269
B,[oscience Reports i, 269-287 (1981)
Printed in G r e a t Britain
Dissections and r e c o n s t r u c t i o n s of
genes and chromosomes
Nobel lecture, 8 December 1980
Paul BERG
Department of Biochemistry, Stanford University School
of Medicine, Stanford, California 94305, U.S.A.
Acknowledgements
T h e N o b e l L e c t u r e a f f o r d s a w e l c o m e o p p o r t u n i t y to express my
g r a t i t u d e and a d m i r a t i o n to the numerous students and colleagues with
w h o m I h a v e w o r k e d and s h a r e d , a l t e r n a t e l y , the elation and disa p p o i n t m e n t of venturing into the u n k n o w n .
Without their genius,
perseverance,
and s t i m u l a t i o n m u c h of o u r w o r k w o u l d not have
flourished. Those who have worked with students and e x p e r i e n c e d the
d i s c o m f o r t of their curiosity, the f r u s t r a t i o n s of t h e i r obstinacy, and
the e x h i l a r a t i o n of their growth know f i r s t - h a n d the magnitude of their
contributions.
Each in our c o m m o n e f f o r t l e f t a mark on the o t h e r ,
and I trust, e a c h r i c h e r f r o m t h e e x p e r i e n c e .
I have also been
fortunate
to have
two
devoted
research
assistants,
Ms. Marianne Dieckmann and 3une Hoshi, who have labored diligently
and e f f e c t i v e l y , a l w a y s w i t h u n d e r s t a n d i n g and s y m p a t h y for my
idiosyncrasies.
I have also been blessed with an amazing group of colleagues at
S t a n f o r d University who have c r e a t e d as stimulating and liberating an
e n v i r o n m e n t as one could long for.
Their many a c h i e v e m e n t s have
been inspirational, and without their h e l p - i n t e l l e c t u a l l y and m a t eriallymy e f f o r t s w o u l d h a v e b e e n s e v e r e l y handicapped.
I am
p a r t i c u l a r l y g r a t e f u l to Arthur Kornberg and C h a r l e s Y a n o f s k y , b o t h
l o n g t i m e c l o s e personal friends, for their unstinting i n t e r e s t , e n c o u r a g e m e n t , support, and c r i t i c i s m of my work, all of which enabled me
t o g r o w and t h r i v e .
And finally, t h e r e is my wife, Millie, without
whom the rare triumphs would have lost their lustre.
Her s t r e n g t h ,
assent, and encouragement freed me to immerse myself in research.
Certainly my work could not have taken place without the generous
and enlightened support of the U.S. National Institutes of Health, the
N a t i o n a l Science Foundation, the American Cancer Society, and
numerous foundations and individuals who invested their wealth in our
research.
9
Nobel
Foundation
1981
270
BERG
Introduction
' A l t h o u g h we are sure not to know
and r a t h e r likely not to know very
can k n o w anything t h a t is known to
may~ with luck and sweat, even find
t h i n g s t h a t h a v e n o t b e f o r e been
everything
much, we
man9 and
out some
known to
man.'
3. Robert
Oppenheimer
Although the concept that genes transmit and control hereditary
characteristics took hold early in this century~ ignorance about the
chemical nature of genes forestalled most inquiries into how they
function.
All of this changed as a result of several d r a m a t i c
developments during the 1940s to 1960s. First, Beadle and Tatum's
researches ( I - 3 ) lent strong support for earlier (#) and widespread
speculations that genes control the formation ol proteins (enzymes);
indeed, the dictum 'one gene - one protein' intensified the search for
the chemical definition oi a gene. The discovery by Avery and his
colleagues (5) and subsequently by Hershey and Chase (6) t h a t
genetic inlormation is encoded in the chemical structure oi deoxyribonucleic acid (DNA) provided the f i r s t clue.
Watson and C r i c k ' s
solution (7) ol the molecular structure ol DNA - the three-dimensional arrangement ol the polymerized nucleotide subunits - revealed
not only the basic design of gene structure but also the outlines ol
how genes are replicated and function.
Suddenly, genes shed t h e i r
purely conceptual and statistical characterizations and acquired defined
chemical identities. Genetic chemistry, or molecular biology as it has
frequently been called, was born.
U n t i l a few years ago~ much of what was known about the
molecular details of gene structure, o r g a n i z a t i o n , and f u n c t i o n had
been learned in studies with prokaryote microorganisms and the viruses
that inhabit them9 particularly the bacterium E s c h e z ' i u h i a c o 2 i and
t h e T and lambdoid bacteriophages.
These organisms were the
favorites of molecular biologists because they can be propagated
readily and rapidly under controllable laboratory conditions.
More
significantly~ utilizing several means of natural genetic exchanges
c h a r a c t e r i s t i c of these organisms and phages, the mapping and
manipulation of their relatively small genomes became routine.
As a
consequence, discrete DNA molecules~ containing one or a few genes~
were isolated in sufficient quantity and p u r i t y to p e r m i t e x t e n s i v e
c h a r a c t e r i z a t i o n s of t h e i r nucleotide sequences and chromosomal
organization.
Moreover, such isolated genetic elements provided the
models~ substrates, and reagents needed to investigate a wide range of
basic questions, the chemical basis of the genetic code; mutagenesis;
the mechanisms of DNA and chromosome replication~ repaiG and
recombination; the details of gene expression and regulation.
The astounding successes in defining the genetic chemistry of
prokaryotes during the 1950s and i960s were both e x h i l a r a t i n g and
challenging. Not surprisingly~ I and others wondered whether the more
complex genetic structures of eukaryote organisms, particularly those
ol mammalian and human cells, were organized and functioned in
RECONSTRUCTING GENES AND CHROMOSOMES
271
a n a l o g o u s ways.
S p e c i f i c a l l y , did the r e q u i r e m e n t s of c e l l u l a r
differentiation and intercellular communication, distinctive c h a r a c t e r =
i s t i c s of m u l t i c e l l u l a r organisms, require the new modes of genome
structure, organization, function, and r e g u l a t i o n ?
