initiate transcription by increasing the enzyme's
affinity for the promotor site. Positive control of gene expression has
been demonstrated for the arabinose operon of E. coli (86). The protein
product of the C gene is a repressor in the absence of the specific in
ducer, arabinose. However, in the presence of arabinose the C gene
protein is converted to an activator which is required for expression of
the arabinose genes.
The sigma subunit of RNA polymerase and the catabolite gene activator
protein (CAP) are examples of protein factors which influence transcription
by increasing the affinity of the RNA polymerase for promoters. Sigma
interacts with the core RNA polymerase and facilitates the binding of the
enzyme to a promoter. CAP, also known as cyclic AMP receptor protein,
plays a role in the regulation of genes subject to catabolite repression.
CAP appears to work in addition to sigma to alter initiation specificity
in the presence of cyclic AMP. Cyclic AMP combines with CAP and produces
an allosteric change in the protein. The cyclic AMP-CAP complex then
binds to the DNA close to the promoter of an inducible operon (i.e.,
lactose, galactose, tryptophan, and histidine) and produces a change in
the DNA so that the RNA polymerase can bind to the specific promoter
region and carry out a round of transcription of the operon in question.
Thus the expression of the lactose operon requires cyclic AMP, CAP, holo-
enzyme and the removal of the repressor from the operator.


13
Alteration of RNA polymerase by phage Infection and sporulation
The structure and selectivity of the bacterial RNA polymerase is
altered under certain conditions of viral infection and sporulation. The
alterations of the selectivity of RNA synthesis occur during lytic growth
of the phage in different stages, giving rise to different transcripts
during the course of phage replication and assembly. The bacteriophage T^
infects E. coli and subsequently host RNA synthesis is inhibited, phage-
specific macromolecular synthesis is initiated and the host cells are
ultimately lysed (87,88). A temporal sequence on phage-specific mRNAs
have been detected (89). The first class of mRNA (immediate early)
appeared immediately after infection, and hybridized exclusively to one
strand of T^ DNA. The second class of mRNA (delayed early) to appear
hybridized to later sequential sites on the same T^ DNA strand. The late
mRNA apparently coded for viral coat protein and it hybridized to the
opposite T^ DNA strand.
The sequence of transcription is mediated by changes in the RNA
polymerase. Sigma factor disappears soon after infection (90) and it is
this loss which is presumably responsible for restriction of the tran
scription of the host genome. There is then a sequential alteration of
each of the subunits of the core RNA polymerase (26,91,92). The subunit
is modified by the covalent attachment of AMP to the alpha subunit. The
RNA polymerase acquires four small T^-specific proteins (93) of 10,000-
25,000 daltons which are coded for by the delayed early genes. Thus the
transcription specificity appears to be induced by modifications in the
polymerase or associated factors.
Bacteriophage lamda can either lysogenize or grow lytically (94,95).
During the lytic growth the host _E. coli RNA polymerase transcribes the


14
phage N and Q genes. The two protein products of these genes then mediate
the further transcription of the lamda DNA (96). It appears that the N
gene product is an antiterminator protein that permits the continued
transcription past a rho dependent termination sequence (97). The Q
gene product is a repressor that binds to the operator region for the
lamda repressor gene, thus preventing the transcription of this gene.
Bacillus subtilis phages SPOL and SP82 show a regulated program of
transcriptional alteration during normal growth (98,99). The infected
cell RNA polymerase contains two proteins not present in the normal RNA
polymerase and the sigma subunit is not required for selectivity (100).
Several observations suggest there are specific changes in the
structure of Bacillus subtilis RNA polymerase during sporulation. The
purified sporulating RNA polymerase lacks sigma factor and contains a
modified 6 subunit of lower molecular weight than the vegetative enzyme
(19,101). In addition, a new polymerase subunit of 60,000-70,000 daltons
appears (102). These modifications appear to be due to the action of
proteases that are present in cells undergoing sporulation, and it is
thought that these changes are related to the alterations in transcrip-
tive specificity that occur during sporulation.
Genetic studies also support a specific role of polymerase in the
sporulation process. Mutants which contain an altered beta subunit fail
to sporulate (101). The evidence form the genetic studies and the bio
chemical studies is consistent with the hypothesis that changes in the
subunit structure of RNA polymerase mediate the changes in transcription
associated with Bacillus subtilis sporulation.


15
Eukaryotic RNA Polymerases
Background
There are several recent review articles on eukaryotic RNA polymerases
(47-52). These may be consulted for discussions that are omitted in this
review. The DNA dependent RNA polymerase activity was first demonstrated
in rat liver nuclei by Weiss in 1955 (103) who partially purified the en
zyme which was firmly attached to DNA. Mans and Novelli (104) were the
first to study a solubilized eukaryotic RNA polymerase in 1964. However
the problem of freeing the RNA polymerase from DNA was more severe for the
other eukaryotic RNA polymerases studied and consequently most studies on
nuclear RNA polymerase activity were carried out on isolated nuclei or
unpurified chromatin until a eukaryotic RNA polymerase was purified in
1965 (105).
Isolated nuclei demonstrated transcription activity which synthesized
mostly GC-rich ribosomal RNA at low ionic strength. This activity was
j |
localized in the nuceolus and stimulated by Mg However, at high ionic
strength mostly DNA-like RNA was synthesized by an activity which was
j |
stimulated by Mn and localized in the nucleoplasm (106-111). The nucleo-
plasmic activity was specifically inhibited by a-amanitin (3).
Multiple forms of nuclear RNA polymerases were demonstrated by Roeder
and Rutter (5) in rat liver and Xenopus laevis. They demonstrated three
types of eukaryotic RNA polymerases which could be separated based on
chromatographic properties, sensitivity to specific inhibitors and sensi
tivity to ions. The first RNA polymerase eluted from a DEAE Sephadex
column (nuclear RNA polymerase I) was found to be of nucleolar orgin,
insensitive to a-amanitin and synthesized a product which hybridized to


16
RNA competitively with ribosomal RNA (rRNA) but not with heterogeneous RNA
(Hn RNA). RNA polymerase II, the second enzyme eluted from the column,
was a nucleoplasmic, a-amanitin sensitive RNA polymerase that synthesized
a product that hybridized to DNA competitively with Hn RNA but not with
r-RNA (32). RNA polymerase III was thought to be nucleoplasmic. Since
then several laboratories have demonstrated that the nucleus, nucleolus
and cellular organelles have unique transcriptional systems (44).
On a protein percentage basis, the amount of RNA polymerase activity
in eukaryotic cells is much lower than in prokaryotes. The RNA polymerases
from calf thymus, which is a tissue rich in the enzyme (112), is only ob
tained in a few milligrams per kilogram of tissue (113-115). This yield,
which is a few orders of magnitude lower than that to Escherichia coli RNA
polymerase (67,70,116) demonstrates one of the difficulties encountered in
studying the regulation of eucaryotic transcription at the molecular level
(studies which require significant quantities of the highly purified enzyme).
The specific activity of the eukaryotic RNA polymerases is in the order of
that of the purified _E. coli RNA polymerase (50,113,114,116).
Mitochondrial transcription
The mitochondrion represents a semi-autonomous organelle within the
eukaryotic cell in the sense that it contains a unique DNA that is geneti
cally active; it is replicated and transcribed within the organelle to
provide products unique from those found elsewhere in the cell. The mito
chondrial DNA has been well characterized physically and chemically (117-
122) and the size of the mitochondrial genome varies from a molecular
weight of 10^ daltons in vertebrates to 11.9 x 10^ daltons in plants
(119,122). DNA with these molecular weights can code for only 5,000 amino


17
acids or about 20 proteins. Therefore, it is impossible for the mitochon
drial genome to code for all of the unique mitochondrial proteins which
must carry out replicative, transcriptive, translative and metabolic func
tions unique to the mitochondrion. All available evidence suggests that
the mitochondrial DNA carries information for the mitochondrial ribosomal
RNA, for at least some mitochondrial tRNAs and for a few mitochondrial
membrane proteins (121,123). Thus mitochondrial DNA is required for the
biogenesis of mitochondria.
The majority of proteins unique to the mitochondrion are coded for
by the nuclear DNA and synthesized on the cytoplasmic ribosomes. Less
than 10% of the mitochondrial proteins are synthesized on mitochondrial
ribosomes; this fraction consists of probably not more than eight species
of hydrophobic proteins that are incorporated into the inner membrane
where they assemble cytochromes, cytochrome oxidase and ATPase (120,
121, 123-126). A few components of the mitochondrial protein synthesizing
system may be coded for by the mitochondrial DNA, although there is no
evidence for the location of the code for the mitochondrial leucyl-tRNA
synthetase (127) and the two mitochondrial peptide chain elongation
factors, G and T (128). However, the prokaryotic characteristics of the
mitochondrial protein synthesizing system makes it difficult to imagine the
code coming from elsewhere in the cell.
The only mitochondrial gene products that have been positively identi
fied are ribosomal RNA and transfer RNA (121,123). Hybridization experi
ments have shown that mitochondrial ribosomal RNA hybridizes specifically
with mitochondrial DNA (129-132) and shares no sequence homology between
some of the mitochondrial 4s transfer RNA and mitochondrial DNA (133,134).
In order to understand the extent to which a mitochondrion depends upon


18
the rest of the cell, a more complete understanding of the mechanisms
which provide unique informational molecules must be examined in detail.
Mitochondrial DNA transcription has been studied in vivo using intact
growing cells and in vitro with isolated mitochondria or solubilized and
partially purified mitochondrial RNA polymerase. These investigations
have provided results which will be useful for the understanding of the
regulatory mechanisms which couple the genetic activities of mitochondria
and nuclei during cell growth and development.
The synthesis of mitochondrial RNA in intact cells has been exten
sively studied in HeLa cells by Attardi and coworkers. Hybridization
experiments of in vivo labeled mitochondrial RNA with the separated H
and L strands of mitochondrial DNA demonstrated that both the H and L
strands are equally transcribed but afterwards the product of the L
strand is almost completely degraded. This means that the problem of
strand selection in mitochondrial transcription could be achieved by
transcribing both strands and rapidly degrading or exporting 98% of the
L-strand transcripts (135-140). The transcription products have been
used to map the mitochondrial DNA; the H-strand contains genes for 9
tRNAs, whereas the L-strand has the two ribosomal RNA genes and also
those for three tRNAs (137-141). Election microscopy of ferritin-
labeled tRNA-mitochondrial DNA hybrids confirmed the location of these
genes (142). Experiments with the HeLa cells also suggest that mito
chondrial messenger RNA is also translated. Mitochondrial RNA containing
poly (A) hybridizes with mitochondrial DNA (142).
Studies of in vitro incorporation of ribonucleotide triphosphates
into high molecular weight RNA have shown that isolated mitochondria


19
are capable, for some time, of carrying out RNA synthesis just as are
mitochondria within the living cell, except that the process appears to
be much slower _in vitro than in vivo. The incorporation of UTP or ATP
generally requires all four ribonucleotide triphosphates and magnesium
(143,144). The incorporation process is considerably faster if the mito
chondria are swollen. Mitochondrial RNA synthesis is inhibited by
actinomycin D, which binds to the DNA template, proving its dependence
on DNA. The product of the Jui vitro incorporation reaction hybridizes
with the H strand of mitochondrial DNA (145). In Xenopus mitochondria
the RNA polymerase initiates at several different sites of the template
predominantly with ATP and 2-fold less frequently with GTP (146).
Studies on mitochondrial RNA synthesis have recently concentrated
on the characterization of the DNA-dependent RNA polymerases. However,
relatively little is known about the properties and functions of the
mitochondrial enzymes as compared to the nuclear RNA polymerases. It
has been difficult to identify and isolate the mitochondrial RNA poly
merase due in part to the difficulty of defining proper conditions for
the solubilization of this enzyme which appears to be firmly bound to
mitochondrial membrane. Studies on mitochondrial RNA polymerases from
a variety of sources have shown that these enzymes exhibit properties
clearly distinguishing them from the respective nuclear RNA polymerases.
The mitochondrial enzyme differs from the nuclear RNA polymerase II
in sensitivity to a-amanitin (11, 147, 148, 149). Low concentrations of
a-amanitin (0.1 to 20 yg/ml) which cause inhibition of nuclear RNA
polymerase II does not affect the mitochondrial RNA polymerase. Although
some functional differences have been described in mitochondrial RNA
polymerases, there is general agreement regarding their size.


20
The molecular weight of the enzyme, consisting of a single polypep
tide chain, seems to be about 60,000 (11-15). The purified mitochondrial
RNA polymerases from Neurospora crassa (11) and rat liver (12,13,147) are
inhibited by rifampicin in contrast to the mitochondrial enzymes from
Xenopus laevis (14), wheat leaf (150) and Ehrlic Ascites (39) which are
resistant to rifampicin. Some investigators have found the yeast mito
chondrial RNA polymerase to be rifampicin resistant (15,148,151-153).
Others describe the isolation of rifampicin-sensitive enzyme (154-155).
The reason for this discrepancy is not clear. It is possible that a
factor or factors needed for conferring sensitivity to rifampicin were
lost from some enzyme preparations.
In contrast to the above studies several laboratories have reported
that the yeast mitochrondrial RNA polymerases are much larger in size and
resemble more closely the nuclear polymerases. Eccleshall and Criddle
demonstrated three RNA polymerases associated with yeast mitochondria
having molecular weights near 500,000 and showing no sensitivity to
rifamycin (153). However, there was no difference between the nuclear
RNA polymerase I and the mitochondrial RNA polymerase I, suggesting that
their mitochondrial preparation was contaminated with nuclear enzyme.
Reports of multiple RNA polymerases associated with yeast mitochondria
having molecular weights near 500,000 and showing no sensitivity to
rifamycin have come from Rogall and Wintersberger (15) and Benson (156).
Scragg has reported a yeast mitochondrial RNA polymerase with molegular
weight greater than 200,000 that does show a rifamycin sensitive enzyme in
yeast (157). However, despite the low molecular weight (67,000) of the
yeast mitochondrial RNA polymerase polypeptide isolated by Rogall and
Wintersberger (15), this enzyme readily forms aggregates with molecular .


21
weights up to about 500,000 in buffers of low ionic strength. The same
is true for the 64,000 molecular weight enzyme purified by Kuntzel and
Schafer (11) and the 60,000 molecular weight enzyme isolated by Scragg
(158). The overall picture of mitochondrial RNA polymerase is one of
either a great diversity of enzyme types associated with different organ
isms or else some differences in the isolation procedures employed in the
various laboratories which give rise to preparations with these differences.
The mitochondrial RNA polymerase consisting of a single polypeptide chain
of molecular weight of 64,000 resembles more the bacteriophate T7 specific
RNA polymerase rather than EA coli or eukaryotic nuclear RNA polymerase.
The T7 enzyme is a product of T7 DNA gene 1. A single polypeptide of
110,000 molecular weight, it generates only eight RNA species in late
transcription of T7 DNA (25).
Chloroplast transcription
The chloroplast also contains unique DNA that is replicated and
transcribed within the organelle. The molecular weight of Euglena
g
chloroplast DNA is approximately 10 daltons (159) which is sufficient
to code for the chloroplast tRNA, rRNA, and mRNA. However, the extent
to which the total potential genetic content is expressed in chloroplast
is open to question. It has been established that the chloroplast DNA
contains cistrons coding for chloroplast ribosomal RNA (160,161) and
transfer RNA (179). Quantitative saturation-hybridization of Euglena
chloroplast rRNA to chloroplast DNA showed that approximately 2% of the
DNA is involved in coding for chloroplast rRNA (160). A higher value (6%)
was obtained by Stutz and Vandrey (161) who isolated Euglena chloroplast
DNA from purified organelles devoid of any nuclear contamination rather


22
than relying on the fractionation of extracted DNA by preparative CsCl
buoyant density centrifugation. The chloroplast ribosomal 23S RNA (1.1 x
10^ daltons) and 16S RNA (0.56 x 10^ daltons) are coded for by separate
cistrons (162) localized on the heavy strand of the chloroplast DNA.
g
Accepting that the molecular weight of Euglena DNA is 10 daltons then
£
2% of this (2 x 10 daltons) is sufficient to code for both the 23S and
16S rRNAs. The value of 6% would allow for three copies of each of the
rRNAs per chloroplast DNA molecule.
The presence of mRNA that can be translated has been demonstrated
in Euglena chloroplast (163). These mRNAs could be the products of
transcription of chloroplast DNA or of nuclear DNA, or of both. However
isolated chloroplasts synthesize RNA and such RNA must necessarily be
chloroplast-DNA coded. Hybridization of RNA synthesized in isolated
tabacco chloroplasts to chloroplast DNA indicated that 21% of the
chloroplast DNA is transcribed (164). This represents a much larger
proportion of the chloroplast DNA than is required to code only for
ribosomal and transfer RNAs and therefore implies that mRNA species are
synthesized on chloroplast DNA.
Chloroplasts of higher plants and algae were shown to contain a
DNA-dependent RNA polymerase as early as 1964 but the enzyme was only
recently solubilized and purified (165). The extreme difficulty with
solubilizing this activity was related to the tight binding of the enzyme-
DNA complex to the thylakoid membranes. This chloroplast enzyme contained
two large polypeptide subunits, which suggested a similarity to the
prokaryotic and the eukaryotic nuclear RNA polymerase rather than the
mitochondrial or bacteriophage T7 RNA polymerases. The chloroplast enzyme
has also been studied in Euglena gracilis (166). The Euglena chloroplast


23
RNA polymerase has not been solubilized and purified mainly due to the
difficulties mentioned above. This prompted the study of chloroplast
transcription complexes consisting of chloroplast membrane fragments,
DNA, and the RNA polymerase activity.
Euglena RNA polymerases
The nuclear RNA polymerase II of Euglene gracilis was partially
purified recently by Congdon and Congdon and Preston (167,168). This
enzyme, defined by its sensitivity to a-amantin, eluted from DEAE
Sepahdex between 0.18 and 0.21 M (NH^^SO^. The RNA polymerase had a
broad salt optimum ranging from 50 mM (NH^^SO^ to 150 mM (NH^^SO^.
Optimal activity was exhibited with optimum MnCl^ concentrations rather
than with MgCl^. The enzyme was more active with poly d(AT) as a
template and preferred denatured over native calf thymus DNA. The
Euglena RNA polymerase, as well as the enzymes from other protists,
were less sensitive to a-amantin than the corresponding fractions from
higher eukaryotes. The Euglena chloroplast and mitochrondrial RNA poly
merases have not been previously solubilized and purified.


MATERIALS AND METHODS
Buffers
All buffers were prepared from analytical reagent grade chemicals
without further purification. Double distilled or deionized water was
used for all solutions. Stock solutions of 1.0 M Tris-HCl pH 7.4, at
25C, 1.0 M Tris-HCl pH 7.9, at 25C, 1.0 M Tris-HCl pH 9.0, at 25C,
0.10 M EDTA pH 7.6, 1.0 M MgCl^ 0.01 M MnCl, 0.10 M dithiothreitol were
diluted to prepare the following buffers:
Buffer STE; 0.25 M sucrose, 0.01 M Tris-HCl pH 7.4 or pH 9.0,
0.10 mM EDTA pH 7.6.
Buffer STM; 0.25 M sucrose, 0.01 M Tris-HCl pH 7.9, 0.01 M
MgCl2.
Lysis buffer; 0.01 M Tris-HCl pH 7.9,20% w/v sucrose. 0.10
M KC1, 5 mM dithiothreitol, 1 mM phenylmethylsulfonylfluoride.
Buffer TGMED; 0.05 M Tris-HCl pH 7.9, 25% glycerol, 5.0 mM
MgCl^j 0.10 mM EDTA pH 7.6, 2.0 mM dithiothreitol.
Ethidium bromide buffer; 1 yg/ml Ethidium bromide, 0.01 M
Tris-HCl pH 7.9, 0.02 M NaCl, and 0.005 M EDTA.
Organism
Premanently bleached mutants of Euglena gracilis klebs strain z,
prepared by treatment with streptomycin (169) were used for these studies.
Growth
The streptomycin bleached mutants were cultured in 2800 ml Fernback
flasks containing 1500 ml of Medium A described by Greenblatt and Schiff (170).
24


25
The cultures were grown at 21 on a gyrotary shaker operating at 120
rev/min. Under these conditions, cells enter stationary phase when the
carbon source is exhausted (171). Growth was measured as turbidity with
a Klett colorimeter with a No. 66 filter.
Cell disruption and subcellular fractionation
Cells from axenic cultures in exponential phase were harvested by
passing the contents of the culture flaks through a DeLaval cream separator
centrifuge operating at room temperature. All further operations were
performed at 0 -4 C.
The cells were washed twice in STE buffer, pH 7.4, then suspended
in at least 5 volumes of STE buffer, pH 9 and passed through an Aminco
2
French Pressure cell at 1000 lb/inch (100) using a power press to give
greater than 90% breakage. Breakage was estimated by microscopic examina
tion. The cell lysate was collected in ice chilled flaks, shakengently
to disperse aggregates, and repeatedly centrifuged at 1000 x jg for 5 min
(Beckman J-21 JA-20 rotor) until only a negligible pellet was produced.
Crude subcellular fractions were collected from the cell free lysate
supernatant by differential centrifugation first at 5000 x jg, 20 min and
then 10,000 x £ for 15 min. The crude 5000 x jg pellet (5P mitochondria)
was washed in STE pH 7.4 buffer.
Mitochondrial purification
Washed 5P mitochondria were purified in gradients of sodium diatri-
zoate (Winthrop Laboratories, New York). Samples containing 5-200 mg
dry weight of mitochondria suspended in the less dense renografin solution
3
(density equals 1.103 g/cm ) containing 0.01 M Tris-Hcl pH 7.4 and 0.10 mM


26
EDTA were layered over the Renografin gradients (density equals 1.103 to
1.30 g/cm^) containing 0.01 M Tris-HCl pH 7.4 and 0.01 mM EDTA. The pre
parations were centrifuged at 20,000 rpm for 2^ hr at 2C (Beckman L-2,
SW 25.1 rotor). Fractions were collected and the mitochondria banding
in the region of the gradient corresponding to a density of 1.21 g/cnP
were washed twice in STE buffer pH 7.4 before use in incorporation studies.
Washed 5P mitochondria were alternatively purified by the enzymatic
digestion of nuclear DNA with 50 yg deoxyribonuclease I (E. C. 3.1.4.5) per
ml. The crude 5P mitochondria were washed in STM buffer pH 7.9 and then
pancreatic DNase I (Sigma Chemical Co., St. Louis, Mo.) was added to a
final concentration of 50 yg per ml in STM buffer. Incubation was carried
out at 4 for 30 min. with slow stirring. The enzymatic digestion was
stopped by the addition of 2 volumes of 10% sucrose, 0.01 M Tris-HCl pH
7.9,0.05 M EDTA pH 7.6. The mitochondria were then washed in STE buffer
pH 7.9.
Succinic dehydrogenase assay
Succinic dehydrogenase activity was measured by the succinate depend
ent reduction of dichlorophenolindophenol (172) in 1 ml reaction mixtures
at room temperature.
Extraction of nucleic acids
DNA was extracted from the mitochondria preparations by the technique
of Bhargava and Halvorson (173).
Characterization of DNA
The method of Preston and Boone (174) was used for the density measure
ment of DNA. The CsCl gradient polyacrylamide gels obtained after the


27
exposure to light were washed with flowing tap distilled water for 2 hr
to remove CsCl. The riboflavin was photooxidized by shaking the gels
under illumination for 16 hr in the presence of 10 ml of 0.01 N NaOH in
50% formamide. The gels were then washed in tap distilled water for 2
hr before adding the gels to ethidium bromide staining buffer. The
staining permitted the quantitative detection of DNA in the gel which
could be scanned in an Aminco Flourometer equipped with a gel scanning
attachment.
DNA standards
DNA was purified from Cytophage P-11 (a strain isolated, identified
and carried at the Department of Microbiology, University of Florida,
Gainesville, FL) as described by Preston and Boone (174). DNA from the
bacteriophage 25 and DNA from Pseudomonas aeruginosa were kindly pro
vided by M. Mandel, M. D. Anderson Tumor Institute, Houston, Texas.
DNA was extracted from isolated nuclei of streptomycin bleached mutants
of Euglena gracilis Klebs strain z by the method of Bhargava and Halvorson
(173).
Preparation of cellular fractions for protein and DNA analysis
Cellular fraction samples were prepared for analysis by Vortex mixing
1 ml of sample with 5 ml of cold 95% ethanol (-20C). The particulate
material was collected by centrifugation at 4000 rpm, 5 min., 2C. The
ethanol extraction was repeated twice more before suspending the pellet in
1 ml of cold double distilled water. The samples were suspended in 1.0 ml
of water then 4.0 ml of cold 0.50 N HC10^ was added and the sample was
mixed on a vortex. The precipitated macromolecules were collected by
centrifugation before being suspended in 4 ml of cold ether-ethanol (1:3)


