Note: Descriptions are shown in the official language in which they were submitted.
115~166
The present invention relates to the isolation of a
specific nucleotide sequence which contains the genetic
information coding for a specific protein, the synthesis
of DNA having this specific nucleotide sequence and
transfer of the DNA to a microorganism host wherein the
DNA may be replicated. More specifically, the present
invention relates to the isolation of the insulin gene,
its purification, transfér and replication in a microbial
host and its subsequent characterization. Novel products
are produced according to the present invention. These
products include a recombinant plasmid containing the
specific nucleotide sequences derived from a higher
organism and a novel microorganism containing as part of
its genetic makeup a specific nucleotide sequence derived
from a higher organism.
The symbols and abbreviations used herein are set
forth in the following table:
DNA - deoxyribonucleic acid A - Adenine
RNA - ribonucleic acid T - Thymine
cDNA - complementary DNA G - Guanine
(enzymatically synthesized C - Cytosine
from an mRNA sequence) Tris - 2-Amino-2-
mRNA - messenger RNA hydroxyethyl-1,3-
tRNA - transfer RNA propanediol
dATP - deoxyadenosine triphosphate EDTA - ethylenediamine
dGTP - deoxyguanosine triphosphate tetraacetic acid
dCTP - deoxycytidine triphosphate ATP - adenosine
triphosphate
TTP - thymidine
triphosphate
DNA is a high molecular weight polymer of biological
origin. The structural units of the polymer are
-1-
~/
11561t~16
deoxyribonucleotides, each of which contains a purine or
pyrimidine base to which is linked deoxyribose having a
phosphate moety esterified at the 3' or 5' hydroxyl of the
deoxyribose. The polymer is constructed by the linking
together of deoxyribonucleotides by the formation of
phosphodiester bonds between the 5' position of one
nucleotide and the 3' of its next neighbor. A linear
polymer of nucleotides is thus formed, sometimes having at
one terminus a free 5' phosphate and/or at the other a free
3' phosphate. In some instances one or both of the phosphate
termini may be removed by hydrolysis leaving free 5' or
3' hydroxyl ends. The significant feature of this linkage
mode is that it results in a polynucleotide strand which is
directional in the sense that one end can be distinguished
from the other.
There are four purine or pyrimidine bases found in
the vast majority of DNA' S which have been analyzed. These
are the purines, adenine and guanine, and the pyrimidines,-
cytosine and thymine (hereinafter A, G, C, and T, respect-
ively). It is the specific sequence of the bases A, G, C,and T which confers the biological functions of DNA as the
repository of genetic information in a living cell.
The native confirmation of DNA is in the form of
paired polynucleotide strands of opposite directionality.
The strands are held together by the cooperative effect of
multiple hydrogen bonding between specific purine-
pyrimidine pairs. The molecular sizes and hydrogen bonding
angles are such that A and T form a specific pair and G and
C for a specific pair. As a result, the base sequence in
one strand of native DNA is mirrored by a complementary
sequence in the other strand, due to the base pairing
--2--
~; .
115~1~i6
relationships just described. By way of illustrating this
relationship, a heptanucleotide having the sequence
ACCGTTG, reading from the 5' end to the 3' end, will be
found paired with a complementary strand having the
sequence CAACGGT from 5' to 3'. By conventlon, however,
the native structure is depicted with one strand in the 5'
to 3' orientation and the complementary strand in the 3'
to 5' orientation:
5' ACCGTTG 31
3' TGGCAAC 5'
DNA may exist in several alternate states in
addition to the native configuration, a linear double
stranded polymer, as described above. It may also exist
as individual single strands and it may exist as a double
stranded molecule for a portion of its length but contain-
ing single stranded gaps or single stranded ends. Of
particular biological significance is the fact that DNA
commonly forms closed rings by the formation of phospho-
diester bonds between its opposite ends. The 5' end of
one strand joins to its 3' end by means of a phosphodiester
linkage, and a similar linkage is formed between the 5'
and 3' ends of the complementary strand. Such rings have
been found ranging in molecular weight from less than 106
to more than 1 x 109. Rings which are not covalently
closed can also be formed. A procedure for forming such
rings from double stranded linear DNA has been developed
in the prior art and will be described in detail below.
In general, the procedure involves the addition of
complementary sequences to either the 5' or 3' ends of the
linear molecule. Such complementary single strand
sequences are termed cohesive ends because they are
1 15~1~6
capable of pairing with each other by means of the
specific hydrogen bonded base pairing relationships des-
cribed. When such pairing occurs, under the appropriate
conditions of temperature, ionic strength and solvent
composition, a double stranded ring can be formed, held
in place by the hydrogen bonding interactions of the cohesive
ends. Similarly a small linear piece may be joined with a
large linear piece provided the two have cohesive ends of
complementary sequence, and the combination can also form
a closed ring if both ends of both molecules are mutually
cohesive. Enzyme reactions can form covalent bonds
joining the ends and stabilizing the structure.
The biological significance of the base sequence
of DNA, as previously stated, is as a repository of genetic
information. It is known that the sequence of bases in DNA
is used as a code specifying the amino acid sequence of all
proteins made by the cell. In addition, portions of the
sequence are used for regulatory purposes, to control the
timing and amount of each protein made. The nature of these
controlling elements is only partially understood. Finally,
the sequence of bases in each strand is used as a template
for the replication of DNA which accompanies cell division.
The manner by which base sequence information in
DNA is used to determine the amino acid sequence of
proteins is a fundamental process which, in its broad
outlines, is universal to all living organisms. It has
been shown that each amino acid commonly found in proteins
is determined by one or more trinucleotide or triplet
sequences. Therefore, for each protein, there is a
corresponding segment of DNA containing a sequence of
triplets corresponding to the protein amino acid sequence.
--4--
1 ~5~ 166
The genetic code is shown in the accompanying table.
In the process of converting the nucleotide sequence
information into amino acid sequence structure, a first
step, termed transcription~ is carried out. In this step,
a local segment of DNA having a sequence which specifies
the protein to be made is first copied with RNA. RNA is a
polynucleotide similar to DNA except that ribose is sub-
stituted for deoxyribose and uracil is used in place of
thymine. The bases in RNA are capable of entering into the
same kind of base pairing relationships that exist with
DNA. Consequently, the RNA transcript of a DNA nucleotide
sequence will be complementary to the sequence copied.
Such RNA is termed messenger RNA (mRNA) because of its
status as an intermidiary between the genetic apparatus
and the protein sythesizing apparatus of the cell.
Isolation of intact mRNA is technically extremely difficult
due to the presence of the enzyme RNase which catalyzes
the hydrolysis of the phosphodiester bonds in the
ribonucleotide sequence. This enzyme is ubiquitous,
extremely stable and highly active. The hydrolysis of
a single phosphodiester bond within the mRNA chain cannot
be tolerated since that would destroy the sequence
continuity necessary to preserve the genetic information.
Within the cell, mRNA is used as a template in a complex
process involving a multiplicity of enzymes and organelles
within the cell, which results in the synthesis of the
specified amino acid sequence. This process is referred
to as the translation of the mRNA.
There are often additional steps, called process-
ing, which are carried out to convert the amino acidsequence synthesized by the translational process into a
--5--
a~
Genetic Code
Phenylalanine(Phe) TTK Histidine(His) CAK
Leucine(Leu) XTY Glutamine(Gln) CAJ
Isoleucine(Ile) ATM Asparagine(Asn) AAK
Methionine(Met) ATG Lysine(Lys) AAJ
Valine(Val) GTL Aspartic acid(Asp) GAK
Serine(Ser) QRS Glutamic acid(Glu) GAJ
Proline(Pro) CCL Cysteine(Cys) TGK
- Threonine(Thr) ACL Tryptophan(Try) TGG
Alanine(Ala) GCL Arginine(Arg) WGZ
Tyrosine(Tyr) TAK Clycine(Gly) GGL
Termination signal TAJ
Termination signal TGA
Key: Each 3-letter triplet represents a trinucleotide of
mRNA, having a 5~ end on the left and a 3' end on the
right. The letters stand for the purine or pyrimidine
bases forming the nucleotide sequence.