Were t h e r e just
variations of the prokaryote theme or wholly new principles waiting to
be discovered in e x p l o r a t i o n s of t h e g e n e t i c c h e m i s t r y of higher
organisms? It seemed important to try to find out.
SV~0's M i n i c h r o m o s o m e
S o m e t i m e d u r i n g 1965-66 I b e c a m e a c q u a i n t e d with R e n a t o
Dulbecco's work on the then newly discovered p o l y o m a virus.
The
g r o w i n g s o p h i s t i c a t i o n of a n i m a l cell culture methods had made it
possible for Dulbecco's laboratory to monitor and quantify t h e virus'
growth cycle in vitro (g).
Particularly significant was the discovery
that the entire virus genome resided in a single, r e l a t i v e l y s m a l l ,
c i r c u l a r DNA m o l e c u l e , one that could accommodate about five to
e i g h t genes ( 9 ) .
I was i n t r i g u e d by the r e s e m b l a n c e b e t w e e n
polyoma's life styles and those of certain bacteriophages. On the one
hand, polyoma resembled lyric bacteriophages in that the virus could
multiply vegetatively, kill its host, and produce large numbers of virus
progeny (g).
There was also a t a n t a l i z i n g s i m i l a r i t y to I y s o g e n i c
bacteriophages, since some infections yielded tumorigenic cells ( t 0 , l t).
The acquisition of new morphologic and growth characteristics, as well
as c e r t a i n virus-specific properties, suggested that tumorigenesis and
cell transformation resulted from covalent i n t e g r a t i o n of viral DNA
into the cell's chromosomal DNA and the consequent perturbation of
cell growth control by the expression of virus genes (12,13).
These d i s c o v e r i e s and provocative speculations, together with an
eagerness to find an e x p e r i m e n t a l model with which to s t u d y t h e
m e c h a n i s m s of m a m m a l i a n gene expression and regulation, prompted
me to spend a y e a r ' s s a b b a t i c a l l e a v e ( 1 9 6 7 - 6 8 ) in D u l b e c c o ' s
laboratory at the Salk Institute.
The work and valuable discussions we
carried on during that time (14) reinforced my c o n v i c t i o n t h a t t h e
tumor virus system would reveal interesting features about mammalian
genetic chemistry.
For s o m e w h a t t e c h n i c a l reasons, when I returned to Stanford, I
adopted SV40, a related virus, to begin our own r e s e a r c h p r o g r a m .
SVL~0 virions are n e a r l y s p h e r i c a l p a r t i c l e s whose c a p s o m e r s are
organized in icosahedral symmetry (Fig. l, upper l e f t ) .
The virions
c o n t a i n t h r e e viral coded polypeptides and a single double-stranded
circular DNA molecule (Fig. 1, upper right) t h a t is n o r m a l l y assoc i a t e d with four h i s t o n e s , H2a, H2b, H3, and H4, in the form of
c o n d e n s e d (Fig. l, lower l e f t ) or b e a d e d (Fig. 1, lower r i g h t )
c h r o m a t i n - l i k e structures. SVt~0 DNA contains 5243 nucleotide pairs
(5.24 kbp)~ the entire sequence of which is known f r o m s t u d i e s in
S.M. W e i s s m a n ' s (15) and W. F i e f s ' (16) l a b o r a t o r i e s .
Coding
information for five (and possibly six) proteins is c o n t a i n e d in t h e
DNA n u c l e o t i d e s e q u e n c e .
T h r e e of the proteins occur in mature
virions, possibly as structural components of the capsid shell, although
one might be associated with the DNA and have a regulatory function
(17).
Of the two non-virion proteins encoded in the DNA sequence,
one is localized in the cell nucleus (large-T antigen) and functions in
viral DNA replication and cell transformation; the other, found in the
BERG
272
Fig. I.
Electron micrographs of: SV40 virions
(u.l.); SV40 DNA (u.r.); 'condensed' SV40 minichromosomes (I.i.); 'relaxed-beaded' SV40 minichromosome
(l.r.). Photo by J. Griffin.
c y t o p l a s m ( s m a l l - t a n t i g e n ) , enhances the efficiency of cell transformation (18). Other proteins related in structure to large-T antigen
have been s p e c u l a t e d a b o u t , but their structures and functions are
unclear.
Restriction endonucleases have played a crucial role in defining the
physical and genetic organization of the SVZt0 genome (19,20).
The
restriction or cleavage sites serve as coordinates for a physical map of
the viral DNA; the availability of such map c o o r d i n a t e s makes it
possible to locate, accurately, particular physical features and genetic
loci. In this system of map coordinates the single EcoRI endonuclease
c l e a v a g e s i t e serves as the r e f e r e n c e marker and is assigned map
position 0/1.0; other positions in the DNA are assigned coordinates in
DNA fractional length units measured clockwise from 0/1.0 (see Fig.
2). At the present time knowledge of the entire nucteotide sequence
has made possible a more precise set of map coordinates: nucleotide
pair number. Thus, nucleotide 0/5243 is placed within ori, t h e s i t e
where DNA replication is initiated, and the other nucleotide pairs are
numbered consecutively in the clockwise direction (see Fig. 2).
RECONSTRUCTING GENES AND CHROMOSOMES
273
o11.o
0.i
A
Eco R l
[1783]
1000
Barn H ]
(2534]
Ie361
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i
i
3000
Hpa]]
[S47]j~
I
I
I
-"
0.7
0/5243
ori
|740]
4000
0.5
Fig. 2. A physical and genetic map of SV40 DNA.
The inner circle symbolizes the closed circular
DNA molecule; indicated within the circle are the
nucleotide-pair map coordinates starting and
ending at 0/5243.
Also shown by small arrows
within the circle are the sites at which five
restriction endonucleases cleave SV40 DNA once.
Arrayed around the outside of the circle are the
map coordinates, expressed in fractional lengths~
beginning at the reference point 0/i.0 (the EcoRI
endonclease
cleavage
site) and proceeding
clockwise around the circle.
The coding regions
for the early and late proteins are shown as
stippled arrows extending from the nucleotide
pair of the first codon to the nucleotide pair
that specifies termination of the protein coding
sequence. Each of the coding regions is embedded
in a mRNA~ the span of which is indicated by
dotted or dashed 5' ends and wavy poly A 3' ends.