28
and centrifuged again. The pellet was suspended in 4 ml of 0.50 M HCIO^
and incubated at 85C for 20 min. to hydrolyze DNA and RNA, cooled and
centrifuged. The top 3 ml of the supernatant was used for DNA analysis.
The pellet was suspended in 3 ml of 1.0 N NaOH and heated in a boiling
water bath until the precipitated protein was dissolved. This solution
was used for analysis of soluble proteins.
Protein determination
The protein concentrations were determined by the Lowry method (175) .
Dissolved bovine serum albumin served as the standard.
DNA determination
DNA concentrations were determined by the indole method of Hubbard
(176). Calf thymus DNA served as a standard. A sample of 0.05 ml was
added to 0.05 ml of 10% trichloroacetic acid and 1 ml of 0.04% indole-2
N HC1 solution. The mixture was shaken and incubated at 97C for 15 min.
and then cooled in ice. The mixture was then extracted with chloroform
three times by adding 2 ml of CHCl^ to the mixture, vortexing and centri
fuging 2000 rpm, 5 min. The chloroform phase was removed after each ex
traction. The absorbance of the aqueous phase at 490 mm was then determined.
RNA polymerase assay
Conditions for the assay of RNA polymerase were similar to those
described by Preston ert al. (177). The routine mixture contained in a
total volume of 0.10 ml: 0.05 M Tris-HCl pH 7.9, 0.005 M MgC^, 0.001
M MnCl2, 0.001 M DTT, 0.01 M KC1, 0.001 M of each unlabeled ribonucleoside
triphosphate, 0.032 mM or 0.004 mM or 0.016 mM (2 Ci/mmole of ^H-labeled
ribonucleoside triphosphate, 100 pg/ml of denatured calf thymus DNA and
enzyme. Assay mixtures were incubated for 10 min. (or the time indicated)


29
at 37C. The reaction was stopped by adding stop bath (0.05 M sodium
pyrophosphate, 2 mg/ml bovine serum albumin, 2 mg/ml Torula RNA and 5 mM
of the unlabeled ribonucleotide which was identical to the labeled NTP)
and chilling in ice (1). Macromolecules were precipitated by adding 2
ml of cold 10% trichloroacetic acid containing 20 mM sodium pyrophosphate
(TCAPP) to each reaction tube. After standing in ice for 15 min. the
precipitate was collected by filtration through a glass fiber disc (What
man GF/C) which had been prewashed with 5 ml of cold TCAPP. Each disc
was washed three times with 5-10 ml TCAPP, then three times with 5-10 ml
ether-ethanol (1:3) 37C, followed by two washes 5-10 ml ether.
Filtration was carried out in perforated porcelain crucibles (Gooch)
adapted to a vaccum manifold with ruber crucible holders (Walter).
Filtration was facilitated by maintaining a partial vaccum (ca. 28 in.
Hg). The GF/C filters were dried and placed in scintillation vials
containing 5 ml of toluene (scintillation grade, Mallinkrodt), 0.4% PPO
(2,5-diphenyloxazole), 0.01% POPOP 1,4-di[2-(5-phenyloxazolyl)]-benzene),
and counted for 10 min. in a liquid scintillation counter (Beckman LS-133).
A unit of enzyme activity is defined as that which will catalyze the
incorporation of 1 pmole of labeled ribonucleoside monophosphate into acid
precipitable material in 10 min. at 37.
Isolation of CTP labeled product
A sample of 0.30 ml of purified mitochondria (0.18 mg/ml protein)
was incubated in a 3.0 ml RNA polymerase reaction mixture (0.05 mM H-CTP,
2 Ci/mmole) for 15 min. The reaction was stopped by adding 3.0 ml of stop
bath (0.05 M sodium pyrophosphate, 1.09 mg/ml _E. coli RNA and 5 mM of
unlabeled CTP) and brought to 0.20 M NaCl and 0.10 M Tris pH9. Triton X


30
100 was added to bring the mixture to a final concentration of 0.5%. The
mixture was then mixed with an equal volume of 80% v/v redistilled aequeous
phenol. The nucleic acids were collected by precipitation with 3 volumes
of cold 95% ethanol (-20C) for 12 hr followed by centrifugation at 4000
rpm 15 min. The nucleic acids were dissolved in buffer (0.20 M NaCl, 0.10
M Tris pH 9) and carried through the ethanol precipitation again. The
final pellet was dissolved in 2 ml of double distilled water.
Column filtration
Biogel P-4 was swollen overnight by mixing 1 g Biogel per 10 ml of
0.10 M NaCl. A 20 ml column was poured and pre-equilibrated by passing
4 volumes of 0.10 M NaCl through it at 4C. Nucleic acid samples in
0.10 M NaCl were added to the column and eluted with 0.10 M NaCl. The
flow rate was 10 drops per min. labeled samples (100 yl) were placed
in 10 ml of Brays scintillation fluid (178) and counted in the liquid
scintillation counter (Beckman LS-133).
Extraction of phospholipids
Phospholipid extraction was performed by the method of Carter and
Kennedy (179) and Raetz and Kennedy (180). To 0.10 ml of the ^h-CTP
labeled product was mixed 1.0 ml of methanol containing 0.10 N HC1. Ten
minutes later 2 ml of chloroform was added and the phases were stirred
with a vortex mixer before adding 4 ml of 2 M KC1. The mixture was then
vigorously mixed for 5 min. and the phases were allowed to separate before
the aqueous phase was removed. The chloroform phase was washed two more
times with 2 M KC1. A sample of each phase following extraction was
placed in a scintillation vial and dried by passing a stream of nitrogen


31
gas over the solution. Then 10 ml of Bray's solution was added to the
vials and counted.
Thin layer chromatography methods
The procedure of Ter Schegget e_t ill. (181) for thin layer chromato
graphy of CDP-diglyceride was followed. Samples were spottedon silica
gel G thin layer plates. Chromatograms were developed with a solvent of
chloroform-methanol-water-ammonia (70:38:2:8). The CDP-diglyceride stand
ard was prepared by the method of Carter and Kennedy (179). Nucleotides
were chromatographed on PEI cellulose by the methods of Randerath and
Randerath (182,183). Ribonucleoside monophosphates were chromatographed
first in 1.0 N acetic acid to a height of 4 cm and then without drying
the plate was transferred to a tank containing 0.30 N LiCl and the solvent
was allowed to rise to 15 cm. The ribonucleoside triphosphates were
separated by developing to 15 cm with 0.50 M ammonium sulfate.
Solubilization and purification of
mitochondrial RNA polymerase
Solubilization of mitochondrial RNA polymerase was achieved by fol
lowing a procedure similar to that of Wintersberger and Rogall (15, 152).
Mitochondria from 150 wet weight of cells were suspended in an equal
volume of lysis buffer. This was added to an equal volume of a lysis
mixture consisting of 1.6% Triton X 100 non ionic detergent, 1.0 M KC1,
and 0.01 M EDTA (pH 7.9). The mixture was mixed gently for 15 min and
then centrifuged for 90 min at 30,000 rmp (Ti 50 rotor) and 0.29 of
solid ammonium sulfate per ml of 30,000 supernatant was added to bring
the extract to 50% saturation with respect to ammonium sulfate. The
precipitate was collected by centrifugation at 40,000 rmp for 90 min


32
(Ti 50 rotor) and dissolved in TGMED containing 0.05 M (NH^^SO^ and
dialyzed against 2 liters of the same buffer for 4 hr. The dialyzate
was applied to a column (0.90 x 20 cm) of DEAE-Sephadex A-25 which had
previously been equilibrated with the same buffer and nonabsorbed protein
was washed off the column with 30 ml of the same buffer. The RNA polymerase
activity was eluted with a 160 ml gradient of 0.05 M to 0.50 M (NH^^SO^
in TG,ED. Fractions (2.50 ml) were collected and 20 pi of each fraction
was assayed for RNA polymerase activity. Concentrations of (NH^^SO^ were
determined with a conductivity bridge.
Chemicals and reagents
The following research grade biochemicals were obtained from Sigma
Chemical Co., St. Louis, No: The ribonucleoside triphosphates GTP, CTP
UTP and ATP as sodium salts; dithiothreitol; phenylmethylsulfonyl flouride;
high molecular weight calf thymus DNA; Torula yeast RNA, Triton X-100; en
zyme grade trizma buffers; chromatographically pure pancreatic ribonuclease
A (EC 2.7.7.16) and deoxyribonuclease I (EC 3.1.4.5): fraction V bovine
serum albumin; cytidylyl (3'->- 5') cytidine (CpC); and snake venom phospho
diesterase (Crotalus atrox venom, Type VII).
The tridium labeled ribonucleoside triphosphates as sodium salts
were purchased from Schwartz/Mann, Orangeburg, NY. These include {5- H}
UTP, (5-^H CTP, (8-^H) ATP, {8-^H} GTP. Ethidium bromide and actinomycin
D were purchased from Calibiochem, LaJolla, CA. Poly d(AT) was obtained
from Miles Laboratories, Kankakee, IL. The rifamycin derivativies AF/05
and AF/013 were the generous gift of Gruppo Lepetit, Milan, Italy. The
a-amanitin was kindly provided by Dr. T. Wieldand, Max-Planck Institute,


33
Heidelberg, West Germany. Sodium diatrizoate was purchased from Winthrop
Laboratories, NY. Nonionic detergent NP-40 was purchased from Shell Oil
Co. Unless specifically noted, all other chemicals were analytical rea
gent grade.


RESULTS
Two experimental approaches were utilized to study the iri vitro mito
chondrial RNA synthesis in Euglena gracilis. The first approach was to
study the incorporation of radioactively labeled ribonucleoside triphos
phates into acid insoluble products by isolated mitochondria which had
been treated with isotonic buffer to assure swelling and permability.
The products of the incorporations were then studied. The second approach
was to study the activity of solubilized and partially purified mitochon
drial DNA dependent RNA polymerase. This approach involved determining
the conditions for solubilizing the enzyme and then for partially purifying
the enzyme. The activity of this enzyme was then characterized.
The Incorporation of Ribonucleoside
Triphosphates by Isolated Mitochondria
The initial research objectives were to determine if RNA synthesizing
activity is retained by mitochondria isolated from Euglena gracilis, and
to look for ways to differentiate between nuclear and mitochondrial RNA
polymerase activities such as sensitivities to inhibitors or special
enzymatic requirements. The approach was to study the RNA synthesizing
activity of isolated mitochondria obtained from a streptomycin bleached
aplastidic mutant of Euglena gracilis, strain z. The mitochondrial
isolation procedure (Figure 1) consisted of rupturing washed cells in the
French pressure cell at 1000 lb/in^ and collecting crude mitochondrial
34


Figure 1. SCHEMA FOR THE ISOLATION OF MITOCHONDRIA


36
ISOLATION PROCEDURE
WASHED CELLS
STE (0.25M Sucrose, O.OIM Tris-HCI pH7.6,
O.ImM EDTA pH 7.4)
2
Fpc IOOO Ib/inch
DISRUPTED CELLS
--I000 Xg, 5 min.
REPEAT I-3X
I 1
IP IS
5000 Xg, 20 min.
r
5P
resusp.
5000Xg, 15 min.
10000 Xg, 15 min.
I 1 I 1
Washed 5P 5S IOP IOS
(Dense Mito.) (Less Dense)
(Mito.)
RENOGRAFIN GRADIENT
PURIFIED MITO. (/>= 1.22 g cm-3)


37
pellets by differential centrifugation, first at 5000 x j and then at 10,000
x for 15 min. The crude 5000 x mitochondrial pellet (5P) containing
the heavier and more enzymatically active mitochondria (33) were used for
these experiments. The 5P mitochondria were washed in buffer and then
subjected to sodium diatrizoate (renografin) gradient centrifugation at
20,000 x for 2^ hr. Fractions were collected and the mitochondria, which
banded in the region of the gradient corresponding to a mean equilibrium
density of 1.22 g/cm were used for the studies reported here.
In order to test the purity of the mitochondrial preparation, the
buoyant density of the DNA in the mitochondrial preparations was routinely
monitored by CsCl gradient centrifugation. The CsCl gradients were genera
ted in the presence of acrylamide and catalyst according to the procedure
of Preston and Boone (174). This method allows the DNA that is banded in
the CsCl gradient to be fixed by exposing the gradient to light in order to
polymerize the acrylamide gels. Ethidium bromide staining permitted a rapid
and sensitive technique to quantitatively detect the DNA in the gels which
could be scanned in an Aminco Fluorometer equipped with a gel scanning
attachment. The linear relationship between the relative position in the
gradient and the buoyant density of the DNA is demonstrated by the plot of
DNA standards of known densities in Figure 2. The purified mitochondrial
preparation yields better than 70% mitochondrial DNA which has a density
of 1.691 g/cm^ and less than 30% nuclear DNA which has a density of 1.707
3
g/cm Although it is evident that the preparation contains some contami
nating nuclear DNA, this method provided the cleanest feasible mitochondria
without treating the mitochondria with an exogenous nuclease.


Figure 2. DENSITY EQUILIBRIA ANALYSIS OF MITOCHONDRIAL DNA
Density equilibria anlaysis of DNA in mitochondria purified on a sodium
diatrizoate (Renografin) gradient were measured by CsCl density gradient
centrifugation as described in the methods section. The markers are
Cytophage P-11 DNA with a density of 1.693 g/cm-*, Psuedomonas aeruginosa
DNA with a density of 1.727 g/cnH, and bacteriophage <(> DNA with a density
of 1.742 g/cm .


FLUORESCENCE
39
CsCI Density Gradient Gel
DNA STAINED WITH ETHIDIUM BROMIDE


40
The renografin purified mitochondria are active biochemically as demon
strated by results in Table 1. Succinic dehydrogenase which is tightly
integrated into the inner membrane of mitochondria has been employed as a
marker enzyme in order to follow the purification of mitochondria. Of the
total cellular succinic dehydrogenase activity 42% is recovered in the
purified mitochondria, with a 27-fold purification per mg of protein and a
70-fold purification per pg of DNA. This demonstrates that the purification
procedure yields mitochondria biochemically active for an enzyme not asso
ciated with the DNA
A Mitochondrial Activity Incorporating Label
From 3h~CTP Into Acid Precipitable Product
The incorporation of label from the four ribonucleoside triphosphates
labeled with tritium by isolated mitochondria was studied by performing
RNA polymerase assays as described in the methods section. The mitochondria
were treated with isotonic buffer to assure swelling and permability. The
relative rate with which label from each ribonucleoside triphosphate was
incorporated into acid insoluble macromolecules was determined by performing
the assays in 0.1 ml reaction mixtures in which only one of the ribonucleo
tides was labeled. It was observed that isolated mitochondria catalyzed
the incorporation of label from ^H-CTP into acid insoluble material at a
rate greater than 10 times that for the other ribonucleotides (Table 2).
It is interesting that ATP and GTP, the ribonucleotides which normally
serve as the best substrate for RNA initiation in procaryotic systems, are
the poorest substrates for the reactions studied here. Figure 3 demonstra
tes the relative rates of incorporation of tritium from ^H-CTP and ^H-UTP,
the two most active substrates. In this experiment the concentration of


TABLE 1
DISTRIBUTION OF SUCCINIC DEHYDRAGENASE ACTIVITY
FRACTION
TOTAL
UNITS
PERCENT
RECOVERY
UNITS/mg
PROTEIN,(X 10~4)
FOLD
PURIFICATION
UNITS/yg
DNA, (X 10-4)
FOLD
PURIFICATION
LYSATE
1.96
100
7
1.0
0.2
1.0
5P MITO
1.78
90
59
8.4
1.1
7.5
WASHED 5P MITO
0.91
46
65
9.3
1.1
7.6
RENO MITO
0.82
42
190
27.0
10.3
70.0
Succinic dehydrogenase activity was measured by the succinate dependent reduction of dichlorphendol-
indophenol (160) in 1 ml reaction mixtures at room temperature. The protein and DNA concentrations
were determined by the procedures described in the methods and procedures. A unit of succinic dehy-
drogenas activity is defined as a ymole of dichlorophenolindophenol reduced per minute per ml of enzyme
sample at 25C.


TABLE 2
NUCLEOTIDE INCORPORATION BY
ISOLATED MITOCHONDRIA
CPM incorporated into acid
insoluble macromolecules.
Labeled
Ribonucleotide
3H-CTP
13860
3h-utp
952
3h-atp
276
3h-ctp
322
The RNA polymerase assay is described in the methods
section. Each reaction mixture contained in a total
volume of 0.1 ml: 1.0 mM of each unlabeled NTP and
0.004 mM of each JH-labeled ribonucleoside triphosphate
with a specific radioactivity of 2 Ci/mmole.


Figure 3. RIBONUCLEOSIDE TRIPHOSPHATES INCORPORATION BY
ISOLATED MITOCHONDRIA
The incorporation of ribonucleoside triphosphates by isolated mitochondria
into acid insoluble products. The RNA polymerase assay described in the
methods section was followed except that the concentration of the radio-
actively labeled ribonucleoside triphosphates was 0.016 mM, 2 Ci/mmole.
The unlabeled ribonucleotides concentration was 0.5 mM each.


fjL/j. Moles of H-CMP Incorporated
44
Time (min.)
5
20
25
//Moles of H-UMP Incorporated


45
the radioactively labeled ribonucleotide was 0.016 mM and that of the
unlabeled ribonucleotides was 0.5 mM each. The other components of the
O
reaction were as described in the methods. Label from JH-CTP was incorpora
ted at a rate greater than 10-fold that for UTP. It should be noted that
the CTP and UTP tritium incorporations are plotted on different scales in
Figure 3. When the incubation is carried out in the presence of RNase A, the
3
product formed with H-CTP incorporation shows no sensitivity whereas the
3
product formed with H-UTP shows some sensitivity to RNase A. This could
be due to the possibility that the CTP product is not RNA or that the product
is complexed with DNA or folded and complexed such that it is RNase insensi
tive. These results also suggest that RNA synthesis could be better followed
by studying the incorporation with labeled UTP.
3
The incorporation of tritium from H-CTP by the isolated mitochondria
was clearly not dependent upon the presence of the other ribonucleoside
triphosphates which appear to inhibit the incorporation by about 10 percent
3
(Table 3). Incorporation of label from H-UTP also shows no dependence
upon added ribonucleotides while the cpm for ATP and GTP were too low to
make any judgment as to their dependency.
The high rate of incorporation with -^H-CTP relative to that with the
other ribonucleoside triphosphates prompted the investigation of the incor-
3
poration with H-CTP. This activity demonstrated no dependency upon added
exogenous DNA or ribonucleoside triphosphates (Table 4). In fact the isolated
3
mitochondria incorporated label from H-CTP at a 10 times greater rate in the
absence of added DNA or nucleoside triphosphates than when they were present.
3
The distribution of the incorporating activity with H-CTP was followed
through the isolation procedure and the results are shown in Table 5. It was


TABLE 3
INCORPORATION OF RIBONUCLEOSIDE TRIPHOSPHATES BY
ISOLATED MITOCHONDRIA.
Labeled
Ribonucleotide CPM/Rx.Mix. Units/g Wet Wt.
3H-CTP
+
NTPs
11,513
2161
-
NTPs
12,792
2401
3h-utp
+
NTPs
616
116
-
NTPs
1,386
260
3h-atp
+
NTPs
206
39
-
NTPs
324
61
3h-gtp
+
NTPs
84
16
NTPs
290
54
The RNA polymerase assay was performed as described in the
methods. 0.1 ml reaction mixtures contained 1.0 mM of each
unlabeled NTP, 0.004 mM ^H NTP, 2 mC/mmole.


TABLE 4
THE DEPENDENCY OF THE MITOCHONDRIAL ACTIVITY
INCORPORATING LABEL FROM JH~CTP UPON ADDED
RIBONUCLEOTIDES AND DNA
Assay Components
Units/mg Protein
Complete Assay with DNA
1,500
-DNA + NTP
1,481
-DNA NTP
11,056
-DNA + UTP
9,611
-DNA + ATP
10,667
-DNA + GTP
8,611
The RNA polymerase assay was performed as described in the
methods. 0.1 ml reaction mixtures contained 1.0 mM of each
unlabeled NTP and 0.004 mM of -^H-CTP with a specific radio
activity of 2 Ci/mmole.


TABLE 5
DISTRIBUTION OF 3H-CMP INCORPORATING ACTIVITY
Fraction
TOTAL
Units
%
Recovery
Units/mg
Protein, X 103
Fold
Purification
Units/yg
DNA
Fold
Purification
Lystate
141,750
-
2.0
-
0.9
-
5P Mito
39,420
28
6.5
3.2
6.5
7.1
Washed 5P Mito
12,320
9
5.9
3.0
7.7
8.0
Reno Mito
18,940
13
17.4
8.7
114.0
125.0
The RNA polymerase assay is described in the methods. Each reaction mixture contained in a
total volume of 0.1 ml; 1.0 mM of each unlabeled NTP and 0.004 mM of 3H-CTP with a specific
radioactivity of 2 Ci/mmole.
CO


49
observed that 13% of the activity was recovered in the purified mitochon
dria with an 8.7-fold purification per mg of protein and 125-fold per yg
of DNA. This suggests that if the activity is associated with DNA, then
it is with mitochondrial DNA and not nuclear DNA, 95% of which has been
removed by the purification procedure.
3
The incorporation of H-CTP into acid insoluble product is inhibited
by actinomycin D (Table 6) at concentrations above 50 pg per ml, suggesting
that a DNA dependent reaction may be involved. However, it is interesting
that relatively high concentrations are required to demonstrate this
inhibition. The possibility of a secondary site of action of actinomycin
D or inhibition by other compounds in the actinomycin D can not be ruled
out. However, pyrophosphate at 2 pmoles per ml inhibited the activity
while inorganic phosphate at 2 pmoles per ml failed to inhibit the incor
poration indicating that the incorporation involves a pyrophosphorolysis.
This indicates the incorporation of CMP and rules against reactions which
incorporate CMP from CDP such as that of nucleotide phosphorylase or the
incorporation of CDP from CTP.
Rifampin and streptovaricin, inhibitors of initiation and elongation
of prokaryotic RNA polymerases respectively, failed to inhibit the activi
ty. Rifamycin AF/013, and inhibitor of initiation with eukaroytic nuclear
RNA polymerases as well as prokaryotic RNA polymerases, also failed to
inhibit the activity. Ethidium bromide, which will bind to DNA, showed
only slight inhibition which is probably not significant.
The acid insoluble product is sensitive to snake venom phosphodies
terase and alkaline hydrolysis (Table 7) under two different conditions,
suggesting that the product contains phosphodiester bonds. Up to this
point the product had demonstrated properties that were similar to those


TABLE 6
INHIBITION OF 3H-CMP INCORPORATION
Addition
CPM
Percent of
Control
h2o
4213
100
1% DMF
4179
100
Actinomycin D,
100 pg/ml
602
14
Actinomycin D,
75 pg/ml
2052
48
Actinomycin D,
50 pg/ml
4708
100
Pyrophosphate,
2 pmoles/ml
456
10
Phosphate,
2 pmoles/ml
4055
96
Rifampin,
100 pg/ml
3538
84
Rifamycin AF/013, 100 pg/ml
4050
96
Ethidium Bromide, 100 pg/ml
3370
80
Streptovaricin,
100 pg/ml
3918
93
The RNA polymerase assay was performed with the addition of
inhibitors to the final concentrations indicated above. Rifam
pin, rifamycin AF/013, ethidium bromide and streptovaricin were
solubilized in 1% N'N' dimethyl formamide.


TABLE 7
CHARACTERIZATION OF PRODUCT
Addition
CPM
Percent
of Control
Control
6412
100
Snake Venom Phosphodiesterase,
10 pg/ml
561
9
0.1M KOH 100C, 20 min.
154
2
H20, 100C, 20 min.
4437
70
0.5M KOH, 37C, 16 hr.
119
2
H20, 37C, 16 hr.
5129
80
The RNA polymerase assay was performed with the addition of
phosphodiesterase (E. C.: 3.1.4.1) from Crotalus atrox venom,
Type VII (Sigma, St. Louis). The alkaline hydrolysis was per
formed by first performing the RNA polymerase assay for 10
minutes and then treating the reaction mixture with KOH for the
indicated times before the addition of stop bath and the precip
itation of macromolecules with 10% trichloroachetic acid.