A = adenine
G = guanine
C = cytosine
T = thymine
X = T or C if Y is A or G
X = C if Y is C or T
Y = A, G, C or T if X is C
Y = A or G if X is T
W = C or A if Z is A or G
W = C if Z is C or T
Z = A, G, C or T if W is C
Z = A or G if W is A
QR = TC if S is A, G, C or T
QR = AG if S is T or C
S = A, G, C or T if QR is TC
S = T or C if QR is AG
J = A or G
K = T or C
L = A, T, C or G
M = A, C or T
-5a-
,
functional protein. An example is provided in the case
of insulin.
The immediate precursor of insulin is a single
polypeptide, termed proinsulin, which contains the two
insulin chains A and B connected by another peptide, C.
See Steiner, D. F., Cunningham, D., Spigelman, L. and
Aten, B., Science 157, 697 (1967). Recently it has been
reported that the initial translation product of insulin
mRNA is not proinsulin itself, but a preproinsulin that
contains more than 20 additional amino acids on the amino
terminus of proinsulin. See Cahn, S. J., Keim, P. and
Steiner, D. F., Proc. Natl. Acad. Sci. USA 73, 1964 (1976)
and Lomedico, P.T. and Saunders, G.F., Nucl. Acids Res. 3,
381 (1976). The structure of the preproinsulin molecule
can be represented schematically as NH2-(pre-peptide)-
B chain-(C peptide)-A chain-COOH.
Many proteins of medical or research significance
are found in or made by the cells of higher organisms such
as vertebrates. These include, for example, the hormone
insulin, other peptide hormones such as growth hormone,
proteins involved in the regulation of blood pressure, and
a variety of enzymes having industrial, medical or
research significance. It is frequently difficult to
obtain such proteins in usable quantities by extraction
from the organism, and this problem is especially acute in
the case of proteins of human origin. Therefore there is
a need for techniques whereby such proteins can be made
by cells outside the organism in reasonable quantity.
In certain instances, it is possible to obtain appropriate
30 cell lines which can be maintained by the techniques of
tissue culture. However, the growth of cells in tissue
--6--
,`.'
1~5~
culture is slow, the medium is expensive, conditions must
be accurately controlled, and yields are low. Moreover,
it is often difficult to maintain a cultured cell line
having the desired differentiated characteristics.
In contrast, microorganisms such as bacteria are
relatively easy to grow in chemically defined media.
Fermentation technology is highly advanced, and can be
well controlled, Growth of organisms is rapid and high
yields are possible. In addition, certain microorganisms
have been thoroughly characterized genetically and in fact
are among the best characterized and best understood
organisms.
Therefore it is highly desirable to achieve the
transfer of a gene coding for a protein of medical signifi-
cance, from an organism which normally makes the protein
to an appropriate microorganism. In this way it is possible
that the protein could eventually be made by the micro-
organism, under controlled conditions of growth, and
obtained in the desired quantities. It is also possible
that substantial reductions in the over-all costs of
producing the desired protein could be achieved by such
a process. In addition, the ability to isolate and
transfer the genetic sequence which determines the pro-
duction of a particular protein into a microorganism having
a well-defined genetic background could provide a research
tool of great value to the study of how the synthesis of
such a protein is controlled and how the protein is
processed after synthesis.
The present invention provides a means for
achieving the above recited goals. A process is disclosed
involving a complex series of steps involving enzyme-
--7--
6 --
catalyzed reactions. The nature of these enzyme reactions
as they are understood in the prior art is described
herewith.
Reverse transcriptase catalyzes the synthesis of
DNA complementary to an RNA template strand in the presence
of the RNA template, an oligodeoxynucleotide primer and the
four deoxynucleoside triphosphates, dATP, dGTP, dCTP, and
TTP. The reaction is initiated by the non-covalent bonding
of the oligo-deoxynucleotide primer to the 3' end of mRNA
followed by stepwise addition of the appropriate
deoxynucleotides, as determined by base pairing relation-
ships with the mRNA nucleotide sequence, to the 3' end of
the growing chain. The product molecule may be described
as a hairpin structure containing the original RNA together
with a complementary strand of DNA joined to it by a single
stranded loop of DNA. Reverse transcriptase is also
capable of catalyzing a similar reaction using a single
stranded DNA template, in which case the resulting product-
is a double stranded DNA hairpin having a loop of single
stranded DNA joining one set of ends. See Aviv, H. and
Leder, P., Proc. Natl. Acad. Sci USA 69, 1408 (1972) and
Efstratiadis, A., Kafatos, F. C., Maxam, A. F. and
Maniatis, T., Cell 7, 279 (1976).
Restriction endonucleases are enzymes capable of
hydrolyzing phosphodiester bonds in double stranded DNA,
thereby creating a break in the continuity of the DNA
strand. If the DNA is in the form of a closed loop, the
loop is converted to a linear structure. The principal
feature of an enzyme of this type is that its hydrolytic
action is exerted on~y at a point where a specific
--8--
nucleotide sequence occurs. Such a sequence is termed
the recognition site for the restriction endonuclease.
Restriction endonucleases from a variety of sources have
been isolated and characterized in terms of the nucleotide
sequence of their recognition sites. Some restriction
endonucleases hydrolyze the phosphodiester bonds on both
strands at the same point, producing blunt ends. Others
catalyze hydrolysis of bonds separated by a few nucleotides
from each other, producing free single stranded regions at
each end of the cleaved molecule. Such single stranded
ends are self-complementary, hence conhesive, and may be
used to rejoin the hydrolyzed DNA. Since any DNA suscept-
ible of cleavage by such an enzyme must contain the same
recognition site, the same cohesive ends will be produced,
so that it is possible to join heterologous sequences of
DNA which have been treated with restriction endonuclease
to other sequences similarly treated. See Roberts, R.J.,
Crit. Rev. Biochem 4, 123 (1976). Restriction sites are
relatively rare, however the general utility of restriction
endonucleases have been greatly amplified by the chemical
synthesis of double stranded oligonucleotides bearing the
restriction site sequence. Therefore virtually any segment
of DNA can be coupled to any other segment simply by
attaching the appropriate restriction oligonucleotide to the
the ends of the molecule, and subjecting the product
to the hydrolytic action of the appropriate restriction
endonuclease, hereby producing the requisite cohesive ends,
See Heyneker, H. L., Shine, J., Goodman, H. M., Boyer, H.W.,
Rosenberg, J., Dickerson, R. E., Narang, S. A., Itakura,
K., Lin, S. and Riggs, A. D., Nature 263, 748 (1976) and
1~
S ~
Scheller, R. H., Dickerson, R. E., Boyer, H. W~, Riggs,
A. D. and Itakura, K., Science 196, 177 (1977).
Sl endonuclease is an enzyme of general specificity
capable of hydrolyzing the phosphodiester bonds of single
stranded DNA or of single stranded gaps or loops in other-
wise double stranded DNA. See Vogt, V. M., Eur. J. Biochem,
33, 192 (1973).
DNA ligase is an enzyme capable of catalyzing the
formation of a phosphodiester bond between two segments
of DNA having a 5' phosphate and a 3' hydroxyl, respectively,
such as might be formed by two DNA fragments held together
by means of cohesive ends. The normal function of the
enzyme is thought to be in the joining of single strand
nicks in an otherwise double stranded DNA molecule.
