Canadian Patents Database / Patent 1341625 Summary

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(12) Patent: (11) CA 1341625
(21) Application Number: 495255
(54) English Title: RECOMBINANT MAMMALIAN GM-CSF, DNA ENCODING MAMMALIAN GM-SCF AND METHODS OF USE THEREOF
(54) French Title: GM-CSF RECOMBINANT DE MAMMIFERE ADN CODENT POUR CELUI-CIET METHODES D'UTILISATION
(52) Canadian Patent Classification (CPC):
  • 530/13
  • 167/103.2
  • 195/1.235
  • 195/1.34
(51) International Patent Classification (IPC):
  • C12N 15/27 (2006.01)
  • A61K 38/19 (2006.01)
  • C07K 14/535 (2006.01)
  • C12N 15/63 (2006.01)
(72) Inventors :
  • DUNN, ASHLEY ROGER (Australia)
  • GOUGH, NICHOLAS MARTIN (Australia)
  • METCALF, DONALD (Australia)
(73) Owners :
  • RESEARCH CORPORATION TECHNOLOGIES, INC. (United States of America)
(71) Applicants :
  • RESEARCH CORPORATION (United States of America)
(74) Agent: OSLER, HOSKIN & HARCOURT LLP
(74) Associate agent:
(45) Issued: 2011-12-20
(22) Filed Date: 1985-11-13
(30) Availability of licence: N/A
(30) Language of filing: English

English Abstract



This invention provides a DNA sequence coding for a
mammalian granulocyte macrophage colony stimulating factor
(GM-CSF), a method cf obtaining same, vectors and hosts
harboring same. The sequence is useful as a probe for
identifying related sequences, selecting GM-CSF encoding mRNA
from a mixture of mRNAs containing same, and a source of
GM-CSF DNA for expression in an appropriate expression
vector. The GM-CSF protein encoded by the sequence is useful
for stimulating the production of granulocytes and
macrophages from their respective progenitor cells.


French Abstract

La présente invention fournit une séquence d'ADN codant pour un facteur de croissance d'une colonie macrophage granulocytaire mammifère (GM-CSF), un procédé permettant d'obtenir celle-ci, des vecteurs et hôtes hébergeant celle-ci. La séquence est utile en tant que sonde pour identifier des séquences apparentées, en sélectionnant le GM-CSF codant l'ARNm à partir d'un mélange d'ARNm les contenant, et une source d'ADN du GM-CSF pour l'expression dans un vecteur d'expression approprié. La protéine GM-CSF codée par la séquence est utile pour stimuler la production de granulocytes et de macrophages à partir de leurs progéniteurs respectifs.


Note: Claims are shown in the official language in which they were submitted.


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THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:
1. A DNA sequence coding for mammalian
granulocyte-macrophage colony stimulating factor (GM-CSF),
wherein said DNA sequence is derived from natural,
synthetic or semi-synthetic sources and further wherein said DNA
sequence comprises a DNA sequence selected from the DNA inserts
of pGM37 and pGM38, said inserts being the DNA inserts of the
recombinant DNA molecules carried by the microorganisms
identified by accession numbers ATCC 53032 and 53036,
respectively, and DNA sequences which hybridize to said DNA
inserts and code for a mammalian GM-CSF.

2. The DNA according to claim 1, wherein the
natural source is a mouse.

3. The DNA according to claim 1, wherein said DNA
is cDNA.

4. The DNA according to claim 1 having the,
following DNA sequence in a 5' to 3' direction
Image


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5. The DNA according to claim 1 having the following
DNA sequence in a 5' or 3' direction

Image
6. A method of producing the DNA coding for the GM-
CSF of claim 1, comprising the steps:
(a) preparing a source of mRNA containing GM-CSF
mRNA;
(b) synthesizing duplex DNA copies of said source
mRNA;
(c) cloning said DNA copies;
(d) providing at least one synthetic GM-CSF probe;
(e) screening clones harboring the DNA copies of step
(b) by colony hybridization with the probes of step (d);
(f) and recovering the clones which hybridize with
said probe.

7. The method according to claim 6, wherein said
source of mRNA is lung mRNA isolated from C57 BL/6 mice
injected with bacterial endotoxin.

8. The method according to claim 6, wherein said
probes are selected from the group consisting of:
(a) 3' ACCTTTGTACACCTTCG 5'
Image
(b) 3' ACCTTTACACAACTTCG 5'

Image


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(c) 3' CTTCGATAATTTCTTCG 5';
Image
(d) 3' CTTCGTTAATTTCTTCG 5'

Image
(e) the sequence of claim 4; and
(f) the sequence of claim 5.

9. A cloning vector comprising the DNA sequence of
claim 1.

10. A cloning vector comprising the DNA sequence of
claim 2.

11. A cloning vector comprising the DNA sequence of
any one of claims 3, 4 or 5.

12. The vector according to claim 9, wherein said
vector is plasmid pGM37.

13. The vector according to claim 9 wherein said
vector is plasmid pGM38.

14. Host cells transformed by the vector of claim 9.
15. Host cells transformed with plasmid pGM37.


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16. The host cells of claim 15 having the identifying
characteristic of ATCC 53032.

17. Host cells transformed with plasmid pGM38.

18. The host cells of claim 17 having the identifying
characteristics of ATCC 53036.

19. A method of producing granulocyte-macrophage
colony stimulating factor (GM-CSF) comprising culturing
host cells transformed by an expression vector comprising
a promoter fragment which functions in said host cells and
a DNA segment as defined in claim 1, the DNA segment being
in an orientation with said promoter such that in the host
cells the GM-CSF DNA is expressed as a non-native GM-CSF
protein.

20. A method of producing granulocyte-macrophage
colony stimulating factor (GM-CSF) comprising culturing
host cells transformed by an expression vector comprising
a promoter fragment which functions in said host cells and
a DNA segment coding for GM-CSF, the DNA segment being, in
an orientation with said promoter such that in the host
cells the GM-CSF DNA is expressed as a non-native GM-CSF
protein, wherein said GM-CSF DNA segment comprises the
sequence of claim 5.

