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LSU Historical Dissertations and Theses
Graduate School
1971
Evidence for the Incorporation of Exogenous Dna
by the Polytene Chromosomes of Living
Drosophila Mojavensis Salivary Gland Cells in
Shortterm Organ Culture.
John Watkins Williams III
Louisiana State University and Agricultural & Mechanical College
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Recommended Citation
Williams, John Watkins III, "Evidence for the Incorporation of Exogenous Dna by the Polytene Chromosomes of Living Drosophila
Mojavensis Salivary Gland Cells in Shortterm Organ Culture." (1971). LSU Historical Dissertations and Theses. 2099.
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WILLIAMS, III, John Watkins, 1942EVIDENCE FOR THE INCORPORATION OF EXOGENOUS
DNA BY THE POLYTENE CHRCMOSCMES OF LIVING
DROSOPHILA MOJAVENSIS SALIVARY GLAND CELLS IN
SFmTmrrrarajcruRE.
The Louisiana State University and Agricultural
and Mechanical College, Ph.D., 1971
Zoology
University Microfilms, A XEROX Company, Ann Arbor, Michigan
EVIDENCE FOR THE INCORPORATION OF EXOGENOUS DNA
BY THE POLYTENE CHROMOSOMES OF LIVING
DROSOPHILA MOJAVENSIS SALIVARY GLAND CELLS
IN SHORT TERM ORGAN CULTURE
A Dissertation
Submitted to the Graduate Facultj' of the
Louisiana State University and
Agricultural and Mechanical College
in partial fulfillment of the
requirements for the degree of
Doctor of Philosophy
in
The Department of Zoology and Physiology
by
John Watkins Williams, III
B.S., University of Southwestern Louisiana, 1965
M.S., Louisiana State University, 1968
August, 1971
PLEASE NOTE:
Some Pages have i n d i s t i n c t
p rin t.
Filmed as re c e iv e d .
UNIVERSITY MICROFILMS
ACKNOWLEDGEMENTS
I wish to extend my appreciation to the graduate
committee of the Department of Zoology and Physiology for
my nomination for a Dissertation Year Fellowship.
I also
want to thank the Graduate School for awarding me the
Fellowship.
My sincerest thanks to Mr. Gary Eberle for
his valuable assistance in preparing some of the photo­
graphs for this manuscript.
To my wife, for her considera­
tion, assistance, and unending patience, I am most grateful.
Most of all, I wish to thank my major professor, Dr. VJ. L.
French, for his guidance, patience, and moral support during
the course of this investigation and the preparation of this
report.
.ii
TABLE 0]? CONTENTS
Chapter
Page
TITLE P A G E ........................................
i
A C K N O W L E D G M E N T S ..................................
ii
TABLE OP C O N T E N T S .............................
ill
LIST OP T A B L E S ............. .'....................
LIST OF FIGURES
ABSTRACT
. .
iv
.............................
............................. ..
v
.
I N T R O D U C T I O N ......................................
vi
1
...............................
...............................
3
17
METHODS AND M A T E R I A L S .............................
31
In-vitro studies
In-vivo studies
Animals
.
....................................
DMA Extraction P r o c e d u r e s .....................
Purification T ..................................
3h -Labelling of D N A ...........................
....................................
Chase DNA
Culture Techniques .............................
Quantitative Techniques
.......................
At-torad 1 ograph 1c T e c h n i q u e s ...................
Acridlne Orange Tagged DNA .. .
..............
Acrldlne Orange - DNAC u l t u r e s ..................
R E S U L T S ............................................
31
32
33
34
36
36
36
38
40
40
42
Extraction, Purification, ^Ii -Labelling
....
42
Quant 1 tat 1 ve Studies on DNA U p t a k e
• 42
Aut or ad i o'graphi c S t u d i e s ......................... - 4 3
Acridine Orange - DNA Complex. Studies
........
44
D I S C U S S I O N ........................................
59
S U M M A R Y ............................................
68
LITERATURE C I T E D .................................... ’ 70
V I T A ...............................................
iii
79
LIST OF TABLES
Table
Page
1.
The Treatment M e a n s ............
54
2.
Analysis of variance and orthogonal
comparisons of the t r e a t m e n t s ...............
54
iv
LIST OF FIGURES
Figure
1.
2.
3.
4.
5.
6.
7.
8.
9.
Page
The U V absorption profile and II -label
distribution of D. melanogaster nucleic
acid MAK f r a c t i o n s .........................
46
A comparison of the TCA soluble and insoluble
fractions of 3h~DNA in Hank's solution with
and without exposure to salivary glands . . .
48
A comparison of the ^H-labelled TCA soluble
and insoluble fractions of the gland homogenates and the relationship of their
size to the cells' total DNA content
. . . .
50
Autoradiograph of chromosomes grown in the
presence of 3H~labelled DNA for four hours
.
52
Autoradiograph of chromosomes grown in the
presence of 311-labelled DNA for four hours
.
52
Chromosomes showing fluorescence of the
incorporated acridine orange- DNA complex, . .
56
Chromosomes showing fluorescence of the
incorporated acridine orange-DNA complex
. .
56
Chromosomes showing fluorescence of the
incorporated acridine orange-DNA complex
. .
58
Acridine orange staining of an injured
salivary gland c e l l .........................
v
58
ABSTRACT
The existing genetic evidence on the incorporation
of exogenous DNA suggests the chromosomal attachment of
incorporated DNA.
This study was designed to give direct
evidence for the chromosomal incorporation of exogenous
DNA.
Autoradiographic and fluorescent studies show that
the exogenous DNA is incorporated into the chromosome.
The number of silver grains on the autoradiographs found
over the chromosomes was four times above background.
This is indicative of the chromosomal incorporation of the
DNA.
Both the autoradiographic and fluorescent data indi­
cate a non-localized chromosomal incorporatiop of the
exogenous DUA.
Quantitative studies shew that the amount
of labelled DNA incorporated by the cells was more than
twice the amount that could be incorporated by cellular
DNA synthesis under the controlled cultural conditions.
These results add support to the existing evidence for the
genomic incorporation of exogenous DNA.
Duplicates of the original manuscript are available
from the Library, the Department of Zoology and Physiology,
and Dr. W. L. French of the Department of Zoology and
Physiology, Louisiana State University, Baton Rouge, Lou­
isiana.
The photographs in these copies are in color.
vi
INTRODUCTION
Griffith's discovery of bacterial transformation in
1928 stands as a landmark in the history of genetics.
It
not only demonstrated the transference of hereditary mater­
ials from one bacterium to another, but it laid founda­
tions for studies which led to the establishment of
deoxyribonucleic acid (DNA) as the hereditary material.
Subsequent work by Dawson and Sia (1 9 3 1 ) showed that
transformation could be effected in a test tube.
Alloway
(1933) demonstrated that transformation could be accom­
plished by cell-free extracts.
These studies led to an
extensive systematic chemical analysis of the transforming
substance by Avery, et al. (1 9 4 4 ).
Their work demonstrated
that the transforming material possessed all the properties
of DNA.
Enzymatic studies by McCarty and Avery (1946a and
b) showed that only deoxyribonuclease (DN'ase) would de­
stroy the biological activity of the substance.
These
studies provided irrefutable evidence that the molecules
involved in transformation we re DNA.
Later experiments
32
showed the irreversible incorporation of J P labelled
transforming DNA into a recipient bacterium (Goodal and
Herriot, 19 57; Lerman and Tolmach, 1 9 5 7 )*
Since the dis­
coveries by these early workers, bacterial transformation
has become a relatively common tool of the bacteriologist.
1
2
DNA-mediated transformations in eukaryotes have been
the subject of increasing numbers of investigations over
the past fifteen years.
Transformation of the eukaryotic
cell or intact organism presents a situation considerably
different from that encountered in bacteria.
In the intact
animal transforming molecules may move great distances
from the point of entry and must cross several cell mem­
branes before reaching a desired point of interaction
with the existing genome.
Once this region is reached*
the chromosomes encountered are much more complex than
those of bacteria; their condition of strandedness is yet
undetermined* and they are closely associated with several
types of proteins.
In addition to these difficulties* the
transforming DNA also faces the possibility of attack by
the organism’s defense mechanisms.
Despite the apparent
difficulties of eukaryotic transformation* evidence for
specific genetic transformation of higher organisms is
beginning to accumulate.
In reviewing existing evidence for uptake and genetic
function of exogenous nucleic acids emphasis is placed on
»t .
studies concerning DNA.
The present literature on uptake
of macromolecules may be divided into two major categories:
those studies carried out on cells in culture and those
conducted on intact living animals.
Investigations in
both of these areas have yielded important information on
some basic questions facing geneticists today.
Studies
in-vitro have been more successful than those in vivo'.
3
This study presents a survey of current literature in both
areas as well as original data on the chromosomal incor­
poration of exogenous DNA.
I.
In-vitro studies
The technique of growing eukaryotic cells in chemi­
cally defined media provides an opportunity for geneticists
to study incorporation of macromolecules by such cells under
conditions similar to those existing in bacterial cultures.
This approach not only provides a method for studying
genetic transformations in the cells of higher organisms,
but is also an efficient method of studying the chemical
and physical mechanisms involved in uptake and incorpora­
tion of macromolecules.
Such studies have yielded infor­
mation regarding the transformation of living cells, the
rate of uptake, the approximate size of the incorporated
DNA, the amount of DNA absorbed, the nature of the process
of incorporation, the fate of the incorporated molecules,
and the interaction of the DNA with the existing genome
(Ledoux, 1965).
A number of transformations warrant consideration.
Krauss (1961) reported the successful transformation of
bone marrow cells from a patient with sickle cell anemia.
Individuals with sickle cell anemia produce a unique hemo­
globin polypeptide,^8 ,
DNA was extracted from cells ob­
tained from an individual who was homozygous for the gene
coding for the normal/3 ^ polypeptide.
Treatment of the /3s
bone marrow cells with DNA extract from normal cells re­
sulted in transformation of the cells into ones capable of
synthesis.
Other early successful transformation studies also
involved biochemical traits.
Kantoch and Bang (1962) in­
duced susceptibility to a virus infection in mouse macro­
phages.
There was some variation in the induced resistance,
dependent on the length of the period of exposure and on
the amount of DNA to which the cells were exposed.
Szybalska and Szybalski (1962), working with IMP-pyrophosphorylase deficient human cells, effected a dosedependent transformation of these cells with DNA extracted
from a cell line that was capable of synthesizing this
enzyme.