The jagged or saw-toothed portions of each mRNA
indicate the portions of the transcript that are
spliced in forming the mature mRNAs.
274
BERG
The SV40 minichromosome is expressed in a regulated temporal
sequence a f t e r i t reaches the nucleus of i n f e c t e d p r i m a t e cells.
Initially~ transcription in the counter-clockwise direction of one strand
(the E strand) of about one half of the DNA (the early region) yields
the early mRNAs (Fig. 2).
These mRNAs~ which encode the large-T
and smaH-t antigen polypeptides (the stippled portion of the mRNAs
i n d i c a t e the p r o t e i n - c o d i n g regions)~ have 5' ends originating from
nucleotide sequences near the site marked ori and 3 ' - p o l y a d e n y l a t e d
(poly A) ends from near map position 0.16. Synthesis of large-T
antigen triggers the i n i t i a t i o n of v i r a l DNA r e p l i c a t i o n at ori~ a
specific site in the DNA (Fig. 2 identifies ori at map position 0.67 or
nucleotide position 0/5243); replication then proceeds bi-directionally,
t e r m i n a t i n g about Ig0 ~ away near map position 0.17~ yielding covalently closed circular progeny DNA.
New viral mRNAs appear in
the, polyribosomes c o n c o m i t a n t w i t h DNA r e p l i c a t i o n ; these are
synthesized in the clockwise direction from the L-strand of the other
half of the virus DNA (the late region) and are referred to as late
mRNAs. Transcription of the late mRNAs~ which code for the virion
proteins VPI~ VP2~ and VP3 (the stippled regions designate the
p r o t e i n - c o d i n g regions of these proteins)~ begins from m u l t i p l e
positions between map positions 0.6g-0.72 and terminates at about map
position 0.16. Finally~ the accumulation of progeny DNA molecules
and v i r i o n proteins Culminates in death of the cell and release of
mature virion particles.
SV#0 possesses an a l t e r n a t i v e l i f e cycle when the virus infects
rodent and other non-primate cells.
The same early events take
place - the E-strand mRNAs and large-T and small-t antigens are
synthesized - but DNA replication does not occur and l a t e - s t r a n d
mRNAs and virion proteins are not made.
Frequently, replication o5
cell DNA and mitosis are induced a f t e r infection, and most i nfect ed
cells survive with little evidence of prior infection. Generally~ a small
proportion of the ceils (less than 10%) acquire the ability to multiply
u n d er c u l t u r e c o n d i t i o n s t h a t r e s t r i c t the growth of normal ceils;
moreover, these transformed cells can produce tumors a f t e r inoculation
into appropriate animals.
Invariably~ the transformed ceils contain all
or p a r t of t h e vi r a l DNA c o v a l e n t l y i n t e g r a t e d i nt o t h e c e l l ' s
chromosomal DNA and produce the mRNAs and proteins coded by the
early genes.
During the 1970s several di f f er e nt approaches, carried on in many
laboratories including my own, clarified the a r r a n g e m e n t of SV40 genes
on the DNA and revealed how they function during the virus life cycle
(21-23).
Initially9 viral genes were mapped on the DNA relative to
r e s t r i c t i o n sites by localizing t he regions from which early and late
mRNAs were transcribed.
Subsequently, m o r e p r e c i s e m a p p i n g was
a c h i e v e d by cor r el at i ng the positions of discrete deletions and other
alterations in the viral DNA with specific physiologic d e f e c t s .
But
with the nucleotide sequence map, the boundaries of each SVt~0 gene
and the n u c l e o t i d e s e g m e n t s codi ng f o r e a c h p o l y p e p t i d e can be
s p e c i f i e d with c o n s i d e r a b l e p r e c i s i o n (Fig. 2).
As expected, the
a v a i l a b i l i t y of a p r e c i s e g e n e t i c a n d p h y s i c a l m a p of S V 4 0 ' s
m i n i c h r o m o s o m e has s h i f t e d the research emphasis to explorations of
t h e m o l e c u l a r m e c h a n i s m s g o v e r n i n g e a c h g e n e ' s e x p r e s s i o n and
f u n c t i o n , t h e r e p l i c a t i o n and m a t u r a t i o n of the viral minichromo-
RECONSTRUCTING GENES AND CHROMOSOMES
275
some, recombination between the viral and host DNA, and how virus
and host gene products interact to cause transformation of normal into
tumorigenic ceils. Excellent and more detailed summaries and analyses
of the molecular biology of SV40 and polyoma containing acknowledgements to the important contributions made by many individuals can be
found in several recent monographs (21-23).
SV~O as a Transducing Virus
The analysis of the o r g a n i z a t i o n , expression, and regulation of
bacterial genomes was greatly aided by the use of bacteriophagem e d i a t e d t r a n s f e r of genes between celts.
Indeed, specialized
transducing phages of k, d~80, P22, and others permitted the cloning
and a m p l i f i c a t i o n of s p e c i f i c segments of bacterial DNA, thereby
making it possible to construct celts w i t h unusual and i n f o r m a t i v e
genotypes and to obtain valuable substrates and probes for exploring
mechanisms of transcription~ translation~ and regulation.
This background led me to consider, soon after beginning work with
the tumor viruses, whether SV40 could be used to transduce new genes
into mammalian ceils.
Initially, I had serious reservations about the
success of such a venture because of the predictably low probability of
generating specific recombinants between virus and cell DNA and the
limited capability for selecting or s c r e e n i n g a n i m a l ceils t h a t had
acquired specific genetic properties.
But it seemed that one possible
way out of this difficulty, at least one worth trying, was to produce
the desired SV40 transducing genomes synthetically. Consequently, in
about 1970~ I began to plan the construction, in vitr% of recombinant
DNA molecules with SV40 and selected non-viral DNA segments. The
goal was to propagate such recombinant genomes in s u i t a b l e a n i m a l
cells, either as autonomously replicating or integrated DNA molecules.