21. An in vitro method for stimulating the
production of granulocytes and macrophages comprising
contacting respective progenitor cells of said
granulocytes and macrophages with a stimulating effective
amount of recombinant granulocyte-macrophage stimulating
factor.

22. A recombinant mammalian granulocyte,
macrophage-colony stimulating factor (GM-CSF) polypeptide
encoded by the DNA of claim 1.


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23. The polypeptide of claim 22, wherein
mammalian is murine.

24. The polypeptide of claim 22, wherein said
GM-CSF polypeptide has an amino acid sequence comprising
Image

25. The polypeptide of claim 24 wherein the
serine amino acid residue 116 is replaced by glycine.

26. Mammalian GM-CSF produced by the method of
claim 19.

27. A method of producing GM-CSF comprising
culturing a host cell transformed by an expression vector
comprising a promoter which functions in said host and a
DNA which hybridizes to the nucleotide sequence:

Image


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wherein said DNA codes for GM-CSF, the DNA being in
orientation with said promoter such that in said host cell
the GM-CSF DNA is expressed as a non-native GM-CSF protein.

28. The method according to claim 27, wherein
said CM-CSF DNA segment comprises the sequence

Image
29. GM-CSF produced by the method of claim 27.
30. A method of producing mammalian GM-CSF
comprising culturing a host cell transformed by an
expression vector which comprises a promoter which
functions in said host and a DNA which hybridizes to the
nucleotide sequence

Image


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wherein said DNA encodes mammalian GM-CSF in said host, and
wherein said DNA is in orientation with said promoter such
that in said host cell the mammalian GM-CSF DNA is
expressed as a non-native protein.


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31. A method of producing mammalian GM-CSF comprising
culturing a host cell transformed by an expression vector
which comprises a promoter which functions in said host
and a DNA which hybridizes to the plasmid pGM37 or pGM38
wherein said DNA encodes mammalian GM-CSF in said host and
wherein said DNA is in orientation with said promoter such
that in said host cell the mammalian GM-CSF DNA is
expressed.

32. The method of claim 31, wherein said GM-CSF protein is
recovered in substantially pure form.

33. A method of producing mammalian GM-CSF comprising
culturing a host cell transformed by an expression vector
which comprises a promoter which functions in said host
and a DNA which hybridizes to the complement of the
nucleotide sequence

Image
wherein Y is C or T; wherein R is A or G; and wherein said DNA
encodes mammalian GM-CSF in said host, and wherein said DNA is
in orientation with said promoter such that in said host cell
the mammalian GM-CSF DNA is expressed.


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34. A method of producing mammalian GM-CSF comprising
transforming a host cell by an expression vector
which comprises a promoter which functions in said
host and a DNA which hybridizes to the complement of
the nucleotide sequence

Image
wherein Y is C or T; wherein R is A or G; and wherein said DNA
encodes mammalian GM-CSF in said host, and wherein said DNA is
in orientation with said promoter such that in said host cell
the mammalian GM-CSF DNA is expressed.

35. An isolated DNA encoding mammalian granulocyte
macrophage colony stimulating factor (GM-CSF).

36. An isolated DNA which hybridizes to the plasmid pGM37
wherein said DNA encodes mammalian GM-CSF.

37. An isolated DNA which hybridizes to the plasmid pGM38
wherein said DNA encodes mammalian GM-CSF.


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38. A method of producing mammalian GM-CSF comprising
culturing a host cell transformed with a DNA which
hybridizes to the nucleotide sequences:

3' ACCTTYGTRCAXCTYCG 5'; or
3' ACCTTYACRCAXCTYCG 5'; or
3' CTYCGRTAZTTYCTYCG 5'; or
3' CTYCGYTAZTTYCTYCG 5'

wherein R is A or G; X is C, T, A or G; Y is C or T; and Z is
A, G or T; and wherein said DNA is in orientation with a
promoter such that said transformed host cell expresses
mammalian GM-CSF.

39. A DNA sequence encoding a recombinant protein having


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mammalian GM-CSF activity wherein said DNA sequence
hybridizes to the complement of the following DNA
sequence:

Image
40. A recombinant vector comprising a DNA sequence which
hybridizes to the complement of the following DNA sequence
and encodes a protein having mammalian GM-CSF activity:

Image
41. A host cell transformed with a recombinant vector
comprising a DNA sequence which hybridizes to the
complement of the following DNA sequence and encodes a
protein having mammalian GM-CSF activity:


Image
42. A process for producing a recombinant protein having
mammalian GM-CSF activity, comprising culturing a host
cell transformed with a recombinant vector comprising a
DNA sequence which hybridizes to the complement of the
following DNA sequence:

Image
and recovering the protein produced.

43. An unglycosylated recombinant protein having mammalian GM-
CSF activity encoded by a DNA sequence which hybridizes to
the complement of the following DNA sequence:


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Image

44. A glycosylated recombinant protein having mammalian GM-CSF
activity, encoded by a DNA sequence which hybridizes to
the complement of the following DNA sequence:

Image
45. Use of an unglycosylated recombinant protein having
mammalian GM-CSF activity, encoded by a DNA sequence which
hybridizes to the complement of the following DNA
sequence, for the preparation of a pharmaceutical
composition:

Image


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96. Use of a glycosylated recombinant protein having mammalian
GM-CSF activity, encoded by a DNA sequence which
hybridizes to the complement of the following DNA
sequence, for the preparation of a pharmaceutical
composition:

Image
47. A pharmaceutical composition comprising an unglycosylated
recombinant protein having mammalian GM-CSF activity,
encoded by a DNA sequence which hybridizes to the
complement of the following DNA sequence, and a
pharmaceutically acceptable carrier:

Image


48. A pharmaceutical composition comprising a glycosylated
recombinant protein having mammalian GM-CSF activity
encoded by a DNA sequence which hybridizes to the
complement of the following DNA sequence, and a
pharmaceutically acceptable carrier:

Image
49. Use of an unglycosylated recombinant protein having
mammalian GM-CSF activity, encoded by a DNA sequence which
hybridizes to the complement of the following DNA
sequence, for increasing growth and differentiation of
circulating granulocytes:

Image
50. Use of a recombinant protein having mammalian GM-CSF
activity, encoded by a DNA sequence which hybridizes to


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the complement of the following DNA sequence, for
increasing growth and differentiation of circulating
granulocytes:

Image

Note: Descriptions are shown in the official language in which they were submitted.