52
reported by Duda and Cherry (184) for a poly C polymerase activity in
sugar beet nuclei.
The RNA polymerase assay reaction mixture was scaled up so that the
3
labeled products of the incorporation of H-CMP could be studied. For
this purpose the reaction mixture contained in a total volume of 3.0 ml,
5.0 mM of each unlabeled ribonucleoside triphosphate and 0.05 mM of
3
H-CTP with a specific radioactivity of 2 Ci/mmold. After incubationof
15 min the products were isolated as described in the methods and
material section by phenol extraction followed by ethanol precipitation
of labeled products from the aqueous phase in the presence of carrier
_E. coli RNA and CTP. Zonal centrifugation (Figure 4) on sucrose gradient
was performed in order to determine the size of the tritium labeled pro
duct. The radioactivity remained at the top of the gradient indicating
the material was smaller than that of _E. coli 5S RNA. Therefore, the
sizes of the isolated products were estimated by Biogel P-4 column
filtration (Figure 5). The _E. coli carrier RNA eluted in the void volume
while two adsorbancy peaks were eluted which contained the labeled cyto
sine. Using the elution position of a standard CpC the molecular weight
of the first peak (BGP-1) was estimated to be approximately 800 which
corresponds to the molecular weight of a trinucleotide of poly C. The
molecular weight of the second peak (BGP-2) was estimated to be about
680 which corresponds to the molecular weight of a dinucleotide. It
should be noted that this smaller entity elutes before CpC indicating
that it is larger than a dinucleotide of this structure.
The components in the two peaks eluted from the P-4 column have
ultraviolet absorbancy spectrums very similar if not identical to that
of CpC, showing the characteristic absorption maximum and minimum at


Figure 4. SUCROSE GRADIENT ZONAL CENTRIFUGATION
The H-CMP-labeled product was isolated from the scaled up RNA polymerase reaction mixture using
carrier EL coli RNA. The tritium-labeled materials (200 pi) obtained from the nucleic acid
extraction of the scaled up reaction mixture were layered over 5 ml sucrose (Schwarz Mann special
density grade) gradients, 5 20% w/v, containing 0.01M Tris-HCl pH 7.6 and 0.02 M NaCl and centri
fuged (SW 39 rotor) 6 hr at 4 C. The E_. coli RNA standard was centrifuged in a separate tube. The
gradient was analyzed by pumping the gradient through the Gilford J cell, delivering the densest
portion first with a bottom probe. The ultraviolet absorbancy was monitored with the Gilford re
corder. Fractions were collected and measured for radioactivity by placing 200 pi samples in Brays
scintillation fluid and counted in the scintillation counter. The ultra-violet absorbancy profiles
for the products ( ) and _E. coli RNA ( ) and the radioactivity profile ('*) are shown


CPM. X I04 (--*-)
2 6 0


Figure 5. BIOGEL P-4 COLUMN CHROMATOGRAPHY
The H-CMP labeled product was isolated from the RNA polymerase reaction mixture using carrier
_E. coli RNA. The product samples dissolved in 0.1 M NaCl were added to a 20 ml Biogel P-4
column previously equilibrated with 0.1 M NaCl. Fractions were eluted with 0.1 M NaCl at 4 C.
The eluted fractions were assayed for radioactivity. The standard was CpC.


260
Ln
o
CPM X 10


57
pH 2 and pH 7 for cytosine (Figure 6). This demonstrates that cytosine
is the only base incorporated into the products, and suggests that the
products are homopolymers of CMP.
The possibility that the product may be CDP-diglyceride was inves
tigated, since it has been reported that crude mitochondrial preparations
from rat liver incorporated label from both CTP and dCTP to CDP digly
ceride and dCTP diglyceride respectively (181). These mitochondria
3
incorporated label from H-dCTP into acid-insoluble material at a much
higher rate than any other deoxyribonucleoside triphosphate. About 95%
3
of the acid-insoluble material labeled after incubation with H-dCTP
could be extracted by chloroform. Therefore, studies were performed to
3
ascertain if Euglena mitochondria incorporated the label from H-CTP into
CDP-diglyceride.
The results of phospholipid extraction are shown in Table 8. A 0.1
3
ml sample of the H-CMP labeled product obtained after nucleic acid
extraction and dissolved in water was mixed with 1.0 ml of methanol for
10 minutes before adding 2.0 ml of chloroform with additional mixing.
Then 4.0 ml of 2M KClwas added with vigorous mixing (Vortex mixer, 5
min., 25C). After the phases separated upon standing, less than 1% of
the tritium label could be accounted for in the chloroform phase whereas
64% of the label could be accounted for in the aqueous phase. Repeated
additions of the aqueous KC1 to the chloroform phase resulted in no
extraction of additional label into the aqueous phase. The difficulty
in accounting for all of the radioactivity was due to the quencing
produced by the aqueous KC1 phase in the Bray's scintillation fluid.
Further evidence that the product is not CDP-diglyceride was pro
vided by the failure of the radioactively labeled material eluted from
the P-4 column to migrate in a silica gel G TLC system (Figure 7) in


Figure 6. ULTRA-VIOLET ABSORBANCY SPECTRA
The ultraviolet absorbancy spectra of the H-CMP labeled products eluted from the
column were performed after adjusting the pH. The spectrum for the standard, C^C
same as before and after column filtrations.
Biogel P-4
was the


Ln
vo


60
TABLE 8
PHOSPHOLIPID EXTRACTION OF THE 3H-CMP LABELED PRODUCT
Sample
Total
CPM
% of Control
3H-CTP product
58.50
X
104
-
First extraction of product
in chloroform solution
Chloroform phase
0.09
X
104
0.2
Aqueous phase
37.20
X
104
64.0
Second extraction of
chloroform phase
104
Chloroform phase
0.13
X
0.2
Aqueous phase
0.09
X
104
0.2
Third extraction of
Chloroform phase
Chloroform phase
0.19
X
io4
0.3
Aqueous phase
0.27
X
104
0.4
A 0.1 ml sample of the 3H-CMP-labeled product (containing 58.5 x 104 cpm)
obtained from nucleic acid extraction was dissolved in water and mixed
with 1.0 ml of methonal containing 0.1N HC1. Ten minutes later 2.0 ml
of chloroform was added to the mixture followed by 4 ml of 2M KC1. This
was vigorously mixed on a vortex mixer for 5 minutes at 25. The phases
were allowed to separate upon standing at room temperature and then the
aqueous phase was drawn off and the chloroform phase was extracted 2 more
times. 100 yl samples of each phase were counted for radioactivity as
described in the methods.


Figure 7. THIN LAYER CHROMATOGRAPHY OF THE 3H-CMP LABELED PRODUCTS
ELUTED FROM THE BIOGEL P-4 COLUMN
Ascending chromatography of the 3H-CMP labeled products eluted from the
Biogel P-4 column was performed according to the method of TerSchegett
ert al. (181) on silica gel G in closed tanks at room temperature. The
solvent was chloroform; methanol; water; ammonia (70:30:2:8, v/v). The
standards were CpC and CDP-diglyceride. Twenty microliter samples of
the 3H-CMP labeled products and standards were spotted 3.0 cm from the
lower edge of the plate and dried before developing in the solvent. The
800 MW 3H-CMP labeled product (BGP-1) spot contained 35,000 cpm and the
680 MW product (BGP-2) spot contained 35,000 cpm. This was determined
by spotting the same volume of each product on a plate and then removing
a 1.0 cm2 section containing the spot and measuring its radioactivity in
toluene scintillation fluid. The CDP-diglyceride was detected by ultra
violet light and by staining with iodine vapor. Radioactivity was
detected by sequentially removing 1 cm2 sections from the silica gel plate
and counting them in toluene scintillation fluid.


Silica Gel G TLC
CHLOROFORM: METHANOL: WATER:
AMMONIA (70:38:2:8 v/v)


63
which CDP-digylceride migrates (181). Ascending chromatography of the
3
H-CMP labeled products was carried out as described in the legend.
The labeled products behaved similarly to CpC in that they both failed
to migrate in the system and they failed to stain with iodine vapor
indicating that they contained no lipid moieties. As noted in Figure 7
100% of the cmp were recovered in the 1 cm square section comprising the
origin. No significant radioactivity was detected in the other 1 cm
squares which were sequentially removed from the plate and counted for
radioactivity. The CDP-diglyceride standard migrated in the system and
stained with iodine indicating that the radioactive products are not
CDP-diglyceride.
Attempts were then made to ascertain the structure of the labeled
products. This involved treating materials eluted from the P-4 column
peaks with nucleases and analyzing the cleavage products by their migra
tion in a PEI cellulose TLC system in which ribonucleoside monophosphates
can be distinguished. Treatment of the labeled material (BGP-1 and
BGP-2) eluted from the P-4 column peaks with snake venom phosphodiesterase
resulted in 93% of the radioactive label migrating to a position correspon
ding to 5'-CMP (Figure 8 and 9), the expected cleavage products of poly C
and CpC. However, when the products were treated with RNase A, the radio
active label remained at the origin as did the untreated products. This
data indicates that the products were insensitive to RNase A. This being
the case, then the products have a chemical structure which is different
from that of a homopolymer of CMP since RNase A cleaves CpC to the expected
2', 3' CMP products. However, the possibility exists that these were
homopolymers which have modified 3' terminus which prevents the RNase A
from binding. The possibility of a small oligonucleotide with a 5'


Figure 8. THIN LAYER CHROMATOGRAPHY OF THE PRODUCTS OF THE ENZYMATIC
CLEAVAGE OF THE 3H-CMP LABELED MATERIAL ELUTED FROM THE P-4
COLUMN
Ascending chromatography of the products of the enzymatic cleavage of
the 3H-CMP labeled material eluted from the P-4 column was performed
on PEI cellulose as described in the methods. The molecules eluted
from the Biogel P-4 column BGP-1 (800 MW) and BGP-2 (680 MW) were
treated separately with each of the digestive enzymes; snake venom
phosphodiesterase, and ribonuclease A. The standards were CpC, 2'-
3'-CMP, and 5'CMP. Twenty microliters of each digest was spotted 3.0
cm from the bottom of the plate and dried. The enzymatic digest spots
contained the cpm indicated in the denominators. Ribonucleoside mono
phosphates were chromatographed as described in the methods. The
nucleotides were detected with ultraviolet light. Radioactivity was
detected by sequentially removing 1.5 cm3 sections of the PEI cellulose
and counting them in toluene scintillation fluid.


65
PEI Cellulose TLC
I.ON ACETIC ACID TO 4CM
0.3 LiCI to 15 CM
SOLVENT FRONT
5456 CPM
5839 CPM
I
Ouv @
5196 CPM 8170 CPM
6954 CPM 8636 CPM
I I
P-4 COL
P-4 COL
P-4 COL
CpC
CpC 2',3-CMP 5-CMP
UN-
PEAKS
+
PEAKS
*t*
+
+
TREATED
S.V.P
RNaseA
S.VP
RNaseA


Figure 9
THE MIGRATION OF THE ENZYMATIC CLEAVAGE PRODUCTS CONTAINING
TRITIUM LABEL ON PEI CELLULOSE
The migration of the enzymatic cleavage products containing tritium
label on PEI cellulose as described in Figure 8.


CPM X 10
67


68
blocked terminus and/or methylated nucleoside moieties can not be ruled
out.
The Solubilization and Partial Purification of
Euglena Mitochondrial RNA Polymerase
The final objectives were to solubilize and purify the mitochondrial
DNA-directed RNA polymerase, to characterize its activity, to determine
its requirements for product synthesis, and to compare it to nuclear RNA
polymerase activities with respect to these requirements. In order to
accomplish this it was necessary to prepare mitochondria which were devoid
of contaminating nuclear DNA. This was achieved by obtaining a crude 5P
mitochondrial pellet, as described in Figure 1, and washing it twice in
STM Buffer before incubating the mitochondria in the presence of deoxyri-
bionuclease I (DNase I) for thirty minutes, zero degrees centigrade.
These mitochondria were shown to be devoid of nuclear DNA by work substan
tiated in this laboratory and no further purification was necessary. The
buoyant densities of the DNA in the mitochondrial preparations in CsCl are
demonstrated in Figure 10. The crude washed 5P mitochondria yield mostly
Euglena nuclear DNA (Tube a) which has a density of 1.707 g/cm but after
five minutes of incubation in DNase I at 4 C, most of the nuclear DNA was
removed (Tube b). Thirty minutes of incubation in DNase removes all of
the contaminating nuclear DNA and only mitochondrial DNA remains, which
3
has a density of 1.691 g/cm (Tube c). Further treatment with DNase for
one hour shows that the mitochondrial DNA is still present and is there
fore protected from digestion by DNase I by the mitochondrial membranes.
The mitochondria purified in this manner demonstrated the same ribonucle
otide incorporating activities as the mitochondria purified on renografin
gradients. Table 9 shows that the DNased treated mitochondria incorpora-


Figure 10. DENSITY EQUILIBRIA ANALYSIS OF DNA FROM DNASED MITOCHONDRIA
The density equilibria analysis of DNA in mitochondria purified by DNase treatment was measured
by CsCl density gradient centrifugation as described in the methods section. The DNA standards
were Cytophaga P-11 DNA with a density of 1.693 g/cm^, Pseudomonas aeruginosa DNA with a density
of 1.727 g/cm^, and bacteriophage $25 DNA with a density of 1.742 g/cm^. Euglena gracilis mito
chondrial DNA has a density of 1.691 g/cm^ and Euglena nuclear DNA has a density of 1.707 g/cm^
in CsCl. The sources of the DNA were washed 5P mitochondria with DNA standards (tube a), the same
mitochondrial preparation after treatment with DNase I for 5 min (tube b), after 30 min. of treat
ment in (tube c), after 1 hr. of treatment (tube d) and the DNA standards (tube e).


CsCi Density Gradient Gels
DMA STAINED WITH ETHIDIUM BROMIDE
ol;.
11
a ii!i
30 rni i
1 h
CJ
b
c
d
p
C
1.6S5
EE:.;
.
r -
i.69 j
_
1.7 0 7

_
1.727
1.7 <9 2
i r 11
DMA STD
o


71
TABLE 9
A COMPARISON OF THE INCORPORATION OF 3H-UTP and 3H-CTP BY
ISOLATED MITOCHONDRIA PURIFIED BY TREATMENT WITH DNASE I
Labeled
Ribonucleotide
Reaction
Mixture
CPM
3H-UTP
Complete
1,624
3h-utp
-DNA
869
3h-ctp
Complete
27,364
3h-ctp
-DNA
16,669
Mitochondria purified by DNase I treatment were treated with isotonic
buffer to assure swelling and permability. The RNA polymerase assay
was performed as described in the methods. Reaction mixtures of 0.2
ml contained 1.0 mM of each unlabeled NTP and 0.016 mM 3H-UTP or 3H-CTP,
2 Ci/mmole.


72
3 3
ted tridum from H-CTP at a ten fold greater rate than H-UTP. However,
3
about fifty percent of the incorporating activities from both H-CTP and
3 3
H-UTP are DNA dependent which is in contrast to the H-CTP incorporating
activities in renografin purified mitochondria which was not DNA dependent.
Solubilization of the Mitochondrial
RNA Polymerase Activity
The difficulty of defining proper conditions for solubilization of
the chloroplast RNA polymerase of Euglena gracilis (166,185) plus the
rapid inactivation of mitochondrial RNA polymerase activities by some
solubilization methods (11), prompted the investigation to determine
optimum conditions for solubilizing the activity from Euglena mitochon
dria (Table 10). An approach used by Rogall and Wintersherger (15) were
used to determine the optimum conditions for solubilization of the
mitochondrial RNA polymerase. A volume of mitochondrial suspension in
lysis buffer was added to an equal volume of the detergent-KCl solution
to bring the mixture to the indicated final concentrations. The mixtures
were centrifuged at 30,000 rpm to sediment non-solubilized material and
the supernatants were assayed for RNA polymerase activities. The
efficiencies of the different treatments in the solubilizations of RNA
polymerase activity from the mitochondria are summarized in Table 10.
It can be seen that the combined treatment with KC1 and Triton X-100
produced the highest yield of activity solubilized. However, NP-40, a
detergent employed by other investigators (15,173), and 0.5 M KC1 was
almost as effective. Treatment with 0.5 M KC1 alone failed to solubi
lize significant RNA polymerase activity. Treatment with either deter
gent alone was significantly less than with the Triton X-100 plus 0.5
M KC1 treatment which completely destroyed the succinic dehydrogenase


73
TABLE 10
RNA POLYMERASE ACTIVITY SOLUBILIZED FROM
MITOCHONDRIA
Treatment of
Mitochondria
in Lysis Buffer
RNA Polymerase
(units/g wet
30KS
Activity
wt.)
30KP
+ Water
4.2
69
+ 0.5 M KC1
4.2
63
+0.8% NP-40
45.5
46
+0.8% Triton X-100
34.0
37
+ 0.8% NP-40 + 0.5 M KC1
56.0
0
+ 0.8% Triton + 0.5 M KC1
68.0
15
+ Water (Sonication)
0
28
Mitochondria purified by DNase treatment were suspended in lysis buffer
(lg wet wt/10 ml). Mitochondrial samples of 1.0 ml each were then added
to an equal volume of the indicated lysis mixture which contained 0.01 M
EDTA plus additions shown. The mixtures were mixed gently for 15 min. at
4C and then centrifuged for 90 min. at 30,000 rpm (Ti 50 rotor). The
supernatants and pellets were dialyzed against TGMED Buffer for 4 hrs.
before analyzing them for RNA polymerase activity as described in the
methods. The concentration of ^H-UTP was 0.016 mM with a specific radio
activity of 2 Ci/mmole.


74
activity (Table 11) in the supernatant. The NP-40 plus 0.5 M KC1 treat
ment produced supernatants that contained succinic dehydrogenase activi
ties as high as they were in the control mitochondrial pellet. This
suggested that Triton X-100 detergent was effective in completely
disrupting the inner membrane thus freeing the succinic dehydrogenase of
the membrane associated lipids which are needed to reconstitute the
activity. The NP-40 detergent does not seem to be able to free the suc
cinic dehydrogenase enzyme from the associated lipid since any loss of
activity in the pellet could be observed as activity in the supernate
indicating membrane components are present in this fraction needed for the
expression of succinic dehydrogenase activity. The NP-40 detergent even
with 0.5 M KC1 does not seem to be able to free the succinic dehydrogenase
enzyme from the associated membrane components. It appears that although
there is 80% as much RNA polymerase activity in the 30,000 rpm supernatant
produced by NP-40 as there is in the triton treated supernatant, the NP-40
supernatant may not contain solubilized RNA polymerase but just small
mitochondrial fragments that fail to sediment at 30,000 rpm in 90 minutes.
Therefore, the 0.8% Triton X-100 treatment with 0.5 M KC1 was used for
solubilizing the mitochondrial RNA polymerase.
The Effect of Ammonium Sulfate Precipitation on
Mitochondrial RNA Polymerase Activity
It has been reported that Neurospora mitochondrial RNA polymerase
(11) activity could not survive ammonium sulfate precipitation, chroma
tography on DEAE-Cellulose, extensive dialysis, concentration by
ultrafiltration, and sucrose gradient centrifugation. Therefore, it was
necessary to determine if Euglena mitochondrial RNA polymerase could
survive ammonium sulfate treatment because the selection of proper salt


75
TABLE 11
SUCCINIC DEHYDROGENASE ACTIVITY IN THE MITOCHONDRIAL
SUPERNATANTS AND PELLETS AFTER SOLUBILIZATION
TREATMENT
Treatment of
Mitochondria
in Lysis Buffer
Succinic Dehydrogenase
(Units x 10-3)
30KS
Activity
30KP
+ Water
0
40
+ 0.5 M KC1
0
49
+0.8% NP-40
22
27
+ 0.8% Triton X-100
0
31
+ 0.8% NP-40 + 0.5 M KC1
34
0
+ 0.8% Triton + 0.5 M KC1
0
0
+ Water (sonication)
0
0
A unit is a pinole of dichlorophenolindophenol reduced per minute per
ml of enzyme sample at 25C.


76
concentration would allow the fractionation of RNA polymerase away from
other mitochondrial proteins, thus effecting another purification step.
The effect of ammonium sulfate on the mitochondrial RNA synthesizing
activity was measured (Table 12) by adding to solubilized mitochondrial
RNA polymerase samples ammonium sulfate to the desired concentration with
mixing followed by centrifugation at 40,000 rpm for 90 minutes. The RNA
polymerase activity was measured in the redissolved and dialysed protein
samples as described in the methods. The results indicated that 90% of
the activity was recovered when the ammonium sulfate concentration was
50%. It was interesting to observe that when the 30,000 rpm super
(control) was centrifuged at 40,000 rpm for 70 min. 19% of the activity
was lost. This could represent the activity that is not completely
solubilized and thus sediments at 40,000 rpm but not at 30,000 rpm.
Solubilization and Purification
Of Mitochondrial RNA Polymerase
The scheme for the purification of the mitochondrial RNA polymerase
is shown in Figure 11. The optimum conditions described in Table 10
were used to solubilize mitochondrial RNA polymerase. The washed mito
chondria obtained from 182g (wet weight) of cells were purified by
treatment with pancreatic deoxyribonuclease (100 pg/ml) for thirty minutes.
The mitochondria obtained after two washings in STE buffer were suspended
in lysis buffer and either used directly for enzyme solubilization or
stored in liquid nitrogen prior to use. An equal volume of lysis mixture
was mixed with mitochondrial suspension to bring the final concentration
to 0.8% Triton X-100, 0.50 M KC1, and 0.005M EDTA. The mixture was
gently stirred for 15 minutes, 4C. The supernatant obtained after cen
trifuging at 30,000 rpm for 90 minutes was brought to 50% saturation with


77
TABLE 12
THE EFFECT OF AMMONIUM SULFATE PRECIPITATION ON MITOCHONDRIAL
RNA POLYMERASE ACTIVITY
Treatment % (NH^^SO^
Saturation
Activity
(CPM/ml)
%
Recovered
in Pellet
30K Super
12420
-
+ 0, 40KP
1915
19
+ 30, 40KP
5660
46
+ 40, 40KP
6640
53
+ 50, 40KP
11220
90
+ 60, 40KP
6220
50
+ 65, 40KP
8830
71
+ 70, 40KP
9880
80
+ 75, 40KP
9090
73
+ 80, 40KP
7520
61
Mitochondria purified by DNase treatment were suspended in lysis
buffer (1 g wet wt/10 ml) and the RNA polymerase activity was
solubilized as described in the methods. Five milliliters of the
mitochondrial suspension were added to an equal volume of the lysis
mixture (1.6% Triton X-100, 1.0 M KC1, and 0.01 M EDTA pH 7.9) and
mixed gently for 15 min. at 4C and then centrifuged for 90 min. at
30,000 rpm (Ti 50 rotor). The supernatants were divided into 1.0
ml aliquots and solid ammonium sulfate was added to each aliquot to
the desired concentration. The ammonium sulfate was dissolved at
4C by gently mixing and the precipitated protein was collected by
centrifugation at 40,000 rpm, 70 min. (Ti 50 rotor). Each pellet was
dissolved in 1.0 ml of TGMED buffer and dialyzed against 2 liters of
TGMED for 4 hrs. at 4C. The ammonium sulfate concentration in each
dialysate was determined with the conductance bridge and the salt
concentration was adjusted to 0.05 M ammonium sulfate. The RNA
polymerase assay was performed as described in the methods except that
KC1 was omitted.


78
Washed 5P Mitochondria
DNased, 30 min, 4C
Rx Stopped w/3 vol. 0.1 M EDTA pH 7.5: 10% Sucrose
cent. 10,000 x £, 20 min (Repeat 2X in STE buffer)
10 KS Purified Mitochondria
Resuspended in lysis buffer
Add equal vol. lysis mixture:
1.6% Triton X-100
1.0 M KC1
0.01 M EDTA
Cent. 30,000 rpm, 90 min, 2C
30 KS 30 KP
50% (NH^^SO^ satuation
Cent. 40,000 rpm, 70 min, 2C
i I
40 KS 40 KP
Dissolved in TGMED Buffer
Dialyze 4 hr against 2 liters
TGMED: 40 mM (NH4)2S04
DEAE-Sephadex A-25
column chromatography
DCI
Figure 11. PROCEDURE FOR THE PARTIAL PURIFICATION OF MITOCHONDRIA
RNA POLYMERASE


79
respect to ammonium sulfate. The precipitation collected at 40,000 rpm
was dissolved in TGMED buffer and dialysed against TGMED containing 50 mM
ammonium sulfate. The dialysate was subjected to DEAE-Sephadex chroma-
3
tography and the eluted fractions were assayed for H-CTP incorporating
activities using the RNA polymerase assay (Figure 12). The void volume
contained an activity which incorporated tritium preferrentially from
3
the substrate H-CTP by a reaction which was not dependent upon added
DNA or the other Ribonucleotide substrates into acid insoluble products
which resisted pancreatic ribonuclease digestion (Table 13). However,
3
an activity which incorporated label from H-UTP into acid insoluble and
RNase digestible products was eluted from the DEAE-Sephadex column
(Figure 12) in a single peak between 0.32M and 0.37M ammonium sulfate in
TGMED buffer. The specific activity of the RNA polymerase after this
purification step was 0.3 nmoles UMP incorporated/mg protein (estimated
from &280 absorbancy)/10 minutes at 37C. This enzyme activity demon
strates a dependence upon added DNA and ribonucleoside triphosphate
3
substrates (Table 14) for the incorporation of label from H-UTP. The
mitochondrial RNA polymerase activity is also inhibited by low concentra
tions (10 pg/ml) of actinomycin D (Table 15). The activity is insensi
tive to rifampin, a-amanitin, streptovaricin, rifamycin AF/013 and
rifamycin AF/05. Attempts to concentrate this activity by rechromato
graphy on a smaller DEAE-Sephadex A-25 column or to further purify the
activity by glycerol gradient centrifugation resulted in the loss of
significant activity.