At the t i m e t h e r e w e r e few if any a n i m a l genes a v a i l a b l e f o r
r e c o m b i n a t i o n with SVt~0 DNA but I anticipated that a variety of
suitable genes would eventually be isolated. Therefore, the first task
was to devise a g e n e r a l way to join t o g e t h e r , in vitr% any two
different DNA molecules.
H e r s h e y and his c o l l e a g u e s had a l r e a d y shown that DNA ligase
promoted circularization or end-to-end joinings of k phage DNA (2t~).
This o c c u r r e d b e c a u s e X phage DNA has cohesive ends, i.e., singlestranded, overlapping, complementary DNA ends ( 2 5 ) . S% it seemed
t h a t if c o h e s i v e ends could be s y n t h e s i z e d onto the ends of DNA
molecules, they could be covalently joined in vitro with DNA ligase.
D u r i n g 1 9 7 t - 7 2 , using t h e n - a v a i l a b l e e n z y m e s and r e l a t i v e l y
straightforward enzymologic procedures, David A. 3ackson, Robert H.
Symons, and I (26) and, independently and concurrently, Peter E.
Lobban and A. D. Kaiser (27) devised a way to synthesize synthetic
cohesive termini on the ends of any DNA molecules, thereby paving
the way for constructing recombinant DNAs in vitro.
Our procedure
(Fig. 3) was developed using as the model 'foreign' DNA a bacterial
plasmid that contained some bacteriophage X DNA and three B. co2i
genes t h a t specify enzymes required for galactose utilization (28).
Circular SV40 DNA (5.24 kbp) and kdvga2 plasmid DNA (about 10
kbp) were each cleaved with a specific endonuclease to convert them
to linear molecules.
Then, after a brief digestion with X-exonuciease
276
BERG
Construction Of Hybrid Genome
Q
Q
1
3' Endonuclease
1
B'
P
P
3'
1
I
dATP
p
~
P
~, exonuclease
3' , P
P B'
Terminal Transferase
(dA)n
I
p
3'
dTTP
~'
(dT)n
(dA)n
nnea/t,~v
p
~,K
(dA)n
(dAln
P'
]]
IdTln
I
DNA polymerose+ 4dTP
DNA ligase + DPN
Exonuclease TIT
(dA)n
L%%%;'J
(dTln
Fig.
DNA.
3.
The construction of SV40-ldvgal recombinant
See text for comment on individual steps.
to remove about f i f t y nucleotides from the 5' termini, short 'tails' of
either deoxyadenylate or deoxythymidylate residues were added to the
3' termini using purified deoxynucleotidyl terminal transferase.
After
mixing and annealing under appropriate conditions, the two DNAs were
joined and cyclized via their complementary 'tails' (Fig. 3). The gaps
that occur where the two DNA molecules are held together were filled
in w i t h DNA polymerase I and its deoxynucleoside triphosphate
substrate% and the resulting molecules were c o v a l e n t l y sealed w i t h
DNA Jigase; exonuclease Ill was present to permit repair of nicks or
gaps created during the manipulations.
RECONSTRUCTING GENES AND CHROMOSOMES
277
The resulting hybrid DNA was approximately three times the size
ol SV40 DNA and, t h e r e f o r e , could not be p r o p a g a t e d as an e n c a p sidated virus. But we intended to test whether the E. e o Z i galactose
genes would be expressed a f t e r introduction into the chromosomes of
cultured animal cells.
M o r e o v e r , s i n c e t h e Xdvgal plasmid could
replicate autonomously in E. c o Z i
(28), we also planned to det erm i ne
if SVg0 DNA would be propagated in E. c o Z i cells and if any SV40
g e n e s would be e x p r e s s e d in t h e b a c t e r i a l h o s t .
Although the
SVgO-Xdvgal
r e c o m b i n a n t DNA shown in Fig. 3 coul d not h a v e
replicated in E. e o Z i - a gene needed for replication of the plasmid
DNA in E. c o Z i had been inactivated by the insertion of the SV40
DNA - a relatively simple modification of the procedure - the use of
XdvgaZ d i m e r i c DNA as accept or for the SV40 DNA insert - could
h a v e c i r c u m v e n t e d this d i f l i c u l t y .
Nevertheless, because many
colleagues expressed concern about the potential risks of disseminating
E. e o l i c o n t a i n i n g SV40 o n c o g e n e s , t h e e x p e r i m e n t s w i t h t h i s
recombinant DNA were discontinued.
S in ce t h a t t i m e t h e r e has b e e n an e x p l o s i v e g r o w t h in t h e
a p p l i c a t i o n of r e c o m b i n a n t DNA m e t h o d s Ior a n u m b e r of novel
purposes and challenging pr obl em s .
This i m p r e s s i v e p r o g r e s s owes
much of its impetus to the growing sophistication about the properties
and use of restriction endonucleases, the development of easier ways
of r e c o m b i n i n g d i f l e r e n t DNA molecules, and, most importantly, the
availability of plasmids and phages that made it possible to propagate
and a m p l i f y r e c o m b i n a n t DNAs in a variety of microbial hosts t s e e
r e l e r e n c e s 29 and 30 for a collection ol notable examples).
By 1975, e x t e n s i v e c l o n i n g e x p e r i m e n t s had produced elaborate
libraries of eukar yot e DNA segments containing single or c l u s t e r s of
genes from many species ol organisms. As expected, studies of their
molecular anatomy and chromosomal a r r a n g e m e n t have p r o v i d e d new
i n s i g h t s a b o u t possible mechanisms of gene regulation in normal and
developmentally interesting animal s y s t e m s .
But, it s e e m e d l i k e l y
Irom the beginning that ways would be needed to assay isolated genes
for their biological activity in vivo. Consequently, I returned to the
o r i g i n a l goal of using SVg0 to introduce cloned genes into cultured
mammalian cells.
But this time we explored a s o m e w h a t d i f f e r e n t
approach.
During 1972-74 J a n e t Mertz and I (31) learned how to propagate
SVg0 deletion mutants by c o m p l e m e n t a t i o n using a p p r o p r i a t e SV40
t e m p e r a t u r e - s e n s i t i v e ( t s ) mutants as helpers.