Transformation of these cells was inhibited by
D N ’ase and by competition with DNA from other mammalian
sources.
Shepley, et al, (1965) altered the cellular
morphology and clonal appearance of hamster cells by
treating them with DNA or RNA extracted from hamster and
rat kidney, polyoma-virus-transformed cells, stilboestrolinduced-kidney-tumor cells, and Bacillus subtllls.
The
greatest effects were achieved with nucleic acids from
the chemically induced tumor cells.
Degradation of nucleic,
acids prior to incubation with the cells destroyed their
transforming properties.
Pope, et al. (1968) transformed
normal human fetal leucocytes to ones resembling those from
patients with leukemia or infectious mononucleosis by
treating them with an extract from another human leucocyte
cell line.
The QJ.MR-WIL cell line, the source of the fil­
trate, carries a herpes-type virus in its genome.
The
physical properties, such as sedimentation values, of the
isolated, heat and ether sensitive, transforming factor
are compatible with those of the herpes virus carried in
the QIMR-WIL line.
In addition, Majumdar and Bose (1968)
transformed an azathymine sensitive strain of human uvular
carcinoma cells.
The drug resistance was mediated by
phenol extracted DNA from a resistant cell line derived
from the same type of carcinoma.
The authors theorized
that the DNA probably replaces only one of the pair of
alleles concerned with drug resistance; this theory is
feasible if drug resistance is a dominant character.
More sophisticated in-vitro transformation experi­
ments produced results which indicated new complex, enzyma­
tic action apparently dependent on the incorporated DNA.
GTiclc and Salim (1967a) obtained a transformation of nonpigmented hamster cells.
An amelanotic melanoma strain of
hamster cells (AM5 ) was exposed to DNA from a melanotic
melanoma cell line (MM2 ).
Pigment production by the nor*i
mally non-pigmented cells was indicative of transformation.
The statistically significant level of transformation ob­
served was ten times as high as that obtained by Szybalska
and Szybalski.
Ottolenghi-Nightingale (1969) also obtained
transformation of non-pigmented cells.
The melanin gran­
ules were proof of the functional state of the incorporated
DNA.
6
Brackett, et_ al. (19 71 ) obtained a unique type of
transformation through the use of rabbit spermatozoa.
The
SV-4 0 virus genome was readily absorbed by spermatozoa in
vitro.
Autoradiographic data showed the incorporated ge­
nome was located in the postacrosomal area.
The intact
SV-4 0 virus also attached to the sperm cells but was not
located by autoradiography.
Enzyme digestion studies gave
indications that the intact virus did not penetrate the
spermatozoon, but remained attached to the surface.
When
sperm cells were exposed to either the SV-4 0 genome or the
SV-4 0 virus and incubated with CV-1 cells, which are sen­
sitive to SV-4 0 virus infection, cytopathic effects were
observed and the production of the SV-4 0 specific T-antigen
was demonstrated.
Microscopic observations showed that
sperm cells penetrated the CV-1 cells.
Sperm cells car­
rying the intact viruses effected the changes within
72
hr,
while those cells carrying the genome alone took 12 days
to initiate the phenotypic changes.
In addition to these
changes, infectious viruses were obtained from invaded
cells.
DNA-treated sperm cells were injected into the
• >,
uterine horns of a female rabbit in which ovulation .was
induced and fertilization occurred.
When these fertilized
ova were cocultivated with CV-1 cells, cytopathic effects
and the presence of T-antigen, specific to the SV-4 0 virus,
were observed.
Such results indicated that the viral genome
was absorbed into sperm cells and1transferred via fertili­
zation to an ovum, where it reproduced.
Bendich, et_ al. (1969) suggested that cellular trans­
formation was a possible mechanism for metastatic' spread
of cancer.
They accomplished the transfer of Ehrlich
ascites tumor DNA from cell to cell as well as uptake of
extracted Ehrlich ascites tumor cell DNA* moreover, uptake
of entire L 1210 chromosomes by cells in-vitro was demon­
strated.
cells.
Specific antigens were detected in the transformed
Passage of the specific antigen reactions through
one hundred generations of cells indicated that the incor­
porated. DNA was being replicated and was capable of func­
tioning in protein synthesis.
Observation of DNA incorporation indicates that the
rate of uptake may vary among different cell lines.
Sirotnak and Hutchingson (1959) detected very rapid uptake
of DNA in L1210 cells that had been maintained intraperitoneally and exposed to
P labelled DNA in vitro.
Maxi­
mum uptake in eight day old cells was achieved in three
minutes.
Gl'ick (1967a) achieved similar results with this
same cell line; the optimal rate of absorption was observed
during the first five minutes.
Studies with Ehrlich•1
Lettre/ ascites carcinoma cells (Meizel, 1 9 6 5 ; Meizel and
Kay, 1965; Kay, 1966) showed that these cells achieve maxi­
mum absorption during the first 15 min.of incubation with
exogenous DNA.
Contrasting results were obtained with two other
abnormal cell lines.
Borenfreund’and Bendich (1961) in
studying uptake of exogenous DNA by HeLa cells, observed
8
that the cells did not achieve maximum uptake until 24 hr
after initiation of the culture.
Hill and Spurna (1968),
working with I. cells, obtained nucleolar labelling after
24 hr of incubation.
From such reports, it seems that
rates of incorporation of exogenous macromolecules may be
a function which is dependent on the strain of cells in­
volved .
If cellular transformation is to be accomplished,
the incorporated DNA must be of a size that would allow
for the coding of at ..least one new specific polypeptide.
This requirement means that a fairly large piece of highly
polymerized DNA must be absorbed by the cell.
Transforma­
tion experiments indicate that incorporation of such large
molecules takes place.
Several studies have given more specific indications
of the size of DNA molecules taken up by living cells.
Gartler (19 59)* using
H-labelled homologous DNA, showed
that the amount of label incorporated by Henle cells, after
1 hr of culture, was not affected by competition with bases,
nucleosides, or nucleotides; only excess cold DNA reduced
the amount of incorporated label. He further demonstrated
lack of DNA breakdown in the culture medium by alcoholic
reprecipitation of the DNA remaining in the medium.
In
i960 Gartler, using Earle’s L cells tagged the donor DNA
with both a radioactive molecule,
cule, 5-bromo-deoxyuridine (5DBU);
14
C, and a heavy mole­
This technique per­
mitted separation of donor and recipient DNAs by CsCl
centrifugation and provided a means of identification and
characterization of each type of DNA.
Any breakdown and
recombination of the molecules would change both the la­
belling and density characteristics of the respective
fractions.
Very few changes were detected, indicating that
the molecules were being absorbed in their macromolecular
form.
Yosida, e_t al. (1968) demonstrated that entire, iso­
lated rat metaphase chromosomes could be incorporated into
embryonic mouse cells in vitro.
The chromosomes usually
showed some degradation but were easily recognizable in
the metaphase configurations.
While these studies did not indicate actual molecular
sizes of the molecules incorporated, they showed that the
molecules were large and were not totally destroyed and
reutilized by the cells.
Halpern, ei; al_. (1968) found
that non-viral tumor growth was inhibited by DNA extracted
from homologous tissues.
Maximal inhibition was achieved
with molecules of the sizes found in DEAE-cellulose (Diethylaminoethylcellulose) centrifuge chromotography Frac­
tions III and VI.
The molecular weights of these fractions
were 5 X lcA and 5 X 10^ respectively (Ledoux,. 1965)/. They
also found that D N ’ase I treatment of Fraction VI enhanced
its ability to inhibit tumor growth.
Digestion of Fraction
VI with D N ’ase I shifted the majority of remaining mole­
cules to Fraction III.
DN'ase I treatment of Fraction III
resulted in the loss of its inhibitory effect.
The evi­
dence presented in these studies indicated that DNA
10
absorbed by cells was of a high molecular weight* at least
h
5 X 10 * was highly polymerized* and maintained its struc­
ture during the process of entering the cell.
If high molecular weight DNA is absorbed into a liv­
ing cell and specific phenotypic changes can be observed*
then a substantial quantity of DNA must be entering the
cell,
A number of investigators have made attempts to
measure the relative amount of exogenous DNA that can be
taken up by a cell in vitro.
Indications of the amount of
DNA incorporated by a cell varied from a high of 10$ of
the cell’s normal DNA compliment (Bendich* 1961 and
Borenfreund and Bendich* 1961) in HeLa cells to a low
of 2$ in L 1210 cells (Glick* 1967a).
These quantities
of DNA are large and should be sufficient to induce trans­
formation* as even 2$ of a mammalian cell’s genome would
contain many genes.
The exact method by which cells take in large macro­
molecules has been the subject of much speculation.
most logical method would be pinocytosis.
The
While actual
pinocytosis of DNA molecules has not been substantially
demonstrated* information concerning the physical and chem­
ical factors controlling the process has been obtained.
Bendich (1961) demonstrated that optimal uptake occurs
at 3 7 °C in mammalian cells.
Glick (1967a) also found that
the process was temperature dependent.
The data obtained
by Meizel and Kay (1965)* Meizel ('1 9 6 5 )* Glick (1967a)*
and Gibb and Kay (1968) indicated that uptake is energy-
11
dependent, optimally occurring at pH 7 «
The general energy
source for the reaction seemed to be glycolysis.
Meizel
and Kay (1965) a^d Warden and Thorne (1969) enhanced DNA
t
uptake by use of polycations, which increase pinocytosis.
Ryser (1967)* reporting on uptake of protein by tumor cells,
found that only polycations enhance protein incorporation
by pinocytosis.
Other environmental changes, such as an
increase in glucose concentration or a lowering of pH,
which stimulates pinocytosis in amoebae, result in cell
destruction and a high degree of albumin fixation.
These
accounts all point to the probability.of pinocytosis as
the mechanism of cellular uptake of DNA.
reported pinocytosis of DNA in the
cell growth.
Adams (19 64)
and S phases of HeLa
While it appears that pinocytosis is the
probable method of incorporation, further evidence is
needed to validate this theory.
Can a high molecular weight DNA fragment maintain
molecular integrity in a culture of growing cells?
it can, it may be absorbed by the cells.
If
After a large
molecule, such as DNA, is incorporated into a living cell,
*\
what happens to it?
It seems that the invasion of'the'
cell by a macromolecule would trigger the cell’s defense
mechanisms against the invader.