Figure 12. ION EXCHANGE CHROMATOGRAPHY ON DEAE-SEPHADEX A-25
3
The procedure is described in the methods. The RNA polymerase activity profile when H-UTP was
substrate is represented by the solid line while the profile when ^h-CTP was the substrate is
represented by the broken line ( A A ). The A280 Pro;file is represented by the double
line while the ammonium sulfate concentration in each fraction is represented by the heavy dashed
line.


FRACTION NUMBER
CTP(epm) o
o o
o o
< 1 j >
3H-UTJ> INCORPORATED '( CPM/20UI)h
M O
o o o
t 1 1 r
8
p
In
ABSORBANCE AT 280 nm
J
xe


TABLE 13
A COMPARISON OF TRITIUM INCORPORATION FROM 3H-UTP and 3H-CTP
BY THE ACTIVITY
IN THE DEAE-SEPHADEX A-25 COLUMN FRACTION 3
Reaction
Mixture
CPM
3H-UTP
Complete
200
3H-CTP
Complete
-DNA
-NTP
+RNase
5428
2581
4389
5316
The RNA polymerase assay reaction mixtures contained 95
pi of the void volume activity in a total volume of 0.20
ml. The concentration of ^h-CTP, were 0.016 mM, 2 Ci/mmole.


83
TABLE 14
THE DEPENDENCY OF THE DEAE COLUMN FRACTION 38 INCORPORATION
OF 3H-UMP UPON ADDED RIBONUCLEOSIDE TRIPHOSPHATES AND DNA
Additions
or
Deletions
CMP
Percent
of
Control
Complete
1488
-
-DNA
187
13
-NTP
398
27
-ATP
168
11
-CTP
238
16
-GTP
178
12
+ Pancreatic RNase
(100 yg/ml)
355
24
The RNA polymerase assay reaction mixtures contained 20 yl of DCI
in a total volume of 0.20 ml. The concentration of 3H-UTP was 0.016
mM, 2 Ci/mmole.


TABLE 15
INHIBITORS OF H-UTP
COLUMN FRACTION 38
INCORPORATION
BY THE DEAE
Inhibitors
CPM
% Activity
Distilled Water
2496
100
1% DMF
2521
100
Actinomycin D, 10 yg/ml
82
3
Rifampin, 100 pg/ml
1898
76
Rifamycin AF/013, 100 yg/ml
2109
85
Rifamycin AF/05, 100 yg/ml
2065
83
Streptovaricin, 100 yg/ml
2369
95
a-Amanitin, 5 yg/ml
1490
60
Pancreatic RNase 100 yg/ml
69
3
The RNA polymerase assay was performed with the addition of
inhibitors to the final concentrations indicated above. The
concentration of %-UTP was 0.016 mM, 2 Ci/mmole. Rifampin,
rifamycin AF/013, rafamycin AF/05, and streptovaricin were sol
ubilized in 1% N'N' dimethyl formamide. Reaction mixtures con
tained 50 ml of the DCI enzyme in a total volume of 0.2 ml.


85
Enzymatic Properties of the
Mitochondrial RNA Polymerase
The dependence of the mitochondrial RNA polymerase on the concen
tration of MnC^ is shown in Figure 13. Low concentration (about 1 mM)
j |
of Mn stimulated the activity of this enzyme and higher concentrations
decreased the activity. However, the enzyme activities were also stimu-
| j
lated by low concentrations of Mg (Figure 14) demonstrating the optimal
concentration at 0.5 mM.
The template requirement of the mitochondrial enzyme is summarized
in Table 16. The mitochondrial enzyme prefers poly d(AT) as a template
which is similar to the rifampicin sensitive mitochondrial RNA polymerase
from Neurospora crassa, described by Kuntzel and Schafer (11). It is
interesting to note that while Euglena nuclear DNA was a more effective
template than denatured calf thymus DNA that it is not necessarily a
more preferred template than Euglena mitochondrial DNA. The mitochondrial
DNA was only available in 1/5 the saturating DNA concentration and a
comparison of the templates was not possible.


Figure 13. DEPENDENCE OF THE ACTIVITY OF THE DEAE FRACTION 38
ON THE CONCENTRATION OF Mn++
Assays were carried out as described in methods. The concentration
of ^H-UTP was 0.016 mM, 2 Ci/mmole. The reaction mixtures contained
20 pi of DCI enzyme in a total volume of 0.2 ml.


87
1
10


Full Text
UNIVERSITY


MITOCHONDRIAL RNA SYNTHESIS AND RIBONUCLEOTIDE INCORPORATION STUDIES
IN Euglena gracilis
by
GEORGE ERWIN BROWN
A DISSERTATION PRESENTED TO THE GRADUATE COUNCIL OF THE UNIVERSITY
OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS
FOR THE DEGREE OF DOCTOR OF PHILOSOPHY
UNIVERSITY OF FLORIDA
1976

This dissertation is dedicated to my wife, Carolyn M. Brown, my parents,
Mr. and Mrs. Marshall V. Brown, and my children, George E. Brown and
Erika N. Brown, for their endless encouragement and personal sacrifices
during the time I spent in graduate school.

ACKNOWLEDGEMENTS
The author wishes to express his gratitude to the members of his
doctoral committee for their advice, guidance and assistance in the
preparation of this dissertation. Special acknowledgements of thanks
are given to the author's major professor and advisor, Dr. J. F. Preston,
for providing the laboratory facilities and supplies for conducting the
research reported here. The laboratory techniques learned in his labora¬
tory have already proved to be rewarding for the author in his pursuit
of other research projects. The use of Euglena gracilis as a test organism
for studying organelle development originated with Dr. Preston. He con¬
tributed much of his normal off duty time offering advice and suggestions
toward the completion of this study.
The author also thanks Dr. R. F. Mans for the personal interest he
took in the author's research and academic development. The advice and
guidance he provided proved to be valuable in pursuing the study of
nucleotide incorporations. The consultations held with Dr. Mans provided
the stimulation to complete this research rather than to terminate it.
In addition the knowledge obtained in the "Rusty Mans Molecular Journal
Club" was most rewarding in terms of the author's academic development.
Expressions of gratitude are also in line for Dr. L. 0. Engram for
his encouragements when things looked bad and for his valuable suggestions
that led to the completion of this research. Dr. Engram provided chemicals
and laboratory facilities for conducting several experiments.
iii

Dr. E. M. Hoffmann will be long remembered as the author's first
graduate instructor and for inspiring the author's interest in microbiology.
The author also thanks him for his assistance in the preparation of this
dissertation and for providing laboratory equipment for conducting this
research.
The author wishes to express his gratitude to Dr. T. W. O'Brien for
serving as the mitochondria expert and for providing advice and guidance
in the initial phase of this research. The consultations held with
Dr. O'Brien provided the author with the assistance and encouragements for
completing this dissertation.
iv

TABLE OF CONTENTS
Page
ACKNOWLEDGMENTS iii
LIST OF TABLES vi
LIST OF FIGURES vii
KEY TO ABBREVIATIONS viii
ABSTRACT x
INTRODUCTION 1
LITERATURE REVIEW 8
MATERIALS AND METHODS 24
RESULTS 34
DISCUSSION 91
CONCLUSIONS 97
REFERENCES 98
BIOGRAPHICAL SKETCH 108
v

LIST OF TABLES
Page
1. DISTRIBUTION OF SUCCINIC DEHYDROGENASE ACTIVITY 41
2. NUCLEOTIDE INCORPORATION BY ISOLATED MITOCHONDRIA 42
3. INCORPORATION OF RIBONUCLEOSIDE TRIPHOSPHATES BY ISOLATED
MITOCHONDRIA 46
4. THE DEPENDENCY OF THE MITOCHONDRIAL ACTIVITY INCORPORATING LABEL
FROM 3H-CTP UPON ADDED RIBONUCLEOTIDES AND DNA 47
5. DISTRIBUTION OF 3H-CMP INCORPORATING ACTIVITY 48
6. INHIBITION OF 3H-CMP INCORPORATION 50
7. CHARACTERIZATION OF PRODUCT 51
O
8. PHOSPHOLIPID EXTRACTION OF THE H-CMP LABELED PRODUCT 60
9. A COMPARISON OF THE INCORPORATION OF 3H-UTP AND 3H-CTP BY
ISOLATED MITOCHONDRIA PURIFIED BY TREATMENT WITH DNASE 1 71
10. RNA POLYMERASE ACTIVITY SOLUBILIZED FROM MITOCHONDRIA 73
11. SUCCINIC DEHYDROGENASE ACTIVITY IN THE MITOCHONDRIAL SUPERNA¬
TANTS AND PELLETS AFTER SOLUBILIZATION TREATMENT 75
12. THE EFFECT OF AMMONIUM SULFATE PRECIPITATION ON MITOCHONDRIAL
RNA POLYMERASE ACTIVITY 77
3 3
13. A COMPARISON OF TRITIUM INCORPORATION FROM H-UTP AND H-CTP
BY THE ACTIVITY IN THE DEAE-SEPHADEX A-25 COLUMN FRACTION 3 82
14. THE DEPENDENCY OF THE DEAE COLUMN FRACTION 38 INCORPORATION OF
3H-UMP UPON ADDED RIBONUCLEOSIDE TRIPHOSPHATES AND DNA 83
15. INHIBITORS OF 3H-UMP INCORPORATION BY THE DEAE COLUMN FRACTION
38 84
16. TEMPLATE SPECIFICITY OF THE DEAE COLUMN FRACTION 38 90

LIST OF FIGURES
Page
1. SCHEMA FOR THE ISOLATION OF MITOCHONDRIA 35
2. DENSITY EQUILIBRIA ANALYSIS OF MITOCHONDRIAL DNA 38
3. RIBONUCLEOSIDE TRIPHOSPHATES INCORPORATION BY ISOLATED
MITOCHONDRIA 43
4. SUCROSE GRADIENT ZONAL CENTRIFUGATION 53
5. BIOGEL P-4 COLUMN CHROMATOGRAPHY 55
6. ULTRA VIOLET ABSORBANCY SPECTRA 58
7. THIN LAYER CHROMATOGRAPHY OF THE 3H-CMP LABELED PRODUCTS ELUTED
FROM THE BIOGEL P-4 COLUMN 61
8. THIN LAYER CHROMATOGRAPHY OF THE PRODUCTS OF THE ENZYMATIC
CLEAVAGE OF THE 3H-CMP LABELED MATERIAL ELUTED FROM THE
P-4 COLUMN 64
9. THE MIGRATION OF THE ENZYMATIC CLEAVAGE PRODUCTS CONTAINING
TRITIUM LABEL ON PEI CELLULOSE 66
10. DENSITY EQUILIBRIA ANALYSIS OF DNA FROM DNASED MITOCHONDRIA 69
11. PROCEDURE FOR THE PARTIAL PURIFICATION OF MITOCHONDRIA RNA
POLYMERASE 78
12. ION EXCHANGE CHROMATOGRAPHY ON DEAE-SEPHADEX A-25 80
13. DEPENDENCE OF THE ACTIVITY OF THE DEAE FRACTION 38 ON THE
CONCENTRATION OF Mn++ 86
14. DEPENDENCE OF THE ACTIVITY OF THE DEAE FRACTION 38 ON THE
CONCENTRATION OF Mg++ 88
vii

KEY TO ABBREVIATIONS
A260
absorbancy at 260 nm
A280
absorbancy at 280 nm
AMP
adenosine monophosphate
ATP
adenosine triphosphate
ATPase
adenosine triphosphatase
BGP- 1
the first peak of tridium labeled material
eluted from Biogel P-4 column
BGP- 2
the second peak of tridium labeled material
eluted from Biogel P-4 column
BSA
bovine serum albumin
CAP
catabolite gene-activator protein
CDP-diglyceride
cytidine diphosphate diglyceride
CMP
cytidine monophosphate
CpC
cytidylyl (3' 5’) cytidine
cpm
counts per minute
CTP
cytidine triphosphate
dCTP
deoxycytidine triphosphate
cyclic AMP
adenosine 3' : 5'-cyclic monophosphoric acid
DEAE
diethylaminoethane
DNA
deoxyribonucleic acid
DNase
deoxyribonuclease (EC 3.1.4.5)
dpm
disintegrations per minute
DTT
dithiothreitol
viii

EDTA
ethylenediamine tetraacetate
&
gravity
GMP
guanosine monophosphate
GTP
guanosine triphosphate
hr
hour
3h
radioactively labeled with tridium
I
Iodine
NTPs
ATP, CTP, GTP, UTP (minus the labeled substrate)
poly d(AT)
alternating copolymers of deoxyadenylic and
deoxythymidylic acid
poly (C)
poly cytidylic acid
POP
2, 5 - diphenyloxazole
POPOP
1, 4 - di- [2-(5-phenyloxazolyl)]-benzene
RNA
ribonucleic acid
Hn RNA
heterogeneous nuclear ribonucleic acid
in RNA
messenger ribonucleic acid
r RNA
ribosomal ribonucleic acid
t RNA
transfer ribonucleic acid
RNase
ribonuclease (EC 2.7.7.16)
S
sedimentation coefficient
SDS
sodium dodecyl sulfate
SSC
standard saline citrate
SVP
snake venom phosphodiesterase
TCA
trichloroacetic acid
tris
tris (hydroxymethyl amino) methane
UMP
uridine monophosphate
UTP
uridine triphosphate
UV
ultra violet
ix

Abstract of Dissertation Presented to the Graduate Council of the
University of Florida in Partial Fulfillment of the Requirements for
the Degree of Doctor of Philosophy
MITOCHONDRIAL RNA SYNTHESIS AND RIBONUCLEOTIDE
INCORPORATION STUDIES IN
Euglena gracilis
By
GEORGE ERWIN BROWN
August, 1976
Chairman: James F. Preston, Ph.D
Major Department: Microbiology
Two experimental approaches were utilized to study the in vitro
mitochondrial RNA synthesis in Euglena gracilis. First the RNA syn¬
thesis activity was examined in isolated and highly purified mitochondria
of Euglena gracilis strain z streptomycin bleached aplastic mutants. The
mitochondria obtained after isopynic centrifugation in sodium diatrizoate
3
gradients yield mitochondrial DNA (p=1.691 g/cm ) and little nuclear DNA
3 3
(p=1.707 g/cm ). The incorporation of label from the H-ribonucleoside
triphosphates into acid insoluble products showed no dependence upon the
presence of the other ribonucleotides or that of added DNA. The preferred
3
substrate was H-CTP from which label was incorporated into acid insoluble
products at a rate 100 times greater than the other labeled ribonucleotides.
This activity incorporates CMP from CTP into acid insoluble products
x

which contain phosphodiester bonds by a pyrophosphorolysis reaction. The
activity was inhibited by pyrophosphate and actinomycin D at 100 mg/ml but
not by inorganic phosphate, a-amanitin, rifampicin or low concentrations of
actinomycin D (50 mg/ml). The products were sensitive to snake venom phos¬
phodiesterase and alkaline hydrolysis but not pancreatic ribonuclease. The
products were isolated by phenol extraction followed by ethanol precipita¬
tion of the aqueous phase. Two classes of molecules with mean molecular
weights of 800 and 680 respectively were isolated. They were found to have
absorbancy spectra identical to that of C^C and were resistant to pancreatic
ribonuclease digestion. However, snake venom phosphodiesterase cleaved the
products into 5'-CMP, the expected cleavage product of CpC or poly C.
Mitochondria, purified by either isopycnic centrifugation or by treat¬
ment with pancreatic deoxyribonuclease, contained an associated activity
3
which incorporated label from H-UTP into acid insoluble products which
were sensitive to pancreatic ribonuclease digestion. This activity was
DNA dependent in the DNase mitochondria which was in contrast to that
observed for sodium diatrizoate purified mitochondria.
The second approach was to. study the activity of solubilized and
partially purified mitochondrial RNA polymerase. An enzyme purification
procedure is described utilizing a solution of Triton X-100 detergent and
KC1, centrifugation, ammonium sulfate precipitation, and DEAE-Sephadex A-25
chromotography that resulted in a partially purified enzyme with a specific
activity of 0.3 nmoles UTP/mg/10 min at 37°. The incorporation of UMP into
acid insoluble material required DNA the four ribonucleoside triphosphates:
GTP, CTP, UTP, ATP, and metal, either Mn"*""1" or Mg"*-1". The product of the
reaction was sensitive to pancreatic ribonuclease digestion. The mito-
xi

chondrial enzyme is distinctly different from the nuclear RNA polymerase II
with respect to chromatographic behavior and antibiotic sensitivity. It
eluted from the DEAE-Sephadex column in a single peak between 0.32 M and
0.37 M ammonium sulfate in TGMED buffer, was stimulated by low concentration
| |
of the metals Mn and was insensitive to a-amanitin and rifampicin.
/
Chairman
xii

INTRODUCTION
Informational macromolecules are those through which genetic in¬
formation actually flows. Genetic information may be defined as that
primarily required to assemble a protein needed to perpetuate biological
order, but the definition applies also to the information for ribonucleic
acids (RNAs) which are not translated into protein. Deoxyribonucleic
acids (DNA) functions as the repository for information or coding se¬
quences ultimately destined to appear either in RNA or protein, and in
noncoding sequences which can act as signals for such things as initiation
and termination of the biosynthesis of a RNA chain. Within the cell the
informational sequences are transmitted on which specific proteins are
synthesized.
The DNA dependent RNA polymerases (E. C. 2.7.7.6.) are the key
enzymes implicated in the first step of genetic information flow from DNA
to protein. The term DNA dependent RNA polymerases is used to designate
enzymic activities which, using DNA templates, catalyze the sequential
assembly of the four ribonucleoside triphosphates into RNA molecules.
The synthetic reaction requires the presence of a DNA or a polydeosy-
ribonucleotide template and a divalent metal ion. The main features of
the transcription reaction consist of the enzyme locating and binding to
specific sites on the DNA, initiating a RNA chain de novo, extending the
chain by moving along the DNA using only one strand as a template to
direct the polymerization of the four ribonucleoside triphosphates into
1

2
a complementary RNA molecule, and finally, in some cases, termination of
the chain with the release of the enzyme from the DNA template. RNA
polymerases are responsible for the synthesis of ribosomal RNA's, transfer
RNA's and messenger RNA's, all of which are needed for translation of the
mRNA template into proteins.
Substantial data have been accumulated on the regulation of RNA
polymerases in prokaryotes, where a single enzyme is responsible for the
synthesis of all types of cellular RNA, but comparatively little is
known in the case of eukaryotes, where the situation is more complex.
Within the last few years, it has been demonstrated that eukaryotic cells
contain several types of RNA polymerases, which differ in their locali¬
zations, structures, and functions.
Nuclei contain several different RNA polymerases. Nuclear RNA
polymerase I is localized in the nucleolus (1). It is primarily con¬
cerned with the transcription of genes specifying the larger (45s)
ribosomal precursor RNA. Nuclear RNA polymerase II is localized in the
nucleoplasm and presumably transcribes messenger RNA. It is inhibited
by a-amanitin (2,3) an octapeptide produced by several species of the
genus Amanita (4). In several organisms a third (5,6) or even a fourth
(7,8) nuclear RNA polymerase activity was found. A separate RNA poly¬
merase III, which is distinct from forms I and II, has been shown to
transcribe (4s) t-RNA and 5s RNA (9,10). In mammals this enzyme is
slightly sensitive to a-amanitin, being 1,000 fold more resisistant than
RNA polymerase II.
RNA polymerase activity has also been found localized in mito¬
chondria and chloroplasts. The mitochondrial enzymes exhibit properties

3
which clearly distinguish them from the nuclear RNA polymerases. The
mitochondrial enzyme is insensitive to a-amanitin and although some
functional differences have been described in mitochondrial RNA poly¬
merase, there is general agreement that the molecular weight of the
enzyme, consisting of a single polypeptide chain, is about 60,000
daltons (11-15).
Control of RNA synthesis for regulating cell
growth, development, and differentiation
If considerations were based purely on energy requirements, it
would be reasonable to exclude DNA replication and mRNA translation as
primary levels where gene expression is controlled. Several reasons may
be given for excluding changes in the kinds or numbers of genes and post-
transcriptional modification as steps at which regulation generally takes
place. Nuclei of cells in different stages of development in the same
individual appear to be genetically identical and changes in the number of
gene copies from which mRNA is transcribed have not been observed. Like¬
wise, there is no good evidence that changes in the kinds of proteins
synthesized in development can be attributed to controls at the trans¬
lational level and in addition eukaryotic cells have no mechanism for
t
excluding the translation of messages characteristic of other cell
types (16). It seems that translational controls are used to make quanti¬
tative adjustments to a pattern of protein synthesis determined primarily
by the synthesis of new messages.
It can be concluded by elimination that transcription must be the
level at which gene expression is primarily controlled. In bacterial
systems, there are two well-characterized examples of programmed trans-

scription: bacteriophage development and bacterial sporulation. These
processes are similar in some ways to differentiation. Therefore, under¬
standing the control of transcription in these processes may provide some
clues to the regulation of transcription in eukaryotic systems. Studies
of transcriptive regulation of phage A show how the expression of specific
genes is regulated by other gene products (17). In both T^ bacteriophage
development and Bacillus subtilis sporulation, significant changes that
are coordinated with development occur in the RNA polymerase molecule
and associated proteins (18,19,20). In _B. subtilis there is evidence
suggesting that these changes may be related to the onset of sporulation.
The primary need for regulating transcription is that, by turning
it on and off, protein synthesis is controlled at the origin. In both
prokaryotic and eukaryotic organisms regulation must enable cells to
cope with fluctuation in their immediate environment. In the prokaryote
most of the genes come into operation sooner or later during each cell
cycle, but this is not true of many genes in the higher eukaryote where
differentiation has resulted in a strategy of regulation unlike that in
the prokaryote. By contrast the lower unicellular eukaryote has a re¬
gulatory resemblance to the prokaryote. Evidence that gene transcription
is controlled in eukaryotic development is provided by the Balbiani rings
of polytene chromosomes. The ring is characteristically expanded and
active in RNA synthesis or unpuffed and inactive in transcription at
different stages of development (19). Also, agents such as actinomycin
which inhibit RNA synthesis but which have little immediate effect on
translation arrest eukaryotic development within a short time. These
effects suggest an essential role for the regulation of transcription in
development.

5
Regulation of transcription by alteration
in molecular properties of RNA polymerase
The regulation of transcription may be affected by control of any
of the components involved in the RNA synthesis: the template, the poly¬
merase, associated factors and other environmental conditions. However,
the multiplicity and different intranuclear localization of DNA-dependent
RNA polymerases have suggested that gene expression in eukaryotic cells
is regulated, in part, by distinct RNA polymerases with different template
specificities. The quantity and quality of RNA synthesis can depend
either on the concentration of RNA polymerase or on the level of activity
of the enzyme. In growing _E. coli cells, there is a pool of excess RNA
polymerase (21) although the polymerase subunits seem to be synthesized
at the same rate as other cellular proteins (22). Therefore, the regula¬
tion of growth depends upon modulation of the RNA polymerase activity
rather than upon the concentration of the enzyme. Although the rRNA
genes account for only about 0.4% of the genome (23), rRNA accounts for
40% of the total RNA synthesized in growing cells and is therefore
preferentially synthesized (24) .
On the other hand, bacteriophage infection results in the inhibition
of host cell transcription presumably by the destruction or alteration of
the host RNA polymerase (25,26). Similar effects on host RNA synthesis
have been found in viral infections in mammalian cells (27,28). This
may be analogous to terminal differentiation where the loss of nuclear
function is involved (29)
Regulation of transcription and control of
organelle development
An understanding of the mechanisms by which a eukaryotic cell
controls organelle development should give valuable insight into many

6
similar cellular control processes such as those involved in differentia¬
tion, morphogenesis and tumor production. Since RNA polymerase has been
implicated in controlling development in prokaryotic systems (18,19,25,
30,31) it could be a major controlling factor in Euglena gracilis organelle
development. Therefore, the research in this laboratory focuses upon the
RNA polymerases found in Euglena, and upon their role in organelle de¬
velopment. Euglena is an excellent organism to study transcription in
organelles since it contains both chloroplasts and mitochondria. The
organism is a unicellular flagellate with 10 to 12 chloroplast per cell,
numerous mitochondria, and a polyploid nucleus. Euglena can be grown
phototrophically, heterotrophically or mixotrophically. These growth
conditions will alter the development of the organelles and therefore
offer a useful system for studying organelle development. Phototrophic
conditions are restrictive for photosynthetic function, but the other
two growth conditions are permissive. In cells grown phototrophically,
mitochondrial development is repressed, while the chloroplasts function
to provide the bulk of the cell's energy. Cells grown mixotrophically
derive energy from functional chloroplasts and mitochondria. However,
wild type cells can be grown in the dark as etiolated heterotrophs
where the proplastids, which have stopped forming chlorophyll and
chloroplast membrane, exist as undeveloped proplastids which are
capable of differentiating into functional mature chloroplasts .upon
exposure to light. The heterotrophic dark grown cells primarily use
mitochondrial oxidative phosphorylation as a source of energy. It has
been shown (32) that cells grown heterotrophically have large active
mitochondria, and that exposure to light, which starts plastid develop-

7
ment, results in a decrease in mitochondrial density and function (33).
Mutants that completely lack plastids (aplastidic) have been produced by
treatment with streptomycin, heat, or ultraviolet light (34,35). These
permanently bleached cells are perfectly viable as long as they are
grown on a carbon source which can be respired. This makes it feasible
to distinguish processes controlling mitochondrial development from those
controlling chloroplast development.
This dissertation concerns itself with studies of mitochondrial RNA
synthesis in Euglena gracilis and is directed towards elucidating the
components of the transcriptional process, in particular, characterizing
the mitochondrial DNA dependent RNA polymerase with a view toward under¬
standing the control of transcription. The RNA synthesizing activity of
isolated mitochondria has been studied and the DNA dependent RNA polymerase
has been solubilized and partially purified. The experimental approach
in this study was to develop a method for preparing highly purified mito¬
chondria, to study the incorporation of labeled ribonucleoside triphosphate
precursors into RNA by isolated mitochondria and to identify the labeled
products. The mitochondrial DNA-directed RNA polymerase was partially
purified by ion exchange chromatography. The enzyme activity was charac¬
terized to determine its requirements for product synthesis and to compare
it to nuclear activities with respect to these requirements.