This advance made it
feasible to consider propagating genomes containing exogenous DNA in
place of specific regions of SV40 DNA. Accordingly, Stephen Goll and
I devised a procedure to cons t r uct such r e c o m b i n a n t s by r e s e c t i n g
d e f i n e d segments ol SVg0 DNA with appropriate restriction endonucleases and replacing them with 'foreign' DNA segments using synthetic
c o h e s i v e ends ( 3 2 , 3 3 ) ( F i g . 4) .
In this e x p e r i m e n t a l design the
recombinant genomes must contain the origin ol SVg0 DNA replication
(ori) so that they can be propagated; also they must be smaller than
5.3 kbp, that is, not more than one mature viral DNA length, to be
i n c o r p o r a t e d i nt o virus p a r t i c l e s .
F u r t h e r m o r e , because the SV40
v ecto r lacks genetic functions coded by the excised DNA segment, the
r e c o m b i n a n t g e n o m e s a r e d e f e c t i v e and must be propagated with a
helper virus that can supply the missing gene product or products. In
278
BERG
/
~1
.
l _o@
\\
\\,
/I
fi
II
/ I
,,'y
"4
II
i
_i
9
+-
\
i/
'-
Fig. 4.
A s c h e m e for c o n s t r u c t i n g SV40
transducing genomes in vitro.
Segments of the
late (upward track) or early (downward track)
regions of SV40 DNA (the dashed circle on the
left) are removed by sequential cleavages with
restriction endonucleases.
Appropriate-sized
segments of any DNA, produced by restriction
enzyme cleavages, enzymatic copying of mRNA, or
chemical synthesis, are inserted in place of
the resected SV40 DNA segment.
Joining, via
natural or synthetic cohesive ends (symbolized
by the jagged lines), is mediated by a DNA
ligase.
Ori indicates the position of the
origin of SV40 DNA replication.
our protocol the r e c o m b i n a n t g e n o m e retains at least one functioning
virus gene and, c o n s e q u e n t l y , can c o m p l e m e n t a d e f e c t i v e gene in the
helper virus.
For e x a m p l e , r e c o m b i n a n t s in which the i n s e r t e d DNA
replaces all or part of SV40's /ate region can be p r o p a g a t e d with SV#O
m u t a n t s t h a t have a d e f e c t i v e early region (e.g. at high t e m p e r a t u r e
RECONSTRUCTING GENES AND CHROMOSOMES
279
with t e m p e r a t u r e ~ s e n s i t i v e early mutants);
similarly, recombinants
having exogenous DNA implants in place of DNA segments in the early
region can be propagated with a helper genome that is defective in its
late region (in this instance, at high temperature with t e m p e r a t u r e sensitive late mutants).
Our i n i t i a l a t t e m p t s (32,33) to obtain expression of cloned DNA
segments as distinct mRNAs and proteins following introduction of the
recombinant genomes into cultured cells were negative. But as soon
as we recognized t h a t e x p r e s s i o n of t h e new g e n e t i c i n f o r m a t i o n
depended upon whether the transcript, originating from SV#0 promoters
and ending at SV#0 s p e c i f i e d poly A s i t e s , could be spliced, our
fortunes changed. The initial success in obtaining expression of added
genetic elements as mRNAs and proteins following t r a n s f e c t i o n into
c u l t u r e d monkey ceils was achieved with a DNA segment coding for
rabbit B-globin (3#).
Soon a f t e r w a r d , a b a c t e r i a l gene ( E c o g p t )
coding for xanthine-guanine phosphoribosyl transferase (XGPRT) (35),
a mouse DNA specifying dihydrofolate reductase (DHFR) (36), and a
bacterial gene (neoN) for aminoglycoside phosphotransferase (37) were
successfully transduced into m a m m a l i a n cells via SV#0-DNA-based
vectors.
G e n e r a l l y , the transduced DNA segments are expressed at
rates comparable to those of the SV~0 genes they replace, but some
anomalies in the RNA processing have been observed.
Hamer and Leder have also c o n s t r u c t e d and p r o p a g a t e d r e c o m b i n a n t s of SVS0 DNA with c l o n e d mouse-genomic B-globin (38) or
c~-globin (39) genes. In certain of their recombinants the transduced
g e n e s are e x p r e s s e d f r o m SV#0 l a t e p r o m o t e r signals, but other
constructions r e v e a l t h a t t r a n s c r i p t i o n can be i n i t i a t e d from t h e
c~-g/obin promoter as well (39).
Their experiments also demonstrate
that proper splicing of the globin intervening sequences and translation
of the resulting mRNAs can occur in a heterologous host.
New Transducing Vectors for Mammalian Cells
In the experiments referred to above, our principal aim had been to
e•
the ability of the recombinant g e n o m e s to r e p l i c a t e in t h e
virus' p e r m i s s i v e host.
For example, following infection of monkey
cells, the SV#0 r e c o m b i n a n t g e n o m e s are a m p l i f i e d a b o u t 104- to
105-fold, thereby ensuring a high yield of the products expressed from
the transduced genes. This system has taught us a great deal about
t h e n e c e s s i t y and m e c h a n i s t i c subtleties of RNA splicing (40), the
rules governing expression of coding sequences i n s e r t e d at d i f f e r e n t
p o s i t i o n s in SV#0 DNA (#1), and some novel features of SV#0 gene
expression i t s e l f ( # 2 ) .
But this e x p e r i m e n t a l design has s e v e r a l
distinct shOrtcomings. During the course of the infection the ceils are
killed, precluding the opportunity to m o n i t o r t h e t r a n s d u c e d g e n e ' s
expression in continuously multiplying cell populations. Moreover, only
ceils which can replicate SV#0 DNA are able to amplify the cotransd u c e d genes.
This constraint excludes many specialized and differentiated animal cells as hosts for the transduced genes.
To circumvent these disadvantages we have developed a new group
of transducing vectors that can be used to introduce and maintain new
genetic information in a variety of mammalian ceils (Fig. 5).
pSV2
(#3) and its d e r i v a t i v e s pSV3 and pSV5 (35,##) c o n t a i n a DNA
280
BERG
AmpR
pBR32
~ 1
E~-gpt
AmpR
pBR32
AmpR
2
or
~'vu i i
"
",\\
p B R 3 2 2 ori
•
psvs.,,
:.,
:!. Hindlll
Pstl~
J/" i/I I
~ /
\\
\
Fig. 5.