Possibly, this is what does happen to the DNA incor­
porated into the cells; at least one group of investigators
has apparently found this to be the case.
Bensch, et a l .
(1966) reacted gelatin coated DNA with colloidal gold.
12
Electron microscope studies were made of L cells exposed
to these particles.
The DNA-protein particles were quickly
phagocytized by the cells and lysosomal reaction with
phagocytic vacuoles was demonstrated cytochemically.
Dis­
persion of the bound colloidal gold as well as its clumping
in ’’empty" vacuoles was observed.
These results were in­
terpreted as demonstrating hydrolysis of the DNA, which
would prevent any transformation reactions of the types
previously described.
However, many investigators disagree with the results
obtained by Bensch.
Hill (1962) tested extracellular break­
down of DNA in culture by growing cells in a culture cham­
ber divided by a diffusable membrane. One side contained
•2
bone marrow cells and "H-labelled DNA from mouse lympho­
cytes and the other side contained only growing bone
marrow cells.
Label was found only in cells growing on
the DNA containing side.
Apparently there was not enough
extracellular breakdown to permit degraded DNA to cross
the membrane barrier and label the cells growing on the
•
other side.
*i
Kay (1961), Meizel and Kay (1965)^ Kay (1966), and
Gibb and Kay (1968), utilizing labelled adenine and thy­
midine in exogenous DNA, compared the specific activities
of the A/T ratio before and after cellular incorporation.
If the exogenous DNA was being broken down and reutilized,
labelled bases should be diluted and specific activity
ratios altered.
All studies showed that the specific
13
activity ratios remained fairly constant, which indicated
the molecular integrity of the incorporated DNA.
Gartler (i960) and Ayad (1968) utilized the differ­
ential CsCl centrifugation properties of labelled DNA
containing heavy molecules, 5BDU (5 -bromo-deoxyuridine)
and lUdR (5 -iodo-2 -deoxyuridine), to separate incorporated
DNA from the endogenous DNA of recipient cells.
In both
cases they obtained a banding pattern which was indicative
of intact incorporation of exogenous DNA.
Doerfler (1968)
showed that viral DNA was incorporated intact into the
cell's genome by locating labelled viral DNA in the DNA
fraction of baby hamster kidney cells.
Gurdon, et al. (1969) injected various types of
vertebrate DNA into the cytoplasm of anucleate eggs of
Xenopus laevis.
This DNA was allowed to replicate and
was extracted and characterized.
The newly replicated
DNA was double stranded and had the same characteristics
as the injected D N A s .
The specificity of the replication
was demonstrated by injecting ribosomal satelite DNA and
then hybridizing the newly replicated DNA to r-KNA.
Such
*i .
data conclusively show that exogenous DNA can be incor­
porated intact by a cell in culture and can utilize the
existing cellular mechanisms for its replication and tran­
scription .
If purified DNA can be incorporated by an intact
growing cell, replicate, produce specific polypeptides,
and be transmitted from one cell generation to the next,
14
it probably has some interrelationship with the cell’s
own genome.
There has been a considerable effort to deter­
mine the nature of this relationship and the action of
incorporated DNA on the cell's synthesizing mechanisms.
Direct visual evidence for interaction of exogenous
DNA with the existing genome was presented by Leuchtenberger, et. al. ('1 9 5 8 )., Frederic., et al. (1962), and LutwakMann, et al.
(1969).
All observed chromosomal modifications
in cells treated with DNA.
Generally, these modifications
assumed the appearance of chromosome damage resulting from
breaks, arm exchanges, additions and deletions.
These
experiments employed high concentrations of DNA, as high
as 0 .1$ in one case.
The chromosomal modifications may
be the results of non-genetic properties of the DNA solu­
tion.
Gartler (i960) and Ayad and Fox (1968) provided more
definitive evidence for interaction of absorbed DNA with
the existing cell genome.
In both experiments, donor DNAs
were tagged with heavy molecules.
CsCl centrifugation of
the treated cell extract yielded a band of intermediate
density.
This fraction, after DN'ase treatment or Boni­
fication, was resolved into two peaks corresponding to
the original DNA densities.
This type of separation indi­
cated incorporation of a piece of heavy DNA into the linear
configuration of the existing genome.
Roth, et al, (1968)
demonstrated an intimate relationship between absorbed
DNA and the normal cell compliment.
By measuring increases
15
in acroflavin-hydrochloride binding to nuclei of cells
treated with exogenous DNA, and following the increase
through subsequent generations, they determined that there
was a statistically valid increase in the cells’ nuclear
content.
These data do not necessarily imply that exo­
genous DNA is integrated into the existing genome, but they
do demonstrate incorporation into the nucleus, which would
place the new DNA in close proximity to the existing genome.
Djordjevic, et al. (1962) demonstrated replacement
of an ultraviolet damaged genome with exogenous, isologous
DNA.
Treatment with DNA reduced the cell death rate from
50$ to less than 10$.
Since ultraviolet treatment would
not have destroyed the entire genome, these results indi­
cate an interaction between the incorporated DNA and the
remaining undamaged genome.
Kohan, et al. (1968) found that exogenous nucleic
acids inhibited the production of a vaccina virus in in­
fected chorionic-allantoic membrane cells.
Hal p e m , et
al. (1966 and 1968) found that autogenous DNA inhibited
the transplantibility of non-viral mammalian tumors.
The
inhibitory effect of the exogenous DNA was species speci­
fic.
Only mouse DNA was effective,* rat and calf thymus
DNA did not inhibit transplantibility of the tumor.
Such
specificity implied an interaction between DNA entering
the cell and the endogenous D N A .
Glick, at a l . presented information which gives some
insight into the possible actions of exogenous DNA within
16
recipient cells.
Exogenous DNA has a cytotoxic effect on
L 1210 cells in a maintenance medium of phosphate-buffered
saline and glucose.
However, if cells were preincubated
in a growth medium and then exposed to exogenous DNA, there
was enhancement of cell viability (Glick, 1967b; Glick,
1970).
While degradation of exogenous DNA was non-specific,
the Inhibitory or cytotoxic effect was species specific
and source specific (must be from normal cells), but not
organ specific (Glick, 1967c; Glick and Salim, 1967b;
Glick, Salim, and Crystal, 1968).
Glick and Goldberg
(1965, 1966) found that DNA treatment without preincuba­
tion reduced the tumor producing ability of L1210 cells
injected into mice following DNA treatment.
Crystal and Glick (1968), using synchronized cells,
found that inhibition was not effective in the S or M
phases of the cell cycle.
Other studies showed that exo­
genous DNA is capable of stimulating DNA or RNA synthesis,
but it inhibits uptake of nucleic acid precursors (Glick
and Sahler, 1967; Mauer, et al., 1968).
This indicates
'
that incorporated DNA is capable of functioning as a part
*t
of the cell1s genome.
Metabolic inhibition studies (Glick and Sahler, 1967)
and temperature studies (Glick, 1967a) indicated that DNA
must penetrate the cell in order to effect inhibition.
Cell death may result from an KNA produced by the incor­
porated DNA.
Glick (1967b, 1 9 7 0 ) also suggested that en­
hancement of cell viability under different incubation
17
conditions may result from an interaction between incor­
porated DNA and a substance secreted by the cells.
Prom the evidence presented, it appears that eukary­
otic cells growing in tissue culture are capable of ab­
sorbing high molecular weight DNA without excessive degra­
dation of the molecules.
After incorporation, exogenous
DNA moves to the nucleus, where it is replicated and tran­
scribed.
While it is evident that there are physiological
interactions between incorporated and endogenous DNA, evi­
dence for linear.integration of exogenous DNA into the
existing genome is not conclusive.
Evidence for such
action can be obtained by recombination and genetic studies
of eukaryotic cells.
At present, such studies are best
achieved in intact animals.
II.
In-vivo studies
The In-vitro studies already discussed provided
information concerning uptake of DNA by eukaryotic cells.
However, occurrences in tissue culture cannot always be
applied to intact organisms.
In an animal or plant, macro­
molecules face a very effective defense system developed
over many years of evolution.
The methods of introducing
exogenous DNA into the intact organism are of great impor­
tance.
In addition, various stages in an organism’s life­
cycle offer different problems and opportunities for the
incorporation of exogenous DNA.
18
The ultimate goal of uptake studies is transforma­
tion of specific genes along directed lines.
Experiments
on intact organisms offer information on other aspects
of DNA uptake such as its fate, its destination within
the cell, and its replicative and synthetic activities.
The few successful attempts at in-vivo transformation
have permitted genetic investigations which yielded much
information on the relationship of the incorporated DNA
to the endogenous genome.
There are few feasible ways of introducing a macro­
molecule into a whole organism.
One method is to treat
an organism when it consists of one, or a fairly small
number of cells, such as a germinating plant seed or an
early embryo of an animal.
Under such conditions, the
organism may be exposed to exogenous DNA directly by soak­
ing; it may be injected; or it may be perfused.
If non-
embryonic stages of an organism are involved, DNA must
either be injected into or fed to the organism.
The prob­
ability of absorption of DNA through the "skin" of an in­
tact organism seems remote.
This is especially true of
»»,
mammals and insects, the two animal groups most commonly
used in such experiments.
If exogenous DNA is introduced by injection or feeding,
it must exist in a hostile environment for a period of time
before entering the cell.
Both the circulatory and diges­
tive systems possess enzymes which should actively degrade
DNA.
This is particularly true of the digestive system;
19
consequentI3' there have been very few attempts at trans­
formation by feeding of exogenous DNA.
Injection is the
most popular and perhaps most effective means of intro­
ducing DNA into an intact organism.
There are several methods which are employed in
injecting exogenous DNA into animals.
In vertebrates the
most widely used method is intravenous injection which
places exogenous DNA directly into the blood stream.
Here
it can be quickly distributed to the various organs which
can absorb and utilize it.
Intraperitoneal injection of
mammals has been attempted and is the method of choice
with insects because it places the DNA in the haemocoel,
where it can be distributed throughout the organism.
The
third possible method of injection, intramuscular, has not
been frequently used.
Studies by Ledoux, et a_l. showed that exogenous DNA
rapidly disappears from the blood stream of mice.
Within
1 hr after injection, the DNA disappeared from the blood
stream and was located primarily in organs of the genital'
tract, particularly in the ovaries and vaginae.
The DNA
found in the cells represented 4 - 6°/o of the cellular DNA
content (Ledoux, 1 9 6 5 ; Ledoux, at al., 19&7 )*
Injection
of pregnant mice with E. coll DNA showed incorporation of
the DNA in the growing embryos within twenty-four hours.