LITERATURE REVEIW
Guides to the Literature
In recent years, several review articles or symposia on RNA poly¬
merases from prokaryotes (20,36-46) and eukaryotes (47-52), on the
regulatory elements (53,54) and on transcription (55-59), have appeared.
There are several introductory texts (60-62) and collected papers (63-66)
which may be consulted for a more historical background discussion.
General Mechanisms of RNA Synthesis
The synthesis of RNA from DNA is mediated by a DNA dependent RNA
polymerase that uses ribonucleoside triphosphates as precursors. In
general the reaction involves binding of enzyme to DNA template and
the asymmetric transcription of the DNA. RNA synthesis appears to proceed
in the following steps: template binding, chain initiation, chain elon¬
gation and termination. The RNA polymerase binds to specific initiation
sites (promoter sites) on the DNA in a specific reaction in which the
strands of the DNA are opened over a short local region. Chain initiation
involves the binding of two ribonucleotide triphosphates to the RNA
polymerase followed by elimination of inorganic pyrophosphate to form a
dinucleotide tetraphosphate. The initial nucleotide retains its triphos¬
phate while each added ribonucleotide triphosphate has its two terminal
phosphates cleaved as pyrophosphate. The direction of elongation is
from 3' hydroxy to 5' phosphate in the DNA, the RNA being made 5' to 3'
8

in the antiparallel direction. The initial product is complementary to
the region of the DNA employed as template and the RNA may be modified
9
post transcriptionally.
Prokaryotic Transcriptions
Background
There are several reviews of prokaryotic transcription available
(35,36,56,57) which may be consulted for discussions of aspects that
will not be considered here. The basic transcriptional studies have
been carried out in bacterial systems with and without phage infection
and these studies have served as a model system for understanding the
transcriptional process and its control. Only the properties of the
bacterial RNA polymerase and the regulation of bacterial RNA synthesis
will be reviewed here.
Prokaryotic RNA polymerase
The bacterial RNA polymerases are large molecules (molecular
weights between 400,000 and 500,000) and have complex subunit structures.
Two enzymatically active forms of the RNA polymerase are currently known:
the holoenzyme and the core enzyme. The holoenzyme contains the following
polypeptide chain subunits: one beta prime (3') subunit; one beta (3)
subunit; two alpha (a) subunits; and either one sigma (o) subunit or one
sigma prime (o') subunit. The subunits have the molecular weights of
approximately 160,000; 155,000; 90,000; and 40,000 respectively. Two
forms of the holoenzyme with the structures ct233'ci(67) and ot233'a'(68)
have been found in Escherichia coli. The core enzyme lacks a sigma
subunit. Therefore, the holoenzyme can be separated into two functional
parts: a core enzyme, which is able to synthesize RNA but lacks the

10
ability to initiate such synthesis specifically; and a sigma subunit,
which acts catalytically to allow the efficient initiation of RNA at
specific promoter sites. Bacterial RNA polymerases from different
species appear to be closely related in subunit structure but show dif¬
ferences in the sizes of the subunits (42). Some preparations of the
Eh coli enzyme contain a minor component a) (69,70) which has a molecular
weight of about 10,000 and is present in the stoichiometry of two u per
holoenzyme. The subunits of the holoenzyme are functional subunits
since it has been demonstrated that these subunits are essential for the
reconstitution of enzymic activity when the enzyme is reformed from
separated subunits (71,72). The m subunit is not required for recon¬
stitution of RNA polymerase holoenzyme activity.
All transcription in Eh coli is sensitive to inhibitors of the
purified enzyme, such as rifampicin and streptolydigin (73,74), which
block RNA synthesis through interaction with the 3 subunit of the
enzyme (75). Thus, all RNA synthesis appears to depend on an enzyme
complex in which the 3 subunit is functional. The beta subunit is also
altered in enzymes from mutants resistant to rifampicin (71) or
streptolydigin (76-78).
General mechanism of prokaryotic transcription
Transcription is a process involving a series of different bio¬
chemical events. The holoenzyme is able to recognize and to bind to
specific regions on the DNA template (the promoters) and to undergo a
conformational change, catalyzing the addition of substrate ribonucleo-
side triphosphates to the binding site (79), with the subsequent for¬
mation of the first internucleotide linkage of ATP or GTP with a second

11
ribonucleoside triphosphate. Inorganic pyrophosphate is eliminated,
resulting in the formation of a dinucleotide tetraphosphate of the general
structure pppPupX. The sigma subunit appears to be released (80,81) and
the core enzyme moves along the DNA template synthesizing the RNA chain
by adding ribonucleoside monophosphates to the 3' hydroxy terminus of the
nascent RNA chain from ribonucleoside triphosphate substrates. When the
RNA polymerase encounters a specific termination site on the DNA template,
RNA chain growth is terminated and the nascent RNA chain and the RNA
polymerase are released from the template. A termination protein factor,
rho, has been implicated in this last step in _E. coli (82). However, the
RNA polymerase can in some instances terminate accurately without rho.
Prokaryotic polymerases and regulation of transcription
Two basic mechanisms, negative control and positive control, appear
to be equally effective in controlling prokaryotic transcription. In
negative control, the regulator gene product (a repressor molecule) in
its active form binds to the operator gene to prevent the transcription
of the operon's structural genes. When the repressor is inactivated by
the specific inducer, transcription of the structural genes may proceed.
Thus the repressor controls RNA synthesis by negatively affecting RNA
polymerase activity with respect to specific sites on DNA. The classic
example of negative control is the lac operon of E. coli (83). The lactose
operon is under the negative control of the lactose repressor which binds
specifically to the lactose operator (84,85) region of the DNA, thus pre¬
venting transcription of the DNA past the operator. The expression of
this operon requires induction by a galactoside. The inducers bind to
the repressor and decrease the repressor's affinity for the lactose operator.

12
The histidine operon is another example of regulation by negative
repression, with histidyl-tRNA as the co-repressor necessary for activa¬
tion of the histidine repressor which binds to the histidine operator
region of the DNA and prevents transcription of the DNA past the operator (86).
In positive control the RNA polymerase has low affinity for the
promoter site, and the controlling molecule in its active form helps the
RNA polymerase to initiate transcription by increasing the enzyme's
affinity for the promotor site. Positive control of gene expression has
been demonstrated for the arabinose operon of E. coli (86). The protein
product of the C gene is a repressor in the absence of the specific in¬
ducer, arabinose. However, in the presence of arabinose the C gene
protein is converted to an activator which is required for expression of
the arabinose genes.
The sigma subunit of RNA polymerase and the catabolite gene activator
protein (CAP) are examples of protein factors which influence transcription
by increasing the affinity of the RNA polymerase for promoters. Sigma
interacts with the core RNA polymerase and facilitates the binding of the
enzyme to a promoter. CAP, also known as cyclic AMP receptor protein,
plays a role in the regulation of genes subject to catabolite repression.
CAP appears to work in addition to sigma to alter initiation specificity
in the presence of cyclic AMP. Cyclic AMP combines with CAP and produces
an allosteric change in the protein. The cyclic AMP-CAP complex then
binds to the DNA close to the promoter of an inducible operon (i.e.,
lactose, galactose, tryptophan, and histidine) and produces a change in
the DNA so that the RNA polymerase can bind to the specific promoter
region and carry out a round of transcription of the operon in question.
Thus the expression of the lactose operon requires cyclic AMP, CAP, holo-
enzyme and the removal of the repressor from the operator.

13
Alteration of RNA polymerase by phage Infection and sporulation
The structure and selectivity of the bacterial RNA polymerase is
altered under certain conditions of viral infection and sporulation. The
alterations of the selectivity of RNA synthesis occur during lytic growth
of the phage in different stages, giving rise to different transcripts
during the course of phage replication and assembly. The bacteriophage T^
infects E. coli and subsequently host RNA synthesis is inhibited, phage-
specific macromolecular synthesis is initiated and the host cells are
ultimately lysed (87,88). A temporal sequence on phage-specific mRNAs
have been detected (89). The first class of mRNA (immediate early)
appeared immediately after infection, and hybridized exclusively to one
strand of T^ DNA. The second class of mRNA (delayed early) to appear
hybridized to later sequential sites on the same T^ DNA strand. The late
mRNA apparently coded for viral coat protein and it hybridized to the
opposite T^ DNA strand.
The sequence of transcription is mediated by changes in the RNA
polymerase. Sigma factor disappears soon after infection (90) and it is
this loss which is presumably responsible for restriction of the tran¬
scription of the host genome. There is then a sequential alteration of
each of the subunits of the core RNA polymerase (26,91,92). The subunit
is modified by the covalent attachment of AMP to the alpha subunit. The
RNA polymerase acquires four small T^-specific proteins (93) of 10,000-
25,000 daltons which are coded for by the delayed early genes. Thus the
transcription specificity appears to be induced by modifications in the
polymerase or associated factors.
Bacteriophage lamda can either lysogenize or grow lytically (94,95).
During the lytic growth the host _E. coli RNA polymerase transcribes the

14
phage N and Q genes. The two protein products of these genes then mediate
the further transcription of the lamda DNA (96). It appears that the N
gene product is an antiterminator protein that permits the continued
transcription past a rho dependent termination sequence (97). The Q
gene product is a repressor that binds to the operator region for the
lamda repressor gene, thus preventing the transcription of this gene.
Bacillus subtilis phages SPOL and SP82 show a regulated program of
transcriptional alteration during normal growth (98,99). The infected
cell RNA polymerase contains two proteins not present in the normal RNA
polymerase and the sigma subunit is not required for selectivity (100).
Several observations suggest there are specific changes in the
structure of Bacillus subtilis RNA polymerase during sporulation. The
purified sporulating RNA polymerase lacks sigma factor and contains a
modified 6 subunit of lower molecular weight than the vegetative enzyme
(19,101). In addition, a new polymerase subunit of 60,000-70,000 daltons
appears (102). These modifications appear to be due to the action of
proteases that are present in cells undergoing sporulation, and it is
thought that these changes are related to the alterations in transcrip-
tive specificity that occur during sporulation.
Genetic studies also support a specific role of polymerase in the
sporulation process. Mutants which contain an altered beta subunit fail
to sporulate (101). The evidence form the genetic studies and the bio¬
chemical studies is consistent with the hypothesis that changes in the
subunit structure of RNA polymerase mediate the changes in transcription
associated with Bacillus subtilis sporulation.

15
Eukaryotic RNA Polymerases
Background
There are several recent review articles on eukaryotic RNA polymerases
(47-52). These may be consulted for discussions that are omitted in this
review. The DNA dependent RNA polymerase activity was first demonstrated
in rat liver nuclei by Weiss in 1955 (103) who partially purified the en¬
zyme which was firmly attached to DNA. Mans and Novelli (104) were the
first to study a solubilized eukaryotic RNA polymerase in 1964. However
the problem of freeing the RNA polymerase from DNA was more severe for the
other eukaryotic RNA polymerases studied and consequently most studies on
nuclear RNA polymerase activity were carried out on isolated nuclei or
unpurified chromatin until a eukaryotic RNA polymerase was purified in
1965 (105).
Isolated nuclei demonstrated transcription activity which synthesized
mostly GC-rich ribosomal RNA at low ionic strength. This activity was
j |
localized in the nuceolus and stimulated by Mg . However, at high ionic
strength mostly DNA-like RNA was synthesized by an activity which was
j j
stimulated by Mn and localized in the nucleoplasm (106-111). The nucleo-
plasmic activity was specifically inhibited by a-amanitin (3).
Multiple forms of nuclear RNA polymerases were demonstrated by Roeder
and Rutter (5) in rat liver and Xenopus laevis. They demonstrated three
types of eukaryotic RNA polymerases which could be separated based on
chromatographic properties, sensitivity to specific inhibitors and sensi¬
tivity to ions. The first RNA polymerase eluted from a DEAE Sephadex
column (nuclear RNA polymerase I) was found to be of nucleolar orgin,
insensitive to a-amanitin and synthesized a product which hybridized to

16
RNA competitively with ribosomal RNA (rRNA) but not with heterogeneous RNA
(Hn RNA). RNA polymerase II, the second enzyme eluted from the column,
was a nucleoplasmic, a-amanitin sensitive RNA polymerase that synthesized
a product that hybridized to DNA competitively with Hn RNA but not with
r-RNA (32). RNA polymerase III was thought to be nucleoplasmic. Since
then several laboratories have demonstrated that the nucleus, nucleolus
and cellular organelles have unique transcriptional systems (44).
On a protein percentage basis, the amount of RNA polymerase activity
in eukaryotic cells is much lower than in prokaryotes. The RNA polymerases
from calf thymus, which is a tissue rich in the enzyme (112), is only ob¬
tained in a few milligrams per kilogram of tissue (113-115). This yield,
which is a few orders of magnitude lower than that to Escherichia coli RNA
polymerase (67,70,116) demonstrates one of the difficulties encountered in
studying the regulation of eucaryotic transcription at the molecular level
(studies which require significant quantities of the highly purified enzyme).
The specific activity of the eukaryotic RNA polymerases is in the order of
that of the purified E. coli RNA polymerase (50,113,114,116).
Mitochondrial transcription
The mitochondrion represents a semi-autonomous organelle within the
eukaryotic cell in the sense that it contains a unique DNA that is geneti¬
cally active; it is replicated and transcribed within the organelle to
provide products unique from those found elsewhere in the cell. The mito¬
chondrial DNA has been well characterized physically and chemically (117-
122) and the size of the mitochondrial genome varies from a molecular
weight of 10^ daltons in vertebrates to 11.9 x 10^ daltons in plants
(119,122). DNA with these molecular weights can code for only 5,000 amino

17
acids or about 20 proteins. Therefore, it is impossible for the mitochon¬
drial genome to code for all of the unique mitochondrial proteins which
must carry out replicative, transcriptive, translative and metabolic func¬
tions unique to the mitochondrion. All available evidence suggests that
the mitochondrial DNA carries information for the mitochondrial ribosomal
RNA, for at least some mitochondrial tRNAs and for a few mitochondrial
membrane proteins (121,123). Thus mitochondrial DNA is required for the
biogenesis of mitochondria.
The majority of proteins unique to the mitochondrion are coded for
by the nuclear DNA and synthesized on the cytoplasmic ribosomes. Less
than 10% of the mitochondrial proteins are synthesized on mitochondrial
ribosomes; this fraction consists of probably not more than eight species
of hydrophobic proteins that are incorporated into the inner membrane
where they assemble cytochromes, cytochrome oxidase and ATPase (120,
121, 123-126). A few components of the mitochondrial protein synthesizing
system may be coded for by the mitochondrial DNA, although there is no
evidence for the location of the code for the mitochondrial leucyl-tRNA
synthetase (127) and the two mitochondrial peptide chain elongation
factors, G and T (128). However, the prokaryotic characteristics of the
mitochondrial protein synthesizing system makes it difficult to imagine the
code coming from elsewhere in the cell.
The only mitochondrial gene products that have been positively identi¬
fied are ribosomal RNA and transfer RNA (121,123). Hybridization experi¬
ments have shown that mitochondrial ribosomal RNA hybridizes specifically
with mitochondrial DNA (129-132) and shares no sequence homology between
some of the mitochondrial 4s transfer RNA and mitochondrial DNA (133,134).
In order to understand the extent to which a mitochondrion depends upon

18
the rest of the cell, a more complete understanding of the mechanisms
which provide unique informational molecules must be examined in detail.
Mitochondrial DNA transcription has been studied in vivo using intact
growing cells and in vitro with isolated mitochondria or solubilized and
partially purified mitochondrial RNA polymerase. These investigations
have provided results which will be useful for the understanding of the
regulatory mechanisms which couple the genetic activities of mitochondria
and nuclei during cell growth and development.
The synthesis of mitochondrial RNA in intact cells has been exten¬
sively studied in HeLa cells by Attardi and coworkers. Hybridization
experiments of in vivo labeled mitochondrial RNA with the separated H
and L strands of mitochondrial DNA demonstrated that both the H and L
strands are equally transcribed but afterwards the product of the L
strand is almost completely degraded. This means that the problem of
strand selection in mitochondrial transcription could be achieved by
transcribing both strands and rapidly degrading or exporting 98% of the
L-strand transcripts (135-140). The transcription products have been
used to map the mitochondrial DNA; the H-strand contains genes for 9
tRNAs, whereas the L-strand has the two ribosomal RNA genes and also
those for three tRNAs (137-141). Election microscopy of ferritin-
labeled tRNA-mitochondrial DNA hybrids confirmed the location of these
genes (142). Experiments with the HeLa cells also suggest that mito¬
chondrial messenger RNA is also translated. Mitochondrial RNA containing
poly (A) hybridizes with mitochondrial DNA (142).
Studies of in vitro incorporation of ribonucleotide triphosphates
into high molecular weight RNA have shown that isolated mitochondria

19
are capable, for some time, of carrying out RNA synthesis just as are
mitochondria within the living cell, except that the process appears to
be much slower _in vitro than in vivo. The incorporation of UTP or ATP
generally requires all four ribonucleotide triphosphates and magnesium
(143,144). The incorporation process is considerably faster if the mito¬
chondria are swollen. Mitochondrial RNA synthesis is inhibited by
actinomycin D, which binds to the DNA template, proving its dependence
on DNA. The product of the Jui vitro incorporation reaction hybridizes
with the H strand of mitochondrial DNA (145). In Xenopus mitochondria
the RNA polymerase initiates at several different sites of the template
predominantly with ATP and 2-fold less frequently with GTP (146).
Studies on mitochondrial RNA synthesis have recently concentrated
on the characterization of the DNA-dependent RNA polymerases. However,
relatively little is known about the properties and functions of the
mitochondrial enzymes as compared to the nuclear RNA polymerases. It
has been difficult to identify and isolate the mitochondrial RNA poly¬
merase due in part to the difficulty of defining proper conditions for
the solubilization of this enzyme which appears to be firmly bound to
mitochondrial membrane. Studies on mitochondrial RNA polymerases from
a variety of sources have shown that these enzymes exhibit properties
clearly distinguishing them from the respective nuclear RNA polymerases.
The mitochondrial enzyme differs from the nuclear RNA polymerase II
in sensitivity to a-amanitin (11, 147, 148, 149). Low concentrations of
a-amanitin (0.1 to 20 yg/ml) which cause inhibition of nuclear RNA
polymerase II does not affect the mitochondrial RNA polymerase. Although
some functional differences have been described in mitochondrial RNA
polymerases, there is general agreement regarding their size.

20
The molecular weight of the enzyme, consisting of a single polypep¬
tide chain, seems to be about 60,000 (11-15). The purified mitochondrial
RNA polymerases from Neurospora crassa (11) and rat liver (12,13,147) are
inhibited by rifampicin in contrast to the mitochondrial enzymes from
Xenopus laevis (14), wheat leaf (150) and Ehrlic Ascites (39) which are
resistant to rifampicin. Some investigators have found the yeast mito¬
chondrial RNA polymerase to be rifampicin resistant (15,148,151-153).
Others describe the isolation of rifampicin-sensitive enzyme (154-155).
The reason for this discrepancy is not clear. It is possible that a
factor or factors needed for conferring sensitivity to rifampicin were
lost from some enzyme preparations.
In contrast to the above studies several laboratories have reported
that the yeast mitochrondrial RNA polymerases are much larger in size and
resemble more closely the nuclear polymerases. Eccleshall and Criddle
demonstrated three RNA polymerases associated with yeast mitochondria
having molecular weights near 500,000 and showing no sensitivity to
rifamycin (153). However, there was no difference between the nuclear
RNA polymerase I and the mitochondrial RNA polymerase I, suggesting that
their mitochondrial preparation was contaminated with nuclear enzyme.
Reports of multiple RNA polymerases associated with yeast mitochondria
having molecular weights near 500,000 and showing no sensitivity to
rifamycin have come from Rogall and Wintersberger (15) and Benson (156).
Scragg has reported a yeast mitochondrial RNA polymerase with molegular
weight greater than 200,000 that does show a rifamycin sensitive enzyme in
yeast (157). However, despite the low molecular weight (67,000) of the
yeast mitochondrial RNA polymerase polypeptide isolated by Rogall and
Wintersberger (15), this enzyme readily forms aggregates with molecular ,

21
weights up to about 500,000 in buffers of low ionic strength. The same
is true for the 64,000 molecular weight enzyme purified by Kuntzel and
Schafer (11) and the 60,000 molecular weight enzyme isolated by Scragg
(158). The overall picture of mitochondrial RNA polymerase is one of
either a great diversity of enzyme types associated with different organ¬
isms or else some differences in the isolation procedures employed in the
various laboratories which give rise to preparations with these differences.
The mitochondrial RNA polymerase consisting of a single polypeptide chain
of molecular weight of 64,000 resembles more the bacteriophate T7 specific
RNA polymerase rather than EA coli or eukaryotic nuclear RNA polymerase.
The T7 enzyme is a product of T7 DNA gene 1. A single polypeptide of
110,000 molecular weight, it generates only eight RNA species in late
transcription of T7 DNA (25).
Chloroplast transcription
The chloroplast also contains unique DNA that is replicated and
transcribed within the organelle. The molecular weight of Euglena
g
chloroplast DNA is approximately 10 daltons (159) which is sufficient
to code for the chloroplast tRNA, rRNA, and mRNA. However, the extent
to which the total potential genetic content is expressed in chloroplast
is open to question. It has been established that the chloroplast DNA
contains cistrons coding for chloroplast ribosomal RNA (160,161) and
transfer RNA (179). Quantitative saturation-hybridization of Euglena
chloroplast rRNA to chloroplast DNA showed that approximately 2% of the
DNA is involved in coding for chloroplast rRNA (160). A higher value (6%)
was obtained by Stutz and Vandrey (161) who isolated Euglena chloroplast
DNA from purified organelles devoid of any nuclear contamination rather

22
than relying on the fractionation of extracted DNA by preparative CsCl
buoyant density centrifugation. The chloroplast ribosomal 23S RNA (1.1 x
10^ daltons) and 16S RNA (0.56 x 10^ daltons) are coded for by separate
cistrons (162) localized on the heavy strand of the chloroplast DNA.
g
Accepting that the molecular weight of Euglena DNA is 10 daltons then
£
2% of this (2 x 10 daltons) is sufficient to code for both the 23S and
16S rRNAs. The value of 6% would allow for three copies of each of the
rRNAs per chloroplast DNA molecule.
The presence of mRNA that can be translated has been demonstrated
in Euglena chloroplast (163). These mRNAs could be the products of
transcription of chloroplast DNA or of nuclear DNA, or of both. However
isolated chloroplasts synthesize RNA and such RNA must necessarily be
chloroplast-DNA coded. Hybridization of RNA synthesized in isolated
tabacco chloroplasts to chloroplast DNA indicated that 21% of the
chloroplast DNA is transcribed (164). This represents a much larger
proportion of the chloroplast DNA than is required to code only for
ribosomal and transfer RNAs and therefore implies that mRNA species are
synthesized on chloroplast DNA.
Chloroplasts of higher plants and algae were shown to contain a
DNA-dependent RNA polymerase as early as 1964 but the enzyme was only
recently solubilized and purified (165). The extreme difficulty with
solubilizing this activity was related to the tight binding of the enzyme-
DNA complex to the thylakoid membranes. This chloroplast enzyme contained
two large polypeptide subunits, which suggested a similarity to the
prokaryotic and the eukaryotic nuclear RNA polymerase rather than the
mitochondrial or bacteriophage T7 RNA polymerases. The chloroplast enzyme
has also been studied in Euglena gracilis (166). The Euglena chloroplast

23
RNA polymerase has not been solubilized and purified mainly due to the
difficulties mentioned above. This prompted the study of chloroplast
transcription complexes consisting of chloroplast membrane fragments,
DNA, and the RNA polymerase activity.
Euglena RNA polymerases
The nuclear RNA polymerase II of Euglene gracilis was partially
purified recently by Congdon and Congdon and Preston (167,168). This
enzyme, defined by its sensitivity to a-amantin, eluted from DEAE
Sepahdex between 0.18 and 0.21 M (NH^^SO^. The RNA polymerase had a
broad salt optimum ranging from 50 mM (NH^^SO^ to 150 mM (NH^^SO^.
Optimal activity was exhibited with optimum MnCl^ concentrations rather
than with MgCl^. The enzyme was more active with poly d(AT) as a
template and preferred denatured over native calf thymus DNA. The
Euglena RNA polymerase, as well as the enzymes from other protists,
were less sensitive to a-amantin than the corresponding fractions from
higher eukaryotes. The Euglena chloroplast and mitochrondrial RNA poly¬
merases have not been previously solubilized and purified.