Structure of plasmid transforming vectors.
The solid black segments in each diagram represent 2.3
kbp of pBR322 DNA sequence that contains the origin of
pBR322 DNA replication and the ampicillinase gene. The
stippled regions represent segments derived from SV40
DNA, the open region (in psv5-gpt) is from polyoma DNA,
and the hatched segment represents Ncogpt DNA containing the gene coding for E. co2i XGPRT.
s e g m e n t (shown as the f i l l e d region in Fig. 5) from an E. c o l i
plasmid (pBR522) that permits these DNAs to propagate in E. c o l i
cells, thereby greatly simplifying the genetic manipulations involved in
their use. Each of the vectors contains a marker gene (shown in Fig.
5 as the hatched segment) flanked at the 5' end with a DNA segment
containing the SV40 early p r o m o t e r and o r i g i n of DNA r e p l i c a t i o n
(ori); another SV40 DNA segment that ensures splicing and polyadenylation of the t r a n s c r i p t is l o c a t e d at t h e 3' end of t h e m a r k e r
segment (the SV40-derived DNA segments are shown stippled in Fig.
5).
Additional DNA segments can also be inserted into t h e v e c t o r
DNAs at any of several unique restriction sites; consequently, a single
DNA molecule can transduce several genes of interest simultaneously.
pSV2 can not replicate in mammalian cells because it and the cell
lack the means to i n i t i a t e DNA r e p l i c a t i o n a t ori.
This can be
r e c t i f i e d by i n s e r t i n g , at pSV2!s single BamHl cleavage site, DNA
segments which contain either a complete early region from SV40 DNA
(pSV3), or polyoma's early region (pSVS) (Fig. 5).
The viral early
regions inserted into pSV3 and pSV5 vectors c o d e for p r o t e i n s t h a t
p r o m o t e DNA r e p l i c a t i o n s f r o m t h e i r respective origins; therefore,
pSV3 DNA can replicate in monkey ceils and pSV5 DNA replicates in
mouse ceils (44).
RECONSTRUCTING GENES AND CHROMOSOMES
281
To date t h r e e m a r k e r DNA s e g m e n t s have been used in conjunction
with the pSV2, -3, and -5 vector DNAs: Ecogpt, a n E. coli gene that
codes for the e n z y m e xanthine-guanine phosphoribosyltransferase
( X G P R T ) (35,44); a mouse c D N A segment that specifies D H F R (36);
neo R, a bacterial plasmid gene specifying an aminoglycoside phosphotransferase that inactivates the antibacterial action of neomycinkanamycin derivatives (37).
Here, I shall only summarize several
recent findings with Ecogpt as the marker gene but, suffice it to say,
transfection of a variety of cells with any of the vector-dhfr or
v e c t o r - n e o R d e r i v a t i v e s results in expression of t h e m o u s e D H F R or
neo R p h e n o t y p e , r e s p e c t i v e l y .
Richard Mulligan isolated the E c o g p t gene (35) to d e t e r m i n e if it
was useful for the d e t e c t i o n and s e l e c t i v e growth of m a m m a l i a n cells
that acquired that gene.
T h e f i r s t p r i o r i t y was to establish t h a t
i n t r o d u c t i o n of E c o g p t i n t o m a m m a l i a n c e i l s w o u l d p r o m o t e t h e
p r o d u c t i o n of E. c o l i XGPRT.
We found t h a t e x t r a c t s from c u l t u r e d
monkey cells exposed to pSV2-gpt, pSV3-g_nt, and p S V 5 - g p t DNAs did
contain two G P R T e n z y m e a c t i v i t i e s (Fig. 6A); one corresponds to the
normal cellular e n z y m e , h y p o x a n t h i n e - g u a n i n e phosphoribosyl t r a n s f e r a s e
(HGPRT)
by its e l e c t r o p h o r e t i c behavior in p o l y a c r y l a m i d e gel and
the o t h e r has t h e s a m e e l e c t r o p h o r e t i c
m o b i l i t y as t h e E. c o l i
XGPRT.
O t h e r c h a r a c t e r i z a t i o n s of XGPRT s y n t h e s i z e d in monkey
cells i n d i c a t e t h a t it is indistinguishable by s e v e r a l c r i t e r i a from t h e
s a m e e n z y m e m a d e in E. c o l l .
E. c o l i
X G P R T is analogous to the m a m m a l i a n e n z y m e H G P R T .
Both use guanine as a s u b s t r a t e for purine n u c l e o t i d e synthesis.
But
E. c o l i
X G P R T d i f f e r s f r o m its m a m m a l i a n c o u n t e r p a r t in t h a t it
uses x a n t h i n e m o r e e f f i c i e n t l y than hypoxanthine as a s u b s t r a t e (45);
t h e m a m m a l i a n e n z y m e can use hypoxanthine but not x a n t h i n e as a
p r e c u r s o r for purine n u c l e o t i d e f o r m a t i o n ( 4 6 ) .
S i n c e E. c o l i X G P R T is p r o d u c e d in monkey cells a f t e r i n t r o duction of the E c o g p t gene, it was i m p o r t a n t to learn if the b a c t e r i a l
e n z y m e could r e p l a c e the cellular H G P R T f u n c t i o n .
This point could
be t e s t e d with human L e s c h - N y h a n cells b e c a u s e t h e y l a c k H G P R T
and, as a c o n s e q u e n c e , can not grow in a c u l t u r e medium containing
a m i n o p t e r i n , hypoxanthin% and t h y m i d i n e ( H A T m e d i u m ) ( 4 7 ) .
It
s e e m e d l i k e l y t h a t t r a n s f e c t i o n of L e s c h - N y h a n cells with pSV-gpt
DNAs would show w h e t h e r the f o r m a t i o n of E. c o l i XGPRT enabled
t h e s e m u t a n t ceils to survive in HAT medium.
As e x p e c t e d , L e s c h - N y h a n ceils did not survive in HAT medium if
t h e y had n o t r e c e i v e d v e c t o r - g p t DNA.