The DNA was not significantly degraded, having survived
transit in the blood stream as well as passage through
placental membranes (Ledoux and Charles, 1967).
20
Rusinova, et al. (1969) traced the fate of exogenous
DNA in rats after both intravenous and intraperitoneal
injection.
DNA rapidly left the blood stream and was
preferentially incorporated by the bone marrow.
DNA in­
jected abdominally was not incorporated effectively.
After
24 hi’ some DNA remained in the abdominal cavity and much
of it was degraded.
Vielkind and Vielkind (1971) observed
the fate of bacterial DNA following injection into yolk of
fish embryos.
It was their opinion that the bacterial
DNA was broken down and resynthesized into fish DNA.
DNA
injected into the yolk of a growing embryo was exposed
to digestive enzymes which caused degradation of the in­
jected DNA.
Apparently DNA injected directly into the
circulatory system is absorbed into the tissues before
circulating DN'ases can act on it.
However, abdominally
injected DNA encounters difficulty in entering the blood
stream and is subjected to degradation.
Direct exposure
of nucleic acids to digestive processes also result in their
degradation.
The study of DNA incorporation into intact organisms
has been facilitated by using embryonic and larval stages
of developing animals and plants.
Prom such studies we
have learned much concerning the fate of incorporated DNA,
its ultimate site of cellular incorporation, and its ability
to replicate and direct cellular synthesis.
Yamada and Nawa (1967, 3968,' 1969) studied the fate
of bacterial ^H-DNA incorporated into Ephestia eggs by
21
soaking the eggs in DNA containing solution.
They utilized
DEAE-cellulose centrifuge chromotography and CsCl density
gradient centrifugation to determine the degree of polymer­
ization and molecular weight of respective DNA fractions,
and to differentiate between bacterial and endogenous DNA.
They found that the amount of DNA incorporated was both time
and concentration dependent and that after ultracentrifuga­
tion the incorporated DNA banded in a different position
than endogenous DNA.
After 6 hr of incubation there was
a shift in molecular weight of the incorporated DNA.
In
samples incubated in the DNA solution for two and four
hour periods, the molecular weight of the incorporated
DNA was about 1 X 10^ - 5 X 10^. In 6 hr samples the size
4
had decreased to 5 X 10 . There was no label in the low
molecular weight fractions.
They also found that 15 hr
after treatment some bacterial DNA had been denatured.
Together, these data implied that the exogenous DNA was
rapidly incorporated in a high molecular weight state
without depolymerization.
Ledoux, et_ al. used similar techniques to elucidate
the fate of exogenous DNA in plant tissue (Ledoux and
Huart, 1968, 1969; Stroun,
et_
1967).
Utilizing simi­
lar centrifugation techniques, they found that germinating
barley roots and tomato shoots incorporated highly polymer­
ized DNA.
In addition they encountered an intermediate
band, which, denaturation and soni'cation studies showed,
apparently represented linear Incorporation of exogenous
22
DNA into the existing genome.
The model proposed closely
resembles the incorporation of exogenous DNA into a bac­
terial genome.
Gibson (1968) determined that Paramecium is capable
of ingesting pieces of DNA and incorporating them into the
macronucleus.
Competition studies showed that the DNA was
incorporated intact, rather than being broken down and
reincorporated.
This was further substantiated by centri­
fugation studies utilizing incorporation of heavy isotopes
into donor DNA.
However, he was unable to effect any
transformation of specific genes.
Desai (1970) injected 3H-DNA from E. coli into larvae,
of D. melanogaster and Chironomus thummi.
five hours,
After four to
label was found in the nuclei of the salivary
gland cells,
specifically over the band regions of polytene
Op
chromosomes. Hill (1961) observed incorporation of
P-DNA
into nuclei of bone marrow cells of irradiated mice fol­
lowing injection of homologous lymphocytes containing
32P-DNA.
The information presented thus far supports the idea
that an intact organism can incorporate large macromole­
cules, particularly DNA, into its cells.
However, no
information on the biological activity of these incorpo­
rated molecules has been presented.
Evidence for biological activity of incorporated DNA
comes from a few reports of successful, directed, genetic
alteration of intact tissues and animals.
Lemperle (1968)
23
achieved prolonged survival of skin allografts by incuba­
tion with recipient DNA or RNA prior to transplantation.
He suggested that absorbed recipient nucleic acids were
active in protein synthesis in the transplanted tissue
and thus made it more antigenically acceptable to the
recipient.
Graffi, et^ al. (1969) induced lymphoma or
leukemia in hamsters of a strain free from skin epitheli­
omas with DNA extracted from viral induced epitheliomas.
These data illustrated biological functions of incorpora­
ted DNA.
Successful transformation of easily visualized phe­
notypic traits has only recently been accomplished in
intact eukaryotic organisms.
Earlier reports employed
methods of introducing the exogenous DNA into the animal
which make the results questionable while other researchers
have obtained results indicating that DNA is mutagenic.
The original transformation of a higher animal was
carried out by Benoit, et al. (1957) •
By daily injection
of small amounts of DNA from Khaki-Campbell ducks into
young Peking ducks, they effected several phenotypic chan•»
ges.
However, subsequent attempts to duplicate this ex­
periment failed.
Subsequent attempts at ln-vlvo transformation of
eukaryotic animals have been generally unsuccessful.
In
addition, Drosophila experiments have indicated that DNA
is mutagenic in vivo.
Fahmy and tfahmy (1962) induced
"minute" mutations by injection of adult Drosophila males
with DNA.
They were not able to induce production of
recessive lethals by this treatment.
In 1965* they repor­
ted induction of point mutations at the garnet locus by
.DNA treatment.
These mutations occurred with about the
same frequency as those induced by X-irradiation.
Small
pieces of DNA were most effective and there appeared to
be a dose response.
Pluorsheim (1962a, 1.962b) observed a
cytotoxic effect of DNA on mice injected with homologous
cells previously treated with DNA to enhance their histo­
compatibility .
Martinovitch, et_ al. (1962) observed teratological
changes in offspring of chickens which, as embryos, had
been treated with exogenous DNA.
The results of the exper­
iment are questionable because the carrier solution, Tyrode
is capable of inducing this type of effect.
There was,
however, a dose response to the quantity of DNA in the
injections.
Mathew (1965) demonstrated induction of recessive
lethals by treatment with DNA.
However, the procedure con­
sisted of feeding calf thymus DNA to D. melanogaster larvae
This method of introducing exogenous DNA into an intact
organism is not the method of choice.
Exposure of high
molecular weight DNA to the conditions existing in
Drosophila culture almost certainly results in degradation
of the molecules.
Gershenzon (1966) reported on specific
DNA directed mutations achieved in' this manner.
Because
of almost certain degradation of the DNA, both studies are
probably reporting the genetic effects of DNA breakdown
products:
bases* nucleosides* nucleotides* ar.d oligonu­
cleotides .
Nawa reported DNA-mediated transformation of specific
phenotypic characteristics in two insects* Epliestia* a
moth* and Bombyx* the silk worm.
The early works of Nawa
and Yamada were carried out on Ephestla* involving treat­
ment of both eggs and larvae.
Nawa (19S 5 ) attempted to
transform scale color of the forewing of Ephestia.
A mu­
tant having scales lacking black pigment was used as a
recipient.
DNA was obtained from both wild type*
2<yfo
black scales, and mutant strain* 100fo black scales.
Larvae
were injected and* following metamorphosis* forewing scales
wer*e examined.
Very few transformed scales were found.
Nawa concluded that transformation was an individual scale
effect.
In 1966, Nawa and Yamada reported the change of an
eye color* mutant in Ephestia. They reported establishment
of breeding stocks of transformed animals as well as segre­
gation of the transformed genes and the biochemical nature
•\
of the trait.
The following discussion is a summary of
those findings (Nawa and Yamada* 1966* 1967» 1968a* and
1968b).
Red eye color is based on the lack of the enzyme
tryptophan pyrrolase and is a recessive trait.
Eggs and
larvae from homozygous recessive*'red-eyed* moths were
treated with DNA extracted from homozygous dominant* black-
26
eyed moths.
One black-eyed individual was obtained from
the treated generation.
The "non-transformed" individuals
were backcrossed to homozygous recessives and gave six
black-eyed offspring in the next generation and nine in
the subsequent generation.
These transformed individuals
were again backcrossed to achieve segregation.
Segrega­
tion of the transformed genes gave several abnormal patterns.
Some individuals appeared to be .heterozygotes as expected.
Others segregated as if they were homozygous for the gene;
this is abnormal in such a cross.
The mutant female found
in the treated generation also gave an odd type of segre­
gation.
These two cases are most easily interpreted on
the basis of the incorporated DNA existing as an episome
which might be lost in some germ cells.
Another individual
showed a segregation pattern indicative of mosaicism.
In
order to determine whether the incorporated DNA was being
read or whether a small mutation had occurred which would
allow activation of the tryptophan pyrrolase enzyme., Nawa
subjected a mixture of wild enzyme and enzyme extracted
•
from transformed animals to starch gel electrophoresis.
»\
Only one band was distinguishable, indicating that the
amino acid sequences of the two enzymes were very similar.
More recently, Nawa, et_ al. (1969* 1971) effected a DNA
mediated transformation in Bombyx.
The results obtained
were similar to those in Ephestia and illustrated aberrant
types of segregation.
These data'indicate that transfor­
mation does occur, but leave questions concerning the
27
actual relationship between incorporated DNA and the
existing genome.
Fox and Yoon described specific genetic effects of
DNA in .Drosophila..
They applied very specific genetic
tests, which can be carried out with this organism, to
elucidate the relationship of incorporated exogenous DNA
to the existing genome.
More recently, Fox, et_ al. pro­
posed a model to illustrate this relationship.
In I966, Fox and Yoon reported on specific changes
induced in Drosophila by DNA extracted from stocks carry­
ing alternate alleles to the treated stock.
They effected
changes from recessive to dominant and from dominant to
recessive by treating eggs of a particular stock with
the appropriate DNA.
No whole body transformations were
obtained; only mosaics were observed.
The authors postu­
lated that the changes might be due to a heritable insta­
bility in the chromosome.
Fox, et al. (1968). raised the question of whether or
not the locus specific effects of DNA in Drosophila involved
its integration into the linear order of the chromosome.