However, under appropriate conditions, DNA ligase is capable
of catalyzing blunt end ligation in which two molecules
having blunt ends are covalently joined. See Sgaramella,
V., Van de Sande, J. H., and Khorana, H. G., Proc. Natl.
Acad. Sci. USA 67, 1468 (1970).
Akaline phosphatase is an enzyme of general
specificity capable of hydrolyzing phosphate esters includ-
ing 5' terminal phosphates on DNA.
A further step in the overall process to be des-
cribed is the insertion of a specific DNA fragment into
a DNA vector, such as a plasmid. Plasmid is the term
applied to any autonomously replicating DNA unit which
might be found in a microbial cell, other than the genome of
the host cell itself. A plasmid is not genetically linkPd
to the chromosome of the host cell. Plasmid DNA's exist
as double stranded ring molcecules generally on the order
of a few million molecular weight, although some are
--10--
,.. .
1 1 5~ ~ 66
greater than 10% molecular weight, and they usually re-
present only a small percent of the total DNA of the cell.
Plasmid DNA is usually separable from host cell DNA by
virtue of the great difference in size between them.
Plasmids can replicate independently of the rate of host
cell division and in some cases their replication rate
can be controlled by the investigator by variations in
the growth conditions. Although the plasmid exists as
a closed ring, it is possible by artificial means to
introduce a segment of DNA into the plasmid, forming a
recombinant plasmid with enlarged molecular size, without
substantially affecting its ability to replicate or to
express whatever genes it may carry. The plasmid therefore
serves as a useful vector for transferring a segment of
DNA into a nèw host cell. Plasmids which are useful for
recombinant DNA technology typically contain genes which
may be useful for selection purposes, such as genes for
drug resistance.
In addition to the specialized techniques of the
20 prior art just described, the present work also entails
the use of numerous conventional techniques known in the
art including chromatography, electrophoresis, centrifuga-
tion, solvent extraction, and precipitation. Reference is
made to such specific techniques in the examples.
For general background see Watson, J.D., The
Molecular Biology of the Gene, 3d Ed., Benjamin, Menlo
Park, California, (1976); Davidson, J.N., The Biochemistry
of the Nucleic Acids, 8th Ed., Revised by Adams, R.L.P.,
Burdon, R.H., Campbell, A.M. and Smellie, R.M.S.,
Academic Press, New York, (1976); and Hayes, W., "The
Genetics of Bacteria and Their Viruses", Studies in
--11--
.~
. S 6
Basic Genetics and Molecular Biology, 2d Ed., Blackwell
Scientific Pub., Oxford tl968).
To illustrate the practice of the present
invention, the isolation and transfer of the rat insulin
gene is described in detail. Insulin was chosen for this
effort because of its central significance from the stand-
point of clinical medicine, and from the standpoint of
basic research. The disclosed procedure is applicable by
those of ordinary skill inthe art to the isolation of the
insulin gene of other organisms, including humans.
Insulin was first isolated in 1922. At the present
time, the use of this hormone in the treatment of diabetes
is well-known. Although slaughterhouses provide beef and
pig pancreases as insulin sources, a shortage of the
hormone is developing as the number of diabetics increases
worldwide. Moreover, some diabetics develop an allergic
reaction to beef and pig insulin, with deleterious effects.
The ability to produce human insulin in quantities suffic-
ient to satisfy world need is therefore highly desirable.
Manufacturing human insulin in bacteria is a technique
which could achieve this desired goal. However, prior to
the present invention, progress toward this desired goal
has been thwarted by the fact that no technique has been
developed to introduce the insulin gene into a bacteria.
The present invention provides such a technique.
Further research is required before it is possible
to make proteins, like insulin, on a commercial scale from
bacteria which have received a specific DNA sequence that
is the genetic determinant of that protein. Whether or
not a gene within a cell makes protein depends on many
factors, including the position and orientation of the
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1 15~1~6
DNA relative to special sequences of the host DNA thattell the host cell when to start and stop making protein.
The first steps, isolating the appropriate gene and trans-
ferring it to bacteria, are now achievable by the processes
of the present invention. These processes are described
in detail for the insulin gene.
In addition to its direct usefulness in the pro-
duction of proteins of therapeutic interest by micro-
organisms, the process of the present invention in
research is designed to gain a further understanding of
the expression of insulin genes in normal and pathological
states such as diabetes. Little is currently known about
the nature of such control. Although insulin is composed
of two polypeptide chains, designated A and B, it is the
product of a single gene. Insulir is produced specifically
by certain endocrine cells, termed B cells, in the
pancreas. The s cells are found as part of certain
histologically distinct structures within the pancreas
known as the islets of Langerhans, where they comprise
the majority of cells.
The ability to obtain DNA having a specific sequence
which is the genetic code for a specific protein makes it
possible to modify the nucleotide sequence by chemical
or biological means such that the specific protein
ultimately produced is also modified. This would make it
possible to produce, for example, a modified insulin
tailored to suit a specific medical need. The genetic
capacity to produce any insulin-related amino acid sequence
having the essential functional properties of insulin may
therefore be conferred upon a microorganism.
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115~1~i6
The ability to transfer the genetic code for a
genetic protein necessary to the normal metabolism of
a particular higher organism to a microorganism such as
a bacterium opens significant possibilities for culture
production of such proteins. This in turn affords
significant possibilities for augmenting or replacing
the output of such proteins with those produced by
microorganisms altered pursuant to this invention,
whenever the ability of the higher organism to function
normally in the production of such proteins has been
impaired, and suggest, e.g., the possibility of
establishing symbiotic relationships between microorganisms
produced pursuant to this invention and human beings with
chronic or acute deficiency diseases, whereby microorganisms
genetically altered as herein taught might be implanted in
or otherwise associated with a human to compensate for the
pathologic deficiency in the metabolism of the latter.
SUMMARY OF THE INVENTION
A process is disclosed for isolating a specific
nucleotide sequence containing genetic information,
synthesis of DNA having the specific nucleotide sequence
and transfer of the DNA to a host microorganism. While
the invention is particularly exemplified by a specific
DNA sequence transferred to a bacterium and containing
the structural gene for rat preproinsulin, it is con-
templated that the method is of good applicability to the
transfer of any desired DNA sequence from a higher organism,
such as a vertebrate, to any microorganism. A higher
organism is here defined as any eucaryotic organism having
differentiated tissues, including but not limited to,
insects, molluscs, plants, vertebrates including mammals,
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1 1 5 ~
the latter category including cattle, swine, primates
and humans. A microorganism, as is understood in the art,
may be any microscopic living organism such as is included
in the term, protist, whether procaryotic or eucaryotic,
including for example bacteria, protozoa, algae and fungi,
the latter category including yeasts. In the process,
a selected cell population is first isolated by an improved
method. Intact mRNA is extracted from the cells by a novel
procedure whereby virtually all RNAse activity is suppress-
ed. Intact messenger RNA is purified from the extract by
column chromatography and subjected to the action of the
enzyme reverse transcriptase acting in the presence of the
four deoxynucleoside triphosphates needed to synthesize a
complementary (cDNA) strand. The product of this first
reaction with reverse transcriptase is subjected to a
procedure which selectively removes the ribonucleotide
sequence. The remaining deoxynucleotide sequence,
complementary to the original mRNA, is incubated in a
second reaction with reverse transcriptase or DNA poly-
merase in the presence of the four deoxynucleoside
triphosphates. The resulting product is a duplex cDNA
structure having its complementary strands joined to-
gether at one end by a single stranded loop. This product
is then treated with single strand specific nuclease which
cleaves the single stranded loop. The resulting double
stranded cDNA is next extended in length by the addition
at both ends of a specific DNA containing a restriction
enzyme recognition site sequence. The addition is cataly-
zed by a DNA ligase enzyme. The extended cDNA is next
treated with a restriction endonuclease, producing self-
complementary single stranded ends at the five-prime
-15-
~ 1 5 ~
termini of each strand in the duplex.