MATERIALS AND METHODS
Buffers
All buffers were prepared from analytical reagent grade chemicals
without further purification. Double distilled or deionized water was
used for all solutions. Stock solutions of 1.0 M Tris-HCl pH 7.4, at
25°C, 1.0 M Tris-HCl pH 7.9, at 25°C, 1.0 M Tris-HCl pH 9.0, at 25°C,
0.10 M EDTA pH 7.6, 1.0 M MgCl^» 0.01 M MnCl, 0.10 M dithiothreitol were
diluted to prepare the following buffers:
Buffer STE; 0.25 M sucrose, 0.01 M Tris-HCl pH 7.4 or pH 9.0,
0.10 mM EDTA pH 7.6.
Buffer STM; 0.25 M sucrose, 0.01 M Tris-HCl pH 7.9, 0.01 M
MgCl2.
Lysis buffer; 0.01 M Tris-HCl pH 7.9,20% w/v sucrose. 0.10
M KC1, 5 mM dithiothreitol, 1 mM phenylmethylsulfonylfluoride.
Buffer TGMED; 0.05 M Tris-HCl pH 7.9, 25% glycerol, 5.0 mM
MgCl^j 0.10 mM EDTA pH 7.6, 2.0 mM dithiothreitol.
Ethidium bromide buffer; 1 yg/ml Ethidium bromide, 0.01 M
Tris-HCl pH 7.9, 0.02 M NaCl, and 0.005 M EDTA.
Organism
Premanently bleached mutants of Euglena gracilis klebs strain z,
prepared by treatment with streptomycin (169) were used for these studies.
Growth
The streptomycin bleached mutants were cultured in 2800 ml Fernback
flasks containing 1500 ml of Medium A described by Greenblatt and Schiff (170).
24

25
The cultures were grown at 27° on a gyrotary shaker operating at 120
rev/min. Under these conditions, cells enter stationary phase when the
carbon source is exhausted (171). Growth was measured as turbidity with
a Klett colorimeter with a No. 66 filter.
Cell disruption and subcellular fractionation
Cells from axenic cultures in exponential phase were harvested by
passing the contents of the culture flaks through a DeLaval cream separator
centrifuge operating at room temperature. All further operations were
performed at 0° -4° C.
The cells were washed twice in STE buffer, pH 7.4, then suspended
in at least 5 volumes of STE buffer, pH 9 and passed through an Aminco
2
French Pressure cell at 1000 lb/inch (±100) using a power press to give
greater than 90% breakage. Breakage was estimated by microscopic examina¬
tion. The cell lysate was collected in ice chilled flaks, shakengently
to disperse aggregates, and repeatedly centrifuged at 1000 x j» for 5 min
(Beckman J-21 JA-20 rotor) until only a negligible pellet was produced.
Crude subcellular fractions were collected from the cell free lysate
supernatant by differential centrifugation first at 5000 x j>, 20 min and
then 10,000 x £ for 15 min. The crude 5000 x pellet (5P mitochondria)
was washed in STE pH 7.4 buffer.
Mitochondrial purification
Washed 5P mitochondria were purified in gradients of sodium diatri-
zoate (Winthrop Laboratories, New York). Samples containing 5-200 mg
dry weight of mitochondria suspended in the less dense renografin solution
3
(density equals 1.103 g/cm ) containing 0.01 M Tris-Hcl pH 7.4 and 0.10 mM

26
EDTA were layered over the Renografin gradients (density equals 1.103 to
1.30 g/cm^) containing 0.01 M Tris-HCl pH 7.4 and 0.01 mM EDTA. The pre¬
parations were centrifuged at 20,000 rpm for 2^ hr at 2°C (Beckman L-2,
SW 25.1 rotor). Fractions were collected and the mitochondria banding
in the region of the gradient corresponding to a density of 1.21 g/cnP
were washed twice in STE buffer pH 7.4 before use in incorporation studies.
Washed 5P mitochondria were alternatively purified by the enzymatic
digestion of nuclear DNA with 50 yg deoxyribonuclease I (E. C. 3.1.4.5) per
ml. The crude 5P mitochondria were washed in STM buffer pH 7.9 and then
pancreatic DNase I (Sigma Chemical Co., St. Louis, Mo.) was added to a
final concentration of 50 yg per ml in STM buffer. Incubation was carried
out at 4° for 30 min. with slow stirring. The enzymatic digestion was
stopped by the addition of 2 volumes of 10% sucrose, 0.01 M Tris-HCl pH
7.9,0.05 M EDTA pH 7.6. The mitochondria were then washed in STE buffer
pH 7.9.
Succinic dehydrogenase assay
Succinic dehydrogenase activity was measured by the succinate depend¬
ent reduction of dichlorophenolindophenol (172) in 1 ml reaction mixtures
at room temperature.
Extraction of nucleic acids
DNA was extracted from the mitochondria preparations by the technique
of Bhargava and Halvorson (173).
Characterization of DNA
The method of Preston and Boone (174) was used for the density measure¬
ment of DNA. The CsCl gradient polyacrylamide gels obtained after the

27
exposure to light were washed with flowing tap distilled water for 2 hr
to remove CsCl. The riboflavin was photooxidized by shaking the gels
under illumination for 16 hr in the presence of 10 ml of 0.01 N NaOH in
50% formamide. The gels were then washed in tap distilled water for 2
hr before adding the gels to ethidium bromide staining buffer. The
staining permitted the quantitative detection of DNA in the gel which
could be scanned in an Aminco Flourometer equipped with a gel scanning
attachment.
DNA standards
DNA was purified from Cytophage P-11 (a strain isolated, identified
and carried at the Department of Microbiology, University of Florida,
Gainesville, FL) as described by Preston and Boone (174). DNA from the
bacteriophage 25 and DNA from Pseudomonas aeruginosa were kindly pro¬
vided by M. Mandel, M. D. Anderson Tumor Institute, Houston, Texas.
DNA was extracted from isolated nuclei of streptomycin bleached mutants
of Euglena gracilis Klebs strain z by the method of Bhargava and Halvorson
(173).
Preparation of cellular fractions for protein and DNA analysis
Cellular fraction samples were prepared for analysis by Vortex mixing
1 ml of sample with 5 ml of cold 95% ethanol (-20°C). The particulate
material was collected by centrifugation at 4000 rpm, 5 min., 2°C. The
ethanol extraction was repeated twice more before suspending the pellet in
1 ml of cold double distilled water. The samples were suspended in 1.0 ml
of water then 4.0 ml of cold 0.50 N HC10^ was added and the sample was
mixed on a vortex. The precipitated macromolecules were collected by
centrifugation before being suspended in 4 ml of cold ether-ethanol (1:3)

28
and centrifuged again. The pellet was suspended in 4 ml of 0.50 M HCIO^
and incubated at 85°C for 20 min. to hydrolyze DNA and RNA, cooled and
centrifuged. The top 3 ml of the supernatant was used for DNA analysis.
The pellet was suspended in 3 ml of 1.0 N NaOH and heated in a boiling
water bath until the precipitated protein was dissolved. This solution
was used for analysis of soluble proteins.
Protein determination
The protein concentrations were determined by the Lowry method (175) .
Dissolved bovine serum albumin served as the standard.
DNA determination
DNA concentrations were determined by the indole method of Hubbard
(176). Calf thymus DNA served as a standard. A sample of 0.05 ml was
added to 0.05 ml of 10% trichloroacetic acid and 1 ml of 0.04% indole-2
N HC1 solution. The mixture was shaken and incubated at 97°C for 15 min.
and then cooled in ice. The mixture was then extracted with chloroform
three times by adding 2 ml of CHCl^ to the mixture, vortexing and centri¬
fuging 2000 rpm, 5 min. The chloroform phase was removed after each ex¬
traction. The absorbance of the aqueous phase at 490 mm was then determined.
RNA polymerase assay
Conditions for the assay of RNA polymerase were similar to those
described by Preston ert al. (177). The routine mixture contained in a
total volume of 0.10 ml: 0.05 M Tris-HCl pH 7.9, 0.005 M MgC^, 0.001
M MnCl2, 0.001 M DTT, 0.01 M KC1, 0.001 M of each unlabeled ribonucleoside
triphosphate, 0.032 mM or 0.004 mM or 0.016 mM (2 Ci/mmole of ^H-labeled
ribonucleoside triphosphate, 100 pg/ml of denatured calf thymus DNA and
enzyme. Assay mixtures were incubated for 10 min. (or the time indicated)

29
at 37°C. The reaction was stopped by adding stop bath (0.05 M sodium
pyrophosphate, 2 mg/ml bovine serum albumin, 2 mg/ml Torula RNA and 5 mM
of the unlabeled ribonucleotide which was identical to the labeled NTP)
and chilling in ice (1). Macromolecules were precipitated by adding 2
ml of cold 10% trichloroacetic acid containing 20 mM sodium pyrophosphate
(TCAPP) to each reaction tube. After standing in ice for 15 min. the
precipitate was collected by filtration through a glass fiber disc (What¬
man GF/C) which had been prewashed with 5 ml of cold TCAPP. Each disc
was washed three times with 5-10 ml TCAPP, then three times with 5-10 ml
ether-ethanol (1:3) 37°C, followed by two washes 5-10 ml ether.
Filtration was carried out in perforated porcelain crucibles (Gooch)
adapted to a vaccum manifold with ruber crucible holders (Walter).
Filtration was facilitated by maintaining a partial vaccum (ca. 28 in.
Hg). The GF/C filters were dried and placed in scintillation vials
containing 5 ml of toluene (scintillation grade, Mallinkrodt), 0.4% PPO
(2,5-diphenyloxazole), 0.01% POPOP 1,4-di[2-(5-phenyloxazolyl)]-benzene),
and counted for 10 min. in a liquid scintillation counter (Beckman LS-133).
A unit of enzyme activity is defined as that which will catalyze the
incorporation of 1 pmole of labeled ribonucleoside monophosphate into acid
precipitable material in 10 min. at 37°.
Isolation of CTP labeled product
A sample of 0.30 ml of purified mitochondria (0.18 mg/ml protein)
was incubated in a 3.0 ml RNA polymerase reaction mixture (0.05 mM H-CTP,
2 Ci/mmole) for 15 min. The reaction was stopped by adding 3.0 ml of stop
bath (0.05 M sodium pyrophosphate, 1.09 mg/ml _E. coli RNA and 5 mM of
unlabeled CTP) and brought to 0.20 M NaCl and 0.10 M Tris pH9. Triton X

30
100 was added to bring the mixture to a final concentration of 0.5%. The
mixture was then mixed with an equal volume of 80% v/v redistilled aequeous
phenol. The nucleic acids were collected by precipitation with 3 volumes
of cold 95% ethanol (-20°C) for 12 hr followed by centrifugation at 4000
rpm 15 min. The nucleic acids were dissolved in buffer (0.20 M NaCl, 0.10
M Tris pH 9) and carried through the ethanol precipitation again. The
final pellet was dissolved in 2 ml of double distilled water.
Column filtration
Biogel P-4 was swollen overnight by mixing 1 g Biogel per 10 ml of
0.10 M NaCl. A 20 ml column was poured and pre-equilibrated by passing
4 volumes of 0.10 M NaCl through it at 4°C. Nucleic acid samples in
0.10 M NaCl were added to the column and eluted with 0.10 M NaCl. The
flow rate was 10 drops per min. labeled samples (100 yl) were placed
in 10 ml of Brays scintillation fluid (178) and counted in the liquid
scintillation counter (Beckman LS-133).
Extraction of phospholipids
Phospholipid extraction was performed by the method of Carter and
Kennedy (179) and Raetz and Kennedy (180). To 0.10 ml of the ^h-CTP
labeled product was mixed 1.0 ml of methanol containing 0.10 N HC1. Ten
minutes later 2 ml of chloroform was added and the phases were stirred
with a vortex mixer before adding 4 ml of 2 M KC1. The mixture was then
vigorously mixed for 5 min. and the phases were allowed to separate before
the aqueous phase was removed. The chloroform phase was washed two more
times with 2 M KC1. A sample of each phase following extraction was
placed in a scintillation vial and dried by passing a stream of nitrogen

31
gas over the solution. Then 10 ml of Bray's solution was added to the
vials and counted.
Thin layer chromatography methods
The procedure of Ter Schegget e_t ill. (181) for thin layer chromato¬
graphy of CDP-diglyceride was followed. Samples were spottedon silica
gel G thin layer plates. Chromatograms were developed with a solvent of
chloroform-methanol-water-ammonia (70:38:2:8). The CDP-diglyceride stand¬
ard was prepared by the method of Carter and Kennedy (179). Nucleotides
were chromatographed on PEI cellulose by the methods of Randerath and
Randerath (182,183). Ribonucleoside monophosphates were chromatographed
first in 1.0 N acetic acid to a height of 4 cm and then without drying
the plate was transferred to a tank containing 0.30 N LiCl and the solvent
was allowed to rise to 15 cm. The ribonucleoside triphosphates were
separated by developing to 15 cm with 0.50 M ammonium sulfate.
Solubilization and purification of
mitochondrial RNA polymerase
Solubilization of mitochondrial RNA polymerase was achieved by fol¬
lowing a procedure similar to that of Wintersberger and Rogall (15, 152).
Mitochondria from 150 wet weight of cells were suspended in an equal
volume of lysis buffer. This was added to an equal volume of a lysis
mixture consisting of 1.6% Triton X 100 non ionic detergent, 1.0 M KC1,
and 0.01 M EDTA (pH 7.9). The mixture was mixed gently for 15 min and
then centrifuged for 90 min at 30,000 rmp (Ti 50 rotor) and 0.29 ¿ of
solid ammonium sulfate per ml of 30,000 supernatant was added to bring
the extract to 50% saturation with respect to ammonium sulfate. The
precipitate was collected by centrifugation at 40,000 rmp for 90 min

32
(Ti 50 rotor) and dissolved in TGMED containing 0.05 M (NH^^SO^ and
dialyzed against 2 liters of the same buffer for 4 hr. The dialyzate
was applied to a column (0.90 x 20 cm) of DEAE-Sephadex A-25 which had
previously been equilibrated with the same buffer and nonabsorbed protein
was washed off the column with 30 ml of the same buffer. The RNA polymerase
activity was eluted with a 160 ml gradient of 0.05 M to 0.50 M (NH^^SO^
in TG,ED. Fractions (2.50 ml) were collected and 20 pi of each fraction
was assayed for RNA polymerase activity. Concentrations of (NH^^SO^ were
determined with a conductivity bridge.
Chemicals and reagents
The following research grade biochemicals were obtained from Sigma
Chemical Co., St. Louis, No: The ribonucleoside triphosphates GTP, CTP
UTP and ATP as sodium salts; dithiothreitol; phenylmethylsulfonyl flouride;
high molecular weight calf thymus DNA; Torula yeast RNA, Triton X-100; en¬
zyme grade trizma buffers; chromatographically pure pancreatic ribonuclease
A (EC 2.7.7.16) and deoxyribonuclease I (EC 3.1.4.5): fraction V bovine
serum albumin; cytidylyl (3'-»- 5') cytidine (CpC); and snake venom phospho¬
diesterase (Crotalus atrox venom, Type VII).
The tridium labeled ribonucleoside triphosphates as sodium salts
were purchased from Schwartz/Mann, Orangeburg, NY. These include {5— H}
UTP, (5-^HÍ CTP, (8-^H) ATP, {8-^H} GTP. Ethidium bromide and actinomycin
D were purchased from Calibiochem, LaJolla, CA. Poly d(AT) was obtained
from Miles Laboratories, Kankakee, IL. The rifamycin derivativies AF/05
and AF/013 were the generous gift of Gruppo Lepetit, Milan, Italy. The
a-amanitin was kindly provided by Dr. T. Wieldand, Max-Planck Institute,

33
Heidelberg, West Germany. Sodium diatrizoate was purchased from Winthrop
Laboratories, NY. Nonionic detergent NP-40 was purchased from Shell Oil
Co. Unless specifically noted, all other chemicals were analytical rea¬
gent grade.

RESULTS
Two experimental approaches were utilized to study the iji vitro mito¬
chondrial RNA synthesis in Euglena gracilis. The first approach was to
study the incorporation of radioactively labeled ribonucleoside triphos¬
phates into acid insoluble products by isolated mitochondria which had
been treated with isotonic buffer to assure swelling and permability.
The products of the incorporations were then studied. The second approach
was to study the activity of solubilized and partially purified mitochon¬
drial DNA dependent RNA polymerase. This approach involved determining
the conditions for solubilizing the enzyme and then for partially purifying
the enzyme. The activity of this enzyme was then characterized.
The Incorporation of Ribonucleoside
Triphosphates by Isolated Mitochondria
The initial research objectives were to determine if RNA synthesizing
activity is retained by mitochondria isolated from Euglena gracilis, and
to look for ways to differentiate between nuclear and mitochondrial RNA
polymerase activities such as sensitivities to inhibitors or special
enzymatic requirements. The approach was to study the RNA synthesizing
activity of isolated mitochondria obtained from a streptomycin bleached
aplastidic mutant of Euglena gracilis, strain z. The mitochondrial
isolation procedure (Figure 1) consisted of rupturing washed cells in the
French pressure cell at 1000 lb/in^ and collecting crude mitochondrial
34

Figure 1. SCHEMA FOR THE ISOLATION OF MITOCHONDRIA

36
ISOLATION PROCEDURE
WASHED CELLS
STE (0.25M Sucrose, O.OIM Tris-HCI pH7.6,
O.ImM EDTA pH 7.4)
2
--Fpc 1000 Ib/inch
DISRUPTED CELLS
--I000 Xg, 5 min.
REPEAT I-3X
I 1
IP IS
5000 Xg, 20 min.
r
5P
resusp.
5000Xg, 15 min.
10000 Xg, 15 min.
I 1 I 1
Washed 5P 5S 10 P IOS
(Dense Mito.) (Less Dense)
(Mito.)
RENOGRAFIN GRADIENT
PURIFIED MITO. {p= 1.22 g cm-3)

37
pellets by differential centrifugation, first at 5000 x j» and then at 10,000
x for 15 min. The crude 5000 x ¿ mitochondrial pellet (5P) containing
the heavier and more enzymatically active mitochondria (33) were used for
these experiments. The 5P mitochondria were washed in buffer and then
subjected to sodium diatrizoate (renografin) gradient centrifugation at
20,000 x ¿ for 2% hr. Fractions were collected and the mitochondria, which
banded in the region of the gradient corresponding to a mean equilibrium
density of 1.22 g/cm were used for the studies reported here.
In order to test the purity of the mitochondrial preparation, the
buoyant density of the DNA in the mitochondrial preparations was routinely
monitored by CsCl gradient centrifugation. The CsCl gradients were genera¬
ted in the presence of acrylamide and catalyst according to the procedure
of Preston and Boone (174). This method allows the DNA that is banded in
the CsCl gradient to be fixed by exposing the gradient to light in order to
polymerize the acrylamide gels. Ethidium bromide staining permitted a rapid
and sensitive technique to quantitatively detect the DNA in the gels which
could be scanned in an Aminco Fluorometer equipped with a gel scanning
attachment. The linear relationship between the relative position in the
gradient and the buoyant density of the DNA is demonstrated by the plot of
DNA standards of known densities in Figure 2. The purified mitochondrial
preparation yields better than 70% mitochondrial DNA which has a density
of 1.691 g/cm^ and less than 30% nuclear DNA which has a density of 1.707
3
g/cm . Although it is evident that the preparation contains some contami¬
nating nuclear DNA, this method provided the cleanest feasible mitochondria
without treating the mitochondria with an exogenous nuclease.

Figure 2. DENSITY EQUILIBRIA ANALYSIS OF MITOCHONDRIAL DNA
Density equilibria anlaysis of DNA in mitochondria purified on a sodium
diatrizoate (Renografin) gradient were measured by CsCl density gradient
centrifugation as described in the methods section. The markers are
Cytophage P-11 DNA with a density of 1.693 g/cm-*, Psuedomonas aeruginosa
DNA with a density of 1.727 g/cnH, and bacteriophage <(> DNA with a density
of 1.742 g/cm .

FLUORESCENCE
39
CsCI Density Gradient Gel
DNA STAINED WITH ETHIDIUM BROMIDE

40
The renografin purified mitochondria are active biochemically as demon¬
strated by results in Table 1. Succinic dehydrogenase which is tightly
integrated into the inner membrane of mitochondria has been employed as a
marker enzyme in order to follow the purification of mitochondria. Of the
total cellular succinic dehydrogenase activity 42% is recovered in the
purified mitochondria, with a 27-fold purification per mg of protein and a
70-fold purification per pg of DNA. This demonstrates that the purification
procedure yields mitochondria biochemically active for an enzyme not asso¬
ciated with the DNA
A Mitochondrial Activity Incorporating Label
From ^H-CTP Into Acid Precipitable Product
The incorporation of label from the four ribonucleoside triphosphates
labeled with tritium by isolated mitochondria was studied by performing
RNA polymerase assays as described in the methods section. The mitochondria
were treated with isotonic buffer to assure swelling and permability. The
relative rate with which label from each ribonucleoside triphosphate was
incorporated into acid insoluble macromolecules was determined by performing
the assays in 0.1 ml reaction mixtures in which only one of the ribonucleo¬
tides was labeled. It was observed that isolated mitochondria catalyzed
the incorporation of label from ^H-CTP into acid insoluble material at a
rate greater than 10 times that for the other ribonucleotides (Table 2).
It is interesting that ATP and GTP, the ribonucleotides which normally
serve as the best substrate for RNA initiation in procaryotic systems, are
the poorest substrates for the reactions studied here. Figure 3 demonstra¬
tes the relative rates of incorporation of tritium from ^H-CTP and ^H-UTP,
the two most active substrates. In this experiment the concentration of

TABLE 1
DISTRIBUTION OF SUCCINIC DEHYDRAGENASE ACTIVITY
FRACTION
TOTAL
UNITS
PERCENT
RECOVERY
UNITS/mg
PROTEIN, (X 10“4)
FOLD
PURIFICATION
UNITS/yg
DNA, (X 10-4)
FOLD
PURIFICATION
LYSATE
1.96
100
7
1.0
0.2
1.0
5P MITO
1.78
90
59
8.4
1.1
7.5
WASHED 5P MITO
0.91
46
65
9.3
1.1
7.6
RENO MITO
0.82
42
190
27.0
10.3
70.0
Succinic dehydrogenase activity was measured by the succinate dependent reduction of dichlorphendol-
indophenol (160) in 1 ml reaction mixtures at room temperature. The protein and DNA concentrations
were determined by the procedures described in the methods and procedures. A unit of succinic dehy-
drogenas activity is defined as a ymole of dichlorophenolindophenol reduced per minute per ml of enzyme
sample at 25°C.

TABLE 2
NUCLEOTIDE INCORPORATION BY
ISOLATED MITOCHONDRIA
CPM incorporated into acid
insoluble macromolecules.
Labeled
Ribonucleotide
3H-CTP
13860
3h-utp
952
3h-atp
276
3h-ctp
322
The RNA polymerase assay is described in the methods
section. Each reaction mixture contained in a total
volume of 0.1 ml: 1.0 mM of each unlabeled NTP and
0.004 mM of each JH-labeled ribonucleoside triphosphate
with a specific radioactivity of 2 Ci/mmole.