*\
Changes from dominant alleles to recessives indicated that
a replacement of the endogenous allele was necessary.
How­
ever, continued lack of whole body transformations does
not fit this theory.
In 1970, Fox, et_ al. began a series of papers which
appeared to answer these questions' (Fox and Yoon, 1970]
Fox, et a l ., 1970] Fox, et al., 1971).
They accomplished
transformation of a number of genes on both the X and
Third chromosomes and established fifty-one breeding stocks
of v+ (the normal allele of the vermillion locus) trans­
formed animals.
type.
The transformations were all of the mosaic
Even after a large number of generations, the traits
still appeared as mosaics.
Attempts to induce chromosome
integration, hence production of whole body transformations,
by X-irradiation were unsuccessful.
Mapping of incorporated genes by genetic crosses
showed that, in two of the three tested stocks, alteration
has occurred at the v+ locus.
In the third, it was pos­
sible that the phenotype produced involved a change in a
non-specific supressor mutation.
In addition, distribu­
tion of transformed traits followed distribution of the
target chi’omosome immediately after treatment.
no apparent delay in chromosomal attachment.
There was
These obser­
vations led Pox. to propose an "exosome" model of chromo­
somal relationship.
That is, the exogenous DNA attached
to the existing chromosome with specific gene for gene
pairing, but remained outside of the linear structure of
»»
the chromosome.
It was capable of replication along'with
the existing genes, but did not replace them.
It was
transmitted through the mitotic and melotic processes as
if part of the chromosome.
At transcription, only one of
the alleles on that chromosome was read and results in
mosaicism.
Loss of this exogenous' segment might occur,
as evidenced by the occasional appearance of a non-
29
transformed individual in the breeding stocks.
In general, P o x ’s theory seems to explain adequately
the results obtained in his laboratory and makes some of
N a w a ’s results understandable.
This is possible because
both are working with an eye color mutant that is depen­
dent on a particular enzyme which can produce the required
phenotypes without being produced by every cell in the
body.
From the data presented in' this discussion, it ap­
pears that transformation of eukaryotic cells and intact
organisms is possible.
Direct and indirect testing indi­
cated that DNA can enter a cell intact, even after dis­
tribution by the circulatory system.
Once inside the cell,
it is incorporated into the nucleus in close association
with the existing chromatin.
Genetic tests indicated that
incorporated DNA is associated with the chromosomes and
that it, in fact, attaches at the site containing its
allele.
The purpose of this study was to demonstrate directly
chromosomal incorporation of exogenous DNA.
A test system
utilizing short term organ culture of intact salivary
glands was employed.
Salivary glands from D. mojavensis
larvae were exposed to labelled D. melanogaster DNA.
The
amount of DNA incorporated into the cells was quantitated.
Autoradiographic and fluorescent techniques were used to
visualize the sites of incorporation.
30
Short terra organ culture
allows direct exposure of
the cells to exogenous DNA and minimizes extracellular
degradation of the molecules.
The cells employed remain
in a highly differentiated condition and retain their
histological integrity.
Salivary gland cells of Drosophila
larvae contain specialized interphase chromosomes.
These
large chromosomes exhibit a high degree of polyteny and
are easily visible with light microscopy.
Such tissues
provide an excellent system for demonstrating the chromo­
somal incorporation of exogenous DNA..
METHODS AMD MATERIALS
Animals
Breeding populations of two species of Drosophila,
Drosophila mojavensis and Drosophila melanogaster, were
maintained on standard corn meal - agar medium at room
temperature (22°C).
The adults of both strains were
transferred to new medium two or three times each week
and the larvae were fed daily on Fleischmann's Active
Dry Yeast.
The D. mojavensis adults were purchased from
Carolina Biological Supply Company, Burlington, North
Carolina, and a strain of Oregon-R D. melanogaster was
obtained from Dr. W. R. Lee's laboratory, Louisiana State
University, Baton Rouge, Louisiana.
D. melanogaster females were allowed to lay eggs
on c o m meal - agar medium for a period of 12 hr before
being removed.
Hatched larvae were maintained until the
majority reached the third instar stage of development.
At this time, the larvae were floated from the medium
with a 30$ sucrose solution, transferred to a large sep­
aratory funnel and washed with increasing dilutions of
the sucrose solution until they were free of the medium.
The larvae were washed into a Buchner Funnel and rinsed
several times with water.
The clean, living larvae were
weighed and frozen with liquid nitrogen.
31
32
DNA Extraction Procedures
Labelled and non-'labelled DNA was obtained by an
extraction procedure which has been developed in this
laboratory over a number of years and which produces an
excellent yield of deproteinized nucleic acids (French,
per. com.).
All procedu.res were carried out in glass
containers at 0° - 4°C.
Frozen Drosophila larvae were
ground to a fine powder in liquid nitrogen using a motar
and pestle.
The ground tissue was suspended in 0.7M NaCl
that contained 0.015M Na^ C^H^O^ and 0.001$ Heparin at
pH 7.4 (15ml/ 4g tissue).
The suspension was placed in
a glass .stoppered flask with an equal, volume of phenol,
saturated with the above saline solution.
The mixture
was shaken for 30min and centrifuged at 12,500 g (20min)
in a Servall RC 2 Refrigerated Centrifuge.
After removing
the saline supernatant, the interphase and phenol layers
were mixed with another equal volume of saline and centri­
fuged as before.
.The two nucleic acid containing saline
supernatants were pooled and were extracted twice with
equal volumes of saline saturated phenol.
Following this,
the saline layer was extracted twice with an equal volume
of chloroform-isoamyl alcohol, 24:1
(V/V).
The mixture
was then shaken for lOmin-and then centrifuged at 10,000 g
to separate the phases.
The nucleic acids were precipi­
tated by slow addition of two volumes of cold, -20°C,
absolute ethanol to the saline solution, 0°C.
The pre­
cipitate was allowed to form overnight at -20° C .
33
Precipitated nucleic acids were collected by centrifuga­
tion at 10,000 g for 10 rnin and dried by vacuum dessication prior to purification.
Purification
The dried nucleic acid precipitates were dissolved
in 0.1M NaCl buffered with 0.05M sodium phosphate at pH
6 .7 . The nucleic acids were loaded on a modified MAK
column, methylated albumin on Kieselguhr (Osawa, 1967),
and fractionated by stepwise elution at 38°C.
A MAK column was prepared by boiling Ig of cellulose
powder in 10ml of~buffered 0.1M N a C l .
After cooling, this
slurry was poured into a 15mm diam jacketed Glenco glass
chromatographic column.
This cellulose layer prevents
blockage of the fritted glass disc at the bottom of the
column.
Ten grams of washed Kieselguhr was boiled in
50ml of the buffered saline solution for 20min, cooled
to room temperature and mixed with 2.5ml of
albumin.
Vfo
methylated
This slurry was poured into the column without
disturbing the cellulose layer.
under 3 psi.
This MAK layer was packed
A layer of buffered saline was maintained
above the packed layer to prevent contact with the air.
The column was then topped with a layer composed of Ig
of Kieselguhr boiled in 10ml of the buffered 0.1M NaCl
solution and a filter paper disc.
Nucleic acid fractionation was accomplished by
loading the MAK column with approximately 7mg of nucleic
34
acids in 25ml of buffered 0.1M NaCl.
This volume was
passed onto the column at a flow rate of lml/min.
Following the loading of the column, it was washed
with 100ml of the 0.1M buffered saline at a flow rate of
3ml/min to remove nucleotides and other low molecular
weight materials.
washes of:
This wash was followed by 200ml aliquot
0.45M NaCl, O. 65M NaCl, and 1.2M NaCl, all
buffered to pH 6.7 with 0.05M sodium phosphate.
These
saline concentrations will elute the 4s and 5s RNA, the
DNA, and the l8s and 28s RNA respectively.
All elutions
from the column were collected in 5ml fractions using a
Canal Company automatic fraction collector equipped with
a drop counter.
The
ultraviolet absorption elution
profile was monitored by a Coleman Hitachi 101 Spectro­
photometer equipped with a continuous flow cell,X =
260nm.
The elution profile was continuously recorded
by a Beckman recorder.
All DNA used in this study was
prepared in this manner.
%-LabeIling of DNA
Tritium labelling of the DNA was accomplished by
modification of the method described by Hitossa and
Spiegelman (1965).
D. melanogaster eggs were collected
after l4hr of egg deposition on corn meal - agar medium
plated in petri dishes.
paper and rinsed with
The eggs were washed onto filter
’’
(Ofi
ethanol.
The filter paper was
cut into small pieces and placed in culture vials.
Each
vial contained:
Ig corn meal, lg sucrose, lg bakers
yeast, O.lg Bacteria grade U.S.P. agar, dissolved in
10ml HgO and boiled for 45min., Following boiling, the
3
vials were inoculated with Imc of H-Thymidine in 0,2ml
sterile II^O (Miles Laboratories).
The larvae were main­
tained in these vials until they reached third instar,
then floated with
30f
sucrose, and frozen for DNA extrac­
tion.
This labelling procedure yielded 9-2g of larvae
3
which had incorporated H-Thymidine into their D N A .
The DNA from these larvae was extracted by the
procedure described above and purified by MAK fractiona­
tion.
Following MAK fractionation, the DNA peak was sub­
jected to RNAase digestion, 4hr at 37°C.
The RNAase was
treated at 100°C for 10 min at pH 3 in order to destroy
other enzymes and was added to give a final concentration
of 150^g/ml.
solution
if
The enzyme was denatured by making' the
with sodium dodecyl sulfate (SDS) and shaking
for lOmin at 45°C.
The denatured proteins were removed
by two successive extractions with water saturated phenol'
followed by two extractions with chloroform-isoamyl alco­
hol.
DNA was then precipitated by slow addition of two
volumes of absolute ethanol (-20°C).
The precipitate was
allowed to form overnight in a freezer (-20°C) and was
collected by centrifugation.
Following vacuum desiccation
at -20°C, it was dissolved in sterile Hank's balanced salt
solution (Harnden, 1965), pH 6 .9 , 'to give a final concen­
tration of 60/jg/ml.
The specific activity of the DNA was
36
r/lcpm^g.
Parity of the preparation was determined by
spectral analysis using a Beckman DBG grating spectropho­
tometer with
X
varying from 300nm to 220nm.
Chase DNA
Commercial salmon sperm DNA was MAK fractionated,
precipitated, and dissolved in Hank's solution at a final
concentration of Img/rnl.