A plasmid DNA having a recognition site for the
same restriction endonuclease is treated with the enzyme,
in order to cleave the polynucleotide strand and produce
self-complementary single strand nucleotide sequences at
the 5' termini. ~he S' terminal phosphate groups on the
single stranded ends are removed to prevent the plasmid
from forming a ring structure capable of transforming
a host cell. The prepared cDNA and plasmid DNA are in-
cubated together in the presence of DNA ligase. Under thereaction conditions described, the formation of a viable
closed ring of plasmid DNA can only occur if a segment of
cDNA is included. The plasmid containing the cDNA sequence
is then introduced into an appropriate host cell. Cells
which have received a viable plasmid are detected by the
appearance of colonies having a genetic trait conferred
by the plasmid, such as drug resistance. Pure bacterial
strains containing the recombinant plasmid having the
incorporated cDNA sequence are then grown up, and the
recombinant plasmid reisolated. Large amounts of re-
combinant plasmid DNA may be prepared in this manner
and the specific cDNA sequence reisolated therefrom by
endonucleolytic cleavage with the appropriate restriction
enzyme.
The basic process of the invention is applicable
to the isolation and transfer to a host microorganism of
any desired nucleotide sequence obtained from a higher
organism, including man. It is contemplated that the
method will be useful in the transfer of a gene coding
for a specific protein which may have medical or industrial
-16-
115~1~6
value. In demonstrating the invention, the nucleotide
sequence coding for insulin has been isolated from rat,
tranferred to a bacteria and replicated herein. The
method is applicable to the transfer of a nucleotide
sequence isolated from a human source.
DETAILED DESCRIPTION OF THE INVENTION
The present invention subsumes a method of the
isolation of a DNA molecule of specific nucleotide sequence
and its transfer to a microorganism, wherein the original
nucleotide sequence of the DNA is found after replication
in the transferee organism.
The sequence of steps comprising the process of
the invention can be classified in four general categories:
1. _e isolation of a desired cell population
from a higher organism. There are two potential sources
of a genetic sequence coding for specific protein: the
DNA of the source organism itself, and an RNA transcript
of the DNA. The current safety requirements of the
National Institute of Health specify that human genes
of any kind can be put into recombinant DNA, and then
into bacteria, only after the genes have been very
carefully purified or in special high-risk (P4) facilities.
See Federal Register, Vol. 41, No. 131, July 7, 1967,
pp. 27902-27943. Therefore, for any procedure having
potential utility for the production of the human protein,
such as the present process, the preferred approach is the
isolation of specific mRNA having a nucleotide sequence
which codes for the desired protein. The adoption of this
strategy has the further advantage that the mRNA can be
- -17-
. . ,
more easily purified than DNA extracted from the cell.In particular, it is possible to take advantage of the
fact that in highly differentiated organisms such as
vertebrates, it may be possible to identify a specific
population of cells having a specific location within
the organism, whose function is primarily devoted to
the production of the protein in question. Alternatively,
such a population may exist during a transient developmental
stage of the organism. In such cell populations, a large
10 portion of the mRNA isolated from the cells will have the
desired nucleotide sequence. Therefore, the choice of cell
population is to isolate, and the method of isolation
employed, can be substantially advantageous from the
standpoint of the initial purity of the mRNA isolated
therefrom.
The process employed herein is a modification of
the procedure of Lacy, P. E., and Kostianovsky, M.,
Diabetes 16, 35 (1967). Details of the present procedure
are given in Example 1. The process is applicable, within
the scope of ordinary skill in the art, to the isolation of
islet cells from human pancreas. Careful attention to
exact procedural detail is essential. The most important
details include the use of silicone-treated glassware, the
use of empirically-selected collagenase, the use of the
proper ratio of incubation volume to container size, the
use of a proper shaking rate and careful visual observation
of the course of collagenase digestion to ensure that the
reaction has proceeded to the optimum extent. The process
of the present invention has the advantage of improved
reproducibility and permits successful isolation of islet
cells on a larger scale than heretofore practicable.
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.~.~
i.A~I .
l 15~6
Insulin-producing cells may be derived from other
sources, such as fetal calf pancreas or cultured islet
tumor cells. The isolation of pure islet cells will be
much simpler in such cases, especially where pure cell
cultures are used. The method of isolating islet cells
described supra would not be needed in such cases, however,
the method remain advantageous because of its general
applicability.
2. Extraction of mRNA. An important feature of the
present invention is the essentially complete removal of
RNase activity in the cell extract. The mRNA to be extract-
ed is a single polynucleotide strand, unpaired with any
complementary strand. Therefore, the hydrolytic cleavage
of a single phosphodiester bond in the sequence would
render the entire molecule useless for the purpose of
transferring an intact genetic sequence to a microorganism.
As stated hereinabove, the enzyme RNase is widely dis-
tributed, active and exceptionally stable. It is found
on the skin, survives ordinary glassware washing techniques
and sometimes contaminates stocks of organic chemicals.
The difficulties are especially acute in dealing with
extracts of pancreas cells, since the pancreas is a
source of digestive enzymes and is, therefore, rich in
RNase. However, the problem of RNase contamination is
pre~ent in all tissues, and the method disclosed hereln
to eliminate RNase activity is applicable to all tissues.
The exceptional effectiveness of the method is demonstrated
in the present invention by the successful isolation of
intact mRNA from isolated islet cells of the pancreas.
--19--
) G
RNase is effectively inhibited during isolation
from intact cells by homogenizing a cell preparation in
guanidinium thiocyanate buffered to low pH and containing
mercaptoethanol. Other guanidinium salts such as
guanidinium hydrochloride may be used, but they are less
effective denaturing agents. The use of guanidinium
hydrochloride has been suggested by Cox, R.A., Methods in
Enzymology 12, Part B, pp. 120-129 (1968), but the present
workers have found this method inadequate for the purpose
herein described. Mercaptoethanol further reduces RNase
activity by disrupting its intermolecular disulfide bonds.
The combination of mercaptoethanol with a patent denaturing
agents such as guanidinum thiocyanate, pursuant to this
invention, tends to enhance the effectiveness of
mercaptoethanol by rendering the inactivation of RNase
essentially irreversible. The pH may be varied in the
range of 5.0-8Ø The preferred pH is 5.0 because of its
effect in inducing a more tightly folded conformation
of the RNA, thereby rendering it more resistant to RNase
activity.
Following the homogenization step, the RNA is
separated from the bulk of the cellular protein and DNA.
A variety of procedures has been developed for this
purpose, any of which is suitable, all of which are well-
known in the art. A common practice in the prior art
is to use an ethanol precipitation procedure which
selectively precipitates RNA. The preferred technique
of the present invention is to bypass the precipitation
step and layer the homogenate directly on a solution of
5.7 M cesium chloride in a centrifuge tube and then to
subject the tube to centrifugation as described in
-20-
. ~
Glisin, V., Crkvenjakov, R., and Byus, C., Biochemistry 13,2633 (1974). This method is preferred because an environ-
ment continuously hostile to RNase is maintained and RNA
is recovered in high yield, free of DNA and protein.
The above recited procedures result in the
purification of total RNA from the cell homogenate. However,
only a portion of such RNA is the desired mRNA. In order
to further purify the desired mRNA, advantage is taken of
the fact that in the cells of higher organisms, mRNA,
after transcription, is further processed in the cell by
the attachment of polyadenylic acid. 5uch mRNA containing
poly A sequences attached thereto may be selectively
isolated by chromatography on columns of cellulose to
which is attached oligo-thymidylate, as described by Aviv,
H., and Leder, P., supra. The foregoing procedures are
sufficient to provide essentially pure, intact, translat-
able mRNA from sources rich in RNase. The purification
of mRNA and subsequent in vitro procedures may be carried
out in essentially the same manner for any mRNA, regardless
of the source organism.