But when L e s c h - N y h a n cell
cultures r e c e i v e d pSV2-gpt or p S V 3 - g p t DNA, surviving colonies w e r e
r e c o v e r e d at a f r e q u e n c y of a b o u t 10 -4 (35).
R e p r e s e n t a t i v e s of the
surviving clones, subcultured in HAT medium for z~0 g e n e r a t i o n s , w e r e
found to contain E. c o l i X G P R T (Fig. 6B). Thus, the acquisition of
E c o g p t DNA and c o n s e q u e n t expression of E. c o l i X G P R T r e c t i f i e d
t h e d e f e c t c a u s e d by t h e a b s e n c e of the cellular H G P R T in t h e s e
cells.
The expression of E c o g p t provides a novel c a p a b i l i t y for m a m m a l i a n
ceils; normal m a m m a l i a n c e i l s do n o t u t i l i z e x a n t h i n e f o r p u r i n e
n u c l e o t i d e f o r m a t i o n , but those t h a t express and make E. c o l i XGPRT
can do so.
The acquisition and expression of E c o g p t can, t h e r e f o r e ,
be made the basis for an e f f e c t i v e dominant s e l e c t i o n for m a m m a l i a n
cells ( 4 8 ) .
BERG
282
ORIGIN
MONKEY
HGPRT
E. coli
XGPRT
/YYY~
ORIGIN ~
HUMAN
HGPRTase
E.coh
XGPRTase
RFCONSTRUCTING GENES AND CHROMOSOMES
283
Purine nucleotides are synthesized de novo or by salvage pathways
(Fig. 7).
In the de novo p a t h w a y , inosinic acid ( I M P ) , t h e f i r s t
n u c l e o t i d e i n t e r m e d i a t e , is c o n v e r t e d to adenylic acid (AMP) via
a d e n y l o s u c c i n a t e and to g u a n y l i c acid (GMP) via x a n t h y l i c acid
(XMP). Salvage of free purines occurs by condensation with phosphoribosyl p y r o p h o s p h a t e ( P R P P ) : a d e n i n e phosphoribosyl t r a n s f e r a s e
( A P R T ) a c c o u n t s for the formation of AMP from adenine (A) and
hypoxanthine-guanine phosphoribosyl t r a n s f e r a s e ( H G P R T ) c o n v e r t s
h y p o x a n t h i n e (Hx) and guanine (G) to IMP and GMP, respectively.
No m a m m a l i a n e n z y m e c o m p a r a b l e to t h e b a c t e r i a l e n z y m e for
converting xanthine (X) to XMP is known.
Mycophenolic acid (MPA), an inhibitor of IMP dehydrogenase (49),
p r e v e n t s t h e f o r m a t i o n of XMP and, t h e r e f o r e , of GMP.
The
inhibition of cell growth by mycophenolic acid can be reversed by the
addition of guanine to the medium, because guanine can be converted
to its mononucleotide by HGPRT.
Since normal mammalian cells do
not c o n v e r t x a n t h i n e to XMP, t h e y can not grow if the medium
containing mycophenolic acid is supplemented with xanthine. However,
cells that contain E. co2i XGPRT should grow under these conditions.
Transfection of monkey cells with pSV2, -3 or -5-gpe DNA and
s u b s e q u e n t t r a n s f e r to a medium containing mycophenolic acid plus
xanthine yields surviving colonies with frequencies ranging from 10-4 to
10-s (48). Omission of the DNAs, or transfection with pSV2~ -3, and
-5 DNA containing other marker s e g m e n t s ( e . g . B-globin or mouse
DHFR DNA), or r e m o v a l of t h e ori segment from pSV2-gpt DNA,
results in fewer than l0 -7 surviving c o l o n i e s .
Ecogpt-transformed
c l o n e s c o n t a i n cells t h a t produce both the normal HGPRT and the
Ecogpe product, XGPRT. Analysis of the genetically transformed cells'
DNA i n d i c a t e s t h a t they have retained one to a few copies of the
pSV-gpe DNA, probably integrated into their chromosomal DNA. The
s t r u c t u r e , o r g a n i z a t i o n , and e x p r e s s i o n of t h e s e i n t e g r a t e d genes
Fig. 6.
Detection of GPRT activity following
electrophoresis of cell extracts in polyacrylamide gels.
a) Extracts of about 5 x I0 ~ CVl
cells harvested 3 days after transfection with 10
~g of pSV2- , -3-, or -5-gpt DNAs were electrophoresed in polyacrylamide gels and assayed for
GPRT activity
in situ by i n c u b a t i o n
with
SH-labeled guanine and detection of the labeled
GMP product by fluorographic autoradiography
(35). The black areas correspond to the location
of guanine phosphoribosyl transferase activity on
the gel, and the arrows i n d i c a t e the k n o w n
positions of the monkey and E. co2i GPRT enzymes.
b) E x t r a c t s
of u n t r e a t e d
(LNSV)
and
pSV-gpt-transformed
L e s c h - N y h a n cells were
analyzed as described above. The track marked E.
coli contained an extract of bacteria containing
XGPRT.
284
BERG
PURINE NUCLEOTIDE
Hx
SYNTHESIS
X
PRPP
G
l
PRPP
Precursors
MTxR
99
> IMP
MPA
9
>
XMP
PRPP
> GMP
AMP
PRPP
A
Fig. 7.
Pathways of purine nucleotide synthesis.
Methotrexate (MtxR) inhibits de novo synthesis of
purines, and mycophenolic acid (MPA) specifically
inhibits the conversion of IMP to XMP (49).
The
arrows between purine bases and their respective
mononucleotides indicate the reactions of the bases
w i t h PRPP c a t a l y z e d by p u r i n e p h o s p h o r i b o s y l
transferase. An arrow also indicates that AMP can be
deaminated to IMP.
the relation of these promoters to their expression are presently being
studied.
Our p r e s e n t s t u d i e s s u g g e s t t h a t the requirements for obtaining
expression of prokaryote genes are not formidable. There is no reason
to b e l i e v e t h a t t h e b a c t e r i a l genes Ecogpt and n e o R are unique in
their ability to be e x p r e s s e d in m a m m a l i a n c e l l s .