Culture Techniques
Late third instar larvae of D. mojavensis were
selected from actively growing stocks.
Each larva was
washed in three changes of 70%> ethanol, followed by two
changes of sterile Hank's solution.
Salivary glands were
pulled into a drop of saline and stripped of excess tissue.
The glands were rinsed twice in fresh saline solution
and transferred by pipette to sterile 0.5ml depression
slides containing 0.4ml of tagged DNA solution and covered
with a siliconized coverslip.
After 45 min of incubation,
0.1ml of Hank's with chase DNA was added.
After 4hrs expo­
sure to exogenous DNA, the glands were removed and rinsed
»;
in two changes of Hank's solution, and either homogenized
for scintillation counting or prepared for microscopic
observation.
Quantitative Techniques
An estimation of the amount of DNA incorporated by
the living glands was accomplished by homogenizing ten
pairs or glands in 0.5ml of cold 5$ TCA (trichloroacetic
acid) .
This homogenate was mlllipore filtered (pore size
The filter was washed with two volumes of cold
TCA and digested in 0.2ml perchloric acid / HgOg.
The
filter digest and 0.2ml of filtrate were placed in sepa­
rate scintillation cocktails containing 4ml of 2-ethoxyethanol, and 10ml of fluor (lOg PDB in 1L toluene).
The
cocktails were counted in a Beckman liquid scintillation
counter.
Vfo
Each sample was counted for 50min or until a
error was rea.ched.
The medium was removed from t h e .culture chambers
and filtered in the manner previously described.
The
filter was washed with two volumes of cold TCA.
Scin­
tillation cocktails were prepared as described above.
Controls were prepared by holding 0 Jbml of the ^H-DNA
solution with 0.1ml of salmon sperm DNA solution' in cul­
ture chambers without salivary glands.
The controls were
treated in the same manner as the medium from the experi­
mental cultures.
The DNA synthesizing ability of the salivary glands it.was tested by preparing a culture medium containing free
thymidine in the same quantity (71.9/<g/ml) and with the
same specific activity (ll^cpm^g) as would have existed
in the culture medium if the DNA was completely degraded.
%-Thymidine (Miles Laboratories) was purified by ascending
thin layer chromatography (Randerath and Randerath, 1967),
using methanol:
IICl: IigO (7 0 : 20: 10) as a solvent.
The
38
-Thymidine spots were eluted onto acid washed Whatman
chromatographic paper wicks with 0.1N HC1.
Dried wicks
were cut into small pieces and eluted overnight with
H a n k ’s.
The solution was centrifuged at 5*000 g for lOmin
for separation of paper debris.
The supernatant solution
was removed and the specific activity was determined.
Non­
labelled thymidine (Car. Bio. Chem.) was used to prepare
a solution of H a n k ’s containing 71.3/tg of thymidine/ml.
The final concentration of the thymidine was determined
spectrophotometrically.
An aliquot of the ^H-Thymidine
was added to this solution in order to achieve a specific
activity of ll4cpm^ug.
Glands were maintained for 4hrs
in this medium.
Autoradiographic Techniques
Salivary glands from third instar larvae were main­
tained in
H-DNA containing cultures.
After 4 hrs the
’
*
,
glands were removed and washed in H a n k ’s solution. Stan­
.
dard chromosome squashes were made in cold 45^ acetic acid
buffered to pH 2.2 with sodium acetate.
The coverslips
were removed by freezing in liquid nitrogen.
The slides
were washed in three changes of cold TCA, air dried, and
covered
with Kodak AR-10 stripping film (Schmid, 1965).
The slides were air dried overnight, placed in bakalite
slide boxes containing a silica gel dessicant and triple
wrapped in
aluminum foil and stored at 4°C.
times were set at four and six weeks.
Exposure
Autoradiographic
controls were prepared by maintaining the glands in nonDNA containing medium.
After exposure, the slides were removed from the
storage boxes, and the backs were painted with clear
fingernail polish to secure the autoradiographic film to
the slides.
The autoradiographs were developed in Kodak
D-19b, 6min at l8°C; washed in water, 30sec; fixed in
Kodak Acid Fixer, lOmin; washed in Best Products indus­
tries #30 Hypo Eliminator, 2min; washed in running water,
lOmin; and air dried.
The slides were stained with dilute
buffered Giemsa, ^-min; dipped twice in distilled water;
and air dried (Schmid, 1965).
Following clearing in two
changes of xylene, lmin each, the preparations were moun­
ted with Permount and a #1 coverslip.
"'The preparations were observed with a Leitz Ortholux.
microscope using bright field optics at approximately
lOOOx magnification and the silver grains over the chro­
mosomes were counted.
Background levels were determined
by counting an area immediately adjacent to the chromo­
somes.
Background grains were counted if they occurred
• 1.
within two chromosome widths on either side of the chro­
mosome .
The total count was divided by four* in order to
arrive at a figure that represented background in an area
approximating that occupied by the chromosome.
The level
of chromosomal label was expressed as the ratio of grains
over the chromosomes to the corresponding background label.
The data were analyzed using an analysis of variance test •■■■>
40
with an orthogonal comparison of the partitioned sums of
the squares in order to compare the difference between
groups.
Photographs of the spreads were taken through the
Ortholux microscope using a Leica 35mm camera and Kodak
Panatomic-X film.
The film was developed in a 1:3 dilu­
tion of Kodak Microdal-X, and prints were made on Kodak
Kodobromide F-5 single weight print paper.
Acrldine Orange Tagged DNA
Non-radioactive DNA was tagged with acridine orange.
Following MAK fractionation of nucleic acids obtained from
D. melanogaster third instar larvae, the DNA peak was
purified enzymatically with RNAase, as previously described.
Following vacuum dessication (-20°C), the DNA was dissolved
in Hank's solution to give a final concentration of 150/^g/
ml.
Tagging was accomplished by adding a very small amount
of ether purified acridine orange to the solution.
Care
was taken not to add excessive dye, for this will precipi­
tate the DNA.
The dye-nucleic acid complex w a s 'separated
from the unbound dye by gel-filtration with Sephadex G-25.
H a n k ’s solution was used as an elutant.
The presence of
the dye-DNA complex in a particular fraction was detected
by short wave ultraviolet illumination.
Acridine Orange - DNA Cultures
Salivary glands were incubated in H a n k ’s containing
the acridine orange-DNA complex.
After 45min, 0.1ml of
4i
salmon sperm DNA solution containing lOO/vg of DNA was
added.
Controls were established by growing glands in
acridine orange in H a n k ’s without DNA.
Purposely injured
glands were maintained in both media containing the acri­
dine ‘orange -DNA complex and acridine orange without DNA.
The glands were maintained in culture for 4hrs and then
spread in cold 45/£ acetic acid buffered to pH 2.2 with
sodium acetate.
The coverslips were removed and the
slides were immersed in buffered 0.1M NaCl containing
20^ glycerol.
All slides were prepared as wet-mounts
and were observed through the Ortholux microscope equipped
with a blue light fluorescence attachment, X = 400nm.
Fluorescing chromosomes were photographed with dark-field
optics, using Kodak High Speed Ektaohrome Type B film.
The photographic slides were copied on Kodak Ektacolor
Professional Film with a Nikon FTN 35*nm camera and slide
copier, and prints were made from these negatives by the
Kodak Company.
RESULTS
Extraction, Purification, and
3
I-I - Labelling
The extraction of nucleic acids from the 9.2g of
larvae obtained from the cultures containing ^j-I-Thymidine
yielded 10ml of solution containing 3mg of nucleic acid
per ml.
MAK fractionation of the nucleic acids showed
that the “\H-label was highly concentrated in the DNA
peak, although there were some traces of label in the
two'ENA peaks.
Separation of the respective nucleic acid
fractions was complete, and there was no evidence of
radioactive material in the intermediate samples.
The
results of the extraction, purification, and ^H-labelling
procedures
are
presented in Figure 1.
Spectral analysis
of enzymatically purified DNA showed a peak absorbance
at 260nm and 280nm/260nm, 230nm/260nm ratios of 0.48,
0.42 respectively.
was 171cpm^g.
Final specific activity of the DNA
This DNA solution was then used as a
culture medium.
Quantitative Studies on DNA Uptake
The data presented were obtained from three repli­
cates involving ten cultures each and an equal number of
controls.
The TCA fractionation of the culture medium
3
showed a much greater proportion of I-I-labelled material
in the TCA soluble (low molecular weight) fraction (Figure
42
2) over that found in the controls.
in the soluble,
3
H-labelled material:
There was an increase
insoluble,
labelled material ratio from 1.3:1 to 6:1.
3
H-
These data
represent the means of the values obtained with three
replicates.
In the TCA fractionation of the gland homogenates
the insoluble fraction of two of the replicates was as
large or larger than the soluble fraction.
In the third,
the reverse was true; the soluble fraction was much larger
than the insoluble fraction (Figure 3)j moreover the
insoluble fraction was smaller than in the other two
experiments.
These data indicate an average uptake of
0.0096/Jg of exogenous DNA per pair of salivary glands,
)
equivalent to approximately 12yo of their DNA content.
,
Glands grown in cultures containing free
H-Thymi-
dine, in the same concentration (71.9yug/ml) and with the
same specific activity (ll4cpm/^ug) as would be present
with total degradation of the exogenous DNA, did not sur­
vive.
The longest survival period under these conditions
was about 1 hr.
These data indicate that free thymidine
at this concentration is toxic to the salivary glands
under these controlled experimental conditions.
Autoradiopjraphic Studies
Observations of the autoradiographs showed that the
silver grains were primarily located over the chromosomes
and nucleoli.
The pattern of labelling was generally
44
uniform (Figure 4) although a few of the spreads showed
some localized labelling (Figure 5)•
Label was found
over a number of cells on each s l i d e .
Statistical analysis of the level of labelling over
the chromosomes in these experiments showed that there
was a highly significant difference between the treatments
(Tables 1 and 2).
An orthogpnal comparison of the two
experimental treatments with the controls showed that
there was a highly significant difference between the
amount of chromosomal labelling found in the controls and
the cells exposed to exogenous ^II-DNA.
a
similar compari­
son of autoradiographs exposed to 3H-labelled chromosomes
for four weeks and those exposed for six weeks showed no
statistically significant difference between these two
treatments (Table 2).
Acrldine Orange - DNA Complex. Studies
Living salivary glands treated' with the acridine
orange - DNA complex for four hours showed intensive
chromosomal fluorescence (Figures 6, 7 j and 8).