Under certain circumstances, for example when
tissue culture cells are used as the mRNA source, RNase
contamination may be sufficiently low that the RNase in-
hibition method just described will not be needed. In
such cases, prior art techniques for reducing RNase
activity may be sufficient.
3. Formation of cDNA. Reference is made to Fig. 1
for a schematic representation of the remaining steps of
the process. The first step in this process is the
formation of a sequence of DNA complementary to the
purified mRNA. The enzyme of choice for this reaction is
-21-
reverse ~ranscriptase, although in principle any enzymecapable of forming a faithful complementary D~A strand
using the mRNA as a template could be used. The reaction
may be carried out under conditions described in the prior
art, using mRNA as a template and a mixture of four
deoxynucleoside triphosphates as precursors for the
DNA strand. It is convenient to provide that one of the
deoxynucleoside triphosphates be labeled with P in the
alpha position in order to monitor the course of the
reaction, provide a tag for recovering the product a~ter
separation procedures such as chromatography and electro-
phoresis, and for the purpose of making quantitative
estimates of recovery. See Efstratiadis, A., et al.,
supra.
As diagrammed in the figure, the product of the
reverse transcriptase reaction is a double stranded hairpin
structure with non-covalent linkage between the RNA strand
and the DNA strand.
The product of the reverse transcriptase reaction
is removed from the reaction mixture by standard technlques
known in the art. It has been found useful to employ a
combination of phenol extraction, chromatography on
Sephadex 1/ G-100 and ethanol precipitation.
Once the cDNA has been enzymatically synthesized,
the RNA template may be removed. Various procedures are
known in the prior art for the selective degradation of
RNA in the presence of DNA The preferred method is
alkaline hydrolysis, which is highly selective and can be
readily controlled by pH adjustment.
_/ Tradmark, Pharmacia Inc., Uppsala, Sweden
-22-
~'
Following the alkaline hydrolysis reaction and
subsequent neutralization, the P labeled cDNA may be
concentrated by ethanol precipitation if desired.
Synthesis of a double stranded hairpin cDNA is
accomplished by the use of an appropriate enzyme, such as
DNA polymerase or reverse transcriptase. Reaction con-
ditions similar to those described previously are employed,
including the use of an ~- P labeled nucleoside tri-
phosphate. Reverse transcriptase is available from a
variety of sources. A convenient source is avian
myeloblastosis virus. The virus is available from
Dr. D.J. Beard, Life Sciences Incorporated, St. Petersburg,
Florida, who produces the virus under contract with the
National Institutes of Health.
Following the formation of the cDNA hairpin, it
may be convenient to purify the DNA from the reaction
mixture. As described previously, it has been found
convenient to employ the steps of phenol extraction,
chromatography on Sephadex G-100 and ethanol precipitation
to purify the DNA product free of contaminating protein.
The hairpin structure may be converted to a
conventional double stranded DNA structure by the removal
of the single stranded loop joining the ends of the
complementary strands. A variety of enzymes capable of
specific hydrolytic cleavage of single stranded regions
of DNA is available for this purpose. A convenient enzyme
for this purpose is the Sl nuclease isolated from
Aspergillus oryzae. The enzyme may be purchased from
Miles Research Products, Elkhart, Indiana. Treatment of
the hairpin DNA structure with Sl nuclease results in a
high yield of cDNA molecules with base paired ends. After
-23-
. ~
1 1 5 ~
the extraction, chromatography and ethanol precipitationas previously described. The use of reverse transcriptase
and Sl nuclease in the synthesis of double stranded cDNA
transcripts of mRNA has been described by Efstratiadis et
al., supra.
The next step in the process involves the treatment
of the ends of the cDNA product to provide appropriate
sequences at each end containing a restriction endonuclease
recognition site. The choice of DNA fragment to be added
to the ends is determined by matters of manipulative
convenience. The sequence which is to be added to the ends
is chosen on the basis of the particular restriction
endonuclease enzyme chosen, and this choice in turn depends
on the choice of D~A vector with which the cDNA is to be
recombined. The plasmid chosen should have at least one
site susceptible to restriction endonuclease cleavage.
For example, the plasmid pMB9 contains one restriction
site for the enzyme Hind III. Hind III is isolated from
Hemophilus influenzae and purified by the method of
Smith, H. O., and Wilcox, K. W., J. Mol.Biol, 51, 379
(1970). The enzyme Hae III, from Hemophilus aegypticus
is purified by the method of Middleton, J.H., Edgell,
M. H., and Hutchison III, C. A., J. Virol, 10, 42 (1972).
An enzyme from Hemophilus suis, designated Hsu I, catalyzes
the same site-specific hydrolysis, at the same recognition
site, as Hind III. These two enzymes are therefore
considered as functionally interchangeable.
It is convenien-t to employ a chemically synthesized
double stranded decanucleotide containing the recognition
sequence for Hind III, for the purpose of attachment to
the ends of the cDNA duplex. The double stranded
-24-
~' .
115~3.~6
decanucleotide has the sequence shown in Figure l. SeeHeynecker, H. L., et al., and Scheller, R. H., et al.,
supra. A variety of such synthetic restriction site
sequences is available to workers in the art, so that it
is possible to prepare the ends of a duplex DNA so as to be
sensitive to the action of any of a wide variety of
restriction endonucleases.
The attachment of restriction site sequences to
the ends of cDNA may be accomplished by any step known to
lO workers in the art. The method of choice is a reaction
termed blunt end ligation, catalyzed by DNA ligase purifed
by the method of Panet, A., et al., Biochemistry 12, 5045
(1973). The blunt end ligation reaction has been described
by Sgaramella, V., et al., supra. The product of the blunt
end ligation reaction between a blunt ended cDNA and a
large molar excess of double stranded decanucleotide
containing the Hind III endonuclease restriction site is
a cDNA having Hind III restriction site sequences at each
end. Treatment of the reaction product with Hind III
20 endonuclease results in cleavage at the restriction site
with the formation of single stranded 5' self-complementary
ends, as shown in figure 1.
4. Formation of a recombinant vector. In
principle, a wide variety of viral and plasmid DNA's could
be used to form recombinants with a cDNA prepared in the
manner just described. The principal requirements are that
the DNA be capable of entering a host cell, undergoing
replication in the host cell and should, in addition,
have a genetic determinant through which it is possible
30 to select those host cells which have received the vector.
For reasons of public safety, however, the range of choice
should be restricted to those vector species deemed
-25-
. '/j
il5~1~6
suitable for the type of experiments employed, in accordingwith the NIH guidelines, supra. The list of approved DNA
vectors is continuously being enlarged, as new vectors are
developed and approved by the NIH Recombinant DNA Safety
Committee, and it is to be understood that this invention
contemplates the use of any viral and plasmid DNA's that
have the described capabilities, including those on which
NIH approval may later be granted. Suitable vectors which
are currently approved for use include a variety of
10 derivatives of bacteriophage lambda (See e.g. Blattner,
F. R., Williams, B. G., Bluckl, A. E., Denniston-Thompson,
K., Faber, H. E., Furlong, L. A., Grunwald, D. J., Kiefer,
D. O., Moore, D. D., Schumm, J. W., Sheldon, E. L., and
Smithies, O., Science 196, 161 (1977) and derivatives
. _
of the plasmid col El, see e.g. Rodriguez, R. L., Bolivar,
S., Goodman, H. M., Boyer, H. W., and Betlach, M. N.,
ICN-UCLA Symposium on Molecular Mechanisms In The Control
of Gene Expression, D. P. Nierlich, W. J. Rutter, C. E.