I foresee that
b a c t e r i a , t h e i r v i r u s e s , and s i m p l e e u k a r y o t e s will provide a rich
source of genes for the modification of mammalian cells.
Prospects
The development and application of recombinant DNA techniques
has opened a new era of scientific discovery, one t h a t promises to
influence our future in myriad ways.
It has already had a dramatic
and far-reaching impact in the field of genetics, indeed in all of
molecular biology.
Molecular cloning provides the means to solve the
organization and detailed molecular structure of extended regions of
R E C O N S T R U C T I N G GENES AND CHROMOSOMES
285
chromosomes
a n d e v e n t u a l l y t h e e n t i r e g e n o m e of a n y o r g a n i s m
including m a n ' s .
A l r e a d y , i n v e s t i g a t o r s h a v e i s o l a t e d a n u m b e r of
m a m m a l i a n and h u m a n genes, and in s o m e i n s t a n c e s d e t e r m i n e d t h e i r
c h r o m o s o m a l a r r a n g e m e n t and e v e n t h e i r d e t a i l e d n u c l e o t i d e s e q u e n c e .
S u c h d e t a i l e d i n f o r m a t i o n has profound i m p l i c a t i o n for the f u t u r e of
medicine.
3ust as our p r e s e n t k n o w l e d g e and p r a c t i c e of m e d i c i n e
relies on a s o p h i s t i c a t e d k n o w l e d g e of h u m a n a n a t o m y , physiology, and
b i o c h e m i s t r y , so will dealing w i t h d i s e a s e in t h e f u t u r e d e m a n d a
detailed understanding
of t h e m o l e c u l a r a n a t o m y , physiology, and
b i o c h e m i s t r y of t h e h u m a n g e n o m e .
T h e r e is no d o u b t t h a t t h e
d e v e l o p m e n t and a p p l i c a t i o n of r e c o m b i n a n t DNA t e c h n i q u e s has put us
at the t h r e s h o l d of new f o r m s of m e d i c i n e .
There are many who
contemplate
the treatment
of c r i p p l i n g g e n e t i c d i s e a s e s t h r o u g h
r e p l a c e m e n t of d e f e c t i v e g e n e s by n o r m a l c o u n t e r p a r t s
o b t a i n e d by
molecular cloning.
S c e n a r i o s a b o u t h o w t h i s c o u l d be d o n e a r e
r a m p a n t , only a f e w of which a r e plausible.
G e n e r e p l a c e m e n t as a
therapeutic
a p p r o a c h has m a n y p i t f a l l s and unknowns, a m o n g s t which
are questions concerning the feasibility and desirability for any
p a r t i c u l a r g e n e t i c disease, to say nothing a b o u t the risks. It s e e m s to
me t h a t if w e a r e e v e r to p r o c e e d along t h e s e lines we shall need a
m o r e d e t a i l e d k n o w l e d g e of how h u m a n genes a r e o r g a n i z e d and how
t h e y f u n c t i o n and a r e r e g u l a t e d .
We shall also need physicians who
a r e as c o n v e r s a n t
with t h e a n a t o m y and physiology of c h r o m o s o m e s
and genes as the c a r d i a c surgeon is with t h e s t r u c t u r e of the h e a r t
and c i r c u l a t o r y t r e e . G e n e t h e r a p y will h a v e to be e v a l u a t e d in t e r m s
of a l t e r n a t i v e and m o r e c o n v e n t i o n a l f o r m s of t r e a t m e n t ,
j u s t as is
now done before undertaking heart
valve replacements
and renal
transplants.
Aside f r o m the s c i e n t i f i c i s s u e s , t h e r e a r e t h e m a n y
contentious
e t h i c a l a n d m o r a l q u e s t i o n s t h a t a r e r a i s e d by such
s t r a t e g i e s . P e r h a p s it is not too soon to i n i t i a t e serious discussions on
the s c i e n t i f i c , m e d i c a l , and e t h i c a l issues t h a t shall c o n f r o n t us.
The a d v e n t and w i d e s p r e a d use of t h e r e c o m b i n a n t DNA t e c h n o l o g y
for basic and m e d i c a l r e s e a r c h and t h e i m p l i c a t i o n s for industrial and
p h a r m a c e u t i c a l a p p l i c a t i o n h a v e also r e v e a l e d , or p e r h a p s c r e a t e d , an
u n d e r l y i n g a p p r e h e n s i o n , an a p p r e h e n s i o n a b o u t probing the n a t u r e of
life i t s e l f , a questioning of w h e t h e r c e r t a i n inquiries a t t h e e d g e of
o u r k n o w l e d g e a n d o u r i g n o r a n c e should c e a s e for f e a r of what we
could d i s c o v e r or c r e a t e .
I p r e f e r t h e m o r e o p t i m i s t i c and uplifting
v i e w e x p r e s s e d by Sir P e t e r M e d a w a r in his essay e n t i t l e d 'On The
E:[fecting of All Things P o s s i b l e ' (The Hope o f P r o g r e s s , McThuen and
Co. Ltd., London, 1972).
'If we imagine
the evolution
of l i v i n g o r g a n i s m s
c o m p r e s s e d i n t o a y e a r of c o s m i c t i m e ,
then the
e v o l u t i o n of man has o c c u p i e d a day.
Only during the
p a s t l0 to 15 m i n u t e s of the h u m a n d a y h a s o u r l i f e
been a n y t h i n g but p r e c a r i o u s .
We a r e still b e g i n n e r s and
m a y hope to i m p r o v e . To deride the hope of p r o g r e s s is
the ultimate
f a t u i t y , t h e last word in the p o v e r t y of
spirit and m e a n n e s s of mind.'
This p a s s a g e s p e a k s of the need to p r o c e e d . The r e c o m b i n a n t DNA
b r e a k t h r o u g h has provided us with a new and p o w e r f u l a p p r o a c h to t h e
BERG
286
q u e s t i o n s t h a t have intrigued and plagued man for centuries.
one, would not shrink from that challenge.
I, for
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