Injured
glands maintained in this culture medium did not show any
fluorescence.
Both types of glands exhibited heavy
staining of both nuclear and background material when
maintained in free acridine orange without DNA (Figure 9)•
Figure 1.
The ultraviolet., 260nm absorption profile
of and ^H-lahel distribution in D.
melanogaster nucleic acid fractions
eluted from a MAK column by stepwise
elution
__________
UV profile
distribution
175
2 .7 5
150
2+
2 .2 5
OD
2.00
125
1.75
O D
100
260m M
1.50
CPM
1.25
[75
1.00
4&5s
DNA
RN A
50
.75
.50
.25
20
30
40
50
Fraction #
FIG.l
60
70
80
Figure
2.
A comparison of the TCA soluble and
insoluble fractions of
H-DNA in H a n k ’s
solution:
A.
Culture medium after four hours
exposure to living salivary glands
B.
Controls
Sol-
Insol-
Sol.
A
Insol.
B
FIG.2
Figure 3.
Right scale:
A comparison of the propor­
tion of the total DNA content of the cell
that was incorporated during the exposure
to the
radioactive exogenous DNA.
The
amount of DNA represented is based on a
specific activity of lTlc p m ^ g as deter­
mined for the intact exogenous DNA.
Left scale:
A comparison of the
3
H-
labelled TCA soluble and insoluble frac­
tions of the gland homogenates.
The values
reported are the specific activity (cpm/
100X) of the filtrate and the millipore
filter digest.
%Cell
DNA
r *................ .
FIG . 3
Figure 4.
Autoradiograph of chromosomes grown in the
presence of
3
H-labelled DNA for four hours
Exposure time was four weeks.
Note relatively uniform label distribution
Magnification:
Figure 5-
1700x.
Autoradiograph of chromosomes grown in the
3
presence of H-labe'iled DNA for four hours
Exposure time was four weeks.
Note generally localized label.
Magnification:
1700x.
Table 1.
The treatment means are presented with their
standard error.
Treatment 1 is from the
control slides; six weeks exposure.
Treat­
ment 2 is from autoradiographs exposed for
four weeks.
Treatment 3 is from autoradio­
graphs exposed for six weeks.
Table 2.
An f test analysis of variance of the treat­
ments.
An orthogonal comparison of the
control vs. the experimental treatments
and an orthogonal comparison of the two
experimental treatments.
** significant at the 0.01 level
+ not significant at the 0.05 level
Samp1e
20
Sample Size
Mean
Treatment 2.
Treatment 1
20
1-5b ± .097
Treatment 3
20
b. 15 + .193
3.9b + .Ib6
Table 1
d.f
Total
Treatment
110.78
3
8 b .10
1
Exp.j vs. Exp . 2
1
57
f
MS
59
Con. vs. Exp.
Error
SS
83.6 3
b2.05
Table 2
7.09
83.63
59.56**
10. 1
.b6
•329+
10. 1
M
26.68
29 .95 **
f.01
1
.bob
Figures 6 & 7.
Chromosome spreads from two slides
showing fluorescence of the incor­
porated acridine orange - DNA complex.
Note yellow-green color.
Photographed with dark-field optics.
Magnification:
400x
Figure 8 . . Chromosomes showing fluorescence of the
incorporated acridine orange - DNA complex
Note yellow-green color.
Photographed with dark-field optics.
Magnification:
Figure 9.
400x
Acridine orange staining of injured sali­
vary gland cell.
Note yellow color and
lack of chromosome structure.
Photographed with dark-field' optics.
Magnification:
400x
DISCUSSION
With the possibility of genetic engineering becoming
a reality, scientists have begun a search for possible
techniques for the introduction of foreign DNA into the
genome of eukaryotic cells.
promising technique.
DNA uptake appears to be a
Ledoux (1965) points out that exoge­
nous DNA apparently can be absorbed by living cells without
serious degradation of the macromolecules.
Genetic and
molecular studies imply that exogenous DNA is incorporated
into the host cell's chromosomes (Fox, et al,, 1970 ;
Ledoux, 1968, 1969; Nawa, 1966, 1967, 1968a, 1968b, 1969,
1971; Yarnada, 1967* 1968, 1969)•
This study provides
direct evidence for the Incorporation of exogenous DNA
within the band regions of polytene chromosomes.
Short term organ culture provided a technique which
assured intimate contact of the cells with the exogenous
DNA, while maintaining cell-to-cell contact as well as
tissue and organ structure.
Cells in-vitro often revert
to a less specialized state of development and frequently
undergo changes in ploidy.
Cytological observation of
the cells employed in these tests revealed no changes in
chromosome structure or arrangement.
Aseptic culture
conditions minimized extracellular degradation.
The chromosomal structure and arrangement in
Drosophila salivary glands strengthens this system.
59
The
6o
glands themselves are in prolonged interphase and no cell
division occurs after differentiation.
DNA synthesis is
active until about the middle of third instar development,
when it begins to decrease.
Chromosomes exhibit a high
degree of polyteny and are easily visible without induction
of metaphase configurations.
Such polyteny also provides
an increased number of binding sites for exogenous DNA,
making visualization by autoradiography possible.
In
addition, heterochromatin which might interfere with
binding is localized in a chromocenter.
Desai (1970) reported observation of autoradiographic
label in the band regions of dipteran ploytene chromosomes
following injection of larvae with labelled bacterial DNA.
However, he did not present photographic evidence of his
observations.
The data presented in this study confirm
chromosomal incorporation and provide clear visual evi­
dence of such interaction between the host cell's genome
and incorporated DNA.
Maximal DNA uptake occurs if the DNA is highly
polymerized and is not denatured.
The presence of pro­
tein on DNA seems to enhance cellular degradation of absorbed molecules.
The elution profile of the
H-DNA em­
ployed in this system illustrates a clean separation of
the nucleic acid fractions and isolation of the radioac­
tive label within the DNA peak (Figure l ) .
Such a profile
indicated the highly polymerized and native condition of
3
the DNA.
Isolation of the H label indicated association
61
of the label within the macromolecular structure of this
fraction.
Low W
absorption at 230nm and 280nm demon­
strated the absence of aromatic amino acids from the pre­
paration.
These data indicated the qualitative nature of
the DNA preparation employed.
3
Salivary glands exposed to this
PI-DNA for 4 hr in
organ culture partially degraded the DNA (Figure 2).
The
TCA soluble and insoluble fractions contained less label
after exposure to the salivary glands.
The ratio of the
TCA soluble fraction to the TCA insoluble fraction was
altered from that observed in the controls.
Some of the
change resulted from absorbance of high molecular weight
DNA by the glands.
However, the increase in the relative
amount of TCA soluble material was large enough to indicate
some degradation of the macromolecules.
These results,
.that some of the exogenous DNA is degraded, were in agree­
ment with the finding of previous investigators (Ledoux,
1965).
The amount of exogenous DNA absorbed by the cells
3
of the salivary glands exposed to PI-DNA was relatively
high.
As stated previously, the amount of exogenous DNA
incorporated by a living cell may vary greatly (Ledoux,
1965).
The average amount of labelled, exogenous DNA
absorbed by the salivary gland cells in this study was
0.0096/<g per pair of salivary glands.
Since the average
DNA content of a pair of salivary glands is 0.08/Ug of DNA
(Patterson, 1952), the additional DNA is equivalent to 12$
62
of the cells' total DNA content.
This value is higher
than those found in the literature.
The question arises as to whether this incorporated
label represents exogenous DNA in its native molecular
configuration or the incorporation of labelled DNA degra­
dation products into newly synthesized endogenous DNA.
The latter case seems highly unlikely for several reasons.
As previously stated, third instar larval salivary glands
have reduced DNA synthesis and increased protein synthesis
with increasing age.
Ploytene chromosomes of very late
third instar larval salivary glands show visible signs of
deterioration.
The larvae used in this study were as old
as possible, without having begun prepupal changes.
If
the entire quantity of DNA in the cultures was completely
degraded to form a free nucleotide pool for DNA synthesis,
the count/min of the TCA insoluble fraction from' the gland
homogenates would represent 0 .012/ug of incorporated thymi­
dine .
Since doubling time for the DNA in Drosophila
polytene chromosomes ranges from 8 to 14 hr (DuPraw, 1970 }'»
only
of a cell's total DNA content could be synthesized
25?o
3
during the 4 hr exposure to
prises
1965).
29f 0
H-Thymidine.
Thymidine com­
of the bases found in Drosophila DNA (Hastings,
Under these conditions, 0.005/<g of thymidine could
be Incorporated into cellular DNA.
one half of that observed.
in Figure
2
This value is less than
In addition, the data presented
showed that not all the DNA in the cultures
was degraded.
Further evidence for the lack of detectable
DNA synthesis can he seen in Figure 3. Replicate 3* which
contained cells having the greatest amount of TCA soluble
material, had the lowest amount of TCA insoluble material.
This indicated very little incorporation of low molecular
weight material into glandular DNA, which would be detec­
table in the insoluble fraction if exogenous DNA degrada­
tion products were being reuti'lized.
Additional evidence that synthesis is not responsible
for the uptake of label comes from cultures grown in a
medium containing free thymidine in the same concentra­
tions as would exist with complete degradation of the
exogenous DNA.
Partial degradation would not supply suf­
ficient label to achieve even one half of the observed
labelling.
The glands in these cultures all died within
the first hour.
Apparently thymidine at these concentra­
tions was toxic to Drosophila cells.
These results show that exogenous DNA was taken up
by the salivary gland cells and was recovered in the TCA
insoluble, high molecular weight, fraction of the cellular
homogenate. The site of incorporation was not clarified
by these resultsj however, autoradiographs of polytene
chromosome spreads prepared from salivary glands grown in
this same culture medium gave evidence of the sites of
incorporation.
The majority of silver grains were located
over the chromosomes and most of the spreads showed the
label to be distributed over the entire chromosome (Figure
4).
None of the spreads showed high silver grain
64
-
concentrations in specific regions.
tions would be ex.pected if the
via DNA synthesis.
Localized concentra-
H were being incorporated
Replication in polytene chromosomes
begins at several specific sites along each chromosome arm
and proceeds from these points.
Tritium labelling would
be concentrated in the areas undergoing replication at the
time of exposure.
This would result in clumping of the
silver grains over specific areas* which was not observed.