Fox, Eds. (Academic Press, NY, 1976), pp. 471-477.
20 Plasmids derived from col El are characterized by being
relatively small, having molecular weights of the order of
a few million, and having the property that the number of
copies of plasmid DNA per host cell can be increased from
20-40 under normal conditions to 1000 or more, by treatment
of the host cells with chloramphenicol. The ability to
amplify the gene dosage within the host cell makes it
possible under appropriate circumstances, under the
control of the investigator, to cause the host cell to
produce primarily proteins coded for by genes carried on
30 the plasmid. Such derivatives of col El are therefore
preferred in the process of the present invention.
-26-
Suitable derivaties of col El include the plasmids pMB-9,
carrying the gene for tetracycline resistance, and pBR-313,
pBR-315, pBR-316, pBR-317 and pBR-322, which contain, in
addition to the tetracycline resistance gene, a gene for
ampicillin resistance. The presence of the drug resistance
genes provides a convenient way for selecting cells which
have been successfully infected by the plasmid, since
colonies of such cells will grow in the presence of the
drug whereas cells which have not received the plasmid will
10 not grow or form colonies. In the expermients described
herein as specific examples of the present invention, a
plasmid derived from col El was used throughout, containing,
in addition to the described drug resistance marker, one
Hind III site.
As with the choice of plasmid, the choice of a
suitable host is in principle very broad but for purposes
of public safety, narrowly restricted. A strain of E.
Coli designated X-1776 has been developed and has received
NIH approval for processes of the type described herein.
20 See Curtiss, III, R., Ann. Rev. Microbiol., 30, 507 (1976).
As in the case of the plasmids, it will be understood that
the invention contemplates use of any host cell strains
having the capability of acting as a transferee for the
chosen vector, including protists other than bacteriaj for
example yeasts, whenever such strains are approved for
use under the NIH guidelines.
Recombinant plasmids are formed by mixing restrict-
ion endonuclease-treated plasmid DNA with cDNA containing
end groups similarly treated. In order to minimize the
30 chance that segments of cDNA will form combinations with
each other, the plasmid DNA is added in molar excess over
-27-
115~
the cDNA. In prior art procedures this has resulted inthe majority of plasmids circularizirg without an inserted
cDNA fragment. The subsequently transformed cells contained
mainly plasmid and not cDNA recombinant plasmids. As a
result, the selection process was very tedious and time
consuming. The prior art solution to this problem has
been to attempt to devise DNA vectors having a restriction
endonuclease site in the middle of a suitable marker gene
such that the insertion of a recombinant divides the gene
10 thereby causing loss of the function coded by the gene.
Preferably, a method for reducing the number of
colonies to be screened for recombinant plasmids is employ-
ed. The method involves treating plasmid DNA cut with the
restriction endonuclease with alkaline phosphatase, an
enzyme commercially available from several sources, such
as Worthington Biochemical Corporation, Freehold, New
Jersey. Alkaline phosphatase treatment removes the 5'-
terminal phosphates from the endonculease generated ends
of the plasmid and prevents self-ligation of the plasmid
20 DNA. Consequently, circle formation, hence transformation,
will be dependent on the insertion of a DNA fragment con-
taining 5'-phosphorylated termini. The described process
reduces the relative frequency of transformation in the
absence of recombination to less than 1 to 10+4.
For the purpose of illustrating the above described
procedures, cDNA coding for rat insulin has been isolated
and recombined with a plasmid. The DNA molecules were
used to transform E. COLl X-1776. Transformants were
selected by growth on medium containing tetracycline.
30 One recombin2nt plasmid DNA obtained from transformed
cells was found to contain an inserted DNA fragment
-28-
1 ~ 5 ~
approximately 410 nucleotides in length. Other recombinants,isolated by similar procedures, were also obtained and
analyzed. The inserted fragments were released from the
plasmid by Hind III or HSU I endonuclease digestion and
were subjected to DNA sequence analysis by the method of
Maxam, A. M. and Gilbert, W., Proc. Natl. Acad. Sci. USA 74,
560 (1977). The nucleotide sequences of the inserted DNA
fragments were found to be overlapping and to contain the
entire coding region for rat proinsulin I, as well as 13 out
10 of 23 amino acids of the prepeptide sequence. A composite
of the nucleotide sequence of this region was constructed
as shown hereinafter.
The process just described is generally applicable
to the isolation and purification of a gene from a higher
organism, including a human gene, and its transfer to and
replication in a microorganism. Novel recombinant plasmids
containing all or a portion of the isolated gene are des-
cribed. Novel microorganisms hitherto unknown in nature are
described, having as part of their genetic makeup a gene
20 from a higher organism. Specific examples detailing each
step of the process as applied to the isolation, purificat-
tion and transfer of the rat insulin gene into E. coli
will next be described, in order to more clearly reveal
the characteristics and utility of the invention. The
following examples characterize recombinant plasmids
containing portions of the rat insulin gene, and novel
microorganisms containing portions of the rat insulin gene.
Example 1
The described procedures demonstrate the extraction
30 and isolation of rat insulin mRNA, the synthesis of a DNA
complementary thereto and the characterization of the
29-
11S~6
complementary DNA. To prepare purified rat islet cells,the pancreas of an anesthetized rat was infused with Hank's
salt solution by retrograde infusion into the pancreatic
duct. Hank's salt solution is a standard salt solution
mixture known in the art and available from a number of
commercial sources, such as Grand Island Biological Supply
Company, Grand Island, New York. The pancreas was then
removed, minced in Hank's solution at 0C and digested
with collagenase and soybean trypsin inhibitor. All
procedures were conducted at 0C-4C unless otherwise
specified. The conditions of the digestion procedure
were extremely critical. Two minced rat pancreases in an
8 ml total volume in Hank's medium were placed in a 30 ml
glass tube. All glass tubes were pretreated with
silicone.-/ The incubation mixture contained 12 mg
collagenase, an enzyme prepared from Clostridium
histolyticum, essentially by the method of Mandl, I.,
Mackennan, J. D. and Howes, E. L., J. Clin. Invest. 32,
1323 (1943), type CLS IV, obtained from Worthington
Biochemical Corporation, Freehold, New Jers~y, and 1 mg
soybean trypsin inhibitor obtained from Sigma Chemical
Company, St. Louis, Missouri. Incubation was carried out
at 37C for 25 minutes with shaking at the rate of 90
strokes per minute. Continuous inspection was required
to insure that the collagenase digestion had proceeded to
an optimal extent. If the incubation was too short, the
islet cells were incompletely released and if the lncubation
too long the islet cells would begin to lyse. Following
.
~/ Slllclad, trademark, Clay- Adams Division, Becton-
Dickinson Inc., Parsippany, New Jersey
--~0--
1 lS~ 1~6
incubation the tube was centrifuged for 1 minute at 200 x G.
The supernatant was decanted and the pellet washed with
Hank's solution, and this procedure was repeated five
times. After the final centrifugation, the pellet was
suspended in 15 ml. Ficoll, 3/ having a density of 1.085.
- A layer of 8 ml. Ficoll of density 1.080 was added, a layer
of 5 ml. Ficoll of density 1.060 was added, and the tube was
centrifuged in a swinging bucket rotor for 5 minutes at
500 x G followed by 5 mintues at 2000 x G. As a result of
10 the foregoing process, acinar cells remined at the bottom
of the tube and islet cells rose in the fradient and formed
a band between the two top layers. The islet cell band
contained contaminating gaglion cells, lymph nodes, and
connective tissue. Large contaminating fragments were
removed from the material in the band. The reminder of the
preparation was placed under a dissecting microscope
where visible contaminating materials were removed by
hand using a micropipette. The cell preparation was then
diluted in Hank's solution and centrifuged. The super-
20 natant was decanted and the cell pellet stored frozen inliquid nitrogen.