Figure 3. RIBONUCLEOSIDE TRIPHOSPHATES INCORPORATION BY
ISOLATED MITOCHONDRIA
The incorporation of ribonucleoside triphosphates by isolated mitochondria
into acid insoluble products. The RNA polymerase assay described in the
methods section was followed except that the concentration of the radio-
actively labeled ribonucleoside triphosphates was 0.016 mM, 2 Ci/mmole.
The unlabeled ribonucleotides concentration was 0.5 mM each.

fjL/j. Moles of H-CMP Incorporated
44
Time (min.)
5
20
25
/¿/¿Moles of H-UMP Incorporated

45
the radioactively labeled ribonucleotide was 0.016 mM and that of the
unlabeled ribonucleotides was 0.5 mM each. The other components of the
O
reaction were as described in the methods. Label from JH-CTP was incorpora¬
ted at a rate greater than 10-fold that for UTP. It should be noted that
the CTP and UTP tritium incorporations are plotted on different scales in
Figure 3. When the incubation is carried out in the presence of RNase A, the
3
product formed with H-CTP incorporation shows no sensitivity whereas the
3
product formed with H-UTP shows some sensitivity to RNase A. This could
be due to the possibility that the CTP product is not RNA or that the product
is complexed with DNA or folded and complexed such that it is RNase insensi¬
tive. These results also suggest that RNA synthesis could be better followed
by studying the incorporation with labeled UTP.
â– 3
The incorporation of tritium from H-CTP by the isolated mitochondria
was clearly not dependent upon the presence of the other ribonucleoside
triphosphates which appear to inhibit the incorporation by about 10 percent
(Table 3). Incorporation of label from H-UTP also shows no dependence
upon added ribonucleotides while the cpm for ATP and GTP were too low to
make any judgment as to their dependency.
The high rate of incorporation with -^H-CTP relative to that with the
other ribonucleoside triphosphates prompted the investigation of the incor-
3
poration with H-CTP. This activity demonstrated no dependency upon added
exogenous DNA or ribonucleoside triphosphates (Table 4). In fact the isolated
3
mitochondria incorporated label from H-CTP at a 10 times greater rate in the
absence of added DNA or nucleoside triphosphates than when they were present.
3
The distribution of the incorporating activity with H-CTP was followed
through the isolation procedure and the results are shown in Table 5. It was

TABLE 3
INCORPORATION OF RIBONUCLEOSIDE TRIPHOSPHATES BY
ISOLATED MITOCHONDRIA.
Labeled
Ribonucleotide CPM/Rx.Mix. Units/g Wet Wt.
3H-CTP
+
NTPs
11,513
2161
-
NTPs
12,792
2401
3h-utp
+
NTPs
616
116
-
NTPs
1,386
260
3h-atp
+
NTPs
206
39
-
NTPs
324
61
3h-gtp
+
NTPs
84
16
NTPs
290
54
The RNA polymerase assay was performed as described in the
methods. 0.1 ml reaction mixtures contained 1.0 mM of each
unlabeled NTP, 0.004 mM ^H NTP, 2 mC/mmole.

TABLE 4
THE DEPENDENCY OF THE MITOCHONDRIAL ACTIVITY
INCORPORATING LABEL FROM JH~CTP UPON ADDED
RIBONUCLEOTIDES AND DNA
Assay Components
Units/mg Protein
Complete Assay with DNA
1,500
-DNA + NTP
1,481
-DNA - NTP
11,056
-DNA + UTP
9,611
-DNA + ATP
10,667
-DNA + GTP
8,611
The RNA polymerase assay was performed as described in the
methods. 0.1 ml reaction mixtures contained 1.0 mM of each
unlabeled NTP and 0.004 mM of -^H-CTP with a specific radio¬
activity of 2 Ci/mmole.

TABLE 5
DISTRIBUTION OF 3H-CMP INCORPORATING ACTIVITY
Fraction
TOTAL
Units
%
Recovery
Units/mg
Protein, X 103
Fold
Purification
Units/yg
DNA
Fold
Purification
Lystate
141,750
-
2.0
-
0.9
-
5P Mito
39,420
28
6.5
3.2
6.5
7.1
Washed 5P Mito
12,320
9
5.9
3.0
7.7
8.0
Reno Mito
18,940
13
17.4
8.7
114.0
125.0
The RNA polymerase assay is described in the methods. Each reaction mixture contained in a
total volume of 0.1 ml; 1.0 mM of each unlabeled NTP and 0.004 mM of 3H-CTP with a specific
radioactivity of 2 Ci/mmole.
CO

49
observed that 13% of the activity was recovered in the purified mitochon¬
dria with an 8.7-fold purification per mg of protein and 125-fold per yg
of DNA. This suggests that if the activity is associated with DNA, then
it is with mitochondrial DNA and not nuclear DNA, 95% of which has been
removed by the purification procedure.
3
The incorporation of H-CTP into acid insoluble product is inhibited
by actinomycin D (Table 6) at concentrations above 50 pg per ml, suggesting
that a DNA dependent reaction may be involved. However, it is interesting
that relatively high concentrations are required to demonstrate this
inhibition. The possibility of a secondary site of action of actinomycin
D or inhibition by other compounds in the actinomycin D can not be ruled
out. However, pyrophosphate at 2 pmoles per ml inhibited the activity
while inorganic phosphate at 2 pmoles per ml failed to inhibit the incor¬
poration indicating that the incorporation involves a pyrophosphorolysis.
This indicates the incorporation of CMP and rules against reactions which
incorporate CMP from CDP such as that of nucleotide phosphorylase or the
incorporation of CDP from CTP.
Rifampin and streptovaricin, inhibitors of initiation and elongation
of prokaryotic RNA polymerases respectively, failed to inhibit the activi¬
ty. Rifamycin AF/013, and inhibitor of initiation with eukaroytic nuclear
RNA polymerases as well as prokaryotic RNA polymerases, also failed to
inhibit the activity. Ethidium bromide, which will bind to DNA, showed
only slight inhibition which is probably not significant.
The acid insoluble product is sensitive to snake venom phosphodies¬
terase and alkaline hydrolysis (Table 7) under two different conditions,
suggesting that the product contains phosphodiester bonds. Up to this
point the product had demonstrated properties that were similar to those

TABLE 6
INHIBITION OF 3H-CMP INCORPORATION
Addition
CPM
Percent of
Control
h2o
4213
100
1% DMF
4179
100
Actinomycin D,
100 yg/ml
602
14
Actinomycin D,
75 yg/ml
2052
48
Actinomycin D,
50 yg/ml
4708
100
Pyrophosphate,
2 ymoles/ml
456
10
Phosphate,
2 ymoles/ml
4055
96
Rifampin,
100 yg/ml
3538
84
Rifamycin AF/013, 100 pg/ml
4050
96
Ethidium Bromide, 100 yg/ml
3370
80
Streptovaricin,
100 yg/ml
3918
93
The RNA polymerase assay was performed with the addition of
inhibitors to the final concentrations indicated above. Rifam¬
pin, rifamycin AF/013, ethidium bromide and streptovaricin were
solubilized in 1% N'N' dimethyl formamide.

TABLE 7
CHARACTERIZATION OF PRODUCT
Addition
CPM
Percent
of Control
Control
6412
100
Snake Venom Phosphodiesterase,
10 pg/ml
561
9
0.1M KOH 100°C, 20 min.
154
2
H20, 100°C, 20 min.
4437
70
0.5M KOH, 37°C, 16 hr.
119
2
H20, 37°C, 16 hr.
5129
80
The RNA polymerase assay was performed with the addition of
phosphodiesterase (E. C.: 3.1.4.1) from Crotalus atrox venom,
Type VII (Sigma, St. Louis). The alkaline hydrolysis was per¬
formed by first performing the RNA polymerase assay for 10
minutes and then treating the reaction mixture with KOH for the
indicated times before the addition of stop bath and the precip¬
itation of macromolecules with 10% trichloroachetic acid.

52
reported by Duda and Cherry (184) for a poly C polymerase activity in
sugar beet nuclei.
The RNA polymerase assay reaction mixture was scaled up so that the
3
labeled products of the incorporation of H-CMP could be studied. For
this purpose the reaction mixture contained in a total volume of 3.0 ml,
5.0 mM of each unlabeled ribonucleoside triphosphate and 0.05 mM of
3
H-CTP with a specific radioactivity of 2 Ci/mmold. After incubationof
15 min the products were isolated as described in the methods and
material section by phenol extraction followed by ethanol precipitation
of labeled products from the aqueous phase in the presence of carrier
_E. coli RNA and CTP. Zonal centrifugation (Figure 4) on sucrose gradient
was performed in order to determine the size of the tritium labeled pro¬
duct. The radioactivity remained at the top of the gradient indicating
the material was smaller than that of _E. coli 5S RNA. Therefore, the
sizes of the isolated products were estimated by Biogel P-4 column
filtration (Figure 5). The _E. coli carrier RNA eluted in the void volume
while two adsorbancy peaks were eluted which contained the labeled cyto¬
sine. Using the elution position of a standard CpC the molecular weight
of the first peak (BGP-1) was estimated to be approximately 800 which
corresponds to the molecular weight of a trinucleotide of poly C. The
molecular weight of the second peak (BGP-2) was estimated to be about
680 which corresponds to the molecular weight of a dinucleotide. It
should be noted that this smaller entity elutes before CpC indicating
that it is larger than a dinucleotide of this structure.
The components in the two peaks eluted from the P-4 column have
ultraviolet absorbancy spectrums very similar if not identical to that
of CpC, showing the characteristic absorption maximum and minimum at

Figure 4. SUCROSE GRADIENT ZONAL CENTRIFUGATION
The H-CMP-labeled product was isolated from the scaled up RNA polymerase reaction mixture using
carrier EL coli RNA. The tritium-labeled materials (200 pi) obtained from the nucleic acid
extraction of the scaled up reaction mixture were layered over 5 ml sucrose (Schwarz Mann special
density grade) gradients, 5 - 20% w/v, containing 0.01M Tris-HCl pH 7.6 and 0.02 M NaCl and centri¬
fuged (SW 39 rotor) 6 hr at 4 C. The E_. coli RNA standard was centrifuged in a separate tube. The
gradient was analyzed by pumping the gradient through the Gilford J cell, delivering the densest
portion first with a bottom probe. The ultraviolet absorbancy was monitored with the Gilford re¬
corder. Fractions were collected and measured for radioactivity by placing 200 pi samples in Brays
scintillation fluid and counted in the scintillation counter. The ultra-violet absorbancy profiles
for the products ( ) and _E. coli RNA ( ) and the radioactivity profile (—'—*—) are shown

CPM. X I04 (-•-*-)
2 6 0

Figure 5. BIOGEL P-4 COLUMN CHROMATOGRAPHY
The H-CMP labeled product was isolated from the RNA polymerase reaction mixture using carrier
_E. coli RNA. The product samples dissolved in 0.1 M NaCl were added to a 20 ml Biogel P-4
column previously equilibrated with 0.1 M NaCl. Fractions were eluted with 0.1 M NaCl at 4 C.
The eluted fractions were assayed for radioactivity. The standard was CpC.

260
CPM X 10

57
pH 2 and pH 7 for cytosine (Figure 6). This demonstrates that cytosine
is the only base incorporated into the products, and suggests that the
products are homopolymers of CMP.
The possibility that the product may be CDP-diglyceride was inves¬
tigated, since it has been reported that crude mitochondrial preparations
from rat liver incorporated label from both CTP and dCTP to CDP digly¬
ceride and dCTP diglyceride respectively (181). These mitochondria
3
incorporated label from H-dCTP into acid-insoluble material at a much
higher rate than any other deoxyribonucleoside triphosphate. About 95%
3
of the acid-insoluble material labeled after incubation with H-dCTP
could be extracted by chloroform. Therefore, studies were performed to
3
ascertain if Euglena mitochondria incorporated the label from H-CTP into
CDP-diglyceride.
The results of phospholipid extraction are shown in Table 8. A 0.1
3
ml sample of the H-CMP labeled product obtained after nucleic acid
extraction and dissolved in water was mixed with 1.0 ml of methanol for
10 minutes before adding 2.0 ml of chloroform with additional mixing.
Then 4.0 ml of 2M KClwas added with vigorous mixing (Vortex mixer, 5
min., 25°C). After the phases separated upon standing, less than 1% of
the tritium label could be accounted for in the chloroform phase whereas
64% of the label could be accounted for in the aqueous phase. Repeated
additions of the aqueous KC1 to the chloroform phase resulted in no
extraction of additional label into the aqueous phase. The difficulty
in accounting for all of the radioactivity was due to the quencing
produced by the aqueous KC1 phase in the Bray's scintillation fluid.
Further evidence that the product is not CDP-diglyceride was pro¬
vided by the failure of the radioactively labeled material eluted from
the P-4 column to migrate in a silica gel G TLC system (Figure 7) in

Figure 6. ULTRA-VIOLET ABSORBANCY SPECTRA
The ultraviolet absorbancy spectra of the H-CMP labeled products eluted from the
column were performed after adjusting the pH. The spectrum for the standard, C^C
same as before and after column filtrations.
Biogel P-4
was the

ÃœSORBANCY
Ln
vo

60
TABLE 8
PHOSPHOLIPID EXTRACTION OF THE 3H-CMP LABELED PRODUCT
Sample
Total
CPM
% of Control
3H-CTP product
58.50
X
104
-
First extraction of product
in chloroform solution
Chloroform phase
0.09
X
104
0.2
Aqueous phase
37.20
X
104
64.0
Second extraction of
chloroform phase
104
Chloroform phase
0.13
X
0.2
Aqueous phase
0.09
X
104
0.2
Third extraction of
Chloroform phase
Chloroform phase
0.19
X
io4
0.3
Aqueous phase
0.27
X
104
0.4
A 0.1 ml sample of the 3H-CMP-labeled product (containing 58.5 x 104 cpm)
obtained from nucleic acid extraction was dissolved in water and mixed
with 1.0 ml of methonal containing 0.1N HC1. Ten minutes later 2.0 ml
of chloroform was added to the mixture followed by 4 ml of 2M KC1. This
was vigorously mixed on a vortex mixer for 5 minutes at 25°. The phases
were allowed to separate upon standing at room temperature and then the
aqueous phase was drawn off and the chloroform phase was extracted 2 more
times. 100 yl samples of each phase were counted for radioactivity as
described in the methods.

Figure 7. THIN LAYER CHROMATOGRAPHY OF THE 3H-CMP LABELED PRODUCTS
ELUTED FROM THE BIOGEL P-4 COLUMN
Ascending chromatography of the 3H-CMP labeled products eluted from the
Biogel P-4 column was performed according to the method of TerSchegett
ert al. (181) on silica gel G in closed tanks at room temperature. The
solvent was chloroform; methanol; water; ammonia (70:30:2:8, v/v). The
standards were CpC and CDP-diglyceride. Twenty microliter samples of
the 3H-CMP labeled products and standards were spotted 3.0 cm from the
lower edge of the plate and dried before developing in the solvent. The
800 MW 3H-CMP labeled product (BGP-1) spot contained 35,000 cpm and the
680 MW product (BGP-2) spot contained 35,000 cpm. This was determined
by spotting the same volume of each product on a plate and then removing
a 1.0 cm2 section containing the spot and measuring its radioactivity in
toluene scintillation fluid. The CDP-diglyceride was detected by ultra¬
violet light and by staining with iodine vapor. Radioactivity was
detected by sequentially removing 1 cm2 sections from the silica gel plate
and counting them in toluene scintillation fluid.

Silica Gel G TLC
CHLOROFORM: METHANOL: WATER:
AMMONIA (70:38:2:8 v/v)

63
which CDP-digylceride migrates (181). Ascending chromatography of the
3
H-CMP labeled products was carried out as described in the legend.
The labeled products behaved similarly to CpC in that they both failed
to migrate in the system and they failed to stain with iodine vapor
indicating that they contained no lipid moieties. As noted in Figure 7
100% of the cmp were recovered in the 1 cm square section comprising the
origin. No significant radioactivity was detected in the other 1 cm
squares which were sequentially removed from the plate and counted for
radioactivity. The CDP-diglyceride standard migrated in the system and
stained with iodine indicating that the radioactive products are not
CDP-diglyceride.
Attempts were then made to ascertain the structure of the labeled
products. This involved treating materials eluted from the P-4 column
peaks with nucleases and analyzing the cleavage products by their migra¬
tion in a PEI cellulose TLC system in which ribonucleoside monophosphates
can be distinguished. Treatment of the labeled material (BGP-1 and
BGP-2) eluted from the P-4 column peaks with snake venom phosphodiesterase
resulted in 93% of the radioactive label migrating to a position correspon¬
ding to 5'-CMP (Figure 8 and 9), the expected cleavage products of poly C
and CpC. However, when the products were treated with RNase A, the radio¬
active label remained at the origin as did the untreated products. This
data indicates that the products were insensitive to RNase A. This being
the case, then the products have a chemical structure which is different
from that of a homopolymer of CMP since RNase A cleaves CpC to the expected
2', 3' CMP products. However, the possibility exists that these were
homopolymers which have modified 3' terminus which prevents the RNase A
from binding. The possibility of a small oligonucleotide with a 5'

Figure 8. THIN LAYER CHROMATOGRAPHY OF THE PRODUCTS OF THE ENZYMATIC
CLEAVAGE OF THE 3H-CMP LABELED MATERIAL ELUTED FROM THE P-4
COLUMN
Ascending chromatography of the products of the enzymatic cleavage of
the 3H-CMP labeled material eluted from the P-4 column was performed
on PEI cellulose as described in the methods. The molecules eluted
from the Biogel P-4 column BGP-1 (800 MW) and BGP-2 (680 MW) were
treated separately with each of the digestive enzymes; snake venom
phosphodiesterase, and ribonuclease A. The standards were CpC, 2'-
3'-CMP, and 5'CMP. Twenty microliters of each digest was spotted 3.0
cm from the bottom of the plate and dried. The enzymatic digest spots
contained the cpm indicated in the denominators. Ribonucleoside mono¬
phosphates were chromatographed as described in the methods. The
nucleotides were detected with ultraviolet light. Radioactivity was
detected by sequentially removing 1.5 cm3 sections of the PEI cellulose
and counting them in toluene scintillation fluid.

65
PEI Cellulose TLC
I.ON ACETIC ACID TO 4CM
0.3 LiCI to 15 CM
SOLVENT FRONT
5456 CPM
5839 CPM
I
Ouv @
5196 CPM 8170 CPM
6954 CPM 8636 CPM
I I
P-4 COL
P-4 COL
P-4 COL
CpC
CpC 2',3-CMP 5-CMP
UN-
PEAKS
+
PEAKS
*t*
+
+
TREATED
S.V.P
RNaseA
S.VP
RNaseA

Figure 9
THE MIGRATION OF THE ENZYMATIC CLEAVAGE PRODUCTS CONTAINING
TRITIUM LABEL ON PEI CELLULOSE
The migration of the enzymatic cleavage products containing tritium
label on PEI cellulose as described in Figure 8.

CPM X 10
67

68
blocked terminus and/or methylated nucleoside moieties can not be ruled
out.
The Solubilization and Partial Purification of
Euglena Mitochondrial RNA Polymerase
The final objectives were to solubilize and purify the mitochondrial
DNA-directed RNA polymerase, to characterize its activity, to determine
its requirements for product synthesis, and to compare it to nuclear RNA
polymerase activities with respect to these requirements. In order to
accomplish this it was necessary to prepare mitochondria which were devoid
of contaminating nuclear DNA. This was achieved by obtaining a crude 5P
mitochondrial pellet, as described in Figure 1, and washing it twice in
STM Buffer before incubating the mitochondria in the presence of deoxyri-
bionuclease I (DNase I) for thirty minutes, zero degrees centigrade.
These mitochondria were shown to be devoid of nuclear DNA by work substan¬
tiated in this laboratory and no further purification was necessary. The
buoyant densities of the DNA in the mitochondrial preparations in CsCl are
demonstrated in Figure 10. The crude washed 5P mitochondria yield mostly
Euglena nuclear DNA (Tube a) which has a density of 1.707 g/cm but after
five minutes of incubation in DNase I at 4 °C, most of the nuclear DNA was
removed (Tube b). Thirty minutes of incubation in DNase removes all of
the contaminating nuclear DNA and only mitochondrial DNA remains, which
3
has a density of 1.691 g/cm (Tube c). Further treatment with DNase for
one hour shows that the mitochondrial DNA is still present and is there¬
fore protected from digestion by DNase I by the mitochondrial membranes.
The mitochondria purified in this manner demonstrated the same ribonucle¬
otide incorporating activities as the mitochondria purified on renografin
gradients. Table 9 shows that the DNased treated mitochondria incorpora-

Figure 10. DENSITY EQUILIBRIA ANALYSIS OF DNA FROM DNASED MITOCHONDRIA
The density equilibria analysis of DNA in mitochondria purified by DNase treatment was measured
by CsCl density gradient centrifugation as described in the methods section. The DNA standards
were Cytophaga P-11 DNA with a density of 1.693 g/cm^, Pseudomonas aeruginosa DNA with a density
of 1.727 g/cm^, and bacteriophage $25 DNA with a density of 1.742 g/cm^. Euglena gracilis mito¬
chondrial DNA has a density of 1.691 g/cm^ and Euglena nuclear DNA has a density of 1.707 g/cm^
in CsCl. The sources of the DNA were washed 5P mitochondria with DNA standards (tube a), the same
mitochondrial preparation after treatment with DNase I for 5 min (tube b), after 30 min. of treat¬
ment in (tube c), after 1 hr. of treatment (tube d) and the DNA standards (tube e).

CsCi Density Gradient Gels
DMA STAINED WITH ETHIDIUM BROMIDE
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71
TABLE 9
A COMPARISON OF THE INCORPORATION OF 3H-UTP and 3H-CTP BY
ISOLATED MITOCHONDRIA PURIFIED BY TREATMENT WITH DNASE I
Labeled
Ribonucleotide
Reaction
Mixture
CPM
3H-UTP
Complete
1,624
3h-utp
-DNA
869
3h-ctp
Complete
27,364
3h-ctp
-DNA
16,669
Mitochondria purified by DNase I treatment were treated with isotonic
buffer to assure swelling and permability. The RNA polymerase assay
was performed as described in the methods. Reaction mixtures of 0.2
ml contained 1.0 mM of each unlabeled NTP and 0.016 mM 3H-UTP or 3H-CTP,
2 Ci/mmole.

72
3 3
ted tridum from H-CTP at a ten fold greater rate than H-UTP. However,
3
about fifty percent of the incorporating activities from both H-CTP and
3 3
H-UTP are DNA dependent which is in contrast to the H-CTP incorporating
activities in renografin purified mitochondria which was not DNA dependent.
Solubilization of the Mitochondrial
RNA Polymerase Activity
The difficulty of defining proper conditions for solubilization of
the chloroplast RNA polymerase of Euglena gracilis (166,185) plus the
rapid inactivation of mitochondrial RNA polymerase activities by some
solubilization methods (11), prompted the investigation to determine
optimum conditions for solubilizing the activity from Euglena mitochon¬
dria (Table 10). An approach used by Rogall and Wintersherger (15) were
used to determine the optimum conditions for solubilization of the
mitochondrial RNA polymerase. A volume of mitochondrial suspension in
lysis buffer was added to an equal volume of the detergent-KCl solution
to bring the mixture to the indicated final concentrations. The mixtures
were centrifuged at 30,000 rpm to sediment non-solubilized material and
the supernatants were assayed for RNA polymerase activities. The
efficiencies of the different treatments in the solubilizations of RNA
polymerase activity from the mitochondria are summarized in Table 10.
It can be seen that the combined treatment with KC1 and Triton X-100
produced the highest yield of activity solubilized. However, NP-40, a
detergent employed by other investigators (15,173), and 0.5 M KC1 was
almost as effective. Treatment with 0.5 M KC1 alone failed to solubi¬
lize significant RNA polymerase activity. Treatment with either deter¬
gent alone was significantly less than with the Triton X-100 plus 0.5
M KC1 treatment which completely destroyed the succinic dehydrogenase

73
TABLE 10
RNA POLYMERASE ACTIVITY SOLUBILIZED FROM
MITOCHONDRIA
Treatment of
Mitochondria
in Lysis Buffer
RNA Polymerase
(units/g wet
30KS
Activity
wt.)
30KP
+ Water
4.2
69
+ 0.5 M KC1
4.2
63
+0.8% NP-40
45.5
46
+0.8% Triton X-100
34.0
37
+ 0.8% NP-40 + 0.5 M KC1
56.0
0
+ 0.8% Triton + 0.5 M KC1
68.0
15
+ Water (Sonication)
0
28
Mitochondria purified by DNase treatment were suspended in lysis buffer
(lg wet wt/10 ml). Mitochondrial samples of 1.0 ml each were then added
to an equal volume of the indicated lysis mixture which contained 0.01 M
EDTA plus additions shown. The mixtures were mixed gently for 15 min. at
4°C and then centrifuged for 90 min. at 30,000 rpm (Ti 50 rotor). The
supernatants and pellets were dialyzed against TGMED Buffer for 4 hrs.
before analyzing them for RNA polymerase activity as described in the
methods. The concentration of ^H-UTP was 0.016 mM with a specific radio¬
activity of 2 Ci/mmole.