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RECOMBINANT MAMMALIAN GM-CSF, DNA ENCODING MAMMALIAN UM-
CSF AND METHODS OF USE..THEF[F,OF
Field of the Invention,.
This invention relates to..DNA sequences,,
recombinant DNA molecules and processes for producing
proteins or polypeptides with the specificity of protein
molecules which control the production of particular blood
cells. More specifically, this invention relates to DNA
sequences and recombinant DNA molecules that are
characterized in that they code for or include fragments that
code for the protein molecule known as granulocyte-macrophage
colony stimulating factor (GM-CSF).
Background of the invention
The production of blood cells such as erythrocytes
(red blood cells), granulocytes, macrophages and lymphocytes
is under the control of a set of protein molecules which
stimulate multipotential precursor or stem cells in the bone
marrow. During hemopoeitic development, these multipotential
cells form cells of limited developmental.potential which are
variously referred to as committed progenitor cells, colony
2p forming cells or CFCs for individual blood cell types.
Although there may be non-specific stimulators of the
precursor stem cells or CFCs such as the so-called multi-CSF
(interleukin-3) as described by rung, M.C., et al., (Nature
307: 233-237 (1984) or Yokota, T., et al., Proc. Nat'l.
Acad. Sci. USA 81: .1070-1074 (1984)), there are specific
regulators for each of the different cell lineages. In
particular, the production of, granulocytes, and macrophages
from their respective CFCs is under the control of
glycoproteins such as granulocyte-macrophage colony
stimulating factor (GIY-CSF) as described by Burgers, A.W. et
al., (J. Fiol. Chem. 252: 1998-2003 (1977)), granulocyte


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1 colony stimulating factor (G-CSF) as described by Stanley,
E.R. and Heard P.M., (J. Biol. Chem. 252: 4305-4312 (1977))
and macrophage colony stimulating factor (M-CSF) as described
by Nicola, N.A. et al., (J. Biol. Chem. 258: 9017-9021
(1983)). Although these glycoproteins are of low abundance
in the body, it has been possible to purify small amounts of
the murine GM-CSF for partial amino acid sequence analysis
and biological characterization.
However, unless an alternative source of these
proteins can be found, these small amounts will be
insufficient for clinical applications. If, however, these
colony stimulating factors can be produced chemically or
biosynthetically, it should be possible to use these factors
to improve blood cell production in vivo, to produce blood
cells in the laboratory for transfusion and to accelerate the
maturation of leukemic cells. For each of these
applications, it is imperative that the types of blood cells
produced be restricted. In particular, it is important to
limit the production or activation of lymphocytes and/or
their precursors. Thus, whilst molecules such as multi-CSF
could find application in some diseases where a general
increase in blood cell production is required, the use of
glycoproteins such as GM-CSF, G-CSF and M-CSF will be of
particular importance since these stimulate only the
production of cells required to fight primary infection or
remove damaged tissue.
Brief Description of the Invention
This invention provides a DNA sequence coding for
mammalian granulocyte-macrophage colony stimulating factor
(GM-CSF) or its single or multiple base substitutions,
deletions, insertions, or inversions, wherein said DNA

w


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3
v
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1 sequence is derived from natural, synthetic or semi-synthetic
sources and is capable of selecting an mRNA species capable
of directing the synthesis of GM-CSF in vitro from a mixture
of mRNAs containing same.
In another embodiment this invention provides
a method of producing the DNA sequence coding for GM-CSF of
comprising the steps of:
(a) preparing a source of mRNA containing GM-CSF
mRNA;
(b) synthesizing duplex DNA copies of said source
mRNA;
(c) cloning said DNA copies;
(d) providing synthetic GM-CSF probes;
(e) screening clones harboring the DNA copies of
step (b) by colony hybridization with the
probes of step (d);
(f) and recovering the clones which hybridized
with said probes.
In another embodiment this invention provides
a cloning vector comprising, in recombinant form, a DNA
sequence having protein encoding portion whose code sequence
has substantially one-to-one correspondence with
granulocyte-macrophage colony stimulating factor (GM-CSF).
In another embodiment this invention provides
a method of producing granulocyte-macrophage colony
stimulating factor (GM-CSF) comprising culturing a host organ
transformed by an expression vector comprising a promoter
fragment which functions in said host and a DNA segment
coding for GM-CSF, the DNA segment being in an orientation
with said promoter such that in the host the GM-CSF DNA is
expressed as a non-native CM-CSF protein.



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In a final embodiment this invention provides
a method for stimulating the production of granulocytes and
macrophages comprising contacting respective progenitor cells
of said granulocytes and macrophages with a stimulating
effective amount of granulocyte-macrophage stimulating
factor.
Detailed Description of the Drawings
Figure 1 shows the synthetic oligonucleotides used
for identifying GM-CSF clones. A portion of the amino acid
sequence of murine GM-CSF (residues 7-16) is shown on the top
line, the possible combinations of nucleotide (mRNA)
sequences that could encode this peptide segment in the
middle, and the four different sets of oligonucleotide probes
complementary to regions of the mRNA sequences below.
Figure 2 ..shows a map of the GM-CSF mRNA and of
clones pGM37 and pGM38. The mRNA is taken to be 1,200
nucleotides in length. The region of the mRNA.encoding the
mature protein is shown as a thick line. The untranslated
regions are designated by UT and a putative precursor peptide
by P. The regions contained within clones pGM37 and pGM38
are indicated with bars. pGM38 extends from nucleotide 14 in
the sequence presented in Figure (b) to the poly(A) tail,
whereas pGM37 extends from 20 nucleotides 5' to the sequence
presented to position 574.
Figure 2 also shows the nucleotide sequence of
GM-CSF mRNA and predicted amino acid sequence of GM-CSF. The
nucleotide sequence given is a composite of sequence derived
from clones pGM37 and pGM38; at three positions where the
pGM37 nucleotide sequence differs from that of pGM38 and
pGM37 alternatives are given below the line. The sequence of
the mRNA-synonymous strand is listed 5' to 3', with the
predicted amino acid sequence of GM-CSF given above; numbers


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1 at the ends of lines indicate the position of the final
residue (amino acid or nucleotide) on that line. The partial
amino acid sequence determined for GM-CSF is indicated above
the sequence derived from the clones; at positions where
there is no discordance between the two, dashes are given.
The first amino acid residue, determined by analysis of the
protein, could not be assigned, and is indicated by a
question mark.
Figure 3 shows a map of vector pJL3. The
SV40-drived portion is shown as a thick line, and that
derived from plasmid pAT153 as a thin line. The multicloning
site is indicated at the top. Numbers given refer to the
sequence of the parental SV40 or pAT153 sequences.
Figure 4 illustrates the detection of GM-CSF mRNA
in various cells by hybridization with 32P-labelled DNA from
pGM38.
Figure 5 illustrates the stimulation of cellular
proliferation in suspension cultures of haematopoietic
progenitor cells from fetal liver (upper panel) and
multi-CSF-dependent 32D C12 cells (lower panel) by medium
from mRNA-injected Xenopus oocytes. Unfractioned LB3 mRNA is
indicated by heavy lines o - o, mRNA selected by
hybridization to pGM38 DNA; o - o, that selected by vector
DNA alone; the three separate curves represent triplicate
experiments.
Figure 6 is a photomicrograph of a four-day
suspension culture of purified fetal liver haematopoietic
progenitor cells after stimulation by medium from oocytes
injected with pGM38-selected.mRNA from LB3cells. Mitotic
activity and the production of maturing granulocytes and
macrophages can be noted.



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Figure 7 illustrates the detection of the GM-CSF
gene from Eco R1 or Pst I digested DNA hybridized with
32P-labelled DNA from pGM38.
Detailed Description of the Invention
The present invention relates to the production and
characterization of a recombinant DNA molecule for the
production of the specific blood cell regulator
granulocyte-macrophage colony stimulating factor (GM-CSF).
In particular, this invention involves the production of DNA
molecule coding for murine GM-CSF which may prove to be of
particular importance in providing the basis for production
of GM-CSF in alternative hosts, such as bacteria and animal
cells. By way of example, the DNA molecule of this invention
may be used as a probe to isolate the gene sequence for the
equivalent human GM-CSF for use in the production of human
granulocytes and macrophages.
in one embodiment, the present invention provides a
DNA sequence characterized in that at least a portion thereof
codes for a protein or polypeptide displaying the biological
activity of murine GM-CSF. A specific nucleotide sequence of
this aspect of the invention is shown in Figure 2y
Clones harboring a nucleotide sequence useful for
practicing the subject invention may be obtained by the
following cloning strategy.
Isolation of a Murine GM-CSF cDNA Clone
One of the best.sources of GM-CSF is stimulated
mouse lung. A library of greater than 105 cDNA clones
complementary to lung mRNA from endotoxin-treated mice was
constructed as detailed in Example I.

35


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Gr:-CSF containing recombinants were identified in
this library using as probes, short synthetic
oligonucleotides complementary to two portions of the GM-CSF
mRNA sequence, as predicted from the partial amino acid
sequences of the protein. Figure 1 shows the region of the
GM-CSF amino acid sequence between residues 7 and 16, the
possible combinations of nucleotide sequences that could
encode this peptide and the oligonucleotides that were used
as hybridization probes. Since the assignment of amino acid
residue 9 was equivocal (being either histidine or cysteine)
two different sets of oligonucleotides encompassing this
region were synthesized; probe 1 assumes a histidine residue
and probe 2 a cvsteine residue at position 9. The second
region within the amino acid sequence required an extremely
degenerate set of oligonucleotides which were synthesized as
two 48-fold degenerate sets, (probes 3 and 4).
In order to identify GM-CSF recombinants a colony
hybridization assay was employed. The library of cDNA clones
was grown as single colonies on agar plates, replicas of the
colonies transferred to nitrocellulose filters on which the
colonies were then lysed and the plasmid DNA immobilized in
situ. These filters were screened by hybridization with a
mixture of all of the synthesized oligonucleotide probes
which had been 3`P-labelled.
After hybridization and washing the filters,
colonies that had hybridized with the probes were identified
by autoradiography. By this means, 22 independent
recombinants that hybridized with the mixture of probes were
identified. Whilst some of these may well represent
irrelevant sequences, with fortuitous homology with one of
the oligonucleotides, it is unlikely that an irrelevant



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1 sequence will hybridize with two different probes
independently, whereas the authentic GM-CSF sequence should
hybridize with both probes 3/4 and either 1 or 2. Therefore,
plasmid DNA from each of the 22 clones was isolated and
electrophoresed on triplicate agarose gels. After the DNA
was transferred to nitrocellulose by the Southern technique
(J. Mol. Biol. 98: 503, (1975)), the three filters were
hybridized with the three different probes independently. Of
the 22 clones examined, 2 (clones 37 and 38) hybridized with
both probes 1 and 3/4 and thus represented strong candidates
for being GM-CSF DNA.
Confirmation of Identity of Clone
Two lines of experimentation demonstrated that
clones 37 and 38 do in fact correspond to the GM-CSF gene
sequence. Firstly, the nucleotide sequence of the clones was
determined and is shown in Figure 2 , along with its
encoded amino acid sequence. The amino acid sequence
predicted by the nucleotide sequence of the clones are
substantially similar to the N-terminal amino acid sequence
determined from analysis of the protein, there being only 4
discrepancies between the two out of 29 positions compared.
Two of the discrepancies occur at positions which were only
tentatively assigned in the protein sequence. Furthermore,
the nucleotide sequence of the clone predicts a peptide with
a molecular weight (13,500 daltons) very close to that
expected for the GM-CSF protein.
Secondly, clone 38 can specifically select
biologically active GM-CSF mRNA. Clone 38 DNA (and also DNA
from the parental plasmid pJL3) was immobilized on
nitrocellulose and was hybridized with RNA from mouse lung
and also from a T cell line (LB3) which makes GM-CSF and a


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1 related (but distinct) regulator, IL3, after the cells have
been stimulated with conA. After hybridization, the filters
were washed to remove non-hybridized RNA and the specifically
hybridized RNA then eluted. This RNA was injected into
Xenopus oocytes (the ability of which to translate exogenous
mRNA has been well documented) and the culture media assayed
3-5 days later for the presence of GM-CSF and IL3. The
results of several experiments demonstrated that: (a) the
vector DNA alone does not select GM-CSF mRNA; (b) clone 38
DNA selects GM-CSF mRNA from both lung RNA and from LB3; and
(c) clone 38 does not select IL3 mRNA from LB3 RNA - an
internal control of specificity (See Example II).
A third line of experiments provides strong
additional support for the identity of clone 38. When used
as a hybridization probe in a Northern blotting experiment,
clone 38 detects an mRNA species of approximately 1.2kb in
length in RNA from cells which synthesize GM-CSF (mouse lung
and conA-stimulated LB3) but not in a range of myeloid of
lymphoid cells which do not synthesize GM-CSF nor in RNA from
LB3 cells which had not been conA-stimulated. Thus, in the
case of LB3, the mRNA corresponding to clone 38 is inducible
along with the GM-CSF protein (See Example III).
Deposit of Strains Useful in Practicing the Invention
A deposit of a biologically pure culture of the
following strains was made with the American Type Culture
Collection, 12301 Parklawn Drive, Rockville, Maryland on
February 21, 1985 the accession number indicated was assigned
after successful viability testing, and the requisite fees
were paid. Access to said culture will be available during
pendency of the patent application to one determined by the
Commissioner to be entitled thereto under 37 C.F.R. 1.14 and
U.S.C. 122. All restriction on availability of said



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1 culture to the public will be irrevocably removed upon the
granting of a patent based upon the application and said
culture will remain permanently available for a term of at
least five years after the most recent request for the
furnishing of a sample and in any case for a period of at
least 30 years after the date of the deposit. Should the
culture become nonviable or be inadvertently destroyed, it
will be replaced with a viable culture (s) of the same
taxonomic description.
Strain/Plasmid ATCC No.
pGM37 53032
pGM38 53036
Utility of pGM37 and pGM38
In addition to providing a convenient source of
clonable gentic information for murine GM-CSF the hybrid DNA
molecules disclosed herein are also useful as probes for the
detection and isolation of related gene sequences from other
mammalian DNA libraries. In their use for the detection of
related gene sequences, the probes are conveniently labelled
with an analytically detectable reagent. The invention,
however, should not be limited to any particular means of
labelling. Although the examples employ a radioactive label
for detection, other detection methods are well-known in the
art and may be easily substituted. Alternative systems,
although not limiting would include biotin-avidin,
fluorescent dyes, protein, immunological assays such as ELISA
where antibodies to derivitized probe molecules are used, or
antibodies to DNA-DNA hybrids themselves are used; and assays
wherein one strand of DNA is labelled with an inactive
subunit of an enzyme and the probe is labelled with a second
inactive subunit such that upon hybridization the subunits
reassociate and enzyme activity is restored.



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1 The cloned sequence may be subcloned into
expression vectors. In situations where the gene sequence
may be missing a partial sequence, such a sequence may be
synthesized chemically, ligated to cloned sequence and
introduced into an expression vector. The choice of an
appropriate expression vector is well within the skill of an
artisan in the field. Minimally, the expression vector will
contain a promoter fragment which functions in the host to be
transformed and a convenient endonuclease cleavage site such
that the gene sequence coding for GM-CSF may be combined
therewith.

20
30


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1 EXAMPLE I
This Example demonstrates the isolation cDNA GM-CSF
clcnes useful for practicing the subject invention.
Isolation of Mouse Luna mRNA
90 C57 BL/6 mice were injected with bacterial
endotoxin (5ug/mouse). After 3 hours the lungs were removed
and incubated in vitro in serum free-Dulbecco's modified
Eagles medium (Sheridan J. & D. Metcalf J. Cell Physiol. 81:
11-24 (1973)). After 0, 5 and 15 hours in vitro, batches of
30 pairs of lungs were homogenized for 60 seconds in 150m1 M
Tris p H 7.5, 0.1 M NaCl, 1mM EDTA; 0.5% SDS, 200ug/ml proteinase K.
After incubation at 37 C for 1 hour, the homogenate was mixed
with an equal volume of 7M urea, 0.35M NaC1, 10mM EDTA, 1%
SDS, l0mM Tris Cl (pH7.4) and then extracted with
phenol/chloroform/isoamvlalcohol. RNA was precipitated from
the aqueous phase by addition of ethanol. Poly A+ RNA was
selected from the total RNA by two rounds of chromatography
on oligo-dT cellulose. The RNA prepared from the three
different times of in vitro culture were pooled.
Synthesis and Construction of cDNA Clones
Duplex DNA copies of the pooled lung mRNA were
synthesized using avian myeloblastcsis virus reverse
transcriptase for synthesis of the first-strand and
Escherichia coli DNA polymerase I (Klenow fragment) for
synthesis of the second strand, using standard techniques
(Efstratiodas, A. et al. cell 7: 279-288 (1976) and
Kaniatis, T., et al. "Molecular Cloning", Cold Spring Harbor
Lab, New York (1982)). After cleavage of the hairpin loop
with S1 nuclease, oligo(dC) tails (aptiroximately 20-30
residues per end) were added using terminal
deoxynucleotidyltransferase (Michelson, A.M. & S. Orkin J.


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1 Biol. Chem. 257: 14773-14782 (1982)). The tailed cDNA was
fractionated by electrophoresis on a 1.5% agarose gel and
molecules greater than 500 bases in length recovered and
annealed to dG-tailed plasmid DNA. The plasmid used (pJL3)
is an SV40-based expression vector that contains the
P -lactamase gene and origin of DNA replication from pAT153,
the SV40 origin of DNA replication and T-antigen coding
sequences and a multicloning site adjacent to the SV40 late
promoter (See Figure 3). E. coli IMIC1061 (Casadabon, M. and
S. Cohen, J. Mol. Biol. 138: 179-207 (1980)) was transformed
with the annealed cDNA-plasmid mixture using a high
efficiency transformation procedure that yields 108
transformants per jug of vector DNA. Forty-six independent
pools containing approximately 2,000-3,000
ampicillin-resistant clones were stored in 10% glycerol at
-70 C.
Screening of cDNA Clones
For screening by colony hybridization,
approximately 4,000 bacterial colonies from each pool were
grown on agar plates (containing 40 pg ml-1 ampicillin),
transferred to nitrocellulose filter disks and plasmid DNA
amplified by incubation of the filter on agar plates
containing 200 ug ml-1 chloramphenicol (Hanahan, D. & M.
Meselson Gene 10: 63-67 (1980)). After regrowth of colonies
on the original plate, a second nitrocellulose filter was
prepared. The master plate was regrown a second time and
then stored at 4 C. Plasmid DNA was released from the
bacterial colonies and fixed to the nitrocellulose filters
(Nicola N. & D. Metcalf, J. cell Phvsiol. 112: 257-264
(1982)). Before hybridization, filters were incubated for
several hours at 37 C in 6xSSC (SSC=0.15 M NaCl, 0.015 M.
sodium citrate), 0.2%*Ficoll, 0.2% polyvinyl-pyrrollidone
*Trade Mark

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1 (PVP) , 0.2% bovine serum albumin (BSA) containing 50 pug ml-
of denatured salmon sperm DNA and 10 pug ml-1 of denatured E.
coli DNA. Hybridization was in the same solution, containing
0.1% NP40, at 37 C for about 18 hours. The synthetic
oligonucleotide probes described above were radioactively
labelled using [o -'2P] ATP and polynucleotide kinase and
each was present in the hybridization reaction at 1.5 nm
ml-1. After hybridization, filters were extensively washed
in 6xSSC, 0.1% SDS at 42 C and then autoradiographed.
Colonies positive on duplicate filters were picked and
rescreened at low density as before.
Screening of --1100,000 cDNA clones yielded 22
positives, of which two (pGM37 and pGM38) hybridized
separately with probe 1 and with a mixture of 3 plus 4 and
were therefore strong candidates to contain sequences coding
for GM-CSF.

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EXAMPLE II
This Example demonstrates the ability of pGM38 to
select mRNA capable of directing the synthesis of GM-CSF.
Assays for GM-CSF
GM-CSF rRNA was identified by translation in
Xenopus oocytes and the culture medium assayed for ability to
stimulate granulocyte/macrophage proliferation in three
microculture system: (1) liquid cultures containing purified
fetal liver haematopoietic progenitor cells, which respond
directly to all four colony stimulating factors (Burgess,
A.W., et al., Blood 60: 1219-23 (1982)); (2) micro-agar
cultures containing bone marrow cells, in which the formation
of morphologically identifiable granulocyte and macrophage
colonies can be stimulated by all four colony stimulating
factors; and (3) liquid cultures containing the
factor-dependent mast cell line 32D C13, which responds to
multi-CSF but not GN-CSF. From this set of assays, GM-CSF
and multi-CSF can be unambiguously distinguished.
Detection of GM-CSF mRNA in various Cells
Initially it was determined whether the abundance
of transcripts corresponding to pGM38 in various cell types
paralleled the ability of those cells to synthesize GM-CSF.
Messenger RNA from mouse lung (which synthesizes GN CSF) and
from various cells which do not synthesize GM-CSF was
fractionated on formaldehyde-agarose gels, transferred to
nitrocellulose and hybridized with a probe derived from pGM38
as detailed below. Figure 4 shows that this probe detected a
low abundance transcript of about 1.2 kilobases (kb) in mRNA
from mouse lung (lane 3), but not in mRNA from several
randomly selected cell lines which do not synthesize GM-CSD,
including the thymoma-derived cell line TIKAUT (lane 1) and
the plasmacvtoma P3 (lane 2). Furthermore, this probe failed


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1 to detect any transcript in mRNA from WEHl-3B D (lane 6),
RIII (lane 7) and L cells (lane 8), which synthesize
multi-CSF, G-CSF and M-CSF respectively. The very low
abundance of the transcript corresponding to pGM38 in lung
mRNA was surprising given the low frequency of pGM38-related
cDNA clones in the lung cDNA library (N 1 in 50,000).
As a more critical test of whether transcripts
complementary to pGM83 correlated with GM-CSF production, a
cloned T lymphocyte cell line (LB3) was used in which the
synthesis of both GM-CSF and multi-CSF is induced by
concanavalin A. Figure 4 shows that the pGM38-drived probe
detected an abundant mRNA in LB3 cells that had been
stimulated with concanavalin A (lane 5 ) but failed to detect
any transcript in mRNA from unstimulated LB3 cells (lane 4).
As the probe used does not hybridize to the multi-CSF mRNA in
WEH1-3B D (lane 6), such is further evidence that pGM38
hybridized to the GM-CSF mRNA in LB3 cells.
The detection of GM-CSF mRNA in the various cells
was accomplished as follows. 5 dug of poly(A) RNA from TIKAUT
(lane 1), P3 (lane 2), mouse lung (lane 3), unstimulated LB3
(lane 4), concanavalin A-stimulated LB3 (lane 5), WEH13B D
(lane 6), RIII (lane 7) and L cells (lane 8) were
electrophoresed on 1% formaldehyde/agarose gels, transferred
to nitrocellulose and hybridized with a fragment of DNA
spanning the entire insert of pGM38 34P-labelled by
nick-translation (Riaby, P.W.J., et al., J. Mol. Biol. 113:
237-251 (1977)). Hybridization was at 65 C for 16 hours in
2xSSC, 0.1% SDS, 0.2% Ficoll, 0.2% PVP, 0.2% BSA containing
50 dug ml -1 of denatured salmon sperm DNA. LB3 (formerly B3)
is a cloned Thy-1+, Lyt-2 , MT4+ T lymphocyte line derived
from BALB/c anti-DBA/2 mixed leukocyte culture, and
maintained by weekly passage with irradiated DBA/2 spleen


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cells (1,500 R) in the presence of interleukin-2.
Concanavalin A-stimulated LB3 cells were prepared by
culturing cells at 106 per ml with 5 dug ml-1 concanavalin A
in tissue culture medium with 5% fetal calf serum for 5
hours. The molecular weight markers used and their presumed
molecular weights were: mammalian 28 and 185 rRNA (4,700 and
1,800 nucleotides) and E. coli 23 and 16S rRNA (2,904 and
1,541 nucleotides). A lower level of hybridization to
residual 28S rRNA is evident after long autoradiographic
exposure (tracks 1-3).
Hybrid Selection of GM-CSF mRNA and Translation
in Xenopus Oocvtes
Hybrid selection was performed essentially as
described by Miller, J.S., et al. (Meth. Enzym. 101: 650-674
(1983)). pGM38 or vector (pJL3) DNA (5-ug aliquots) were
bound to small squares of nitrocellulose and incubated with
1-2 pg aliquots of concanavalin A-stimulated LB3 mRNA in 50%
formamide, 0.5 M Tris-HC1 pH 7.5, 0.75 M NaCl, 0.002 M EDTA,
0.4% SDS, 10 ug ml E. coli tRNA in a reaction volume of 30
ul at 37 C for 16 hours. After incubation, filters were
exhaustively washed in hybridization buffer at 37 C and then
in 10 ma! Tris-HCI pH 7.5, 2 mM EDTA at 52 C. Bound RNA was
eluted by boiling for 1 minute in 300 }zl 10 mM Tris-HC1 pH
7.5, 2 mM EDTA containing 1.5 ug of E. coli tRNA. RNA was
precipitated by addition of sodium acetate and ethanol and
chilling at -20 C for 16 hours. After centrifugation,
precipitated RNA was washed with cold 70% ethanol, dried and
redissolved in 1.5 jhl 1mM Tris-HC1 pH 7.5, 0.1 mM EDTA. For
each RNA sample, groups of 30 Xenopus oocytes were injected
with 50 nl of RNA per egg. Unfractionated LB3 mRNA was at 1
ug ml-1 for injection and 50 nl was injected per egg. After
incubation of injected oocytes for 4 days, oocyte culture

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1 medium; was diluted 1:2 in medium containing 5% fetal calf
serum, filtered and 5F1 volumes assayed in serial dilutions
in 15-u1 cultures containing 200 fluorescence-activated cell
sorter fractionated 14 day CBA fetal liver progenitor cells
for 200 32D C13 cells. Each point in Figure 5 represents the
mean cell count from duplicate cultures after 2 days of
incubation.
Initially, a test was performed to determine
whether pGM38 DNA could select GM-CSF mRNA from mouse lung
RNA. After injection of unfractionated lung mRNA or mRNA
selected by hybridization to pGM38 DNA into Xenopus oocytes,
very low levels of CSF activity could be detected in the
culture media, a finding that was anticipated from the very
low abundance of mRNA corresponding to pGM38 in mouse lung
(Figure 4). By contrast, mRNA from concanavalin A-stimulated
LB3 cells (which synthesize both GM- and multi-CSF) is rich
in mRNA corresponding to pGM38 (Figure 4) and on injection
into Xenoous oocytes, reproducibly directed the synthesis of
high levels of material stimulating the proliferation of
fetal liver and 32D C13 cells (Figure 5). Triplicate filters
containing either pGM38 or vector DNAs were therefore
challenged with concanavalin A-stimulated LB3 mRNA in a
hybrid-selection experiment as described above. Figure 5
shows that medium from oocytes injected with mRNA selected by
pGM38 DNA strongly stimulated fetal projenitor cell
proliferation with the development of maturing granulocytes
and macrophages (Figure 6). These oocyte-conditioned media
also stimulated typical granulocyte-macrophage colony
formation in micro-agar cultures (18% granulocyte, 53% mixed
granulocyte-macrophage and 29% macrophage colonies). In
neither assay was there evidence that these oocyte-conditioned
media stimulated the proliferation of erythroid,



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1 megakaryocytic or eosinophilic cells, hence the active factor
appeared to be GM-CSF rather than multi-CSF. In confirmation
of this conclusion, these oocyte-conditioned media were found
to be incapable of stimulating proliferation of 32D C13 cells
(Figure 5). Vector DNA alone failed to select mRNA
corresponding to GM-CSF or multi-CSF (Figure 5a, b).
Taken together, these data demonstrate that pGM38
DNA can specifically select a mRNA encoding a factor with the
unique biological characteristics of GM-CSF from a mixture
containing both GM-CSF and multi-CSF mRNAs.

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I EXAMPLE III
This Example provides the nucleotide sequence of
GM-CSF gene.
Nucleotide sequence analysis of the cDNA portion of
pGM37 and pGM38 revealed that these two clones, which contain
sequences complementary to overlapping portions of the same
mRNA, contain the putative GM-CSF mRNA sequence. The
relationship between the cDNA portion of the two clones and
the mRNA is shown in Figure 2 . The nucleotide sequence
given in Figure 2 is a composite derived from both clones.
A stretch of 20 adenosine residues, corresponding to the
poly(A) tail of the mRNA, preceded by the hexanucloetide
AATAAA (found toward the 3' terminus of most eukaryotic
mRNAs) is present at one end of pGM38. This allowed
orientation of the sequences of the cDNA clones with that of
the mRNA.
The sequences presented in Fig. 2 contains a
single large open reading frame of 354 nucleotides; the amino
acid sequence predicted by this region is presented from the
first residue of the mature protein and is given above the
nucleotide sequence. From residues 2 to 29, the amino acid
sequence is identical (but for four residues) with the
partial NH2-terminal amino acid sequence for GM-CSF, which is
shown above that predicted by the cDNA. Of the four
discrepancies, two of the residues (20 and 24) had been only
tentatively assigned in the sequence of the protein. The
first residue on the mature peptide, which could not be
determined in the protein sequence, is predicted by the
nucleotide sequence to be isoleucine. The primary structure
of the protein deduced from the nucleotide sequence has a
molecular weight of 13,500, which is in good agreement with


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1 the apparent molecular weight of 16,800 for GM-CSF
extensively deglycosylated with endoglycosidase F. Two
potential N-glycosylation sites (Asn-X-Thr) which occur
within the predicted amino acid sequence, are indicated in
Figure 2b by asterisks.
pGM37 extends 20 nucleotides 5' to the first
position presented in Figure 2 and does not contain an
initiation codon; several hydrophobic amino acids are
contained within this region (not shown), a characteristic of
signal peptides of secreted proteins. The size of the GM-CSF
mRNA is -1,200 nucleotides (Figure 4), of which ti 150
nucleotides are probably contributed by the poly(A) tail.
The 3' untranslated region is 319 nucleotides and the region
encoding the mature protein 354 nucleotides (Figure 2').
Thus some 350 nucleotides remain for the putative
(NH2-terminal signal peptide an the 5' untranslated region.
There are three discrepancies between the
nucleotide sequence derived from the two cDNA clones
(positions 237, 346 and 507), one of which (position 346)
causes an amino acid sequence ambiguity (Gly or Ser at amino
acid residue 116). As the mice (C57BL/6) from which the cDNA
clones were isolated are highly inbred and hence should be
homozygous at the GM-CSF locus, and as there appears to be
only one gene encoding GM-CSF in the mouse germ line, it is
likely that these three sequences ambiguities reflect
artefacts created during cDNA synthesis, and indeed reverse
transcriptase has an error frequency on synthetic
polynucleotides of approximately one in 600 nucleotides.
Standard methods were employed for the
determination of the sequence presented in Figure 2.
Briefly, the DNA fragments subcloned in M13 vectors were
035


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1 sequenced by the chain-termination method using
dideoxy-nucleoside trisphosphates (Sanger, F. et al., Proc.
Nat'l. Acad. Sci. USA 74: 5463-67 (1977)). Sequencing
reactions were electrophoresed on thermostatically
controlled, 0.2 mm thick, 8% (w:v) polyacrylamide gels. As
mentioned above in reference to Figure 2, the sequence of the
mRNA-synonymous strand is listed 5' to 3', with the predicted
amino acid sequence of GM-CSF given above; numbers at the
ends of lines indicated the position of the final residue
(amino acid or nucleotide) on that line. The partial amino
acid sequence determined for GM-CSF is indicated above the
sequence derived from the clones; at positions where there is
no discordance between the two, dashes are given. The first
amino acid residue, determined by analysis of the protein,
jr, could not be assigned, and is indicated by a question mark.
Potential N-glycosylation sites are indicated with asterisks,
and the putative polyadenylation signal is underlined.

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EXAMPLE IV
This Example demonstrates that GM-CSF is encoded by
a unique gene.
As there are known to be multiple molecular forms
of GM-CSF isolated from various mouse organs, how many genes
the GM-CSF cDNA could detect in the murine genome was tested.
BALE/c embryo or C57BL/6 liver DNA digested with various
restriction endonucleases was fractionated on agarose gels,
transferred to nitrocellulose and hybridized with a fragment
containing the entire cDNA insert in pGM38. In both mouse
strains, a single EcoRI fragment (Figure 7, lanes 1,2), a
single PstI fragment (lanes 3, 4) and a single BamHI fragment
(not shown) were detected. Since the PstI fragment is only
2.5 kb in length it is unlikely that this fragment could
accommodate more than one gene. it is concluded therefore
that there is only one gene encoding the lung-type GM-CSF in
the mouse germ line, barring the unlikely possibility that
two (or more) genes are flanked by identically positioned
EcoRI, PstI and BamHI restriction sites.
Figure 7 also reveals that the GM-CSF gene is
contained within the same EcoRI fragment in DNA from
concanavalin A-stimulated LB3 cells (lane 6) as in embryo DNA
(lane 5). No additional bands were evident in LB3 DNr. nor
was the intensity of hybridization different from that of
embryo DNA, from which it is inferred that there are no
alterations in the copy number or gross changes in the
context of the GM-CSF gene in cells which synthesize large
quantities of GM-CSF.

35

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(22) Filed 1985-11-13
(45) Issued 2011-12-20

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Current owners on record shown in alphabetical order.
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RESEARCH CORPORATION TECHNOLOGIES, INC.
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Past Owners on Record
DUNN, ASHLEY ROGER
GOUGH, NICHOLAS MARTIN
METCALF, DONALD
RESEARCH CORPORATION
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