Islet cells pooled from 200 rats was homogenized
in 4 M guanidinium thiocyanate -/ containing 1 M~
mercaptoethanol buffered to pH 5.0 at 4C. The homogenate
was layered over 1.2 ml, 5.7 M CsCl containing 100 mM EDTA
and centrifuged for 18 hours at 37,000 rpm in the SW 50.1
rotor of a Beckman Ultracentrifuge at 15C (Beckman
Instrument Company,Fullerton, California). RNA traveled
to the bottom of the tube.
_/ Trademark, Pharmacia Chemical Company, Uppsala, Sweden
4/ Tridom, trademark, Fluka AG Chemische Fabrik, Buchs,
Switzerland. -31-
Polyadenylated RNA was isolated by chromatography
of the total RNA preparation on oligo(dT)-cellulose
according to the procedure of Aviv, H., and Leder, P.,
supra.
Avian myeloblastosis virus reverse transcriptase,
provided by D. J. Beard, Life Science Inc., St. Petersburg,
Florida, was used to transcribe total polyadenylated RNA
from rat islets of Langerhans into cDNA. The reactions
were carried out in 50 mM Tris-HCl, pH 8.3, 9mM MgC12,
30 mM NaCl, 20 mM beta-mercaptoethanol, 1 mM each oE 3
nonradioactive deoxyribonucleoside triphosphates, 250 ~M of
the fourth deoxynucleoside triphosphate labeled with
~-3 P, specific activity 50-200 curies per mole, 20
~g/ml oligo-dT12 18 from Collaborative Research, TA7altham,
Massachusetts, 100 ug/ml polyadenylated RNA and 200 units/ml
reverse transcriptase. The mixture was incubated at 45C
for fifteen minutes. After addition of EDTA-Na2 to 25mM,
the solution was extracted with an equal volume of water-
saturated phenol, followed by chromatography of the
aqueous phase on a Sephadex G-100 column, 0.3 cm in
diameter by 10 cm in height in 10 mM Tris-HCl, pH 9.0,
100 mM NaCl, 2mM EDTA. Nucleic acid eluted in the void
volume was precipitated with ethanol after addition of
ammonium acetate, pH 6.0, to 0.25M. The precipitate
was collected by centrifugation, the pellet was dissolved
in 50 ~1 of freshly prepared 0.lM NaOH and incubated at
70C for 20 minutes to hydrolyze the RNA. The mixture
was neutralized by the addition of lM sodium acetate,
pH 4.5, and the P-cDNA product was precipitated with
ethanol and redissolved in water. Aliquots of single
stranded cDNA were analyzed on native polyacrylamide gels
-32-
.~, . .
by the method of Dingman, C.W., and Peacock, A.C.,Biochemistry 7, 659 (1968). The gels were dried and the
P DNA detected by autoradiography using Kodak No-Screen
NS-2T-/ film. The cDNA was heterodisperse, as judged
by the electrophoresis pattern. It contained at least
one prominent cDNA species of about 450 nucleotides, as
judged by comparison with known standards.
Example 2
The synthesis and characterization of double
stranded cDNA containing the sequence of rat insulin is
described. The single stranded cDNA product of Example 1
was treated with reverse transcriptase to synthesize the
complementary strand. Reaction mixture contained 50 mM
Tris-HCL, pH 8.3, 9mM MgC12, 10 mM dithiothreitol, 50 mM
each of three unlabeled deoxyribonucleoside triphosphates,
lmM of an alpha- P-labeled nucleoside triphosphate of
specific activity 1-10 curies per millimole, 50 ~g/ml
cDNA and 220 units/ml of reverse txanscriptase. The
reaction mixture was incubated at 45C for 120 minutes.
The reaction was stopped by addition of EDTA-Na2 to 25 mM,
extracted with phenol and chromatographed on Sephadex
G-100 followed by ethanol precipitation. An aliquot of
the reaction product having 500 cpm to 1,000 cpm was
analyzed by gel electrophoresis as described in Example 1.
A heterodisperse band centering around 450 nucleotides
in length was observed, as determined by comparison with
5/ Trademark, Eastman Kodak Corporation, Rochester,
New York.
-33-
llS~66
standard samples. Aliquots of the DNA reaction products
of Example 1 and Example 2 were separately treated by
digestion with an excess of restriction endonuclease Hae
III and similarly analyzed by gel electrophoresis. Both
the products were cleaved by the endonuclease so that two
- bands of radioactivity were observed on gel electrophoresis.
The bands resulting from the cleavage of double stranded
cDNA represented essentially the same length cleavage
products as did those resulting from the cleavage of the
single stranded cDNA.
Example 3
The blunt-end ligation of Hind III decanucleotide
linkers to rat islet double stranded cDNA of Example 2
is described. The double stranded reaction product of
Example 2 at a concentration of 2-5 ~g/ml was treated
with 30 units of Sl nuclease having an activity of 1200
units/ml, obtained from Miles Laboratories, Elkhart,
Indiana, in 0.03 M sodium acetate, pH 4.6, 0.3 M sodium
chloride, 4.5 mM ZnC12 at 22C for 30 minutes incubation
followed by an additional 15 minutes incubation at 10C.
Addition of Tris-base to 0.1 M final concentration, EDTA
to 25 mM, and E. Coli tRNA, prepared by the method of Von
Ehrenstein, G., Methods in_Enzymology, S.P. Colowick and
N.O. Kaplan, Eds., Vol. 12A, p. 588 (1967), to 40 ~g/ml
was used to stop the digestion. After phenol extraction
of the reaction mixture and Sephadex G~100 chromatography,
the P-cDNA eluted in the void volume was precipitated
with ethanol. This treatment resulted in a high yield of
cDNA molecules with base-paired ends necessary for the
blunt-end ligation to chemically synthesized decanucleo-
tides. Hind III decamers were prepared by the method of
Scheller, R. H., Dickerson, R. E., Boyer, H. W., Rigss,
-34-
ll~B~66
A. D and Itakura, ~., Science 196, 177 (1977). Ligation
= =
of Hind III decamers to cDNA was carried out by incubationat 14C in 66 mM Tris-HCl, pH 7.6, 6.6 mM MgC12, 1 mM ATP,
10 mM dithiothreitol, 3 mM Hind III decamers having
cpm/pmol and T4 DNA ligase, approximately 500 units/ml,
for one hour. The reaction mixture was then heated to
65C for 5 minutes to inactivate the ligase. KCl to 50mM
final concentration, beta-mercaptoethanol to 1 mM final
concentration, and EDTA to 0.1 mM final concentration
were added prior to digestion with 150 units/ml Hsu I or
Hind III endonculease for 2 hours at 37C. Hind III and
Hae III endonuclease are commercially available from New
England Bio-Labs, Beverly, MassachusettsO The reaction
product was analyzed by gel electrophoresis as in Example 1
and a peak corresponding to a sequence of approximately
450 nucleotides was observed, in addition to fragments
of cleaved Hind III decamers.
Example 4
The formation of a recombinant plasmid and its
20 characterization after replication is described. Plasmid
pMB-9 DNA, prepared as described by Rodriguez, R. L.,
Boliver, F., Goodman, H. M., Boyer, H. W., and Betlach,
M., in ICN-UCLA Symposium on Molecular and Cellular
Biology, D. P. Wierlich, W. J. Rutter, and C. F. Fox,
Eds., (Academic Press, New York 1976) pp. 471-477, was
cleaved at the Hind III restriction site with Hsu I
endonuclease, then treated with alkaline phosphatase,
type BAPF, Worthington Biochemical Corporation, Freehold,
New Jersey. The enzyme was present in the reaction
30 mixture at the level of 0.1 unit/microgram DNA and the
reaction mixture was incubated in 25 mM Tris-HCl, for
-35-
l 15~1~6
pH 8 for 30 minutes at 65C, followed by phenol extractionto remove the phosphatase. After ethanol precipitation,
the phosphatase treated plasmid DNA was added to cDNA
containing Hind III cohesive termini at a molar ratio of
3 moles plasmid to 1 mole cDNA. The mixture was incubated
in 66 mM Tris, pH 7.6, 6.6 mM MgC12, 10 mM dithiothreitol,
and 1 mM ATP for one hour at 14C in the presence of
50 units/ml of T4 DNA ligase.
The ligation mixture was added directly to a
suspension of E. Coli X-1776 cells prepared for transfor-
mation as follows: Cells were grown to a cell density
of about 2 x 10 cells/ml in 50 ml of medium containing
Tryptone 10g/1, yeast extract 5 g/l, NaCl 10 g/l, NaOH
2mM, diaminopimelic acid 100 ~g/ml and thymine 40 ~g/ml,
at 37C. Cells were harvested by centrifugation for 5
minutes at 5,000 x G at 5C, resuspended in 20 ml cold
NaCl 10mM, centrifuged as before and resuspended in 20
ml transformation buffer containing 75 mM CaC12, 140 mM
NaCl and 10 mM Tris pH 7.5, and allowed to remain 5 minutes
in ice. The cells were then centrifuged and resuspended
in 0.5 ml transformation buffer. Transformation was
carried out by mixing 100 ~1 of the cell suspension with
50 ~1 recombinant DNA (1 ~g/ml). The mixtl~re was incubated
at 0C for 15 minutes, then transferred to 25C for 4
minutes, then at 0C for 30 minutes. The cells were
then transferred to agar plates for growth under selection
conditions.
Screening for recombinant plasmids was carried out
at 5 micrograms/ml tetracycline for transformation into the
Hind III site. A selected recombinant, designated pAU-l,
was isolated. Crude plasmid preparations of 2 ~g - 5 ~g
ll~ul~B
DNA isolated from pUA-l were digested with an excess of
Hsu I endonuclease. EDTA-Na2 lQ mM, and sucrose 10% w/v
(i.e., weight to volume), final concentration were then
added and the mixture resolved on an 8% polyacrylamide gel.
The DNA was found at a position corresponding to about
410 base pairs in length. In a similar experiment, plasmid
pBR 322 was employed as the transfer vector. All conditions
were as described except final selection of recombinant
clones was carried out on plates containing 20 yg/ml
10 ampicillin.
Example 5
The DNA from pAU-l as described in Example 4 was
further purified by electrophoresis on a 6% polyacrylamide
gel. After elution from the gel the DNA was labeled by
incubation with y-32P-ATP and the enzyme polynucleotide
kinas under conditions described by Maxam and Gilbert,
supra. The enzyme catalyzes the transfer of a radioactive
phosphate group from y- P-ATP to the 5'-ends of the DNA.
The enzyme was obtained from E. coli by the method of
20 Panet, A., et al., Biochemistry 12, 5045 (1973). The DNA
thus labeled was cleaved with Hae III endonuclease as
described in Example 2, and the two labeled fragments,
about 265 and 135 base pairs respectively, were separated
on a polyacrylamide gel under the conditions described in
Example 1. The isolated fragments were subjected to
specific cleavage reactions and sequence analysis accord-
ing to the method of Maxam and Gilbert, supra. The
sequence below is based upon a composite of the findings
from this series of experiments and those of a similar
30 series of cDNA using plasmid vectors derived from col El
such as pMB 9 and pBR 322. In the sequence at the 5' end,
a sequence estimated between 50-120 nucleotides in length
-37-
1 6 6
is undetermined and the poly dA segment at the 3'-end is
of varying length. This sequence is provided as represent~
ing the best information presently available, with the
understanding that ongoing studies may reveal additional
details or may indicate a need for slight revision in some
areas. The corresponding amino acid sequence of rat
proinsulin I begins at the triplet position marked 1 and
ends at triplet position marked 86. Some uncertainty
remains with respect to the sequence underlined with a
10 dashed line.
Example 6
A nucleotide sequence coding for human insulin is
isolated, purified and incorporated in a plasmid essentially
as described in Examples 1-4, starting from human pancreas
tissue isolated from a suitable human source such as a
donated pancreas or a fresh cadaver or a human insulinoma~
A microorganism is produced, essentially as described in
Example 4, having a nucleotide sequence coding for the
human insulin A chain and B chain. The known amino acid
20 sequence of human insulin A chain is:
Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-
Leu-Tyr-Glu-Leu-Glu-Asn-Tyr-Cys-Asn
The known amino acid sequence of the human insulin B chain
1 s :
Phe-Val-Asn-Glu-His-Leu-Cys-Gly-Ser-His-Leu-Val-
Glu-Ala-Leu-Tyr-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-
Phe-Tyr-Thr-Pro-Lys-Thr
The amino acid sequences are numbered from the end having
a free amino group. See Smith, L.F., Diabetes 21
(suppl. 2), 458 (1972).
~ ¢
C~ U ¢
E~O ~~ O ~¢
U~ ~ ¢~ c.~
¢ ~ ¢ ~:
~ U ~ E~
U ~ U U 'C
`¢C¢~ ¢ U
O
~,C:l U
C
E~ U o c, U
3 ¢
U C~ -
¢ ~
Uu ~ ~ E~
¢ <1 C~ ¢ ~
E~ ¢ U ~ ~ C
UC~
¢
C~ o U o
E~ ~ ¢ ~ ;~
U~ C~ ~ U
U~ C
3 ~ ¢
u ¢ ¢ ~
c~ ¢ ~ u u o
U ~a
¢ E~
o U C~ ¢l ~
E~ U~;r U Ul ¢
EU~ 3 ¢j n
U U C~
¢ a~
u~c., ~ c~ v~
c~ u u u a
u ~cu~ 0 ¢¢ O
~~ ¢ 3o ~ ~ ~
U C~ ~ ~
E~ ¢ C~ U
¢
c
--39--
.ft
GENERAL CONCLUDING REMARKS
rr~ith the process of the present invention it has
become possible for the irst time to isolate a nucleotide
sequence coding for a specific regulatory protein from a
higher organism such as a vertebrate, and transfer the
genetic information contained therein to a microorganism
where it may be replicated indefinitely. The disclosed
process may be applied to the isolation and purification
of the human insulin gene, and to its transfer to a
microorganism. A novel combinant plasmid is disclosed,
containing within its nueleotide sequence a subsequence
having the structure of and transcribed from a gene of
a higher organism. A novel mieroorganism is disclosed,
modified to contain a nucleotide sequenee having the
structure of and transeribed from a gene of a higher
organism. The practice of the invention has been
illustrated by demonstrating the transfer of the rat
gene for the proinsulin I to a strain of Escherichia eoli.
The sequence of the main portion of the transferred gene
20 has been determined and has been found to contain the
entire amino acid sequence of rat proinsulin I, as deter-
mined by referenee to the known genetic code which is
common to all forms of life.
~ hile the invention has been described in connection
with speeifie embodiments thereof, it will be understood
that it is eapable of further modifications and this
application is intended to cover any variations, uses,
or adaptations of the invention following, in general,
the principles of the invention and including such departures
30 from the present diselosure as come within known or customary
practice within the art to which the invention pertains
-40-
115~
or which would be readily apparent to those skilled in saidart. With that understanding, the invention is not to be
limited except to the extent required by the appended claims.
-41-