74
activity (Table 11) in the supernatant. The NP-40 plus 0.5 M KC1 treat¬
ment produced supernatants that contained succinic dehydrogenase activi¬
ties as high as they were in the control mitochondrial pellet. This
suggested that Triton X-100 detergent was effective in completely
disrupting the inner membrane thus freeing the succinic dehydrogenase of
the membrane associated lipids which are needed to reconstitute the
activity. The NP-40 detergent does not seem to be able to free the suc¬
cinic dehydrogenase enzyme from the associated lipid since any loss of
activity in the pellet could be observed as activity in the supernate
indicating membrane components are present in this fraction needed for the
expression of succinic dehydrogenase activity. The NP-40 detergent even
with 0.5 M KC1 does not seem to be able to free the succinic dehydrogenase
enzyme from the associated membrane components. It appears that although
there is 80% as much RNA polymerase activity in the 30,000 rpm supernatant
produced by NP-40 as there is in the triton treated supernatant, the NP-40
supernatant may not contain solubilized RNA polymerase but just small
mitochondrial fragments that fail to sediment at 30,000 rpm in 90 minutes.
Therefore, the 0.8% Triton X-100 treatment with 0.5 M KC1 was used for
solubilizing the mitochondrial RNA polymerase.
The Effect of Ammonium Sulfate Precipitation on
Mitochondrial RNA Polymerase Activity
It has been reported that Neurospora mitochondrial RNA polymerase
(11) activity could not survive ammonium sulfate precipitation, chroma¬
tography on DEAE-Cellulose, extensive dialysis, concentration by
ultrafiltration, and sucrose gradient centrifugation. Therefore, it was
necessary to determine if Euglena mitochondrial RNA polymerase could
survive ammonium sulfate treatment because the selection of proper salt

75
TABLE 11
SUCCINIC DEHYDROGENASE ACTIVITY IN THE MITOCHONDRIAL
SUPERNATANTS AND PELLETS AFTER SOLUBILIZATION
TREATMENT
Treatment of
Mitochondria
in Lysis Buffer
Succinic Dehydrogenase
(Units x 10-3)
30KS
Activity
30KP
+ Water
0
40
+ 0.5 M KC1
0
49
+0.8% NP-40
22
27
+ 0.8% Triton X-100
0
31
+ 0.8% NP-40 + 0.5 M KC1
34
0
+ 0.8% Triton + 0.5 M KC1
0
0
+ Water (sonication)
0
0
A unit is a pinole of dichlorophenolindophenol reduced per minute per
ml of enzyme sample at 25°C.

76
concentration would allow the fractionation of RNA polymerase away from
other mitochondrial proteins, thus effecting another purification step.
The effect of ammonium sulfate on the mitochondrial RNA synthesizing
activity was measured (Table 12) by adding to solubilized mitochondrial
RNA polymerase samples ammonium sulfate to the desired concentration with
mixing followed by centrifugation at 40,000 rpm for 90 minutes. The RNA
polymerase activity was measured in the redissolved and dialysed protein
samples as described in the methods. The results indicated that 90% of
the activity was recovered when the ammonium sulfate concentration was
50%. It was interesting to observe that when the 30,000 rpm super
(control) was centrifuged at 40,000 rpm for 70 min. 19% of the activity
was lost. This could represent the activity that is not completely
solubilized and thus sediments at 40,000 rpm but not at 30,000 rpm.
Solubilization and Purification
Of Mitochondrial RNA Polymerase
The scheme for the purification of the mitochondrial RNA polymerase
is shown in Figure 11. The optimum conditions described in Table 10
were used to solubilize mitochondrial RNA polymerase. The washed mito¬
chondria obtained from 182g (wet weight) of cells were purified by
treatment with pancreatic deoxyribonuclease (100 pg/ml) for thirty minutes.
The mitochondria obtained after two washings in STE buffer were suspended
in lysis buffer and either used directly for enzyme solubilization or
stored in liquid nitrogen prior to use. An equal volume of lysis mixture
was mixed with mitochondrial suspension to bring the final concentration
to 0.8% Triton X-100, 0.50 M KC1, and 0.005M EDTA. The mixture was
gently stirred for 15 minutes, 4°C. The supernatant obtained after cen¬
trifuging at 30,000 rpm for 90 minutes was brought to 50% saturation with

77
TABLE 12
THE EFFECT OF AMMONIUM SULFATE PRECIPITATION ON MITOCHONDRIAL
RNA POLYMERASE ACTIVITY
Treatment % (NH^^SO^
Saturation
Activity
(CPM/ml)
%
Recovered
in Pellet
30K Super
12420
-
+ 0, 40KP
1915
19
+ 30, 40KP
5660
46
+ 40, 40KP
6640
53
+ 50, 40KP
11220
90
+ 60, 40KP
6220
50
+ 65, 40KP
8830
71
+ 70, 40KP
9880
80
+ 75, 40KP
9090
73
+ 80, 40KP
7520
61
Mitochondria purified by DNase treatment were suspended in lysis
buffer (1 g wet wt/10 ml) and the RNA polymerase activity was
solubilized as described in the methods. Five milliliters of the
mitochondrial suspension were added to an equal volume of the lysis
mixture (1.6% Triton X-100, 1.0 M KC1, and 0.01 M EDTA pH 7.9) and
mixed gently for 15 min. at 4°C and then centrifuged for 90 min. at
30,000 rpm (Ti 50 rotor). The supernatants were divided into 1.0
ml aliquots and solid ammonium sulfate was added to each aliquot to
the desired concentration. The ammonium sulfate was dissolved at
4°C by gently mixing and the precipitated protein was collected by
centrifugation at 40,000 rpm, 70 min. (Ti 50 rotor). Each pellet was
dissolved in 1.0 ml of TGMED buffer and dialyzed against 2 liters of
TGMED for 4 hrs. at 4°C. The ammonium sulfate concentration in each
dialysate was determined with the conductance bridge and the salt
concentration was adjusted to 0.05 M ammonium sulfate. The RNA
polymerase assay was performed as described in the methods except that
KC1 was omitted.

78
Washed 5P Mitochondria
DNased, 30 min, 4°C
Rx Stopped w/3 vol. 0.1 M EDTA pH 7.5: 10% Sucrose
cent. 10,000 x £, 20 min (Repeat 2X in STE buffer)
10 KS Purified Mitochondria
Resuspended in lysis buffer
Add equal vol. lysis mixture:
1.6% Triton X-100
1.0 M KC1
0.01 M EDTA
Cent. 30,000 rpm, 90 min, 2°C
30 KS 30 KP
50% (NH^^SO^ satuation
Cent. 40,000 rpm, 70 min, 2°C
i I
40 KS 40 KP
Dissolved in TGMED Buffer
Dialyze 4 hr against 2 liters
TGMED: 40 mM (NH4)2S04
DEAE-Sephadex A-25
column chromatography
DCI
Figure 11. PROCEDURE FOR THE PARTIAL PURIFICATION OF MITOCHONDRIA
RNA POLYMERASE

79
respect to ammonium sulfate. The precipitation collected at 40,000 rpm
was dissolved in TGMED buffer and dialysed against TGMED containing 50 mM
ammonium sulfate. The dialysate was subjected to DEAE-Sephadex chroma-
3
tography and the eluted fractions were assayed for H-CTP incorporating
activities using the RNA polymerase assay (Figure 12). The void volume
contained an activity which incorporated tritium preferrentially from
3
the substrate H-CTP by a reaction which was not dependent upon added
DNA or the other Ribonucleotide substrates into acid insoluble products
which resisted pancreatic ribonuclease digestion (Table 13). However,
3
an activity which incorporated label from H-UTP into acid insoluble and
RNase digestible products was eluted from the DEAE-Sephadex column
(Figure 12) in a single peak between 0.32M and 0.37M ammonium sulfate in
TGMED buffer. The specific activity of the RNA polymerase after this
purification step was 0.3 nmoles UMP incorporated/mg protein (estimated
from &280 absorbancy)/10 minutes at 37°C. This enzyme activity demon¬
strates a dependence upon added DNA and ribonucleoside triphosphate
3
substrates (Table 14) for the incorporation of label from H-UTP. The
mitochondrial RNA polymerase activity is also inhibited by low concentra¬
tions (10 pg/ml) of actinomycin D (Table 15). The activity is insensi¬
tive to rifampin, a-amanitin, streptovaricin, rifamycin AF/013 and
rifamycin AF/05. Attempts to concentrate this activity by rechromato¬
graphy on a smaller DEAE-Sephadex A-25 column or to further purify the
activity by glycerol gradient centrifugation resulted in the loss of
significant activity.

Figure 12. ION EXCHANGE CHROMATOGRAPHY ON DEAE-SEPHADEX A-25
3
The procedure is described in the methods. The RNA polymerase activity profile when H-UTP was
substrate is represented by the solid line while the profile when ^h-CTP was the substrate is
represented by the broken line ( A A ). The A280 Pro;file is represented by the double
line while the ammonium sulfate concentration in each fraction is represented by the heavy dashed
line.

-03
-0.1
ABSORBANCE AT 280 nm

TABLE 13
A COMPARISON OF TRITIUM INCORPORATION FROM 3H-UTP and 3H-CTP
BY THE ACTIVITY
IN THE DEAE-SEPHADEX A-25 COLUMN FRACTION 3
Reaction
Mixture
CPM
3H-UTP
Complete
200
3H-CTP
Complete
-DNA
-NTP
+RNase
5428
2581
4389
5316
The RNA polymerase assay reaction mixtures contained 95
pi of the void volume activity in a total volume of 0.20
ml. The concentration of ^h-CTP, were 0.016 mM, 2 Ci/mmole.

83
TABLE 14
THE DEPENDENCY OF THE DEAE COLUMN FRACTION 38 INCORPORATION
OF 3H-UMP UPON ADDED RIBONUCLEOSIDE TRIPHOSPHATES AND DNA
Additions
or
Deletions
CMP
Percent
of
Control
Complete
1488
-
-DNA
187
13
-NTP
398
27
-ATP
168
11
-CTP
238
16
-GTP
178
12
+ Pancreatic RNase
(100 yg/ml)
355
24
The RNA polymerase assay reaction mixtures contained 20 yl of DCI
in a total volume of 0.20 ml. The concentration of 3H-UTP was 0.016
mM, 2 Ci/mmole.

TABLE 15
INHIBITORS OF H-UTP
COLUMN FRACTION 38
INCORPORATION
BY THE DEAE
Inhibitors
CPM
% Activity
Distilled Water
2496
100
1% DMF
2521
100
Actinomycin D, 10 yg/ml
82
3
Rifampin, 100 pg/ml
1898
76
Rifamycin AF/013, 100 yg/ml
2109
85
Rifamycin AF/05, 100 yg/ml
2065
83
Streptovaricin, 100 yg/ml
2369
95
a-Amanitin, 5 yg/ml
1490
60
Pancreatic RNase 100 yg/ml
69
3
The RNA polymerase assay was performed with the addition of
inhibitors to the final concentrations indicated above. The
concentration of %-UTP was 0.016 mM, 2 Ci/mmole. Rifampin,
rifamycin AF/013, rafamycin AF/05, and streptovaricin were sol¬
ubilized in 1% N'N' dimethyl formamide. Reaction mixtures con¬
tained 50 ml of the DCI enzyme in a total volume of 0.2 ml.

85
Enzymatic Properties of the
Mitochondrial RNA Polymerase
The dependence of the mitochondrial RNA polymerase on the concen¬
tration of MnC^ is shown in Figure 13. Low concentration (about 1 mM)
1 |
of Mn stimulated the activity of this enzyme and higher concentrations
decreased the activity. However, the enzyme activities were also stimu-
| j-
lated by low concentrations of Mg (Figure 14) demonstrating the optimal
concentration at 0.5 mM.
The template requirement of the mitochondrial enzyme is summarized
in Table 16. The mitochondrial enzyme prefers poly d(AT) as a template
which is similar to the rifampicin sensitive mitochondrial RNA polymerase
from Neurospora crassa, described by Kuntzel and Schafer (11). It is
interesting to note that while Euglena nuclear DNA was a more effective
template than denatured calf thymus DNA that it is not necessarily a
more preferred template than Euglena mitochondrial DNA. The mitochondrial
DNA was only available in 1/5 the saturating DNA concentration and a
comparison of the templates was not possible.

Figure 13. DEPENDENCE OF THE ACTIVITY OF THE DEAE FRACTION 38
ON THE CONCENTRATION OF Mn++
Assays were carried out as described in methods. The concentration
of ^H-UTP was 0.016 mM, 2 Ci/mmole. The reaction mixtures contained
20 pi of DCI enzyme in a total volume of 0.2 ml.

87
1
10

Figure 14. DEPENDENCE OF THE ACTIVITY OF THE DEAE FRACTION 38
ON THE CONCENTRATION OF Mg++
Assays were carried out as described in methods. The concentration of
^H-UTP was 0.016 mM, 2 Ci/mmole. The reaction mixtures contained 20 yl
of DCI enzyme in a total volume of 0.2 ml.

C PM

TABLE 16
TEMPLATE SPECIFICITY OF THE DEAE COLUMN
FRACTION 38
DNA Template
Units/ml
Percent
of
Control
Standard Assay
Denatured calf thymus
DNA
19
100
Poly d(AT)
45
236
Native Euglena nuclear
DNA
23
121
Native E. coli DNA
14
73
Assays were carried out as described in materials and
methods. Results are expressed as percent of the activi¬
ty measured in the standard assay mixture with denatured
calf thymus DNA as template. The DNA concentration was
50 yg per ml in each reaction. The %-UTP concentration
was 0.016 mM, 2 Ci/mmole.

DISCUSSION
This study was undertaken with the intentions of examining the
mechanism by which Euglena gracilis, a eukaryotic cell, controls mito¬
chondrial development. Since the DNA dependent RNA polymerase has been
implicated in controlling development in prokaryotic systems it was felt
that the mitochondrial RNA polymerase could be a major factor controlling
development of the organelle. Therefore, the mitochondrial RNA synthesiz¬
ing activities were studied with a view toward elucidating the components
of the transcriptional process, in particular characterizing the mitochon¬
drial DNA dependent RNA polymerase. The RNA synthesizing activity was
first studied in isolated mitochondria because it was felt that it was
necessary to characterize such general properties as sensitivity to in¬
hibitors, types of RNA being synthesized, precursor requirements, metallic
ion requirements, and temperature optimum. The streptomycin bleached
aplastidic mutant was used for these studies because it provided a means
by which processes controlling mitochondrial development could be distin¬
guished from those controlling chloroplast development.
Mitochondria were isolated by a procedure that selected for the most
enzymatically active organelles (33) based on succinic dehydrogenase
activity. This procedure also provided purified mitochondria that con¬
tained mostly mitochondrial DNA (70%) based on the buoyant densities in
CsCl (Figure 2). Although some contaminating nuclear DNA was in the
preparation it represented less than 1% of the total nuclar DNA originally
91

92
present in the cell. This procedure provided the cleanest mitochondria
feasible without treating them with an exogenous nuclease.
3
The incorporation of label from H-ribonucleoside triphosphates into
acid insoluble products showed no dependence upon the presence of the
other ribonucleotides or that of added DNA. The cpm incorporated with
3 3
H-ATP or H-GTP as substrates were so low that it made it difficult to
determine the dependence of their incorporation upon the presence of the
other ribonucleotides. The presence of mitochondrial DNA and mitochondrial
pools of endogenous ribonucleotides could account for these results. How-
3
ever, H-CTP was the preferred substrate compared to the other tritium
labeled ribonucleoside triphosphates. The kinetics of the incorporation
(Figure 3) allow the presence of nuclease activity in the mitochondria as
3
demonstrated by departure from linearity after 5 to 10 min. With H-UTP
as substrate these mitochondria are capable of carrying out the synthesis
of a product which demonstrates some sensitivity to RNase. The failure
3
of the labeled product, obtained upon incubation with H-CTP, to be de¬
graded by pancreatic RNase A may be due to either the failure of the nuclease
to get into the mitochondria or the alteration of the RNA 3' terminus thus
preventing the RNase A from binding to the products. It is also possible
that the product is complexed with DNA such that it is RNase insensitive.
The results in Figure 3 also suggest that RNA synthesis based upon at
least partial sensitivity of product to RNase could be better studied by
following the incorporation of UTP by the isolated mitochondria rather
than CTP.
3
The H-CTP incorporating activity appears to be associated with
mitochondrial DNA in that the activity is enriched 125 fold per yg of

93
DNA in the purified mitochondria (Table 2). If the activity is associated
with DNA, then it is with mitochondrial DNA and not nuclear DNA, 95% to
99% of which has been removed by the purification procedure. This does
not eliminate the possibility that the activity is associated with the
mitochondrial membrane since the activity is enriched 8.7 fold per mg
of protein in the purified mitochondria. This would be probable because
the crude mitochondria may be contaminated more with nuclear DNA than
with exogenous protein and enrichment observed may just reflect the extent
to which the contaminating molecules have been removed.
The inhibition of the CMP incorporating activity by actinomycin D
(Table 3) suggests that a DNA dependent reaction may be involved which
would implicate either the RNA or the DNA polymerases. The use of ribo¬
nucleotide precursors eliminate DNA synthesis as a consideration since
it is difficult to imagine the conversion of these precursors to deoxyri-
bonucleotides in the period of the assay. The inhibition of the CMP
incorporating activity by pyrophosphate indicates that the reaction in¬
volves a pyrophosphorolysis, implicating a RNA polymerase type reaction.
However, phospholipid biosynthesis could not be ruled out because a
pyrophosphorolysis reaction is involved in its utilization of CTP.
The acid insoluble CMP incorporating product is sensitive to snake
venom phosphodiesterase as well as to alkaline hydrolysis. The speci¬
ficity of the enzyme for attacking the phosphodiester bond to yield 5'
nucleoside monophosphates and also the sensitivity of the phosphodiester
bond to alkaline hydrolysis certainly indicates that the CTP product
contained phosphodiester bonds. The properties demonstrated by the
product were similar to those reported by Cherry et al. (184) for a poly
C RNA polymerase activity in sugar beet nuclei. This activity was in-

94
hibited by actinomycin D and pyrophosphate while the product was insensi¬
tive to pancreatic RNase digestion yet it was sensitive to snake venom
phosphodiesterase and alkaline hydrolysis. However, they were able to
show by nearest neighbor analysis that the product was poly C.
The products of the CTP incorporating activity for this study were
isolated by phenol extraction followed by ethanol precipitation of the
aqueous phase. The size of the isolated products as determined by their
elution position from a Biogel P-4 column indicated they comprised two
classes of molecules with mean molecular weights of 800 and 680 respect¬
ively (Figure 4). These molecules have absorbancy spectra very similar
if not identical to that of CpC which suggests that cytosine is the only
base incorporated into the products. The snake venom phosphodiesterase
clevage product of both molecular weight classes is 5'-CMP, the expected
clevage product of poly C of CpC. The CTP incorporating products showed
no sensitivity to pancreatic RNase A. The possibility exists that these
molecules are di- or trinucleotides of CMP which have modified 3' terminus
which prevents the RNase A from binding to the molecules. All attempts
, 3
to demonstrate that the products of the H-CTP incorporating activity could
be CDP-diglyceride gave negative results.
Recent studies have demonstrated the presence of an unusual structure
7 5 1 5 » m JJJ
( G ppp N pMp) at the 5' termini of a wide variety of eukaryotic and
viral messenger RNA's in which the 5' end of the messenger RNA is blocked
by a 7-methyl guanosin linked to a 2'0-methylated nucleotide, Nm, through
a 5’—5* pyrophosphate bond (186,187). The 2'-0-methylated nucleotide Nm
is linked to the adjacent nucleotide Mm by a 3'5' phosphodiester bond.
These blocked, methyoated 5'-termini ("caps") are resistant to digestion
by ribonuclease. The possibility exists that the labeled products obtained

95
after incubating Euglena mitochondria with H-CTP may have similar structures
with cytidine as the methylated nucleoside. It is impossible to determine
the assigned role of these small C-labeled molecules in the mitochondria
without knowing their precise structures.
3
An activity which incorporated lable from H-CTP was separated from
the mitochondrial RNA polymerase when it eluted in the void volume during
DEAE-Sephadex chromatography. This activity was similar to the activity
observed in the isolated mitochondria in that it preferentially incorpor-
3
ated label from H-CTP by a reaction which was not DNA dependent and did
not require the other ribonucleotide substrates of RNA synthesis. The
products of this activity were insensitive to pancreatic ribonuclease
digestion. This data provides evidence that this activity is a mito¬
chondrial associated activity; however, it may not be a RNA polymerase
activity but this conclusion must be reserved until the identity of the
product is determined.
The mitochondrial RNA polymerase of Euglena had not previously been
solubilized and this study represents the first time that an RNA synthesizing
activity has been solubilized and partially purified from a cytoplasmic
organelle of this organism. The mitochondria used for solubilization of
the mitochondrial enzyme were treated with DNase I to remove all contami¬
nating nuclear DNA (185) and its associated proteins. The results demon¬
strated that it was possible to define optimum conditions for solubilizing
the RNA synthesizing activities. The Triton X-100 detergent KC1 treatment
was effective in distrupting the mitochondrial membranes and releasing the
RNA polymerase activity (Table 9) which could be fractionated by ammonium
sulfate precipitation. The enzyme activity is also able to survive dialysis
and DEAE-Sephadex chromatography, eluting in a single peak between 0.32

96
and 0.37 M (NH.)„SO. (Figure 11). This enzyme is different from the
4 L 4
Euglena nuclear RNA polymerase II which elutes between 0.18 and 0.21
M (NH,)„S0, and is sensitive to a-amanitin. The mitochondrial enzyme is
4 2 4
insensitive to a-amanitin and rifampicin and demonstrates a DNA depend¬
ence bases upon actinomycin D inhibition and the requirements for added
DNA and all of the ribonucleotide substrates. Poly d(AT) is the pre¬
ferred template and optimal activity is observed with low concentrations
| | | |
of Mn and Mg
Attempts to concentrate the mitochondrial enzyme by rechromatography
on a smaller DEAE-Sephadex column or to further purify the activity by
gylcerol gradient centrifugation resulted in the loss of significant
activity. This loss was probably due to the prolonged time at 4°C
necessary for these additional purification steps.

CONCLUSIONS
Euglena mitochondria contain a RNA polymerase activity which can be
solubilized and then partially purified by DEAE-Sephadex chromatography.
This enzyme is DNA dependent, requires the four ribonucleoside triphos¬
phates and is not inhibited by rifampin. The mitochondrial enzyme is
i
distinctly different from the nuclear RNA polymerase II (167,168) based
on its elution from DEAE-Sephadex chromatography and sensitivity to inhibi¬
tors. The stability and yield of the mitochondrial enzyme is such that
large preparations of mitochondria are required for further enzyme puri¬
fication and characterization of the activity and molecular substructure
(30 liters of log phase cells yield 53 units of mitochondrial activity
per milliter of partially purified enzyme with a specific activity of
0.3 nmoles/mg protein).
Euglena mitochondria also contain an enzyme activity which pre¬
ferentially incorporates CMP from CTP into small molecules of unknown
structure, with molecular weights between 800 and 600 daltons. These
molecules contain phosphodiester bonds but they are resistant to cleavage
by pancreatic ribonuclease.
97

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BIOGRAPHICAL SKETCH
George Erwin Brown was born August 12, 1939 in Houston, Texas. He
lived with his parents, Marshall Vernon Brown and Libbie Nickerson Brown;
two sisters, Ida and Doris; and a brother, Marshall Jr., until he graduated
from Prairie View High School, Prairie View, Texas. He received the Bachelor
of Science Degree in Agricultural Education from Prairie View A&M College,
Prairie View, Texas, May 1960. He did post graduate work in biology at
Prairie View until he entered the U. S. Army as a commissioned officer in
June 1961 where he served as a signal corps officer until July 1963. He
taught High School Biology in the Hull-Daisetta School District and then the
Royal Independent School District, both in Texas, until August 1966 when he
assumed the position of a county agricultural agent for the Texas Agricultural
Extension Service. He remained in this capacity until February 1969 when he
became Director of an Adult Education Center for Harris County Department
of Education. In September 1969, he entered the University of Florida on a
Rockefeller Foundation Fellowship and in June 1970, he entered the Graduate
School of the University of Florida, as a teaching assistant in the Depart¬
ment of Microbiology. This is when he met Dr. James F. Preston who has
kept him busy every since. He received the Master of Science Degree in
June 1972 and is now a candidate for the Doctor of Philosophy Degree in
August 1976.
George is married to the former Carolyn M. Rogers and they have two
children, a son, George Jr., age 5; and a daughter, Erika, age 2. George
108

109
is a member of the American Society for Microbiology, the National Insti¬
tute of Science, and Beta Beta Beta Scientist Society. He is presently
an Assistant Professor of Biology at Prairie View A&M University, Prairie
View, Texas.

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosophy.
/James F. Preston, III, Chairman
Associate Professor of Microbiology
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosophy.
d
1 certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosophy.
7 /
i ?/ .
Edward M. Hoffmann.-'’ v
Associate Professor of¿M'^trobiology
c ,/ i /// / / / /
cgicn iA///-
I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosophy.
Thomas W. O'Brien
Associate Professor of Biochemistry

I certify that I have read this study and that in my opinion it
conforms to acceptable standards of scholarly presentation and is
fully adequate, in scope and quality, as a dissertation for the
degree of Doctor of Philosophy.
Lonnie 0. Ingram ~7
Associate Professor of- Microbiology
This dissertation was submitted to the Graduate Faculty of the
Department of Microbiology in the College of Arts and Sciences
and to the Graduate Council, and was accepted as partial fulfillment of
the requirements for the degree of Doctor of Philosophy.
August, 1976 . ,
/ 1. - \ ‘ <1
Dean, Graduate School
' / • ^
/
/ ,
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