Occasionally areas of higher concentration were found
but even in these cases the label was still distributed
over the spread (Figure 5)•
The highly significant degree
of labelling over the treated chromosomes must be attri­
buted to the presence of radioactive molecules incorporated
into their structure.
The degree of labelling achieved
could not result from DNA -synthesis and therefore must be
attributed to the chromosomal incorporation of exogenous
3H-DNA.
Tagging of DNA with a fluorescent molecule* acridine
orange* provided a rapid and sensitive method of visual!-'
zing the chromosomal incorporation of exogenous DNA.
The
chromosome spreads prepared from salivary glands grown in
the medium containing the acridine orange - DNA complex
showed a characteristic yellow-green fluorescence in short
wave blue light (400nm) (Figures 6* 7* 8).
The fluorescent
color indicates that acridine orange is bound to DNA.
De­
spite the specificity of this color reaction* the unlikely
possibility exists that the dye-DNA complex underwent
dissociation with acridine orange, alone, entering the
cells.
Previously reported evidence on the stability of
DNA in cultures (Ledoux., 1965) makes it doubtful that
enough fluorescent ds^e could be liberated during the pre­
scribed incubation period to produce the intensity of
staining demonstrated (Figures 6, 7 > 8)•
In addition, it was found that injured cells did not
show chromosomal fluorescence after incubation with the
dye-DNA complex.
However, both normal and injured cells
incubated with free acridine orange exhibited intense
fluorescence of both nuclear and background material
(Figure 9 )•
The injured cells in such preparations showed .
extensive deterioration and lack of chromosomal structure.
The normal cells showed very compact nuclei, indicative cf
cell damage.
In contrast to these effects, the acridine
orange-DNA complex entered only living cells and'did not
result in damage.
This suggests that the lack of fluo­
rescence in injured cells incubated with the dye-DNA com­
plex was not due to a change in membrane permiability to
the free dye, but that the acridine orange-DNA complex
was stable under the culture conditions used in this study.
The complex was capable of entering living cells and binding
to the host cells’ chromosomes.
This study demonstrates that under controlled condi­
tions exogenous DNA is absorbed by living cells and binds
to the host cell’s chromosomes.
Whether incorporated DNA
was linearly integrated into the chromosome or existed as
an "exosome" configuration as described by Fox (Fox, et a l ♦
1970)* was not determined.
Most genetic evidence points
to at least a temporary "exosome" arrangement as evidenced
by the aberrent segregation patterns observed by Nawa
(1968).
Since polytene chromosomes are composed of many
copies of the genome arranged in a parallel configuration,
the chromosomal incorporation of relatively large amounts
of exogenous DNA, as reported in this study, would not
necessitate genomic replacement.
This arrangement would
be compatible with the "exosome" model of chromosomal
incorporation.
However, if whole body transformations are
to be accomplished, linear integration of the exogenous
DNA into the host's genome seems to be necessary.
There
is a possibility of transcription of both endogenous and
incorporated DNA, but evidence presented thus far makes
this seem unlikely.
While all the facts about the incor­
poration of exogenous DNA by eukaryotic cells are yet to
be clarified, it is apparent that such a phenomenon is a
reality.
The chromosomal incorporation of exogenous DNA,
demonstrated in this study, enhances the possibility of
practical applications of this phenomenon.
The addition
of selected genes to an existing genome offers many oppor­
tunities for alleviation of genetic disorders.
Repression
of cancer by treatment with exogenous DNA has already
become an area of very active investigation.
The possi­
bility of using DNA uptake as a method for in-vivo nucleic
acid hybridization is under investigation.
Investigation
on alteration of genetic metabolic disorders, such as
sickle cell anemia, by DNA treatment are becoming more
common.
While caution should be taken not to think of
this phenomenon as a cure-all for every genetic disorder
troubling mankind, it appears that DNA uptake may indeed
provide the answer to some of these problems.
SUMMARY
A comprehensive review of the literature on the
uptake of DNA by eukaryotic cells was presented.
Prom the
evidence presented in previous studies, it can be concluded
that living cells are capable of absorbing exogenous DNA
intact and that this DNA can function in the transcription
of genetic information.
Both in-vitro and in-vivo studies
have yielded information on the manner of DNA uptake, the
size of the molecules incorporated, the amount of DNA ab­
sorbed by a cell, the fate of the absorbed DNA, and its
eventual biological activity within the cell.
Recent successful transformations of intact animals
have enabled investigators to suggest that the incorporated
molecules are bound to the host cell's chromosomes rather
than existing as a free episome.
This study provides direct evidence for the chromo­
somal incorporation of exogenous DNA by salivary glands
maintained in short term organ culture.
Quantitative
studies provide evidence that the amount of DNA incor­
porated into the cells could not have occurred through
synthesis.
The quantity of DNA taken up by the cells
was equivalent to about
12%
of their genome.
graphs showed a significant incorporation of
the chromosomes.
Autoradio-
3
H-DNA into
The labelled DNA seemed to be distri­
buted over a cell's entire chromosomal compliment.
68
Further
evidence of chromosomal incorporation was obtained by
using DNA tagged with acridine orange.
Fluorescent micro
copy demonstrated the presence of the acridine orange-DNA
complex in the chromosomes after four hours of culture.
These data provide excellent direct evidence for the
chromosomal incorporation of absorbed exogenous DNA.
This system seems to offer a possible method of
altering many of the genetically mediated malfunctions
of living organisms.
LITERATURE CITED
Adams, J., YJ. Martin, and C. Pomerat (1964). Incorporation
of DNA by HeLa cells during various phases of the
cell cycle. J. Cell B i o l . 23: 3A.
Alloway, J. L. (1933)* Further observations on the use
of pneumococcus extracts in affecting transforma­
tion of type in vitro. J. Expt 1 . M e d ...57: 265 .
Avery, 0. T., C. M. MacLeod, and M. McCarty (1944).
Studies on the chemical nature of the substance
inducing transformation of pneumococcal types. I.
Induction of transformation by a deoxyribonucleic
acid fraction isolated from pneumococcus type III.
J. Ex.ptl. Med. 79*. 137Ayad, S. R. and M. Fox (1968). DNA uptake by a mutant
strain of Lymphoma cells. Nature. 220:35
Bendich, A. (1961). Studies on the biological activity
of the deox.yribnucleic acids. Diseases Nervous
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Bendich, A.. E. Borenfreund, Y. Honda, and M. Steinglass
(1969). Cell transformation and the genesis of
cancer. Arch. Environ. Health. 19:'157*
Benoit, J. P. Leroy, R. Vendrely, and R. Vendrely (1957)•
Des mutations somatiques dirige'es sont-elles
possibles chez les oiseaux? Compt.Rend. Acad.
Sci. 244: 2321.
Bensch, K., G. Gordon, and L. Miller (1966). Electron
microscopic and cytochemical studies on DNA-containing
particles phagocytized by mammalian cells. Trans.
N.Y. Acad. S c i . 28: 715 .
Borenfreund, E. and A. Bendich (1961). A study of the
penetration of mammalian cells by desoxyribonucleic
acids. J. Blophys. Biochem. Cytol. 9: 81.
Brackett, B. G., W. Baranska, V/. Sawlcki, and H. Kaprowski
(1971). Uptake of heterologous genome by mammalian
spermatozoa and its transfer to ova through fertili­
zation.
P.N.A.S. U.S. 68: 353.
70
71
Crystal^, H. A. and J. L. Glick (1968). Influence of par­
tial synchronization of cell growth on inhibition
of L1210 cell viability by mouse thymus DNA.
Experientla. 24: 380,
Dawson, M. H. and R. H. P. Sia (1931)i A technique for
inducing transformation of pneumococcal types in
vitro. J. Exptl. Med. 54: 681.
Desai, L. S. (1970). Incorporation of macromolecules
into the salivary gland cells of Dipteran larvae.
Experientla. 15: 27.
Djordjevic, 0., L. Kostic, and D. Kanazir (1962). Recovery
of ultraviolet-irradiated L strain cells by means
of highly polymerized deoxyribonucleic acid. Nature.
195: 6l4.
Doerfler, W. (1968). The fate of the DNA .of adenovirus
type 12 in baby hamster kidney cells. P.N.A.S. U.S.
60 : 636 .
DuPraw, E. J. (1970). DNA and Chromosomes. New York:
Holt, Rinehart, and Winston, Inc., p. 126.
Fahmy, 0. G. and M. J. Fahmy (1962). Mutagenic activity
of cellular macromolecules in Drosophila melanogaster.
Nature. 196 : 873 .
Fahmy, 0. G. and M. J. Fahmy (1965). Genetic properties
of exogenous DNA at various levels of degradation
in Drosophila melanogaster. Nature. 207: 507 *
Flousheim, G. L. (1962a). Cytotoxic'action of nucleic
acids. Experientla. 18: 328.
Flousheim, G. L. (1962b). System for the recognition of '
genetic transformation in haemopoietic cells.
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VITA
John Watkins Williams, III was born in Alexandria,
Louisiana on 11 March 1942.
After graduating from Riverside
Military Academy in Gainsville, Georgia, in June, i960, he
began his undergraduate studies at Spring Hill College in
Mobile, Alabama in the Pall of i960 .
In September, 1961,
he transferred to the University of Southwestern Louisiana
in Lafayette, Louisiana and in June, 1965> received a
Bachelor of Science degree with a major in Pre-medicine.
Entering the Louisiana State University Graduate School
in September, 1965, in the'Department of Zoology and Phys- .
iology, he served as a graduate teaching assistant from
September, 1965, to January, 1966, and from September,
1966, to June, 1969.
In August, 1968, he was awarded a
Master of Science degree with a major in Zoology.
The
following February, 19^9* he was employed as a research
assistant on a Public Health Service Grant awarded to
Dr. W. L. French.
He was awarded a Graduate School Disser­
tation Year Fellowship for the 1970-1971 academic year,
and is presently a candidate for the Doctor of Philosophy
degree.
79
EXAMINATION AND THESIS REPORT
Candidate:
John Watkins W i l l i a m s ,
M ajor Field:
Title of Thesis:
III
General Zoology
Evidence f o r the I n c o r p o r a t i o n o f Exogenous DNA by the
Polytene Chromosomes o f L i v i n g Drosophila Mojavensis
S a l i v a r y Gland Ce ll s in Short Term Organ Cu ltu re
Approved:
Major Professor and Chairman
Dean of the Graduate School
EXAMINING COMMITTEE:
Date of Examination: