U.S. patent application number 10/796486 was filed with the patent office on 2004-09-02 for growth hormone and growth hormone releasing hormone compositions.
Invention is credited to Morsey, Mohamad A., Sheppard, Michael G..
Application Number | 20040171574 10/796486 |
Document ID | / |
Family ID | 32599528 |
Filed Date | 2004-09-02 |
United States Patent
Application |
20040171574 |
Kind Code |
A1 |
Morsey, Mohamad A. ; et
al. |
September 2, 2004 |
Growth hormone and growth hormone releasing hormone
compositions
Abstract
The present invention relates to methods and compositions of
growth hormone and/or growth hormone releasing hormone that promote
of the release and the elevation of growth hormone when
administered to animals. The present invention further relates to
methods and compositions of growth hormone and/or growth hormone
releasing hormone for treatment of diseases or disorders resulting
from growth hormone related deficiencies. The invention also
provides methods for producing novel growth hormone releasing
hormone variants and their uses thereof.
Inventors: |
Morsey, Mohamad A.;
(Niantic, CT) ; Sheppard, Michael G.; (North
Stonington, CT) |
Correspondence
Address: |
Kohn & Associates, PLLC
Suite 410
30500 Northwestern Hwy.
Farmington Hills
MI
48334
US
|
Family ID: |
32599528 |
Appl. No.: |
10/796486 |
Filed: |
March 8, 2004 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10796486 |
Mar 8, 2004 |
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09628730 |
Jul 28, 2000 |
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6759393 |
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09628730 |
Jul 28, 2000 |
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09546411 |
Apr 10, 2000 |
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60128830 |
Apr 12, 1999 |
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Current U.S.
Class: |
514/44R ;
435/456; 536/23.5 |
Current CPC
Class: |
C07K 14/61 20130101;
A61K 48/00 20130101; C07K 2319/00 20130101; A61K 38/00 20130101;
C07K 14/60 20130101 |
Class at
Publication: |
514/044 ;
536/023.5; 435/456 |
International
Class: |
A61K 048/00; C12N
015/861; C07H 021/04 |
Claims
1. A method for the treating growth hormone related disorders
characterized by growth hormone deficiencies in an animal,
comprising supplying the animal with a polynucleotide sequence that
encodes growth hormone releasing hormone or modified growth hormone
releasing hormone.
2. A method for improving the growth and performance of an animal,
comprising supplying the animal with a polynucleotide sequence that
encodes growth hormone releasing hormone or modified growth hormone
releasing hormone.
3. The method of claim 1 or 2 wherein a polynucleotide sequence
encoding growth hormone releasing hormone or modified growth
hormone releasing hormone is contained in pharmaceutically
acceptable carrier and is administered to an animal.
4. The method of claim 3 in which the carrier is a DNA vector, a
viral vector, a liposome or lipofectin.
5. The method of claim 4 in which the DNA vector is an expression
vector.
6. The expression vector of claim 5 containing a polynucleotide
sequence of growth hormone releasing hormone or modified growth
hormone releasing hormone in operative association with a
nucleotide regulatory sequence that controls expression of the
polynucleotide.
7. The expression vector of claim 6, wherein said regulatory
element is selected from the group consisting of the
cytomegalovirus hCMV immediate early gene, the early or late
promoters of SV40 adenovirus, and the swine alpha-skeletal actin
promoter.
8. The method of claim 2 in which the animal is a cat, dog, cow,
pig, horse, or chicken.
9. A method for the treating growth hormone related disorders
characterized by growth hormone deficiencies in an animal,
comprising supplying the animal with a polynucleotide sequence that
encodes growth hormone or modified growth hormone.
10. A method for improving the growth and performance of an animal,
comprising supplying the animal with a polynucleotide sequence that
encodes growth hormone or modified growth hormone.
11. The method of claim 9 or 10 wherein a polynucleotide sequence
encoding growth hormone or modified growth hormone is contained in
pharmaceutically acceptable carrier and is administered to an
animal.
12. The method of claim 11 in which the carrier is a DNA vector, a
viral vector, a liposome or lipofectin.
13. The method of claim 12 in which the DNA vector is an expression
vector.
14. The method of claim 13, wherein the expression vector includes
a polynucleotide sequence of growth hormone or modified growth
hormone in operative association with a nucleotide regulatory
sequence that controls expression of the polynucleotide.
15. The method of claim 14, wherein the regulatory element is
selected from the group consisting of the cytomegalovirus hCMV
immediate early gene, the early or late promoters of SV40
adenovirus, and the swine alpha-skeletal actin promoter.
16. The method of claim 15 in which the animal is a dog, cat, cow,
pig, horse, or chicken
17. A growth hormone releasing hormone (GHRH) variant comprising
the addition of one amino acid to the amino terminus of a 29 amino
acid amino terminal fragment of GHRH, in a pharmaceutical
formulation suitable for delivery to a human or livestock.
18. The growth hormone releasing hormone of claim 17 wherein the
amino acid is a hydrophobic residue or tyrosine.
19. A growth hormone releasing hormone variant comprising the
addition of two or three amino acids to the amino terminus, of a 29
amino acid amino terminal fragment of GHRH wherein the second amino
acid is not proline or alanine, and in a pharmaceutical formulation
suitable for delivery to a human or livestock.
20. The growth hormone releasing hormone variant of claim 19
comprising the addition of more than three amino acids to the amino
terminus of a 29 amino acid amino terminal fragment of GHRH,
wherein the addition does not interfere with the functional
activity of growth hormone lreleasing hormone.
21. The growth hormone releasing hormone variant of claim 17, 19,
or 20, further comprising a substitution of glycine with alanine at
residue 15.
22. The growth hormone releasing hormone variant of claim 17, 19,
or 20 further comprising a substitution of leucine with alanine at
residue 22.
23. The growth hormone releasing hormone variant of claim 17, 19,
or 20, further comprising substitutions of glycine with alanine at
residue 15 and leucine with alanine at residue 22.
24. The growth hormone releasing hormone variant of claim 17, 19,
or 20, further comprising the addition of glycine and arginine at
the carboxy-terminus.
25. The growth hormone releasing hormone variant of claim 18, 19,
or 21 in which the amino acids are naturally occurring.
26. A polynucleotide sequence encoding the growth hormone releasing
hormone variant of claim 17, 19, or 20.
27. A nucleotide vector containing the polynucleotide sequence of
claim 26.
28. An expression vector containing the polynucleotide sequence of
claim 26 in operative association with a nucleotide regulatory
sequence that controls expression of the polynucleotide sequence in
a host cell.
29. The expression vector of claim 28, wherein said regulatory
element is selected from the group consisting of the
cytomegalovirus hCMV immediate early gene, the early or late
promoters of SV40 adenovirus, and the swine alpha-skeletal actin
promoter.
30. A genetically engineered host cell that contains the
polynucleotide sequence of claim 26.
31. A genetically engineered host cell that contains the
polynucleotide sequence of claim 26 in operative association with a
nucleotide regulatory sequence that controls expression of the
polynucleotide sequence in the host cell.
32. A method for the treating growth hormone related disorders
characterized by growth hormone deficiencies in an animal
comprising supplying the animal with a polynucleotide sequence that
encodes the growth hormone releasing hormone variant of claim 17,
19, or 20.
33. A method for improving the growth and performance of an animal,
comprising supplying the animal with a polynucleotide sequence that
encodes a growth hormone releasing hormone variant of claim 17, 19,
or 20.
34. A purified polypeptide of the growth hormone releasing hormone
variant of claim 17, 19, or 20.
35. A method for the treating growth hormone related disorders
characterized by growth hormone deficiencies in an animal,
comprising supplying the animal with an effective amount of a
polypeptide of claim 35.
36. A method for improving the growth and performance of an animal,
comprising supplying the animal with an effective amount of a
polypeptide of claim 34.
37. A pharmaceutical composition for promoting the expression and
elevation of growth hormone in an animal, comprising administering
to said animal an effective amount of the growth hormone releasing
hormone variant of claim 17, 19, or 20.
38. A pharmaceutical composition for the treatment of growth
hormone related disorders characterized by growth hormone
deficiencies in an animal, comprising administering to said animal
an effective amount of the growth hormone releasing hormone variant
of claim 17, 19, or 20.
39. A pharmaceutical composition for the improvement of growth and
performance of an animal, comprising administering to said animal
an effective amount of a growth hormone releasing hormone variant
of claim 17, 19, or 20.
Description
[0001] The present invention relates to novel variants of growth
hormone releasing hormone (GHRH) that have enhanced resistance to
enzymatic degradation and polynucleotides encoding said GHRH
variants. The present invention relates to methods and therapeutic
compositions for the treatment of growth hormone related
deficiencies comprising administrating to humans, companion
animals, livestock or poultry, plasmid compositions comprising
polynucleotides encoding GHRH or variants thereof, alone or in
combination with polynucleotides encoding growth hormone or
modified growth hormone. The present invention further relates to
methods and compositions that promote the release and expression of
growth hormone in order to enhance the growth and performance of
companion animals, livestock or poultry comprising the
administration plasmid compositions encoding GHRH variants, GHRH or
modified GHRH, and/or GH or modified GH.
BACKGROUND OF THE INVENTION
[0002] Growth hormone-releasing hormone ("GHRH") is a peptide
hormone secreted from the hypothalamus. Following secretion, GHRH
enters the portal circulation connecting the hypothalamus to
pituitary gland. GHRH then interacts with its receptors on the
pituitary gland and induces the release of growth hormone ("GH").
GH secreted from the pituitary gland enters the general circulation
and from there it reaches various organs and tissues of the body
where it interacts with specific receptors and induces a wide range
of developmental effects.
[0003] GHRH peptides have been isolated and characterized from
several species including humans, porcine, ovine and bovine. In
each of these species, GHRH is a small polypeptide consisting of 44
amino acids (GHRH(1-44)-NH.sub.2). However, it has been also shown
that smaller fragments, most notably those consisting of the first
(amino terminal) 29 amino acids (referred to as GHRH1-29 fragment)
retain the same intrinsic biological activity as the full length
parent molecule.
[0004] GHRH is synthesized as a precursor polypeptide consisting of
107 or 108 amino acids depending on the species. Following
synthesis, the precursor GHRH polypeptide undergoes sequential
processing. First, the 31 amino acid signal peptide (Met.sup.-30 to
Arg.sup.0) of the GHRH precursor polypeptide is cleaved (Smith et
al., 1992, Biotechnology 10:315-319). Subsequently, the GHRH
precursor polypeptide is cleaved at position 46-47 and at position
45-46 by a trypsin-like endopeptidase and a carboxypeptidase,
respectively, resulting in generation of GHRH(1-45)-OH and a 30
amino acid peptide (amino acids 77-107) designated GCTP (Brar, A.
K. et al., 1991, Endocrinology 129: 3274-3280). The GHRH(1-45)-OH
polypeptide is further processed by peptidyl glycine
.alpha.-amidating monooxygenase ("PAM"), which transfers an amide
group from Gly.sup.45 to Leu.sup.44 and results in the formation of
GHRH(1-44)-NH.sub.2, the full length form of GHRH (Brar, A. K. et
al., 1991, Endocrinology 129: 3274-3280). The GCTP is also
undergoes processing by PAM, which results in the transfer of an
amide group from Gly.sup.77 to Gln.sup.76. Although the role of
GHRH(1-44)-NH.sub.2 in inducing the release of GH is well
established, the role of the GCTP peptide is less clear. One report
has implicated the GCTP peptide in the control of feeding behavior
(Arase, K. et al., 1987, Endocrinology 121:1960-1965).
[0005] GH has been identified and its gene cloned from many species
including human, porcine, bovine, and equine. Unlike GHRH, there
exists natural variants of GH within a given species. For example,
bovine GH is released from the pituitary gland in one of four
variants which differ from one another by one or more amino acids
and some studies suggest that these variants differ in their
potency (e.g., in terms of their ability to increase milk yield).
Several studies have also identified amino acid substitutions that
lead to an increase in the affinity of GH to its receptors and/or
enhanced stability to enzymatic degradation. Studies have also
shown that immunization against specific peptides from GH (e.g., a
peptide consisting of amino acids 35 to 53 of GH) leads to
production of antibodies that bind growth hormone and increase the
efficacy of GH treatment, presumably because the antibodies delay
the clearance of GH from circulation, thus, increasing half-life of
GH, and/or protect GH from proteolytic degradation (Bomford, F. and
Aston, P., 1990, Endocrinology 125:31-38).
[0006] Significant research efforts have focused on the structural
attributes of GH and GHRH, as well as their biological and
developmental activities. A number of groups have attempted to
exploit GH and GHRH in a manner that could provide important
therapeutic and economic benefits as a result of their use in
humans and animals. For example, the traditional treatment of
GH-deficient children has been the administration of growth hormone
isolated from human pituitary glands, however these preparations
are no longer available in the United States due to
virus-contaminated samples (Vance, 1990, Clin. Chem 36/3: 415-420).
Recombinantly expressed and purified GH have been shown to have
some benefits in treating GH-deficient children, however the
combination of recombinantly expressed GH and GHRH in the treatment
of GH-deficient children has provided conflicting results. (Vance,
supra). Further, purified GH and GHRH must be administered at very
high quantities to be effective as the exposure of these
polypeptides to serum results in their rapid degradation to a
polypeptide which exhibits considerably different biological and
pharmacokinetic properties. (Fronman et al., 1989, J. Clin. Invest.
83:1533-1540).
[0007] Other studies have shown that GH or GHRH administered as
purified polypeptides have significant impact on animal growth
(muscle and bone growth), average daily gain, milk production, feed
efficiency (the ratio of feed consumed to body weight gain),
adipose tissue accretion and others. For example, it has been shown
that daily administration of maximally effective doses of GH
administered to growing pigs for 33-77 days can increase average
daily gain -10-20%, improves feed efficiency 13-33%, decrease
adipose tissue accretion by as much as 70%, and stimulates protein
deposition (muscle growth) by as much as 62%. (Etherton et
al.,1998, Physiological Reviews 78:745-761). Furthermore, when GH
was administered to dairy cows, milk yields were increased by
10-15% (.quadrature.4-6kg/day) (Etherton et al.,1998, Physiological
Reviews 78:745-761).
[0008] A major impediment to fulfilling the therapeutic and
economic potential of GHRH peptides is their susceptibility to
cleavage (and subsequent conversion to inactive forms) by specific
tissue and plasma proteolytic enzymes; most notably
dipeptidylpeplidase IV ("DPPIV"). A number of researchers have
focused on manipulating GHRH in order to develop compounds with
significant therapeutic potential. Consequently, a wide variety of
synthetic GHRH peptide analogues have been produced. They consist
of GHRH polypeptides in which one or more amino acids have been
chemically modified or replaced with other L- or D-amino acids.
These modifications or substitutions are designed to yield
analogues with biological properties superior to those of the
parent molecule in terms of potency, stability and resistance to
chemical and enzymatic degradation. However, these chemically
modified polypeptides are not easily or efficiently produced in a
suitable form to be administered to humans or animals.
[0009] In spite of the significant therapeutic and economic
benefits of GH or GHRH alluded to above, exogenous supplementation
of animals with GH or GHRH proteins have not been widely adopted as
a component of routine management practices to enhance the quality
of meat from animals and/or enhance the productivity of livestock.
This is because in order to get these benefits, animals have to be
repeatedly administered GH or GHRH polypeptides (often daily, but
typically in a slow release formulation given every 7-10 days).
(Etherton, T. D., 1997, Nature Biotechnology 15:12.sup.48) This
situation is labor intensive, time consuming, expensive, and does
not fit current management practices where animals are reared in
large numbers and are handled very infrequently, it is apparent
therefore that in order to realize the therapeutic and economic
benefits of GH and/or GHRH administration, much improved
formulations for delivery of these hormones must be developed to
overcome the current limitations of their use; namely the need for
repeated administration.
SUMMARY OF THE INVENTION
[0010] The present invention relates to novel variants of GHRH that
have enhanced resistance to enzymatic degradation and
polynucleotides encoding said variants. The present invention also
relates to pharmaceutical formulations comprising polynucleotide
sequences encoding GHRH variants alone or in combination with
polynucleotide sequences encoding GHRH, modified GHRH, GH and/or
modified GH. The present invention also relates to pharmaceutical
formulations comprising GHRH variant peptides alone or in
combination with GHRH polypeptides, modified GHRH polypeptides, GH
polypeptides and/or modified GH polypeptides. The present invention
further relates to pharmaceutical formulations comprising canine or
feline GHRH peptides alone or in combination with GHRH variant
polypeptides, modified GHRH polypeptides, GH polypeptides and/or
modified GH polypeptides.
[0011] The present invention relates to therapeutic methods and
compositions for the treatment of growth hormone related
deficiencies comprising growth hormone ("GH") and/or growth
hormone-releasing hormone ("GHRH") in human, companion animals,
livestock and poultry. The invention also relates to methods for
the improvement in the health of humans, companion animals,
livestock and poultry. The invention also relates to methods for
the treatment of obesity and frailty of companion animals. The
invention further relates to methods for the enhancement of the
growth and performance of companion livestock and poultry. The
methods of the present invention comprise pharmaceutical
compositions which enhance the expression of growth hormone or
promote the release of growth hormone or both when administered to
humans, companion animals, livestock or poultry. According to the
present invention, the term "GHRH" relates to the full length
wildtype form of GHRH which is 44 amino acids (aa) or a precursor
form of GHRH. In accordance with the present invention, the term
"modified GHRH" refers to any amino terminal polypeptide fragment
of GHRH from 29 amino acids to 107 or 108 amino acids in length and
any mutant of GHRH, including additions, deletions or substitutions
at the nucleotide or amino acid level, which retains at least the
level of activity of wildtype GHRH, that is, the ability to induce
GH gene transcription at levels comparable to wildtype GHRH.
[0012] In accordance with the present invention, the term "GHRH
variant" relates to a GHRH polypeptide to which one or more amino
acids have been attached to the carboxy or amino terminus of the
polypeptide, or a wildtype GHRH polypeptide that contains a
substitution of one or more amino acids, so that the GHRH variant
retains at least equal or enhanced wildtype GHRH activity and has
enhanced resistance to enzymatic degradation relative to the
wildtype GHRH. In accordance with the present invention, wildtype
GHRH activity is measured by its ability to induce GH gene
transcription. In accordance with the present invention, resistance
to enzymatic degradation is determined by the ability of the
polypeptide to resist degradation caused by dipeptidylpeptidase
type IV.
[0013] According to the present invention, the term "GH" refers to
the full length wildtype form of GH, which is 191 amino acids, and
"modified GH" refers to any fragment of GH and any mutant including
additions, deletions or substitutions at the nucleotide or amino
acid level, which retains at least the level of wildtype activity
of GH, that is, the ability to induce insulin growth factor (IGF)
gene transcription at levels comparable to wildtype GH, or mimic
the anti-adipogenic and lipolytic effects of GH.
DESCRIPTION OF THE FIGURES
[0014] FIG. 1 is a map of the pGHRH4 construct (SEQ ID No.47).
[0015] FIG. 2 is a map of the pGHRH1-44SK construct (SEQ ID
No.48).
[0016] FIG. 3 is a map of the pGHRH1-44WTSK685 construct (SEQ ID
No. 49).
[0017] FIG. 4 is a map of the pGHRH1-44WTSK2014 construct (SEQ ID
No. 50).
[0018] FIG. 5 is a graph depicting the GHRH expression levels
detected in supernatants from pGHRH4 transfected cells.
DETAILED DESCRIPTION OF THE INVENTION
[0019] The present invention relates to novel variants of GHRH that
have enhanced resistance to enzymatic degradation and
polynucleotides encoding said GHRH variants. The present invention
relates to pharmaceutical compositions which promote the release
and/or expression of GH. In particular, the pharmaceutical
compositions of the present invention comprise polynucleotide
sequences encoding GHRH variants alone or in combination with
polynucleotide sequences encoding GHRH, modified GHRH, GH and/or
modified GH or any combination thereof. In another embodiment, the
pharmaceutical compositions of the present invention comprise GHRH
variant polypeptides alone or in combination with GHRH
polypeptides, GHRH modified polypeptides, GH polypeptides or GH
modified polypeptides or any combination thereof.
[0020] The present invention relates to methods of treating
disorders related to GH related deficiencies in humans, companion
animals, livestock and poultry, comprising administering
pharmaceutical formulations which enhance GH expression and/or
release. The present invention further relates to methods of
treating livestock and poultry in order to enhance growth and
performance comprising administering pharmaceutical formulations
which enhance GH expression and/or release.
[0021] The pharmaceutical formulations to be administered in
accordance with the methods of the present invention encompass
plasmid compositions comprising (a) polynucleotide sequences
encoding GHRH variants; (b) polynucleotide sequences encoding GHRH
or modified GHRH; (c) polynucleotide sequences encoding GH or
modified GH; or any combination thereof, wherein the polynucleotide
sequences are operably linked to a promoter or regulatory element,
preferably one that is transcriptionally active in muscle tissue.
The pharmaceutical formulations to be administered in accordance
with the methods of the present invention also include: i) plasmid
compositions comprising polynucleotides encoding for GH or modified
or variant GHRH gene; ii) plasmid compositions comprising
polynucleotides encoding for both GH and GHRH genes; iii) plasmid
compositions comprising polynucleotides encoding for a GHRH gene, a
GH gene or a gene encoding a fusion protein consisting of a peptide
from GH and a carrier protein for induction of GH potency-enhancing
antibodies; (iv) recombinant proteins, peptides, fragments or
derivatives thereof comprising canine GHRH or feline GHRH; (v)
recombinant proteins, peptides, fragments or derivatives thereof of
the GHRH variants of the present invention; and (vi) recombinant
fusion proteins, peptides, fragments or derivatives thereof
comprising GH and GHRH.
[0022] In one embodiment, the pharmaceutical compositions of the
present invention comprise polynucleotide sequences encoding canine
or feline GHRH alone or in combination with polynucleotide
sequences encoding GHRH variant, modified GHRH, GH and/or modified
GH or any combination thereof. In another embodiment, the
pharmaceutical compositions of the present invention comprise
canine or feline GHRH polypeptides alone or in combination with
GHRH variant polypeptides, modified GHRH, GH polypeptides or GH
modified polypeptides or any combination thereof. The
pharmaceutical compositions of the present invention are in
suitable formulation to be administered to humans, companion
animals, livestock or poultry for the treatment of growth hormone
related deficiencies or the enhancement of growth and performance
of livestock and poultry. The pharmaceutical compositions of the
present invention are also in suitable formulation for the
treatment of obesity and frailty of companion animals or the
improvement in the health of humans, companion animals, livestock,
and poultry.
[0023] The present invention relates to therapeutic methods and
compositions for the treatment of growth hormone related
deficiencies comprising growth hormone ("GH"); modified GH; growth
hormone releasing hormone ("GHRH"); GHRH variants; modified GHRH or
any combination thereof. The therapeutic compositions of the
invention are administered to animals, preferably to mammals, more
preferably to companion animals (e.g., dogs, cats and horses),
livestock (e.g., cows and pigs) and poultry (e.g., chickens and
turkeys), and most preferably to humans. The invention also relates
to methods and compositions for the enhancement of the growth and
performance of animals, more preferably mammals, and most
preferably livestock (e.g., cows and pigs) and poultry (e.g.,
chickens and turkeys) with the proviso that such compositions are
not to be administered to mice, rats, rodents, guinea pigs, or
rabbits. The invention also relates to methods and compositions for
the treatment of obesity and frailty of animals, preferably to
mammals, more preferably to companion animals (e.g., dogs, cats and
horses). The invention further relates to methods and compositions
for the improvement in the health of animals, preferably to
mammals, more preferably to companion animals (e.g., dogs, cats and
horses), livestock (e.g., cows and pigs) and poultry (e.g.,
chickens and turkeys), and most preferably to humans.
[0024] The present invention is based in part of the discovery of
recombinantly engineered GHRH variants which retain at least the
level of activity wildtype GHRH, that is the ability to induce GH
gene transcription at levels comparable to wildtype GHRH, and which
have enhanced resistance to enzymatic degradation relative to the
wildtype GHRH. The GHRH variants of the present invention may be
recombinantly expressed at high levels in host cells and easily
isolated and purified in a form suitable for administration to
humans and animals. Thus, the GHRH variants of the present
invention may be efficiently produced and isolated at high levels
as opposed to modified GHRH polypeptides in the art which are
modified using traditional chemistry methods to introduce
modifications in the native GHRH sequence.
[0025] In one embodiment, a GHRH variant of the present invention
comprises the addition of one amino acid, preferably a hydrophobic
residue and more preferably a tyrosine residue, to the amino
terminus (position 1) of GHRH. In another embodiment, a GHRH
variant comprises the addition of two amino acids, wherein the
second amino acid is not proline or alanine, to the amino terminus
(position 1) of GHRH. In another embodiment, a GHRH variant
comprises the addition of three amino acids, wherein the second
amino acid is proline or alanine, to the amino terminus (position
1) of GHRH. In another embodiment, a GHRH variant comprises the
addition of more than three amino acids to the amino terminus
(position 1) of GHRH, wherein the addition does not interfere with
the functional activity of GHRH, that is, the ability of GHRH to
induce GH gene transcription. In a preferred embodiment of the
present invention, a GHRH variant comprises the addition of a
tripeptide to the amino terminus, wherein the tripeptide is
diprotin A or diprotin B or a peptide with a structure analogous to
diprotin A or diprotin B. In yet another embodiment, a GHRH variant
comprises the addition of glycine and arginine at the
carboxy-terminus. This addition results in the amidation of GHRH;
the glycine and arginine is cleaved off and the last amino acid
before the added glycine is amidated.
[0026] In one embodiment, a GHRH variant comprises any of the amino
acid additions described above and the substitution of glycine with
alanine at residue 15. In another embodiment, a GHRH variant
comprises any of the amino acid additions described above and the
substitution of leucine with alanine at residue 22. In another
embodiment, a GHRH variant comprises any of the amino acid
additions described above and the substitutions of glycine with
alanine at residue 15 and leucine with alanine at residue 22. In
another embodiment, a GHRH variant comprises any of the amino acid
additions at the amino terminus and the amino acid additions at the
carboxy-terminus. In another embodiment, a GHRH variant comprises
any of the amino acid additions at the amino terminus described
above, the amino acid additions at the carboxy-terminus described
above, and the substitution of glycine to alanine at residue 15. In
another embodiment, a GHRH variant comprises any of the amino acid
additions at the amino terminus described above, the amino acid
additions at the carboxy-terminus described above, and the
substitution of leucine to alanine at residue 22. In yet another
embodiment, a GHRH variant comprises any of the amino acid
additions at the amino terminus described above, the amino acid
additions at the carboxy-terminus described above, and the
substitutions of glycine to alanine at residue 15 and leucine to
alanine at residue 22. The term "GHRH precursor variant" as used
herein refers to a precursor form of the full length wildtype GHRH
polypeptide to which one or more amino acids have been attached to
the amino terminus of the polypeptide and/or contains a
substitution of one or more amino acids, so that the GHRH precursor
variant retains at least equal or enhanced wildtype GHRH activity
and has enhanced resistance to enzymatic degradation relative to
wildtype GHRH. In one embodiment of the present invention, a GHRH
precursor variant comprises the amino acid additions and/or the
amino acid substitutions described above. The present invention
also encompasses a fusion variant comprising any of the GHRH
variants described above and GH or modified GH. The term "fusion
variant" as used herein refers to a fusion protein comprising GHRH
variants or modified GHRH and GH or modified GH. In one embodiment
of the present invention, a fusion variant comprises any of the
GHRH variants described above, which consist of amino acid
additions at the amino terminus and/or amino acid substitutions,
and GH or modified GH.
[0027] The modifications and/or substitutions of GHRH described
herein are made to GHRH polypeptides which retain the biological
activity at least equal to the full length wildtype GHRH,
preferably a precursor form of GHRH, more preferably the sequence
of GHRH consisting of about 29 amino acids to about 44 amino acids.
The polynucleotide sequences encoding the GHRH variants described
herein are also within the scope of the present invention. The
present invention provides that the polypeptides are encoded by the
nucleic acid fragments of the present invention or by degenerate
variants of the nucleic acid fragments of the present invention. By
"degenerate variant" is intended nucleotide fragments which differ
from a nucleic acid fragment of the present invention (e.g., an
ORF) by nucleotide sequence but, due to the degeneracy of the
genetic code, encode an identical polypeptide sequence. Preferred
nucleic acid fragments of the present invention are the ORFs that
encode proteins. The present invention encompasses GHRH variants
encoded by the polynucleotide sequence any species.
[0028] The present invention encompasses polynucleotide sequences
encoding precursor forms of GHRH, full length wildtype GHRH,
modified GHRH, GHRH variants, and fragments of GHRH from 29 amino
acids to 44 amino acids in length for any species, which retain at
least the activity of wildtype GHRH. For example, the
polynucleotide sequences encoding human, swine, and bovine growth
hormone releasing hormone disclosed in Genbank accession number
SEG_HSGHRH, accession number U90275, and accession number U29611,
respectively, are incorporated herein by reference. The present
invention also encompasses polynucleotide sequences encoding GHRH
polypeptides disclosed for any species (e.g., the polynucleotide
sequence encoding the human GHRH precursor polypeptide disclosed in
Genbank accession number P01286 is incorporated herein by
reference). The present invention further encompasses
polynucleotide sequences encoding full length wildtype GH or
modified GH for any species. For example, the polynucleotide
sequences encoding human, swine, and bovine growth hormone
disclosed in Genbank accession number J03071, accession number
U19787, and accession number E00293, respectively, are incorporated
herein by reference. The present invention also encompasses
polynucleotide sequences encoding GH polypeptides disclosed for any
species (e.g., the polynucleotide sequence encoding the bovine GH
polypeptide disclosed in Genbank accession number STBO is
incorporated herein by reference).
[0029] The polynucleotide sequence encoding GHRH, modified GHRH or
GHRH variants can be inserted into an appropriate expression
vector, i.e., a vector which contains the necessary elements for
the transcription and translation of the inserted protein-coding
sequence. The polynucleotide sequence encoding GH or modified GH
can be inserted into an appropriate expression vector, i.e., a
vector which contains the necessary elements for the transcription
and translation of the inserted protein-coding sequence. The
necessary transcriptional and translational signals can also be
supplied by the native GH or native GHRH genes or its flanking
regions. A variety of host-vector systems may be utilized to
express the protein-coding sequence. These include but are not
limited to mammalian cell systems infected with virus (e.g.,
vaccinia virus, adenovirus, etc.); insect cell systems infected
with virus (e.g., baculovirus); microorganisms such as yeast
containing yeast vectors, or bacteria transformed with
bacteriophage, DNA, plasmid DNA, or cosmid DNA. The expression
elements of vectors vary in their strengths and specificities.
Depending on the host-vector system utilized, any one of a number
of suitable transcription and translation elements may be used. In
one embodiment, the wildtype or modified human GH gene is
expressed. In another embodiment, the wildtype, modified or variant
human GHRH is expressed. In yet another embodiment, the wildtype or
modified human GH and the wildtype, modified or variant human GHRH
gene are expressed.
[0030] Any of the methods previously described for the insertion of
DNA fragments into a vector may be used to construct expression
vectors containing a chimeric gene, comprising GH or modified GH
and GHRH, modified GHRH or GHRH variants, consisting of appropriate
transcriptional and translational control signals and the protein
coding sequences. These methods may include in vitro recombinant
DNA and synthetic techniques and in vivo recombinants (genetic
recombination). Expression of the nucleic acid sequence encoding GH
or modified GH may be regulated by a second nucleic acid sequence
so that the GH or modified GH is expressed in a host transformed
with the recombinant DNA molecule. Expression of the nucleic acid
sequence encoding GHRH, modified GHRH or GHRH variant may be
regulated by a second nucleic acid sequence so that the GHRH
modified GHRH or GHRH variant is expressed in a host transformed
with the recombinant DNA molecule. For example, expression of GH or
GHRH may be controlled by any promoter or enhancer element known in
the art. Promoters which may be used to control GH and/or GHRH gene
expression include, but are not limited to, the Cytomeglovirus
(CMV) immediate early promoter region, the SV40 early promoter
region (Bernoist and Chambon, 1981, Nature 290:304-310), the
promoter contained in the 3' long terminal repeat of Rous sarcoma
virus (Yamamoto, et al., 1980, Cell 22:787-797), the herpes
thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad.
Sci. USA 78:1441-1445), the regulatory sequences of the
metallothionein gene (Brinster et al., 1982, Nature 296:3942);
prokaryotic expression vectors such as the .beta.-lactamase
promoter (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. Sci. USA
75:3727-3731), or the tac promoter (DeBoer et al., 1983, Proc.
Natl. Acad. Sci. USA 80:21-25); see also "Useful proteins from
recombinant bacteria" in Scientific American, 1980, 242:74-94;
plant expression vectors comprising the nopaline synthetase
promoter region (Herrera-Estrella et al., Nature 303:209-213) or
the cauliflower mosaic virus 35S RNA promoter (Gardner et al.,
1981, Nucl. Acids Res. 9:2871), and the promoter of the
photosynthetic enzyme ribulose biphosphate carboxylase
(Herrera-Estrella et al., 1984, Nature 310:115-120); promoter
elements from yeast or other fungi such as the Gal 4 promoter, the
ADC (alcohol dehydrogenase) promoter, PGK (phosphoglycerol kinase)
promoter, alkaline phosphatase promoter, and the following animal
transcriptional control regions, which exhibit tissue specificity
and have been utilized in transgenic animals: elastase I gene
control region which is active in pancreatic acinar cells (Swift et
al., 1984, Cell 38:639-646; Ornitz et al., 1986, Cold Spring Harbor
Symp. Quant Biol. 50:399409; MacDonald, 1987, Hepatology
7:425-515); insulin gene control region which is active in
pancreatic beta cells (Hanahan, 1985, Nature 315:115-122),
immunoglobulin gene control region which is active in lymphoid
cells (Grosschedl et al., 1984, Cell 38:647-658; Adames et al.,
1985, Nature 318:533-538; Alexander et al., 1987, Mol. Cell. Biol.
7:1436-1444), mouse mammary tumor virus control region which is
active in testicular, breast, lymphoid and mast cells (Leder et
al., 1986, Cell 45:485-495), albumin gene control region which is
active in liver (Pinkert et al., 1987, Genes and Devel. 1:268-276),
alpha-fetoprotein gene control region which is active in liver
(Krumlauf et al., 1985, Mol. Cell. Biol. 5:1639-1648; Hammer et
al., 1987, Science 235:53-58; alpha 1-antitrypsin gene control
region which is active in the liver (Kelsey et al., 1987, Genes and
Devel. 1:161-171), beta-globin gene control region which is active
in myeloid cells (Mogram et al., 1985, Nature 315:338-340; Kollias
et al., 1986, Cell 46:89-94; myelin basic protein gene control
region which is active in oligodendrocyte cells in the brain
(Readhead et al., 1987, Cell 48:703-712); myosin light chain-2 gene
control region which is active in skeletal muscle (Sani, 1985,
Nature 314:283-286), swine alpha-skeletal actin control region
which is active in muscle (Reecy, M. et al., 1998, Animal
Biotechnology 9:101-120) and gonadotropic releasing hormone gene
control region which is active in the hypothalamus (Mason et al.,
1986, Science 234:1372-1378).
[0031] In a specific embodiment, a vector is used that comprises a
promoter operably linked to GH- or modified GH-encoding nucleic
acid, one or more origins of replication, and, optionally, one or
more selectable markers (e.g., an antibiotic resistance gene). In
another embodiment, a vector is used that comprises a promoter
operably linked to GHRH-, modified GHRH- or GHRH variant-encoding
nucleic acid, one or more origins of replication, and, optionally,
one or more selectable markers (e.g., an antibiotic resistance
gene). In yet another embodiment, a vector is used that comprises a
promoter operably linked to GH or modified GH and GHRH, modified
GHRH or GHRH variant-encoding nucleic acids, one or more origins of
replication, and, optionally, one or more selectable markers (e.g.,
an antibiotic resistance gene).
[0032] Expression vectors containing gene inserts can be identified
by three general approaches: (a) nucleic acid hybridization, (b)
presence or absence of "marker" gene functions, and (c) expression
of inserted sequences. In the first approach, the presence of the
GH- or modified GH-encoding polynucleotides and GHRH-, modified
GHRH- or GHRH variant-encoding polynucleotides inserted in an
expression vector(s) can be detected by nucleic acid hybridization
using probes comprising sequences that are homologous to the
inserted genes. In the second approach, the recombinant vector/host
system can be identified and selected based upon the presence or
absence of certain "marker" gene functions (e.g., thymidine kinase
activity, resistance to antibiotics, transformation phenotype,
occlusion body formation in baculovirus, etc.) caused by the
insertion of the gene(s) in the vector(s). For example, if the GH
gene is inserted within the marker gene sequence of the vector,
recombinants containing the GH gene insert can be identified by the
absence of the marker gene function. In the third approach,
recombinant expression vectors can be identified by assaying the
gene product expressed by the recombinant. Such assays can be
based, for example, on the physical or functional properties of the
GH and GHRH in in vitro assay systems, e.g., binding of GH with
anti-GH antibody or binding of GHRH with anti-GHRH antibody.
[0033] Once a particular recombinant DNA molecule is identified and
isolated, several methods known in the art may be used to propagate
it. Once a suitable host system and growth conditions are
established, recombinant expression vectors can be propagated and
prepared in quantity. As previously explained, the expression
vectors which can be used include, but are not limited to, the
following vectors or their derivatives: human or animal viruses
such as vaccinia virus or adenovirus; insect viruses such as
baculovirus; yeast vectors; bacteriophage vectors (e.g., lambda),
and plasmid and cosmid DNA vectors, to name but a few.
[0034] In addition, a host cell strain may be chosen which
modulates the expression of the inserted sequences, or modifies and
processes the gene product in the specific fashion desired.
Expression from certain promoters can be elevated in the presence
of certain inducers; thus, expression of the genetically engineered
may be controlled. Furthermore, different host cells have
characteristic and specific mechanisms for the translational and
post-translational processing and modification (e.g.,
glycosylation, phosphorylation of proteins). Appropriate cell lines
or host systems can be chosen to ensure the desired modification
and processing of the foreign protein expressed. For example,
expression in a bacterial system can be used to produce an
unglycosylated core protein product. Expression in yeast will
produce a glycosylated product. Expression in mammalian cells can
be used to ensure "native" glycosylation of a heterologous protein.
Furthermore, different vector/host expression systems may effect
processing reactions to different extents.
[0035] For long-term, high-yield production of recombinant
proteins, stable expression is preferred. For example, cell lines
which stably express the differentially expressed or pathway gene
protein may be engineered. Rather than using expression vectors
which contain viral origins of replication, host cells can be
transformed with DNA controlled by appropriate expression control
elements (e.g., promoter, enhancer, sequences, transcription
terminators, polyadenylation sites, etc.), and a selectable marker.
Following the introduction of the foreign DNA, engineered cells may
be allowed to grow for 1-2 days in an enriched media, and then are
switched to a selective media. The selectable marker in the
recombinant plasmid confers resistance to the selection and allows
cells to stably integrate the plasmid into their chromosomes and
grow to form foci which in turn can be cloned and expanded into
cell lines. This method may advantageously be used to engineer cell
lines which express the differentially expressed or pathway gene
protein. Such engineered cell lines may be particularly useful in
screening and evaluation of compounds that affect the endogenous
activity of the differentially expressed or pathway gene
protein.
[0036] A number of selection systems may be used, including but not
limited to the herpes simplex virus thymidine kinase (Wigler, et
al., 1977, Cell 11:223), hypoxanthine-guanine
phosphoribosyltransferase (Szybalska & Szybalski, 1962, Proc.
Natl. Acad. Sci. USA 48:2026), and adenine
phosphoribosyltransferase (Lowy et al., 1980, Cell 22:817) genes
can be employed in tk.sup.-, hgprt.sup.- or aprt.sup.- cells,
respectively. Also, antimetabolite resistance can be used as the
basis of selection for dhfr, which confers resistance to
methotrexate (Wigler et al., 1980, Natl. Acad. Sci. USA 77:3567;
O'Hare et al., 1981, Proc. Natl. Acad. Sci. USA 78:1527); gpt,
which confers resistance to mycophenolic acid (Mulligan & Berg,
1981, Proc. Natl. Acad. Sci. USA 78:2072); neo, which confers
resistance to the aminoglycoside G-418 (Colberre-Garapin et al.,
1981, J. Mol. Biol. 150:1); and hygro, which confers resistance to
hygromycin (Santerre et al., 1984, Gene 30:147) genes.
[0037] The present invention provides for the treatment or
prevention of GH associated diseases or disorders, including those
disorders characterized by GH deficiency, comprising the
administration of a pharmaceutical formulation to a human,
companion animal, livestock or poultry which enhances GH expression
and/or release. The present invention also provides for methods and
protocols to enhance the growth and performance of livestock and
poultry, comprising the administration of a pharmaceutical
formulation to a companion animal, livestock or poultry which
enhances GH expression and/or release. The present invention also
provides for the treatment of obesity and frailty, comprising the
administration of a pharmaceutical formulation to a companion
animal which enhances or modulates GH expression and/or release.
The present invention further provides for methods for the
improvement in health, comprising the administration of a
pharmaceutical formulation to a human, companion animal, livestock
or poultry which enhances GH expression and/or release. In
accordance with the invention, methods of the present invention
encompass the administration of pharmaceutical formulations
comprising: (a) polynucleotide sequences encoding GHRH variants
alone or in combination with polynucleotide sequences encoding
GHRH, modified GHRH, GH, modified GH or any combination thereof,
wherein the polynucleotide sequences are operably linked to a
promoter or regulatory element, preferably one that is
transcriptionally active in muscle tissue; (b) polynucleotide
sequences encoding canine or feline GHRH alone or in combination
with polynucleotide sequences encoding GHRH variants, modified
GHRH, GH, modified GH or any combination thereof, wherein the
polynucleotide sequences are operably linked to a promoter or
regulatory element, preferably one that is transcriptionally active
in muscle tissue; (c) polynucleotide sequences encoding GHRH,
modified GHRH, GH, modified GH or any combination thereof wherein
the polynucleotide sequences are operably linked to a promoter or
regulatory element, preferably one that is transcriptionally active
in muscle tissue; (c) variant GHRH polypeptides alone, expressed as
a fusion protein, or in combination with GHRH, modified GHRH, GH or
modified GH polypeptides or any combination thereof; or (d) canine
or feline GHRH polypeptides alone, expressed as a fusion protein,
or in combination with GHRH variants, modified GHRH, GH or modified
GH polypeptides or any combination thereof.
[0038] Generally, administration of products of a species origin or
species reactivity (in the case of antibodies) that is the same
species as that of the patient is preferred. Thus, in a preferred
embodiment, human GH and/or GHRH genes, gene fragments or
derivatives thereof are administered to a human patient for therapy
or prophylaxis.
[0039] In a specific embodiment, nucleic acids comprising sequences
encoding GH and/or GHRH or functional derivatives thereof, are
administered to promote the release and/or elevation of growth
hormone, by way of gene therapy. Gene therapy refers to therapy
performed by the administration to a subject of an expressed or
expressible nucleic acid. In this embodiment of the invention, the
nucleic acids produce their encoded protein that mediate a
therapeutic effect by promoting the function of GH.
[0040] Any of the methods for gene therapy available in the art can
be used according to the present invention. Exemplary methods are
described below.
[0041] For general reviews of the methods of gene therapy, see
Goldspiel et al., 1993, Clinical Pharmacy 12:488-505; Wu and Wu,
1991, Biotherapy 3:87-95; Tolstoshev, 1993, Ann. Rev. Pharmacol.
Toxicol. 32:573-596; Mulligan, 1993, Science 260:926-932; and
Morgan and Anderson, 1993, Ann. Rev. Biochem. 62:191-217; May,
1993, TIBTECH 11(5):155-215). Methods commonly known in the art of
recombinant DNA technology which can be used are described in
Ausubel et al. (eds.), 1993, Current Protocols in Molecular
Biology, John Wiley & Sons, NY; and Kriegler, 1990, Gene
Transfer and Expression, A Laboratory Manual, Stockton Press,
NY.
[0042] In a preferred aspect, the compound comprises nucleic acid
sequences encoding GH or modified GH and GHRH, modified GHRH or
GHRH variants, said nucleic acid sequences being part of expression
vectors that express GH or modified GH and GHRH, modified GHRH or
GHRH variants in a suitable host. In particular, such nucleic acid
sequences have promoters operably linked to the GH or modified GH
and GHRH, modified GHRH or GHRH variants coding regions, said
promoters being inducible or constitutive, and, optionally,
tissue-specific. In another particular embodiment, nucleic acid
molecules are used in which the GH or modified GH and GHRH,
modified GHRH or GHRH variants coding sequences and any other
desired sequences are flanked by regions that promote homologous
recombination at a desired site in the genome, thus providing for
intrachromosomal expression of the GH or modified GH and GHRH,
modified GHRH or GHRH variants nucleic acids (Koller and Smithies,
1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijistra et al.,
1989, Nature 342:435-438).
[0043] Delivery of the nucleic acids into a patient may be either
direct, in which case the patient is directly exposed to the
nucleic acid or nucleic acid-carrying vectors, or indirect, in
which case, cells are first transformed with the nucleic acids in
vitro, then transplanted into the patient. These two approaches are
known, respectively, as in vivo or ex vivo gene therapy.
[0044] In a specific embodiment, the nucleic acid sequences are
directly administered in vivo, where it is expressed to produce the
encoded product. This can be accomplished by any of numerous
methods known in the art, e.g., by constructing them as part of an
appropriate nucleic acid expression vector and administering it so
that they become intracellular, e.g., by infection using defective
or attenuated retrovirals or other viral vectors (see U.S. Pat. No.
4,980,286), or by direct injection of naked DNA, or by use of
microparticle bombardment (e.g., a gene gun; Biolistic, Dupont), or
coating with lipids or cell-surface receptors or transfecting
agents, encapsulation in liposomes, microparticles, or
microcapsules, or by administering them in linkage to a peptide
which is known to enter the nucleus, by administering it in linkage
to a ligand subject to receptor-mediated endocytosis (see, e.g., Wu
and Wu, 1987, J. Biol. Chem. 262:4429-4432) (which can be used to
target cell types specifically expressing the receptors), etc. In
another embodiment, nucleic acid-ligand complexes can be formed in
which the ligand comprises a fusogenic viral peptide to disrupt
endosomes, allowing the nucleic acid to avoid lysosomal
degradation. In yet another embodiment, the nucleic acid can be
targeted in vivo for cell specific uptake and expression, by
targeting a specific receptor (see, e.g., PCT Publications WO
92/06180 dated April 16, 1992 (Wu et al.); WO 92/22635 dated Dec.
23, 1992 (Wilson et al.); WO92/20316 dated Nov. 26, 1992 (Findeis
et al.); WO93/14188 dated Jul. 22, 1993 (Clarke et al.), WO
93/20221 dated Oct. 14, 1993 (Young)). Alternatively, the nucleic
acid can be introduced intracellularly and incorporated within host
cell DNA for expression, by homologous recombination (Koller and
Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra
et al., 1989, Nature 342:435-438).
[0045] In a specific embodiment, viral vectors that contain nucleic
acid sequences encoding GH or modified GH and/or GHRH, modified
GHRH or GHRH variants are used. For example, a retroviral vector
can be used (see Miller et al., 1993, Meth. Enzymol. 217:581-599).
These retroviral vectors have been to delete retroviral sequences
that are not necessary for packaging of the viral genome and
integration into host cell DNA. The nucleic acid sequences encoding
the GH or modified GH and GHRH, modified GHRH or GHRH variants to
be used in gene therapy are cloned into one-or more vectors, which
facilitates delivery of the gene into a patient. More detail about
retroviral vectors can be found in Boesen et al., 1994, Biotherapy
6:291-302, which describes the use of a retroviral vector to
deliver the mdr1 gene to hematopoietic stem cells in order to make
the stem cells more resistant to chemotherapy. Other references
illustrating the use of retroviral vectors in gene therapy are:
Clowes et al., 1994, J. Clin. Invest. 93:644-651; Kiem et al.,
1994, Blood 83:1467-1473; Salmons and Gunzberg, 1993, Human Gene
Therapy 4:129-141; and Grossman and Wilson, 1993, Curr. Opin. in
Genetics and Devel. 3:110-114.
[0046] Adenoviruses are other viral vectors that can be used in
gene therapy. Adenoviruses are especially attractive vehicles for
delivering genes to respiratory epithelia. Adenoviruses naturally
infect respiratory epithelia where they cause a mild disease. Other
targets for adenovirus-based delivery systems are liver, the
central nervous system, endothelial cells, and muscle. Adenoviruses
have the advantage of being capable of infecting non-dividing
cells. Kozarsky and Wilson, 1993, Current Opinion in Genetics and
Development 3:499-503 present a review of adenovirus-based gene
therapy. Bout et al., 1994, Human Gene Therapy 5:3-10 demonstrated
the use of adenovirus vectors to transfer genes to the respiratory
epithelia of rhesus monkeys. Other instances of the use of
adenoviruses in gene therapy can be found in Rosenfeld et al.,
1991, Science 252:431-434; Rosenfeld et al., 1992, Cell 68:143-155;
Mastrangeli et al., 1993, J. Clin. Invest. 91:225-234; PCT
Publication WO94/12649; and Wang, et al., 1995, Gene Therapy
2:775-783. In a preferred embodiment, adenovirus vectors are used.
Adeno-associated virus (AAV) has also been proposed for use in gene
therapy (Walsh et al., 1993, Proc. Soc. Exp. Biol. Med.
204:289-300; U.S. Pat. No. 5,436,146).
[0047] Another approach to gene therapy involves transferring a
gene to cells in tissue culture by such methods as electroporation,
lipofection, calcium phosphate mediated transfection, or viral
infection. Usually, the method of transfer includes the transfer of
a selectable marker to the cells. The cells are then placed under
selection to isolate those cells that have taken up and are
expressing the transferred gene. Those cells are then delivered to
a patient.
[0048] In this embodiment, the nucleic acid is introduced into a
cell prior to administration in vivo of the resulting recombinant
cell. Such introduction can be carried out by any method known in
the art, including but not limited to transfection,
electroporation, microinjection, infection with a viral or
bacteriophage vector containing the nucleic acid sequences, cell
fusion, chromosome-mediated gene transfer, microcell-mediated gene
transfer, spheroplast fusion, etc. Numerous techniques are known in
the art for the introduction of foreign genes into cells (see,
e.g., Loeffler and Behr, 1993, Meth. Enzymol. 217:599-618; Cohen et
al., 1993, Meth. Enzymol. 217:618-644; Cline, 1985, Pharmac. Ther.
29:69-92) and may be used in accordance with the present invention,
provided that the necessary developmental and physiological
functions of the recipient cells are not disrupted. The technique
should provide for the stable transfer of the nucleic acid to the
cell, so that the nucleic acid is expressible by the cell and
preferably heritable and expressible by its cell progeny.
[0049] The resulting recombinant cells can be delivered to a
subject by various methods known in the art. Recombinant blood
cells (e.g., hematopoietic stem or progenitor cells) are preferably
administered intravenously. The amount of cells envisioned for use
depends on the desired effect, subject=s state, etc., and can be
determined by one skilled in the art.
[0050] Cells into which a nucleic acid can be introduced for
purposes of gene therapy encompass any desired, available cell
type, and include but are not limited to epithelial cells,
endothelial cells, keratinocytes, fibroblasts, muscle cells,
hepatocytes; blood cells such as T lymphocytes, B lymphocytes,
monocytes, macrophages, neutrophils, eosinophils, megakaryocytes,
granulocytes; various stem or progenitor cells, in particular
hematopoietic stem or progenitor cells, e.g., as obtained from bone
marrow, umbilical cord blood, peripheral blood, fetal liver,
etc.
[0051] In a preferred embodiment, the cell used for gene therapy is
autologous to the subject.
[0052] In an embodiment in which recombinant cells are used in gene
therapy, nucleic acid sequences encoding GH or modified GH and/or
GHRH, modified GHRH or GHRH variants are introduced into the cells
such that they are expressible by the cells or their progeny, and
the recombinant cells are then administered in vivo for therapeutic
effect. In a specific embodiment, stem or progenitor cells are
used. Any stem and/or progenitor cells which can be isolated and
maintained in vitro can potentially be used in accordance with this
embodiment of the present invention (see e.g. PCT Publication WO
94/08598, dated Apr. 28, 1994; Stemple and Anderson, 1992, Cell
71:973-985; Rheinwald, 1980, Meth. Cell Bio. 21A:229; and Pittelkow
and Scott, 1986, Mayo Clinic Proc. 61:771).
[0053] In a specific embodiment, the nucleic acid to be introduced
for purposes of gene therapy comprises an inducible promoter
operably linked to the coding region, such that expression of the
nucleic acid is controllable by controlling the presence or absence
of the appropriate inducer of transcription.
[0054] The polypeptides of the invention include polypeptides which
comprise the amino acid sequence of canine or feline GHRH. The
polypeptides of the invention also include polypeptides which
comprise the amino acid sequence of a GHRH variant of the present
invention. The polypeptides of the invention further include
polypeptides which comprise the amino acid sequence of GH or
modified and GHRH or modified GHRH. Protein compositions of the
present invention may further comprise an acceptable carrier, such
as a hydrophilic, e.g., pharmaceutically acceptable, carrier.
[0055] The invention also relates to methods for producing a
polypeptide comprising growing a culture of the cells of the
invention in a suitable culture medium, and purifying the protein
from the culture. For example, the methods of the invention include
a process for producing a polypeptide in which a host cell
containing a suitable expression vector that includes a
polynucleotide of the invention is cultured under conditions that
allow expression of the encoded polypeptide. The polypeptide can be
recovered from the culture, conveniently from the culture medium,
and further purified.
[0056] The present invention further provides isolated polypeptides
encoded by the nucleic acid fragments of the present invention or
by degenerate variants of the nucleic acid fragments of the present
invention. By "degenerate variant is intended nucleotide fragments
which differ from a nucleic acid fragment of the present invention
(e.g., an ORF) by nucleotide sequence but, due to the degeneracy of
the genetic code, encode an identical polypeptide sequence.
Preferred nucleic acid fragments of the present invention are the
ORFs that encode proteins. A variety of methodologies known in the
art can be utilized to obtain any one of the isolated polypeptides
or proteins of the present invention. At the simplest level, the
amino acid sequence can be synthesized using commercially available
peptide synthesizers. This is particularly useful in producing
small peptides and fragments of larger polypeptides. Fragments are
useful, for example, in generating antibodies against the native
polypeptide. In an alternative method, the polypeptide or protein
is purified from bacterial cells which naturally produce the
polypeptide or protein. One skilled in the art can readily follow
known methods for isolating polypeptides and proteins in order to
obtain one of the isolated polypeptides or proteins of the present
invention. These include, but are not limited to,
immunochromatography, HPLC, size-exclusion chromatography,
ion-exchange chromatography, and immuno-affinity chromatography.
See, e.g., Scopes, Protein Purification: Principles and Practice,
Springer-Verlag (1994); Sambrook, et al., in Molecular Cloning: A
Laboratory Manual; Ausubel et al., Current Protocols in Molecular
Biology.
[0057] The polypeptides and proteins of the present invention can
alternatively be purified from cells which have been altered to
express the desired polypeptide or protein. As used herein, a cell
is said to be altered to express a desired polypeptide or protein
when the cell, through genetic manipulation, is made to produce a
polypeptide or protein which it normally does not produce or which
the cell normally produces at a lower level. One skilled in the art
can readily adapt procedures for introducing and expressing either
recombinant or synthetic sequences into eukaryotic or prokaryotic
cells in order to generate a cell which produces one of the
polypeptides or proteins of the present invention. The purified
polypeptides can be used in in vitro binding assays which are well
known in the art to identify molecules which bind to the
polypeptides. These molecules include but are not limited to, for
e.g., small molecules, molecules from combinatorial libraries,
antibodies or other proteins.
[0058] The protein of the invention may also be expressed as a
product of transgenic animals, e.g., as a component of the milk of
transgenic cows, goats, pigs, or sheep which are characterized by
somatic or germ cells containing a nucleotide sequence encoding the
protein.
[0059] The protein may also be produced by known conventional
chemical synthesis. Methods for constructing the proteins of the
present invention by synthetic means are known to those skilled in
the art. The synthetically-constructed protein sequences, by virtue
of sharing primary, secondary or tertiary structural and/or
conformational characteristics with proteins may possess biological
properties in common-therewith, including protein activity. Thus,
they may be employed as biologically active or immunological
substitutes for natural, purified proteins in screening of
therapeutic compounds and in immunological processes for the
development of antibodies.
[0060] The protein may also be produced by operably linking the
isolated polynucleotide of the invention to suitable control
sequences in one or more insect expression vectors, and employing
an insect expression system. Materials and methods for
baculovirus/insect cell expression systems are commercially
available in kit form from, e.g., Invitrogen, San Diego, Calif.,
U.S.A. (the MaxBat.RTM. kit), and such methods are well known in
the art, as described in Summers and Smith, Texas Agricultural
Experiment Station Bulletin No. 1555 (1987), incorporated herein by
reference. As used herein, an insect cell capable of expressing a
polynucleotide of the present invention is "transformed."
[0061] The protein of the invention may be prepared by culturing
transformed host cells under culture conditions suitable to express
the recombinant protein. The resulting expressed protein may then
be purified from such culture (i.e., from culture medium or cell
extracts) using known purification processes, such as gel
filtration and ion exchange chromatography. The purification of the
protein may also include an affinity column containing agents which
will bind to the protein; one or more column steps over such
affinity resins as concanavalin A-agarose, heparin-toyopearl.RTM.
or Cibacrom blue 3GA Sepharose.RTM.; one or more steps involving
hydrophobic interaction chromatography using such resins as phenyl
ether, butyl ether, or propyl ether; or immunoaffinity
chromatography.
[0062] Alternatively, the protein of the invention may also be
expressed in a form which will facilitate purification. For
example, it may be expressed as a fusion protein, such as those of
maltose binding protein (MBP), glutathione-S-transferase (GST) or
thioredoxin (TRX). Kits for expression and purification of such
fusion proteins are commercially available from New England BioLab
(Beverly, Mass.), Pharmacia (Piscataway, N.J.) and In Vitrogen,
respectively. The protein can also be tagged with an epitope and
subsequently purified by using a specific antibody directed to such
epitope. One such epitope ("Flag") is commercially available from
Kodak (New Haven, Conn.).
[0063] Finally, one or more reverse-phase high performance liquid
chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media,
e.g., silica gel having pendant methyl or other aliphatic groups,
can be employed to further purify the protein. Some or all of the
foregoing purification steps, in various combinations, can also be
employed to provide a substantially homogeneous isolated
recombinant protein. The protein thus purified is substantially
free of other mammalian proteins and is defined in accordance with
the present invention as an "isolated protein."
[0064] The compounds of the invention are preferably tested in
vitro, and then in vivo for the desired therapeutic or prophylactic
activity, prior to use in humans. For example, in vitro assays
which can be used to determine whether administration of a specific
compound is indicated, include in vitro cell culture assays in
which a patient tissue sample is grown in culture, and exposed to
or otherwise administered a compound, and the effect of such
compound upon the tissue sample is observed.
[0065] The expression of GH or modified GH and GHRH, modified GHRH
or GHRH variants can be assayed by the immunoassays, gel
electrophoresis followed by visualization, or any other method
known to those skilled in the art.
[0066] In various specific embodiments, in vitro assays can be
carried out with representative cells of cell types involved in a
patient's disorder, to determine if a compound has a desired effect
upon such cell types. In accordance with the present invention, the
functional activity of GHRH can be measured by its ability to
induce GH gene transcription in vitro. In accordance with the
present invention, the functional activity of GHRH can be measured
by its ability to induce IGF gene transcription in vitro.
[0067] Compounds for use in therapy can be tested in suitable
animal model systems prior to testing in humans, including but not
limited to pigs, chicken, cows or monkeys.
[0068] The invention provides methods of treatment (and
prophylaxis) by administration to a subject of an effective amount
of a compound of the invention. In a preferred aspect, the compound
is substantially purified (e.g., substantially free from substances
that limit its effect or produce undesired side-effects). The
subject is preferably an animal, including but not limited to
animals such as cows, pigs, horses, chickens, cats, dogs, etc., and
is-preferably a mammal, and most preferably human. In a specific
embodiment, a non-human mammal is the subject.
[0069] Formulations and methods of administration that can be
employed when the compound comprises a nucleic acid are described
above; additional appropriate formulations and routes of
administration can be selected from among those described herein
below.
[0070] Various delivery systems are known and can be used to
administer a compound of the invention, e.g., encapsulation in
liposomes, microparticles, microcapsules, recombinant cells capable
of expressing the compound, receptor-mediated endocytosis (see,
e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), construction
of a nucleic acid as part of a retroviral or other vector, etc.
Methods of introduction include but are not limited to
intratumoral, intradermal, intramuscular, intraperitoneal,
intravenous, subcutaneous, intranasal, epidural, and oral routes.
The compounds may be administered by any convenient route, for
example by infusion or bolus injection, by absorption through
epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and
intestinal mucosa, etc.) and may be administered together with
other biologically active agents. Administration can be systemic or
local. In addition, it may be desirable to introduce the
pharmaceutical compositions of the invention into the central
nervous system by any suitable route, including intraventricular
and intrathecal injection; intraventricular injection may be
facilitated by an intraventricular catheter, for example, attached
to a reservoir, such as an Ommaya reservoir. Pulmonary
administration can also be employed, e.g., by use of an inhaler or
nebulizer, and formulation with an aerosolizing agent.
[0071] In a specific embodiment, it may be desirable to administer
the pharmaceutical compositions of the invention locally to the
area in need of treatment; this may be achieved by, for example,
and not by way of limitation, local infusion during surgery,
topical application, e.g., in conjunction with a wound dressing
after surgery, by injection, by means of a catheter, by means of a
suppository, or by means of an implant, said implant being of a
porous, non-porous, or gelatinous material, including membranes,
such as sialastic membranes, or fibers. In one embodiment,
administration can be by direct injection at the site (or former
site) of a malignant tumor or neoplastic or pre-neoplastic
tissue.
[0072] In another embodiment, the compound can be delivered in a
vesicle, in particular a liposome (see Langer, Science
249:1527-1533 (1990); Treat et al., in Liposomes in the Therapy of
Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.),
Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp.
317-327; see generally ibid.)
[0073] In yet another embodiment, the compound can be delivered in
a controlled release system. In one embodiment, a pump may be used
(see Langer, supra; Sefton, CRC Crit. Ref. Biomed. Eng. 14:201
(1987); Buchwald et al., Surgery 88:507 (1980); Saudek et al., N.
Engl. J. Med. 321:574 (1989)). In another embodiment, polymeric
materials can be used (see Medical Applications of Controlled
Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla.
(1974); Controlled Drug Bioavailability, Drug Product Design and
Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger
and Peppas, J. Macromol. Sci. Rev. Macromol. Chem. 23:61 (1983);
see also Levy et al., Science 228:190 (1985); During et al., Ann.
Neurol. 25:351 (1989); Howard et al., J. Neurosurg. 71:105 (1989)).
In yet another embodiment, a controlled release system can be
placed in proximity of the therapeutic target, i.e., the brain,
thus requiring only a fraction of the systemic dose (see, e.g.,
Goodson, in Medical Applications of Controlled Release, supra, vol.
2, pp. 115-138 (1984)).
[0074] Other controlled release systems are discussed in the review
by Langer (Science 249:1527-1533 (1990)).
[0075] In a specific embodiment where the compound of the invention
is a nucleic acid encoding a protein, the nucleic acid can be
administered in vivo to promote expression of its encoded protein,
by constructing it as part of an appropriate nucleic acid
expression vector and administering it so that it becomes
intracellular, e.g., by use of a retroviral vector (see U.S. Pat.
No. 4,980,286), or by direct injection, or by use of microparticle
bombardment (e.g., a gene gun; Biolistic, Dupont), or coating with
lipids or cell-surface receptors or transfecting agents, or by
administering it in linkage to a homeobox-like peptide which is
known to enter the nucleus (see e.g., Joliot et al., 1991, Proc.
Natl. Acad. Sci. USA 88:1864-1868), etc. Alternatively, a nucleic
acid can be introduced intracellularly and incorporated within host
cell DNA for expression, by homologous recombination.
[0076] The present invention also provides pharmaceutical
compositions. Such compositions comprise a therapeutically
effective amount of a compound, and a pharmaceutically acceptable
carrier. In a specific embodiment, the term "pharmaceutically
acceptable" means approved by a regulatory agency of the Federal or
a state government or listed in the U.S. Pharmacopeia or other
generally recognized pharmacopeia for use in animals, and more
particularly in humans. The term "carrier" refers to a diluent,
adjuvant, excipient, or vehicle with which the therapeutic is
administered. Such pharmaceutical carriers can be sterile liquids,
such as water and oils, including those of petroleum, animal,
vegetable or synthetic origin, such as peanut oil, soybean oil,
mineral oil, sesame oil and the like. Water is a preferred carrier
when the pharmaceutical composition is administered intravenously.
Saline solutions and aqueous dextrose and glycerol solutions can
also be employed as liquid carriers, particularly for injectable
solutions. Suitable pharmaceutical excipients include starch,
glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk,
silica gel, sodium stearate, glycerol monostearate, talc, sodium
chloride, dried skim milk, glycerol, propylene, glycol, water,
ethanol and the like. The composition, if desired, can also contain
minor amounts of wetting or emulsifying agents, or pH buffering
agents. These compositions can take the form of solutions,
suspensions, emulsion, tablets, pills, capsules, powders,
sustained-release formulations and the like. The composition can be
formulated as a suppository, with traditional binders and carriers
such as triglycerides. Oral formulation can include standard
carriers such as pharmaceutical grades of mannitol, lactose,
starch, magnesium stearate, sodium saccharine, cellulose, magnesium
carbonate, etc. Examples of suitable pharmaceutical carriers are
described in "Remington's Pharmaceutical Sciences" by E. W. Martin.
Such compositions will contain a therapeutically effective amount
of the compound, preferably in purified form, together with a
suitable amount of carrier so as to provide the form for proper
administration to the patient. The formulation should suit the mode
of administration.
[0077] In a preferred embodiment, the composition is formulated in
accordance with routine procedures as a pharmaceutical composition
adapted for intravenous administration to human beings. Typically,
compositions for intravenous administration are solutions in
sterile isotonic aqueous buffer. Where necessary, the composition
may also include a solubilizing agent and a local anesthetic such
as lignocaine to ease pain at the site of the injection. Generally,
the ingredients are supplied either separately or mixed together in
unit dosage form, for example, as a dry lyophilized powder or water
free concentrate in a hermetically sealed container such as an
ampoule or sachette indicating the quantity of active agent. Where
the composition is to be administered by infusion, it can be
dispensed with an infusion bottle containing sterile pharmaceutical
grade water or saline. Where the composition is administered by
injection, an ampoule of sterile water for injection or saline can
be provided so that the ingredients may be mixed prior to
administration.
[0078] The compounds of the invention can be formulated as neutral
or salt forms. Pharmaceutically acceptable salts include those
formed with free amino groups such as those derived from
hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and
those formed with free carboxyl groups such as those derived from
sodium, potassium, ammonium, calcium, ferric hydroxides,
isopropylamine, triethylamine, 2-ethylamino ethanol, histidine,
procaine, etc.
[0079] The amount of the compound of the invention which will be
effective in the treatment of cancer can be determined by standard
clinical techniques. In addition, in vitro assays may optionally be
employed to help identify optimal dosage ranges. The precise dose
to be employed in the formulation will also depend on the route of
administration, and the seriousness of the disease or disorder, and
should be decided according to the judgment of the practitioner and
each patient's circumstances. However, suitable dosage ranges for
intravenous administration are generally about 20-500 micrograms of
active compound per kilogram body weight. Suitable dosage ranges
for intranasal administration are generally about 0.01 pg/kg body
weight to 1 mg/kg body weight. Effective doses may be extrapolated
from dose-response curves derived from in vitro or animal model
test systems.
[0080] Suppositories generally contain active ingredient in the
range of 0.5% to 10% by weight; oral formulations preferably
contain 10% to 95% active ingredient.
[0081] The invention also provides a pharmaceutical pack or kit
comprising one or more containers filled with one or more of the
ingredients of the pharmaceutical compositions of the invention.
Optionally associated with such container(s) can be a notice in the
form prescribed by a governmental agency regulating the
manufacture, use or sale of pharmaceuticals or biological products,
which notice reflects approval by the agency of manufacture, use or
sale for human administration.
EXAMPLES
Cloning of Canine and Feline GHRH
[0082] 1. Cloning of Canine GHRH
[0083] In order to clone the canine GHRH, the canine genomic
library (Clonetech Lab) is screened with the radioactively labeled
fragment encoding the porcine GHRH (SEQ ID No. 1) following
protocols known to one of ordinary skill in the art. The fragment
encoding the porcine GHRH (SEQ ID No. 1) is labeled using
commercially available DNA labeling kits as recommended by the
manufacturer. Clones identified that contain the gene coding for
the canine precursor GHRH are isolated and sequenced by methods
known to one of ordinary skill in the art. The sequence results
obtained from sequencing both DNA strands of a clone is compared
with sequences of known GHRH species and the canine GHRH is
subcloned into appropriate plasmid vectors (e.g., pVR1012; Vical,
San Diego, Calif.) according to protocols known to one of ordinary
skill in the art.
[0084] 2. Cloning of Feline GHRH
[0085] In order to clone the feline GHRH, the feline genomic
library (Clonetech Lab) is screened with the radioactively labeled
fragment encoding the porcine GHRH (SEQ ID No. 1) following
protocols known to one of ordinary skill in the art. The fragment
encoding the porcine GHRH (SEQ ID No. 1) is labeled using
commercially available DNA labeling kits as recommended by the
manufacturer. Clones identified that contain the gene coding for
the feline precursor GHRH are isolated and sequenced by methods
known to one of ordinary skill in the art. The sequence results
obtained from sequencing both DNA strands of a clone is compared
with sequences of known GHRH species and the feline GHRH is
subcloned into appropriate plasmid vectors (e.g., pVR1012; Vical,
San Diego, Calif.) according to protocols known to one of ordinary
skill in the art.
Synthesis of GHRH Constructs
[0086] 3. pGHRH-4 (pGHRH1-44WTCMV)
[0087] In order to construct a plasmid containing the gene that
codes for the natural porcine GHRH polypeptide and to have the
latter secreted into the blood circulation when such plasmids are
injected into animals, primers designated GHRH-1 (SEQ ID No. 61),
GHRH-2 (SEQ ID No. 62), GHRH-4 (SEQ ID No. 66), and GHRH-7 (SEQ ID
No. 67) were synthesized. The primers were used in reverse
transcription polymerase chain reactions (RT-PCRs) to amplify the
human GHRH signal sequence from human mRNA and the porcine GHRH
protein sequence from porcine mRNA. The resulting PCR-amplified
human GHRH signal sequence and porcine GHRH protein sequence were
digested with Bgl II and Bam HI, respectively. Then the fragments
were ligated together and cloned into the Bam HI site of the
plasmid pVR1012 (Vical, San Diego, Calif.) to produce a plasmid
designated pGHRH-4 (FIG. 1). The expression of the GHRH
oligonucleotide (SEQ ID Nos. 1 and 2), which encodes a 75 amino
acid polypeptide comprising the porcine GHRH protein sequence (44
amino acids) preceded by the signal sequence from human GHRH
protein (31 amino acids), is driven by Cytomegalo-virus immediate
early (CMV IE) promoter/enhancer element.
[0088] 4. pGHRH1-44WTSK685:
[0089] A plasmid containing the polynucleotide sequences encoding
the 75-amino acid GHRH protein described above driven by a 685 bp
fragment derived from the swine a-skeletal actin promoter (SEQ ID
No. 3) was constructed as described below.
[0090] The oligonucleotide fragment encoding the 75 amino acid GHRH
protein (SEQ ID No. 1) was PCR-amplified from plasmid pGHRH4 using
a primer designated p97-S1 containing a Hind III site SEQ ID No. 4)
and a primer designated p97-A258, containing an Xba I site (SEQ ID
No. 5). The PCR-amplified sequence was then cloned into the Hind
III-Xba I site of plasmid pGL3 basic (Promega) to produce a plasmid
designated GHRH1-44 SK (FIG. 2). A 685 bp fragment corresponding to
a portion of the porcine a-skeletal actin promoter designated SK685
was PCR-amplified from swine genomic DNA using primer designated
SK-3, containing a Kpn I site (SEQ ID No. 6) and primer designated
SK4, containing a Hind III site; (SEQ ID No. 7). The PCR-amplified
SK685 promoter fragment was then cloned into plasmid GHRH1-44SK
digested with Kpn I and HindIII enzymes to produce a plasmid
designated GHRH1-44WTSK685 (FIG. 3).
[0091] 5. pGHRH1-44WTSK2014
[0092] A plasmid containing the polynucleotide sequences encoding
the 75-amino acid GHRH protein described above operatively linked
to a fragment derived from the swine a-skeletal actin promoter
approximately 2014 bp (SEQ ID No. 8) was constructed as described
below.
[0093] An approximately 2014 bp fragment designated SK 2014
corresponding to a portion of the porcine .alpha.-skeletal actin
promoter was PCR-amplified from swine genomic DNA using primer
designated SK-7; containing a Kpn I site (SEQ ID No. 9) and primer
designated SK-8; containing a Hind III site; (SEQ ID No. 10). The
PCR-amplified SK2014 promoter fragment was then cloned into plasmid
GHRH1-44SK which was digested with Kpn I and Hind III enzymes to
produce a plasmid designated GHRH1-44WTSK2014 (FIG. 4).
[0094] 6. pGHRH1-29WTCMV
[0095] A plasmid containing the polynucleotide sequences encoding
the signal sequence derived from human GHRH polynucleotides and
encoding amino acids 1-29 of swine GHRH protein was produced as
described below.
[0096] An approximately 189 bp DNA fragment was PCR-amplified from
plasmid pGHRH-4 using primer designated GHRH-5, containing a Bam HI
site; (SEQ ID No. 11) and primer designated GHRH-6, containing a
Bgl II site; (SEQ ID No. 12). The PCR-amplified fragment was
digested with Bam HI and Bgl II enzymes and cloned into plasmid
pVR1012 (Vical, San Diego, Calif.) which was digested with Bam HI
and Bgl II enzymes to produce a plasmid designated GHRH1-29WTCMV
(SEQ ID No. 51) in which expression of the GHRH 1-29 protein is
driven by CMV IE promoter/enhancer sequences.
[0097] 7. pGHRH1-29YWVTCMV
[0098] In order to produce novel variants of GHRH protein with
enhanced stability to enzymatic degradation (e.g. DPPIV enzyme
degradation) a plasmid containing polynucleotide sequences encoding
the signal sequence of human GHRH and an altered version of the
1-29 porcine GHRH protein was produced. The alteration consisted of
the addition of an extra tyrosine residue just preceding the first
tyrosine residue of the natural porcine GHRH 1-29 sequence. This
modification alters the amino terminal in such away that it is no
longer recognized or cleaved by DPPIV enzyme. A plasmid containing
the gene for this variant GHRH protein was produced as described
below.
[0099] A set of overlapping oligonucleotides (SEQ ID Nos. 13-25)
were synthesized, mixed, and GHRH was amplified using the PCR
method. The PCR reaction resulted in the formation of a fragment of
approximately 192 bp encoding amino acids 1-29 of GHRH in which
nucleotides encoding a tyrosine residue were inserted immediately
5' to the coding sequence of GHR(1-29) and further containing a Bam
HI site (5' end) and an Bgl II site (3' end ). The 192 bp fragment
was then digested with Bam HI and Bgl II enzymes and cloned into
plasmid pVR1012 which was digested with Bam HI and Bgl II enzymes
to produce plasmid designated GHRH1-29YWTCMV (SEQ ID No. 52) in
which expression of GHRH1-29 (now 30) is driven by CMV IE promoter
enhancer elements.
[0100] 8. pGHRH1-29YVTSK685
[0101] A plasmid containing the 192 bp fragment described above in
Section 5 under the control of the SK685 promoter fragment was
produced as described below.
[0102] The 192 bp fragment was amplified with two primers
designated p99-S1, containing a 5' end Hind III site; (SEQ ID No.
26) and p99-A214, containing a 3' end Xba I site; (SEQ ID No. 27).
The PCR-amplified fragment was then digested with Hind III and Xba
I enzymes and cloned into plasmid GHRH1-29 Yala1522SK685 (see
below) also digested with Hind III and Xba I enzymes to produce
plasmid GHRH1-29YWTSK685 (SEQ ID No. 53).
[0103] 9. pGHRH1-29YWTSK2014
[0104] A plasmid containing the 192 bp fragment described above in
Section 5 under the control of the SK2014 promoter fragment was
produced as described below.
[0105] The 192 bp fragment was amplified with two primers
designated p99-Si containing a 5' end Hind III site; (SEQ ID No.
28) and p99-A214 containing a 3' end Xba I site; (SEQ ID No. 29).
The PCR-amplified fragment was then digested with Hind III and Xba
I enzymes and cloned into plasmid GHRH1-29 YAla 1522SK2014 (see
below) also digested with Hind III and Xba I enzymes to produce
plasmid GHRH1-29YWTSK2014 (SEQ ID No. 54).
[0106] 10. pGHRH1-29YAla1522CMV
[0107] In order to produce novel variants of GHRH protein with
enhanced stability to enzymatic degradation (e.g., due to DPPIV
enzyme) and enhanced potency, a plasmid containing the signal
sequence of human GHRH and an altered version of the 1-29 porcine
GHRH protein was produced. The alteration consisted of the addition
of an extra tyrosine residue just preceding the first tyrosine
residue of the natural porcine GHRH 1-29 sequence and replacement
of glycine 15 and leucine 22 with alanine. These modifications
alter the amino terminal end of GHRH1-29 in such away that it is no
longer by recognized or cleaved by the DPP IV enzyme and the
modified protein has enhanced potency relative to the 29 or the 44
amino acid GHRH protein. A plasmid containing the gene for this
variant protein was produced as described below.
[0108] A set of overlapping oligonucleotides were synthesized (SEQ
ID Nos. 30-42), mixed and amplified using the PCR method. The PCR
reaction resulted in the formation of a fragment of approximately
192 bp containing a Bam Hi site (5' end) and an Bgl II site (3'
end) and in which an extra three nucleotides encoding for tyrosine
is immediately 5' to the nucleotide sequence encoding the natural
tyrosine at position 1 of the GHRH1-29 sequence. Furthermore, the
alteration included replacement of the 3 nucleotides encoding
glycine 15 with three nucleotides encoding alanine and replacement
of three nucleotides encoding leucine 22 with three nucleotides
encoding alanine. The 192 bp fragment was then digested with Bam HI
and Bgl II enzymes and cloned into plasmid pVR1012 which was
digested with Bam HI and Bgl II enzymes to produce plasmid
designated GHRH1-29YAla 1522CMV in which expression of the variant
GHRH1-29 (now 30) is driven by CMV IE promoter enhancer elements
(SEQ ID No. 55).
[0109] 11. pGHRH1-29YAla15225K685
[0110] A plasmid containing the GHRH1-29YAla1522 fragment described
above under the control of the SK685 promoter fragment was produced
as described below.
[0111] An approximately 192 bp fragment as described above in
Section 8 was PCR-amplified with primers designated p99-S1,
containing a Hind III at 5' end (SEQ ID No. 26) and p99-A214
containing a Xba I site at 3' end; (SEQ ID No. 27). The
PCR-amplified fragment was digested with Hind III and Xba I enzymes
and cloned into plasmid pGL3 (Promega) also digested with Hind III
and Xba I to produce plasmid GHRH1-29YAla 1522SK (SEQ ID No. 56). A
685 bp fragment corresponding to a portion of the porcine
a-skeletal actin promoter was PCR-amplified from swine genomic DNA
using primers SK-3 (Kpn I site) and SK-4 (Hind III site). This
fragment was designated SK685. The PCR-amplified SK685 promoter
fragment was then cloned into plasmid GHRH1-29YAla1522SK digested
with KpnI and HindIII enzymes to produce a plasmid designated
GHRH1-29YA1a1522SK685 (SEQ ID No. 57).
[0112] 12. pGHRH1-29Yala1522SK2014
[0113] A plasmid containing the GHRH1-29YAla1522 fragment described
above under the control of the SK2014 promoter fragment was
produced as described below.
[0114] An approximately 192 bp fragment as described above in
Section 8 was PCR amplified with primers designated p99-S1
containing a Hind III at 5' end; (SEQ ID No. 26) and p99-A214
containing a Xba I site at 3' end; (SEQ ID No. 27). The
PCR-amplified fragment was digested with Hind III and Xba I enzymes
and cloned into plasmid pGL3 (Promega) also digested with HindIII
and Xba I to produce plasmid GHRH1-29YAla1522SK. An 2014 bp
fragment corresponding to a portion of the porcine a-skeletal actin
promoter was PCR-amplified from swine genomic DNA using primers
SK-7 containing a Kpn I site and SK-8 containing a Hind III site.
This fragment was designated SK2014. The PCR-amplified SK2014
promoter fragment was then cloned into plasmid GHRH1-29YAla1522SK
digested with Kpn I and Hind III enzymes to produce a plasmid
designated GHRH1-29YAla1522SK2014 (SEQ ID No. 58).
[0115] 13. pGHRH 1-44YWTCMV
[0116] In order to produce novel variants of GHRH protein with
enhanced stability to enzymatic degradation (e.g., due to DPPIV
enzyme) a plasmid containing the signal sequence of human GHRH and
an altered version of the 1-44 porcine GHRH protein was produced.
The alteration consisting of the addition of an extra tyrosine
residue immediately 5' to the nucleotide sequence encoding the
first tyrosine residue of the natural porcine GHRH 1-44 sequence.
This modification will alter the amino terminal end in such away
that it is no longer by recognized or cleaved by DPPIV enzyme. A
plasmid containing the gene for this modified GHRH protein is
produced as described below.
[0117] A set primers designated GHRH-1 (SEQ ID No. 61) and GHRH-3
(SEQ ID No. 65) were used in a PCR reaction amplify the human GHRH
signal sequence and porcine GHRH from pGHRH1-29YWTCMV. The
resulting GHRH fragment was digested with Bam HI and Bgl II, and
cloned into the Bam HI site of plasmid pVR1012 (Vical, San Diego,
Calif.) to produce the plasmid designated GHRH1-44YWTCMV (SEQ ID
No. 59).
[0118] 14. Synthesis of GH Constructs
[0119] In order to provide plasmid constructs containing GH genes
suitable for treatment of growth hormone maladies or to enhance
animal health and productivity, the construction of plasmids of the
invention and their methods of use are described below in
detail.
[0120] The canine GH gene was cloned into plasmids vectors suitable
for the various aspects of the present invention using the
following procedures. Total RNA was prepared from the pituitary
gland of a dog using the RNAzol B method using reagents and
procedures from Biotecx Laboratories, Houston Tex. Briefly, about
0.15 mg of the tissue was homogenized in 2 ml of RNAzol B solution
in a RNase-free glass homogenizer. The material was then divided
into two equal halves, and RNA extracted by chloroform and ethanol
precipitation. The nucleic acid pellet was dried and taken up in
RNase-free water. Reverse transcription (RT) of total RNA was done
in a 20 ml volume using 0.02 mg of RNA, 138 pmol of oligo #2 (SEQ
ID No. 43), 1 mM MnCl.sub.2, and the recommended amounts of dNTPs
and rTth enzyme from the RT-PCR kit purchased from Perkin-Elmer,
Norwalk, Conn. The reaction was incubated at 70.quadrature. for 11
min in a Perkin-Elmer Thermal cycler. The completed (RT) reaction
was then subjected to PCR following addition of 66 pmol of oligo #1
(SEQ ID No. 44), 2.5 mM MgCl.sub.2, and chelating buffer from the
RT-PCR reaction kit. The PCR conditions were as follows: 94 C, 1
min; 55 C, 1 min; and 72 C, 2 min; for 32 cycles. The
.quadrature.0.7 kb PCR-amplified DNA fragment obtained was cloned
into plasmid pCRScript purchased from Stratagene, La Jolla, Calif.
and used according to manufacturer's recommendations. The
recombinant plasmid thus generated was termed cCG-SP. The insert
fragment was partially sequenced and confirmed to contain the
growth hormone ("GH") DNA sequences. cGH-SP plasmid DNA was then
used as a template to PCR-amplify using oligonucleotides oligo #3
(SEQ ID No. 45) and oligo #4 (SEQ ID No. 46) using reagents from
the PCR system kit from Perkin-Elmer using standard procedures and
following cycling conditions: 94 C, 1 min 1 cycle; 94 C, 30 sec; 55
C, 30 sec; 72 C, 1 min; 30 cycles. The .quadrature.0.7 kb
PCR-amplified DNA fragment obtained was subjected to column
purification (Qiagen, Chatsworth, Calif.), and digested with
restriction enzymes EcoRV and BgIII by standard protocols (Sambrook
et al., 1989). The digested PCR fragment was ligated to EcoRV-BgIII
digested pCMV-MCS, a plasmid derivative of pCMVb (Clontech, Palo
Alto, Calif.), engineered to contain multiple cloning sites in
place of lacZ gene. The ligation product was used to transform E.
coli, and transformants were selected for resistance to ampicillin
(pCMV-MCS-enooded marker). Transformants were analyzed by plasmid
DNA preparation and restriction site analysis, and a clone of the
GH DNA sequences in pCMV-MCS was isolated (termed pCGH#9). The
insert sequences were completely sequenced by standard procedures
to confirm presence of GH DNA sequences. The EcoRV-BgIII GH
fragment from pCGH#9 has also been sub-cloned into gene therapy
plasmid VR1012 (obtained from Vical, San Diego, Calf.). This clone
referred to as pC51.
[0121] 15. Synthesis of GH-GHRH Constructs
[0122] A GH-GHRH fusion protein, comprising the carboxy terminal 20
amino acids of GH and full length wildtype GHRH is produced. The
full length GHRH gene is PCR amplified from plasmid pGHRH-4 using
two primers designated GHRH-1 containing a Bgl II site (SEQ ID No.
61) and GHRH-2 containing a Bam HI site (SEQ ID No. 62). The
PCR-amplified fragment is then cloned into the Bam HI site of the
pVR1012 plasmid. Two complementary oligonucleotides encoding the
carboxy terminal amino acids 172-191 of GH (GH-1 oligo.; SEQ ID No.
62 and GH-2 oligo.; SEQ ID No. 63) are synthesized. The GH-1 and
GH-2 oligonucleotides are annealed and cloned into the Bam HI site
of the pVR1012 plasmid containing the full length GHRH gene to
produce pGHRH1-44WTGHpep (SEQ ID No. 60).
[0123] 16. In Vitro Studies Assessing GHRH Expression Levels
[0124] In order to assess the expression level of GHRH from
pGHRH-4, this plasmid or. pVR1012 was transfected into C2C12 mouse
myoblasts using the fugene reagent according to the manufacturer=s
recommendations (Boehringer Ingelheim). Supernatant harvested from
transfected and non-transfected cells at various time points were
assayed for the presence of GHRH using a commerically available
radioimmune assay kit (Pennisula Laboratories). The results
depicted in FIG. 5 indicate that GHRH production could be detected
24 hours post-transfection.
[0125] 17. The Effect of GH Plasmid Injection on Swine Growth
[0126] In order to evaluate the effect of a single injection of
plasmids containing GH gene on swine growth, experiments addressing
the effect GH treatment on swine of different ages were carried out
according to the experimental design described below.
[0127] Materials & Methods
[0128] Thirty six 3-week old (weaned) cross-bred (Yorkshire X
landrace) pigs of mixed sex were brought into experimental barns
and maintained for an acclimation period of 3 weeks. Animals were
kept in pens (2/pen) according to treatment group. Animals were fed
daily a non-medicated commercial pig diet ad libitum (16% protein
pellet until approximately 45 Kg body weight and then a 14% protein
pellet until slaughter) and fresh water was available ad libitum.
Animals were randomized into one of four treatment groups (A-D;
table 1) according to weight, sex and litter. Eight animals in
group A were injected once with plasmids containing GH gene at 6
weeks of age. Controls (10 animals; group C) were injected with
blank plasmid vector when they were also 6 weeks of age. Eight
animals in group B were injected once with plasmids containing GH
gene at 13 weeks of age. Controls (group D; 10 animals) were
injected with blank plasmid vector when they were also 13 weeks of
age. Food consumption was recorded daily for each pen and animals
were weighed on two consecutive days each week until slaughter.
Loin eye area and back fat were quantified at slaughter.
1TABLE 1 Experimental Design Weight at Number of Age at time of
time of Group Animals Plasmid dose injection injection A 8 4.6 mg 6
weeks 12.4 B 8 4.6 mg 13 weeks 4.2 C 10 Placebo 6 weeks 13.8 D 10
Placebo 13 weeks 41.9
[0129] Results
[0130] As shown in table 2, treatment of 6 week-old pigs with
plasmids encoding GH gene results in enhanced performance. This is
evident by the 5% increase in weight gain and 5.1% increase in
Average daily gain (ADG) achieved in GH injected animals versus
placebo injected pigs. Moreover, the data shows that GH plasmid
injection resulted in an improvement of 3.3 % in feed efficiency
(ratio of feed consumed relative to weight gain) as well as an
increase of 7.2% in loin eye area of GH plasmid injected
animals.
[0131] The data in table 3 shows that treatment of 13 week-old pigs
with plasmids encoding GH gene results in an even higher magnitude
of increase in animal performance relative to age placebo injected
controls than treatment of 6 week-old pigs. This is evident by the
9.5% increase in weight gain and 9.3% increase in Average daily
gain (ADG) achieved in GH injected animals versus placebo injected
pigs. Moreover, the data shows that GH plasmid injection resulted
in an improvement of 6.7 % in feed efficiency (ratio of feed
consumed relative to weight gain) as well as an increase of 6.8% in
loin eye area of GH plasmid injected animals. Thus treatment of
animals with plasmids containing GH gene results in enhanced animal
growth and performance.
2TABLE 2 The effect of GH plasmid administration of weight gain
Group A C % Improvement Weight at injection 12.4 13.8 time (Kg)
Weight gain.sup.a 77.7 73.8 5.0% ADG.sup.b .79 .75 5.1% total feed
473.3 457.6 3.4% intake/pen.sup.c Feed 4.8 4.7 2.1%
intake/pen/day.sup.d feed to gain.sup.e 3.03 3.13 3.3% Loin eye
area at 39.51 36.83 7.2% Slaughter (Cm.sup.2) .sup.a.fwdarw.e= from
time of injection to time of slaughter
[0132]
3TABLE 3 The effect of GH plasmid administration of weight gain
Group B D % Improvement Weight at injection 42.0 41.9 N/A time (Kg)
Weight gain.sup.a 52.1 47.6 9.5% ADG.sup.b 1.06 .97 9.3% total feed
309.6 300.7 3.0% intake/pen.sup.c Feed 6.3 6.1 3.3%
intake/pen/day.sup.d feed to gain.sup.e 3.0 3.2 6.7% Loin eye area
at 41.61 38.96 6.8% Slaughter (Cm.sup.2) a.fwdarw.e = from time of
injection to time of slaughter
[0133] A number of references have been cited and the entire
disclosures of which are incorporated herein by reference.
[0134] The present invention is not to be limited in scope by the
specific embodiments described which are intended as single
illustrations of individual aspects of the invention, and
functionally equivalent methods and components are within the scope
of the invention. Indeed various modifications of the invention, in
addition to those shown and described herein will become apparent
to those skilled in the art from the foregoing description and
accompanying drawings. Such modifications are intended to fall
within the scope of the appended claims.
Sequence CWU 1
1
67 1 246 DNA Artificial Sequence Description of Artificial Sequence
oligonucleotide 1 ggatccgcca ccatgccact ctgggtgttc ttctttgtga
tcctcaccct cagcaacagc 60 tcccactgct ccccacctcc ccctttgacc
ctcaggatgc ggcggtatgc agatgccatc 120 ttcaccaaca gctaccggaa
ggtgctgggc cagctgtccg cccgcaagct gctccaggac 180 atcatgagca
ggcagcaggg agagagaaac caagagcaag gagcaagggt gcggctttga 240 agatct
246 2 75 PRT Artificial Sequence Description of Artificial Sequence
GHRH protein 2 Met Pro Leu Trp Val Phe Phe Phe Val Ile Leu Thr Leu
Ser Asn Ser 1 5 10 15 Ser His Cys Ser Pro Pro Pro Pro Leu Thr Leu
Arg Met Arg Arg Tyr 20 25 30 Ala Asp Ala Ile Phe Thr Asn Ser Tyr
Arg Lys Val Leu Gly Gln Leu 35 40 45 Ser Ala Arg Lys Leu Leu Gln
Asp Ile Met Ser Arg Gln Gln Gly Glu 50 55 60 Arg Asn Gln Glu Gln
Gly Ala Arg Val Arg Leu 65 70 75 3 685 DNA Artificial Sequence
Description of Artificial Sequence obligonucleotide 3 ggtaccatcg
ctggggagct gggggagggg tcgccttcct gccctaccca ggactccggg 60
tgcgaccgct cctctatctc tccagcccac caccactcca ccacttggac acgtctccct
120 cctccctgga gtcgctctag agggtttggg ggtctgagta aagaacccga
agtagggata 180 cagtgtggcg gcaccttcca gaggccccgg gcgcagggta
gaccggggcg gggcggcccg 240 cggacaggtg cagccccagg cgcaggcgca
ctcgcgcctc ccggcgcagg cggtgaacct 300 cgccccaccc cagcccctcc
ggggggcagc tgggccgggt cgggaggggc ccaccagccc 360 gggagacact
ccatatacgg ccaggcccgc tttacctggg ctccggccag gccgctcctt 420
ctttggtcag cacaggggac ccgggcgggg gcccaggccg ctaacccgcc gggggagggg
480 gctccagtgc ccaacaccca aatatggctc gagaagggga gcgacattcc
agtgaggcgg 540 ctcgggggga gaacccgcgg gctatataaa acctgagcgt
ggggaccagc ggccaccgca 600 gcggacagcg ccgagagaag cctcgcttcc
ctcccgcggc gaccagggcc ccagccggag 660 agcagcaggt gtagccacca agctt
685 4 30 DNA Artificial Sequence Description of Artificial Sequence
primer 4 aatcccaagc ttgccaccat gccactctgg 30 5 30 DNA Artificial
Sequence Description of Artificial Sequence Primer 5 tattgctcta
gatcaaagcc gcacccttgc 30 6 39 DNA Artificial Sequence Description
of Artificial Sequence Primer 6 cgggtaccat cgctggggga gctggggcag
gggtcgcct 39 7 40 DNA Artificial Sequence Description of Artificial
Sequence Primer 7 cccgcttggt ggctacacct gctgctctcc ggctggggcc 40 8
2014 DNA Artificial Sequence Description of Artificial Sequence SK
2014 8 ggtaccgcta taggagagaa aagagctgca ctgagcaccc tccttcccct
ttaaatgtca 60 acagattagg agtcagtgaa tgacagcaca cctcttgcta
ccttagagac caaaatttaa 120 gctactcccc ttaagctata gctagagtgc
acctgccagt gtctttagtc cccactgatg 180 gaacaggacc caaggtattg
aagatggaac atagttattc attcatcctc taatttaaaa 240 agctggatat
gctgtacagc agaaattgac ggaacaatgt aaatcaacta taacagaaga 300
aataaaaacc tggggggaaa gaagctgact atgaaacccc aggagctttc tacatgggcc
360 tggactcacc aaactcttta ttttgtaatg gacttctgac atttttagga
agggctgtcc 420 tgatgtgggc tatagaagag ggtttcacat gcttcttcaa
gaggacccac actgtcccag 480 ttgctgagtc ccaccaccag atgctagtgg
cagctatttg gggaacactt aggcactaca 540 aaaaaatgag tgattccatt
ctggctcaca ccatatccct gatgtacccc ttaaagcatg 600 tcactgagtt
catcacagaa aattgtttcc cctgtgcctt ccacaacaag gttagagctg 660
tccttggggc caggggaagg gggcagggag tgagaagcac caactggata acctcctctg
720 acccccactc caccttacca taagtagatc caaatccttc tagaaaatta
ggaaggcata 780 tccccatata tcagcgatat aaatagaact gcttcagcgc
tctggtagac ggtgactctc 840 caaggtggac tgggaggcag cctggccttg
gctgggcatc gtcctctaaa tagaaagatg 900 aacttgttca gcctttccag
aaggaaaact gctgcccagc ctacagtgca acgtccttgt 960 cttccatctg
gaggaagcac gggtgacata tcatctagta agggcacctc tctgtttcca 1020
cctccaggtc gaggggtgtg acccttactt ctcagcctca agggagggac actcaacycc
1080 ccaaaaagac atgagggcgc tcagctcggc ccaccgcacc ccggaccgga
gccgtcaccc 1140 cccgaaattc actcccttca caagccccca agcgcgttct
ctggtgcgga ctgctccggg 1200 gccctggctt tgtgcccagc gttgtcagag
ccaccgccct gagcctgtcc ccgggagccc 1260 cgcgcctcct cccaccgctc
crctctcgcg ccccgcggcc agttgtctgc cccgagacag 1320 ctgcgcgccc
tcccgctgcc ggtggccctc tccggtgggg gtggggaccg acagggtcag 1380
ccctccggat ccggggcgct ccgggtagcg gggagaagtg atcgctgggg agctggggga
1440 ggggtcgcct tcctgcccta cccaggactc cgggtgcgac cgctcctcta
tctctccagc 1500 ccaccaccac tccaccactt ggacacgtct ccctcctccc
tggagtcgct ctagagggtt 1560 tgggggtctg agtaaagaac ccgaagtagg
gatacagtgt ggcggcacct tccagaggcc 1620 ccgggcgcag ggtagaccgg
ggcggggcgg cccgcggaca ggtgcagccc caggcgcagg 1680 cgcactcgcg
cctcccggcg caggcggtga acctcgcccc accccagccc ctccgggggg 1740
cagctgggcc gggtcgggag gggcccacca gcccgggaga cactccatat acggccaggc
1800 ccgctttacc tgggctccgg ccaggccgct ccttctttgg tcagcacagg
ggacccgggc 1860 gggggcccag gccgctaacc cgccggggga gggggctcca
gtgcccaaca cccaaatatg 1920 gctcgagaag gggagcgaca ttccagtgag
gcggctcggg gggagaaccc gcgggctata 1980 taaaacctga gcgtggggac
cagcggccaa gctt 2014 9 30 DNA Artificial Sequence Description of
Artificial Sequence Primer 9 cccaagcttg gccgctggtc cccacgctca 30 10
38 DNA Artificial Sequence Description of Artificial Sequence
Primer 10 caggtaccgc tataggagac aaaagagtgc actgagca 38 11 46 DNA
Artificial Sequence Description of Artificial Sequence Primer 11
agatatcccg gccgctctag accaggcccc tggatccgcc accatg 46 12 44 DNA
Artificial Sequence Description of Artificial Sequence Primer 12
gaagatctct acctgctcat gatgtcctgg agcagcttgc gggc 44 13 20 DNA
Artificial Sequence Description of Artificial Sequence Primer 13
gtcattccga gattcggata 20 14 40 DNA Artificial Sequence Description
of Artificial Sequence Primer 14 gtcattccga gattcggata cacaggatcc
gccaccatcc 40 15 40 DNA Artificial Sequence Description of
Artificial Sequence Primer 15 cactctgggt gttcttcttt gtgatcctca
ccctcagcaa 40 16 40 DNA Artificial Sequence Description of
Artificial Sequence Primer 16 cagctcccac tgctccccac ctcccccttt
gaccctcagg 40 17 40 DNA Artificial Sequence Description of
Artificial Sequence Primer 17 atgcggcggt attatgcaga tgccatcttc
accaacagct 40 18 40 DNA Artificial Sequence Description of
Artificial Sequence Primer 18 accggaaggt gctgggccag ctgtccgccc
gcaagctcct 40 19 40 DNA Artificial Sequence Description of
Artificial Sequence Primer 19 ccaggacatc atgagcaggt agagatctga
taagcgttat 40 20 20 DNA Artificial Sequence Description of
Artificial Sequence Primer 20 ataacgctta tcagatctct 20 21 40 DNA
Artificial Sequence Description of Artificial Sequence Primer 21
acctgctcat gatgtcctgg accagcttgc gggcggacag 40 22 40 DNA Artificial
Sequence Description of Artificial Sequence Primer 22 ctggcccagc
accttccggt agctgttggt gaagatggca 40 23 40 DNA Artificial Sequence
Description of Artificial Sequence Primer 23 tctgcataat accgccgcat
cctgagggtc aaacccccag 40 24 40 DNA Artificial Sequence Description
of Artificial Sequence Primer 24 gtggggagca gtgggagctg ttgctgaggg
tgaggatcac 40 25 40 DNA Artificial Sequence Description of
Artificial Sequence Primer 25 gtggggagca gtgggagctg ttgctgaggg
tcaggatcac 40 26 30 DNA Artificial Sequence Description of
Artificial Sequence Primer 26 gagctcaagc ttgccaccat gccactctgg 30
27 28 DNA Artificial Sequence Description of Artificial Sequence
Primer 27 aagatctaga ctacctgctc atgatgtc 28 28 30 DNA Artificial
Sequence Description of Artificial Sequence Primer 28 gagctcaagc
ttgccaccat gccactctgg 30 29 28 DNA Artificial Sequence Description
of Artificial Sequence Primer 29 aagatctaga ctacctgctc atgatgtc 28
30 20 DNA Artificial Sequence Description of Artificial Sequence
Primer 30 gtcattccga gattcggata 20 31 40 DNA Artificial Sequence
Description of Artificial Sequence Primer 31 gtcattccga gattcggata
cacaggatcc gccaccatcc 40 32 40 DNA Artificial Sequence Description
of Artificial Sequence Primer 32 cactctgggt gttcttcttt gtgatcctca
ccctcagcaa 40 33 39 DNA Artificial Sequence Description of
Artificial Sequence Primer 33 cagctccact gctccccacc tccccctttg
accctcagg 39 34 40 DNA Artificial Sequence Description of
Artificial Sequence Primer 34 atgcggcggt attatgcaga tcccatcttc
accaacagct 40 35 40 DNA Artificial Sequence Description of
Artificial Sequence Primer 35 accggaaggt gctggcccag ctgtccgccc
gcaaggccct 40 36 40 DNA Artificial Sequence Description of
Artificial Sequence Primer 36 ccaggacatc atgagcaggt agagatctga
taagcgttat 40 37 20 DNA Artificial Sequence Description of
Artificial Sequence Primer 37 ataacgctta tcagatctct 20 38 40 DNA
Artificial Sequence Description of Artificial Sequence Primer 38
acctgctcat gatgtcctgg agggccttgc gggcccacag 40 39 40 DNA Artificial
Sequence Description of Artificial Sequence Primer 39 ctgggccagc
accttccggt acctgttggt gaagatggca 40 40 40 DNA Artificial Sequence
Description of Artificial Sequence Primer 40 tctccataat accgccgcat
cctgagggtc aaagggggag 40 41 40 DNA Artificial Sequence Description
of Artificial Sequence Primer 41 gtggggagca gtgggagctg ttgctgaggg
tgaggatcac 40 42 40 DNA Artificial Sequence Description of
Artificial Sequence Primer 42 gtggggagca gtgggagctc ttgctgaggg
tgaggatcac 40 43 25 DNA Artificial Sequence Description of
Artificial Sequence Primer 43 ctagaaggca cagctgcttt ccacg 25 44 25
DNA Artificial Sequence Description of Artificial Sequence Primer
44 atggctgcag gcccccggac ctctg 25 45 27 DNA Artificial Sequence
Description of Artificial Sequence Primer 45 aaagatatca tggctgcagg
cccccgg 27 46 27 DNA Artificial Sequence Description of Artificial
Sequence Primer 46 aaaagatctc tagaaggcac agctgct 27 47 5185 DNA
Artificial Sequence Description of Artificial Sequence pGHRH-4
construct 47 gctgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc
ttccttgacc 60 ctggaaggtg ccactcccac tgtcctttcc taataaaatg
aggaaattgc atcgcattgt 120 ctgagtaggt gtcattctat tctggggggt
ggggtggggc aggacagcaa gggggaggat 180 tgggaagaca atagcaggca
tgctggggat gcggtgggct ctatgggtac ccaggtgctg 240 aagaattgac
ccggttcctc ctgggccaga aagaagcagg cacatcccct tctctgtgac 300
acaccctgtc cacgcccctg gttcttagtt ccagccccac tcataggaca ctcatagctc
360 aggagggctc cgccttcaat cccacccgct aaagtacttg gagcggtctc
tccctccctc 420 atcagcccac caaaccaaac ctagcctcca agagtgggaa
gaaattaaag caagataggc 480 tattaagtgc agagggagag aaaatgcctc
caacatgtga ggaagtaatg agagaaatca 540 tagaatttct tccgcttcct
cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg 600 agcggtatca
gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc 660
aggaaagaac atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt
720 gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc
gacgctcaag 780 tcagaggtgg cgaaacccga caggactata aagataccag
gcgtttcccc ctggaagctc 840 cctcgtgcgc tctcctgttc cgaccctgcc
gcttaccgga tacctgtccg cctttctccc 900 ttcgggaagc gtggcgcttt
ctcatagctc acgctgtagg tatctcagtt cggtgtaggt 960 cgttcgctcc
aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt 1020
atccggtaac tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc
1080 agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag
agttcttgaa 1140 gtggtggcct aactacggct acactagaag aacagtattt
ggtatctgcg ctctgctgaa 1200 gccagttacc ttcggaaaaa gagttggtag
ctcttgatcc ggcaaacaaa ccaccgctgg 1260 tagcggtggt ttttttgttt
gcaagcagca gattacgcgc agaaaaaaag gatctcaaga 1320 agatcctttg
atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg 1380
gattttggtc atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg
1440 aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt
accaatgctt 1500 aatcagtgag gcacctatct cagcgatctg tctatttcgt
tcatccatag ttgcctgact 1560 cggggggggg gggcgctgag gtctgcctcg
tgaagaaggt gttgctgact cataccaggc 1620 ctgaatcgcc ccatcatcca
gccagaaagt gagggagcca cggttgatga gagctttgtt 1680 gtaggtggac
cagttggtga ttttgaactt ttgctttgcc acggaacggt ctgcgttgtc 1740
gggaagatgc gtgatctgat ccttcaactc agcaaaagtt cgatttattc aacaaagccg
1800 ccgtcccgtc aagtcagcgt aatgctctgc cagtgttaca accaattaac
caattctgat 1860 tagaaaaact catcgagcat caaatgaaac tgcaatttat
tcatatcagg attatcaata 1920 ccatattttt gaaaaagccg tttctgtaat
gaaggagaaa actcaccgag gcagttccat 1980 aggatggcaa gatcctggta
tcggtctgcg attccgactc gtccaacatc aatacaacct 2040 attaatttcc
cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg agtgacgact 2100
gaatccggtg agaatggcaa aagcttatgc atttctttcc agacttgttc aacaggccag
2160 ccattacgct cgtcatcaaa atcactcgca tcaaccaaac cgttattcat
tcgtgattgc 2220 gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac
aattacaaac aggaatcgaa 2280 tgcaaccggc gcaggaacac tgccagcgca
tcaacaatat tttcacctga atcaggatat 2340 tcttctaata cctggaatgc
tgttttcccg gggatcgcag tggtgagtaa ccatgcatca 2400 tcaggagtac
ggataaaatg cttgatggtc ggaagaggca taaattccgt cagccagttt 2460
agtctgacca tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac
2520 aactctggcg catcgggctt cccatacaat cgatagattg tcgcacctga
ttgcccgaca 2580 ttatcgcgag cccatttata cccatataaa tcagcatcca
tgttggaatt taatcgcggc 2640 ctcgagcaag acgtttcccg ttgaatatgg
ctcataacac cccttgtatt actgtttatg 2700 taagcagaca gttttattgt
tcatgatgat atatttttat cttgtgcaat gtaacatcag 2760 agattttgag
acacaacgtg gctttccccc cccccccatt attgaagcat ttatcagggt 2820
tattgtctca tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt
2880 ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat
tatcatgaca 2940 ttaacctata aaaataggcg tatcacgagg ccctttcgtc
ctcgcgcgtt tcggtgatga 3000 cggtgaaaac ctctgacaca tgcagctccc
ggagacggtc acagcttgtc tgtaagcgga 3060 tgccgggagc agacaagccc
gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg 3120 gcttaactat
gcggcatcag agcagattgt actgagagtg caccatatgc ggtgtgaaat 3180
accgcacaga tgcgtaagga gaaaataccg catcagattg gctattggcc attgcatacg
3240 ttgtatccat atcataatat gtacatttat attggctcat gtccaacatt
accgccatgt 3300 tgacattgat tattgactag ttattaatag taatcaatta
cggggtcatt agttcatagc 3360 ccatatatgg agttccgcgt tacataactt
acggtaaatg gcccgcctgg ctgaccgccc 3420 aacgaccccc gcccattgac
gtcaataatg acgtatgttc ccatagtaac gccaataggg 3480 actttccatt
gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt ggcagtacat 3540
caagtgtatc atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc
3600 tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta
catctacgta 3660 ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt
acatcaatgg gcgtggatag 3720 cggtttgact cacggggatt tccaagtctc
caccccattg acgtcaatgg gagtttgttt 3780 tggcaccaaa atcaacggga
ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa 3840 atgggcggta
ggcgtgtacg gtgggaggtc tatataagca gagctcgttt agtgaaccgt 3900
cagatcgcct ggagacgcca tccacgctgt tttgacctcc atagaagaca ccgggaccga
3960 tccagcctcc gcggccggga acggtgcatt ggaacgcgga ttccccgtgc
caagagtgac 4020 gtaagtaccg cctatagact ctataggcac acccctttgg
ctcttatgca tgctatactg 4080 tttttggctt ggggcctata cacccccgct
tccttatgct ataggtgatg gtatagctta 4140 gcctataggt gtgggttatt
gaccattatt gaccactccc ctattggtga cgatactttc 4200 cattactaat
ccataacatg gctctttgcc acaactatct ctattggcta tatgccaata 4260
ctctgtcctt cagagactga cacggactct gtatttttac aggatggggt cccatttatt
4320 atttacaaat tcacatatac aacaacgccg tcccccgtgc ccgcagtttt
tattaaacat 4380 agcgtgggat ctccacgcga atctcgggta cgtgttccgg
acatgggctc ttctccggta 4440 gcggcggagc ttccacatcc gagccctggt
cccatgcctc cagcggctca tggtcgctcg 4500 gcagctcctt gctcctaaca
gtggaggcca gacttaggca cagcacaatg cccaccacca 4560 ccagtgtgcc
gcacaaggcc gtggcggtag ggtatgtgtc tgaaaatgag cgtggagatt 4620
gggctcgcac ggctgacgca gatggaagac ttaaggcagc ggcagaagaa gatgcaggca
4680 gctgagttgt tgtattctga taagagtcag aggtaactcc cgttgcggtg
ctgttaacgg 4740 tggagggcag tgtagtctga gcagtactcg ttgctgccgc
gcgcgccacc agacataata 4800 gctgacagac taacagactg ttcctttcca
tgggtctttt ctgcagtcac cgtcgtcgac 4860 acgtgtgatc agatatcgcg
gccgctctag accaggcgcc tggatccgcc accatgccac 4920 tctgggtgtt
cttctttgtg atcctcaccc tcagcaacag ctcccactgc tccccacctc 4980
cccctttgac cctcaggatg cggcggtatg cagatgccat cttcaccaac agctaccgga
5040 aggtgctggg ccagctgtcc gcccgcaagc tgctccagga catcatgagc
aggcagcagg 5100 gagagagaaa ccaagagcaa ggagcaaggg tgcggctttg
aagatcttag tagtagtagg 5160 cggccgctct agaggatcca gatct 5185 48 3369
DNA Artificial Sequence Description of Artificial Sequence
pGHRH1-44SK construct 48 ggtaccgagc tcttacgcgt gctagcccgg
gctcgagatc tgcgatctaa gtaagcttgc 60 caccatgcca ctctgggtgt
tcttctttgt gatcctcacc ctcagcaaca gctcccactg 120 ctccccacct
ccccctttga ccctcaggat gcggcggtat gcagatgcca tcttcaccaa 180
cagctaccgg aaggtgctgg gccagctgtc cgcccgcaag ctgctccagg acatcatgag
240 caggcagcag ggagagagaa accaagagca aggagcaagg gtgcggcttt
gatctagagt 300 cggggcggcc ggccgcttcg agcagacatg ataagataca
ttgatgagtt tggacaaacc 360 acaactagaa tgcagtgaaa aaaatgcttt
atttgtgaaa tttgtgatgc tattgcttta 420 tttgtaacca ttataagctg
caataaacaa gttaacaaca acaattgcat tcattttatg 480 tttcaggttc
agggggaggt
gtgggaggtt ttttaaagca agtaaaacct ctacaaatgt 540 ggtaaaatcg
ataaggatcc gtcgaccgat gcccttgaga gccttcaacc cagtcagctc 600
cttccggtgg gcgcggggca tgactatcgt cgccgcactt atgactgtct tctttatcat
660 gcaactcgta ggacaggtgc cggcagcgct cttccgcttc ctcgctcact
gactcgctgc 720 gctcggtcgt tcggctgcgg cgagcggtat cagctcactc
aaaggcggta atacggttat 780 ccacagaatc aggggataac gcaggaaaga
acatgtgagc aaaaggccag caaaaggcca 840 ggaaccgtaa aaaggccgcg
ttgctggcgt ttttccatag gctccgcccc cctgacgagc 900 atcacaaaaa
tcgacgctca agtcagaggt ggcgaaaccc gacaggacta taaagatacc 960
aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt tccgaccctg ccgcttaccg
1020 gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct ttctcaatgc
tcacgctgta 1080 ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg
ctgtgtgcac gaaccccccg 1140 ttcagcccga ccgctgcgcc ttatccggta
actatcgtct tgagtccaac ccggtaagac 1200 acgacttatc gccactggca
gcagccactg gtaacaggat tagcagagcg aggtatgtag 1260 gcggtgctac
agagttcttg aagtggtggc ctaactacgg ctacactaga aggacagtat 1320
ttggtatctg cgctctgctg aagccagtta ccttcggaaa aagagttggt agctcttgat
1380 ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt ttgcaagcag
cagattacgc 1440 gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc
tacggggtct gacgctcagt 1500 ggaacgaaaa ctcacgttaa gggattttgg
tcatgagatt atcaaaaagg atcttcacct 1560 agatcctttt aaattaaaaa
tgaagtttta aatcaatcta aagtatatat gagtaaactt 1620 ggtctgacag
ttaccaatgc ttaatcagtg aggcacctat ctcagcgatc tgtctatttc 1680
gttcatccat agttgcctga ctccccgtcg tgtagataac tacgatacgg gagggcttac
1740 catctggccc cagtgctgca atgataccgc gagacccacg ctcaccggct
ccagatttat 1800 cagcaataaa ccagccagcc ggaagggccg agcgcagaag
tggtcctgca actttatccg 1860 cctccatcca gtctattaat tgttgccggg
aagctagagt aagtagttcg ccagttaata 1920 gtttgcgcaa cgttgttgcc
attgctacag gcatcgtggt gtcacgctcg tcgtttggta 1980 tggcttcatt
cagctccggt tcccaacgat caaggcgagt tacatgatcc cccatgttgt 2040
gcaaaaaagc ggttagctcc ttcggtcctc cgatcgttgt cagaagtaag ttggccgcag
2100 tgttatcact catggttatg gcagcactgc ataattctct tactgtcatg
ccatccgtaa 2160 gatgcttttc tgtgactggt gagtactcaa ccaagtcatt
ctgagaatag tgtatgcggc 2220 gaccgagttg ctcttgcccg gcgtcaatac
gggataatac cgcgccacat agcagaactt 2280 taaaagtgct catcattgga
aaacgttctt cggggcgaaa actctcaagg atcttaccgc 2340 tgttgagatc
cagttcgatg taacccactc gtgcacccaa ctgatcttca gcatctttta 2400
ctttcaccag cgtttctggg tgagcaaaaa caggaaggca aaatgccgca aaaaagggaa
2460 taagggcgac acggaaatgt tgaatactca tactcttcct ttttcaatat
tattgaagca 2520 tttatcaggg ttattgtctc atgagcggat acatatttga
atgtatttag aaaaataaac 2580 aaataggggt tccgcgcaca tttccccgaa
aagtgccacc tgacgcgccc tgtagcggcg 2640 cattaagcgc ggcgggtgtg
gtggttacgc gcagcgtgac cgctacactt gccagcgccc 2700 tagcgcccgc
tcctttcgct ttcttccctt cctttctcgc cacgttcgcc ggctttcccc 2760
gtcaagctct aaatcggggg ctccctttag ggttccgatt tagtgcttta cggcacctcg
2820 accccaaaaa acttgattag ggtgatggtt cacgtagtgg gccatcgccc
tgatagacgg 2880 tttttcgccc tttgacgttg gagtccacgt tctttaatag
tggactcttg ttccaaactg 2940 gaacaacact caaccctatc tcggtctatt
cttttgattt ataagggatt ttgccgattt 3000 cggcctattg gttaaaaaat
gagctgattt aacaaaaatt taacgcgaat tttaacaaaa 3060 tattaacgtt
tacaatttcc cattcgccat tcaggctgcg caactgttgg gaagggcgat 3120
cggtgcgggc ctcttcgcta ttacgccagc ccaagctacc atgataagta agtaatatta
3180 aggtacggga ggtacttgga gcggccgcaa taaaatatct ttattttcat
tacatctgtg 3240 tgttggtttt ttgtgtgaat cgatagtact aacatacgct
ctccatcaaa acaaaacgaa 3300 acaaaacaaa ctagcaaaat aggctgtccc
cagtgcaagt gcaggtgcca gaacatttct 3360 ctatcgata 3369 49 3976 DNA
Artificial Sequence Description of Artificial Sequence
pGHRH1-44WTSK685 construct 49 ggtaccatcg ctggggagct gggggagggg
tcgccttcct gccctaccca ggactccggg 60 tgcgaccgct cctctatctc
tccagcccac caccactcca ccacttggac acgtctccct 120 cctccctgga
gtcgctctag agggtttggg ggtctgagta aagaacccga agtagggata 180
cagtgtggcg gcaccttcca gaggccccgg gcgcagggta gaccggggcg gggcggcccg
240 cggacaggtg cagccccagg cgcaggcgca ctcgcgcctc ccggcgcagg
cggtgaacct 300 cgccccaccc cagcccctcc ggggggcagc tgggccgggt
cgggaggggc ccaccagccc 360 gggagacact ccatatacgg ccaggcccgc
tttacctggg ctccggccag gccgctcctt 420 ctttggtcag cacaggggac
ccgggcgggg gcccaggccg ctaacccgcc gggggagggg 480 gctccagtgc
ccaacaccca aatatggctc gagaagggga gcgacattcc agtgaggcgg 540
ctcgggggga gaacccgcgg gctatataaa acctgagcgt ggggaccagc ggccaccgca
600 gcggacagcg ccgagagaag cctcgcttcc ctcccgcggc gaccagggcc
ccagccggag 660 agcagcaggt gtagccacca agcttgccac catgccactc
tgggtgttct tctttgtgat 720 cctcaccctc agcaacagct cccactgctc
cccacctccc cctttgaccc tcaggatgcg 780 gcggtatgca gatgccatct
tcaccaacag ctaccggaag gtgctgggcc agctgtccgc 840 ccgcaagctg
ctccaggaca tcatgagcag gcagcaggga gagagaaacc aagagcaagg 900
agcaagggtg cggctttgat ctagagtcgg ggcggccggc cgcttcgagc agacatgata
960 agatacattg atgagtttgg acaaaccaca actagaatgc agtgaaaaaa
atgctttatt 1020 tgtgaaattt gtgatgctat tgctttattt gtaaccatta
taagctgcaa taaacaagtt 1080 aacaacaaca attgcattca ttttatgttt
caggttcagg gggaggtgtg ggaggttttt 1140 taaagcaagt aaaacctcta
caaatgtggt aaaatcgata aggatccgtc gaccgatgcc 1200 cttgagagcc
ttcaacccag tcagctcctt ccggtgggcg cggggcatga ctatcgtcgc 1260
cgcacttatg actgtcttct ttatcatgca actcgtagga caggtgccgg cagcgctctt
1320 ccgcttcctc gctcactgac tcgctgcgct cggtcgttcg gctgcggcga
gcggtatcag 1380 ctcactcaaa ggcggtaata cggttatcca cagaatcagg
ggataacgca ggaaagaaca 1440 tgtgagcaaa aggccagcaa aaggccagga
accgtaaaaa ggccgcgttg ctggcgtttt 1500 tccataggct ccgcccccct
gacgagcatc acaaaaatcg acgctcaagt cagaggtggc 1560 gaaacccgac
aggactataa agataccagg cgtttccccc tggaagctcc ctcgtgcgct 1620
ctcctgttcc gaccctgccg cttaccggat acctgtccgc ctttctccct tcgggaagcg
1680 tggcgctttc tcaatgctca cgctgtaggt atctcagttc ggtgtaggtc
gttcgctcca 1740 agctgggctg tgtgcacgaa ccccccgttc agcccgaccg
ctgcgcctta tccggtaact 1800 atcgtcttga gtccaacccg gtaagacacg
acttatcgcc actggcagca gccactggta 1860 acaggattag cagagcgagg
tatgtaggcg gtgctacaga gttcttgaag tggtggccta 1920 actacggcta
cactagaagg acagtatttg gtatctgcgc tctgctgaag ccagttacct 1980
tcggaaaaag agttggtagc tcttgatccg gcaaacaaac caccgctggt agcggtggtt
2040 tttttgtttg caagcagcag attacgcgca gaaaaaaagg atctcaagaa
gatcctttga 2100 tcttttctac ggggtctgac gctcagtgga acgaaaactc
acgttaaggg attttggtca 2160 tgagattatc aaaaaggatc ttcacctaga
tccttttaaa ttaaaaatga agttttaaat 2220 caatctaaag tatatatgag
taaacttggt ctgacagtta ccaatgctta atcagtgagg 2280 cacctatctc
agcgatctgt ctatttcgtt catccatagt tgcctgactc cccgtcgtgt 2340
agataactac gatacgggag ggcttaccat ctggccccag tgctgcaatg ataccgcgag
2400 acccacgctc accggctcca gatttatcag caataaacca gccagccgga
agggccgagc 2460 gcagaagtgg tcctgcaact ttatccgcct ccatccagtc
tattaattgt tgccgggaag 2520 ctagagtaag tagttcgcca gttaatagtt
tgcgcaacgt tgttgccatt gctacaggca 2580 tcgtggtgtc acgctcgtcg
tttggtatgg cttcattcag ctccggttcc caacgatcaa 2640 ggcgagttac
atgatccccc atgttgtgca aaaaagcggt tagctccttc ggtcctccga 2700
tcgttgtcag aagtaagttg gccgcagtgt tatcactcat ggttatggca gcactgcata
2760 attctcttac tgtcatgcca tccgtaagat gcttttctgt gactggtgag
tactcaacca 2820 agtcattctg agaatagtgt atgcggcgac cgagttgctc
ttgcccggcg tcaatacggg 2880 ataataccgc gccacatagc agaactttaa
aagtgctcat cattggaaaa cgttcttcgg 2940 ggcgaaaact ctcaaggatc
ttaccgctgt tgagatccag ttcgatgtaa cccactcgtg 3000 cacccaactg
atcttcagca tcttttactt tcaccagcgt ttctgggtga gcaaaaacag 3060
gaaggcaaaa tgccgcaaaa aagggaataa gggcgacacg gaaatgttga atactcatac
3120 tcttcctttt tcaatattat tgaagcattt atcagggtta ttgtctcatg
agcggataca 3180 tatttgaatg tatttagaaa aataaacaaa taggggttcc
gcgcacattt ccccgaaaag 3240 tgccacctga cgcgccctgt agcggcgcat
taagcgcggc gggtgtggtg gttacgcgca 3300 gcgtgaccgc tacacttgcc
agcgccctag cgcccgctcc tttcgctttc ttcccttcct 3360 ttctcgccac
gttcgccggc tttccccgtc aagctctaaa tcgggggctc cctttagggt 3420
tccgatttag tgctttacgg cacctcgacc ccaaaaaact tgattagggt gatggttcac
3480 gtagtgggcc atcgccctga tagacggttt ttcgcccttt gacgttggag
tccacgttct 3540 ttaatagtgg actcttgttc caaactggaa caacactcaa
ccctatctcg gtctattctt 3600 ttgatttata agggattttg ccgatttcgg
cctattggtt aaaaaatgag ctgatttaac 3660 aaaaatttaa cgcgaatttt
aacaaaatat taacgtttac aatttcccat tcgccattca 3720 ggctgcgcaa
ctgttgggaa gggcgatcgg tgcgggcctc ttcgctatta cgccagccca 3780
agctaccatg ataagtaagt aatattaagg tacgggaggt acttggagcg gccgcaataa
3840 aatatcttta ttttcattac atctgtgtgt tggttttttg tgtgaatcga
tagtactaac 3900 atacgctctc catcaaaaca aaacgaaaca aaacaaacta
gcaaaatagg ctgtccccag 3960 tgcaagtgca ggtgcc 3976 50 5325 DNA
Artificial Sequence Description of Artificial Sequence
pGHRH1-44WTSK2014 construct 50 ggtaccgcta taggagagaa aagagctgca
ctgagcaccc tccttcccct ttaaatgtca 60 acagattagg agtcagtgaa
tgacagcaca cctcttgcta ccttagagac caaaatttaa 120 gctactcccc
ttaagctata gctagagtgc acctgccagt gtctttagtc cccactgatg 180
gaacaggacc caaggtattg aagatggaac atagttattc attcatcctc taatttaaaa
240 agctggatat gctgtacagc agaaattgac ggaacaatgt aaatcaacta
taacagaaga 300 aataaaaacc tggggggaaa gaagctgact atgaaacccc
aggagctttc tacatgggcc 360 tggactcacc aaactcttta ttttgtaatg
gacttctgac atttttagga agggctgtcc 420 tgatgtgggc tatagaagag
ggtttcacat gcttcttcaa gaggacccac actgtcccag 480 ttgctgagtc
ccaccaccag atgctagtgg cagctatttg gggaacactt aggcactaca 540
aaaaaatgag tgattccatt ctggctcaca ccatatccct gatgtacccc ttaaagcatg
600 tcactgagtt catcacagaa aattgtttcc cctgtgcctt ccacaacaag
gttagagctg 660 tccttggggc caggggaagg gggcagggag tgagaagcac
caactggata acctcctctg 720 acccccactc caccttacca taagtagatc
caaatccttc tagaaaatta ggaaggcata 780 tccccatata tcagcgatat
aaatagaact gcttcagcgc tctggtagac ggtgactctc 840 caaggtggac
tgggaggcag cctggccttg gctgggcatc gtcctctaaa tagaaagatg 900
aacttgttca gcctttccag aaggaaaact gctgcccagc ctacagtgca acgtccttgt
960 cttccatctg gaggaagcac gggtgacata tcatctagta agggcacctc
tctgtttcca 1020 cctccaggtc gaggggtgtg acccttactt ctcagcctca
agggagggac actcaacccc 1080 ccaaaaagac atgagggcgc tcagctcggc
ccaccgcacc ccggaccgga gccgtcaccc 1140 cccgaaattc actcccttca
caagccccca agcgcgttct ctggtgcgga ctgctccggg 1200 gccctggctt
tgtgcccagc gttgtcagag ccaccgccct gagcctgtcc ccgggagccc 1260
cgcgcctcct cccaccgctc cgctctcgcg ccccgcggcc agttgtctgc cccgagacag
1320 ctgcgcgccc tcccgctgcc ggtggccctc tccggtgggg gtggggaccg
acagggtcag 1380 ccctccggat ccggggcgct ccgggtagcg gggagaagtg
atcgctgggg agctggggga 1440 ggggtcgcct tcctgcccta cccaggactc
cgggtgcgac cgctcctcta tctctccagc 1500 ccaccaccac tccaccactt
ggacacgtct ccctcctccc tggagtcgct ctagagggtt 1560 tgggggtctg
agtaaagaac ccgaagtagg gatacagtgt ggcggcacct tccagaggcc 1620
ccgggcgcag ggtagaccgg ggcggggcgg cccgcggaca ggtgcagccc caggcgcagg
1680 cgcactcgcg cctcccggcg caggcggtga acctcgcccc accccagccc
ctccgggggg 1740 cagctgggcc gggtcgggag gggcccacca gcccgggaga
cactccatat acggccaggc 1800 ccgctttacc tgggctccgg ccaggccgct
ccttctttgg tcagcacagg ggacccgggc 1860 gggggcccag gccgctaacc
cgccggggga gggggctcca gtgcccaaca cccaaatatg 1920 gctcgagaag
gggagcgaca ttccagtgag gcggctcggg gggagaaccc gcgggctata 1980
taaaacctga gcgtggggac cagcggccaa gcttgccacc atgccactct gggtgttctt
2040 ctttgtgatc ctcaccctca gcaacagctc ccactgctcc ccacctcccc
ctttgaccct 2100 caggatgcgg cggtatgcag atgccatctt caccaacagc
taccggaagg tgctgggcca 2160 gctgtccgcc cgcaagctgc tccaggacat
catgagcagg cagcagggag agagaaacca 2220 agagcaagga gcaagggtgc
ggctttgatc tagagtcggg gcggccggcc gcttcgagca 2280 gacatgataa
gatacattga tgagtttgga caaaccacaa ctagaatgca gtgaaaaaaa 2340
tgctttattt gtgaaatttg tgatgctatt gctttatttg taaccattat aagctgcaat
2400 aaacaagtta acaacaacaa ttgcattcat tttatgtttc aggttcaggg
ggaggtgtgg 2460 gaggtttttt aaagcaagta aaacctctac aaatgtggta
aaatcgataa ggatccgtcg 2520 accgatgccc ttgagagcct tcaacccagt
cagctccttc cggtgggcgc ggggcatgac 2580 tatcgtcgcc gcacttatga
ctgtcttctt tatcatgcaa ctcgtaggac aggtgccggc 2640 agcgctcttc
cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg ctgcggcgag 2700
cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg gataacgcag
2760 gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag
gccgcgttgc 2820 tggcgttttt ccataggctc cgcccccctg acgagcatca
caaaaatcga cgctcaagtc 2880 agaggtggcg aaacccgaca ggactataaa
gataccaggc gtttccccct ggaagctccc 2940 tcgtgcgctc tcctgttccg
accctgccgc ttaccggata cctgtccgcc tttctccctt 3000 cgggaagcgt
ggcgctttct caatgctcac gctgtaggta tctcagttcg gtgtaggtcg 3060
ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc tgcgccttat
3120 ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca
ctggcagcag 3180 ccactggtaa caggattagc agagcgaggt atgtaggcgg
tgctacagag ttcttgaagt 3240 ggtggcctaa ctacggctac actagaagga
cagtatttgg tatctgcgct ctgctgaagc 3300 cagttacctt cggaaaaaga
gttggtagct cttgatccgg caaacaaacc accgctggta 3360 gcggtggttt
ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag 3420
atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca cgttaaggga
3480 ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat
taaaaatgaa 3540 gttttaaatc aatctaaagt atatatgagt aaacttggtc
tgacagttac caatgcttaa 3600 tcagtgaggc acctatctca gcgatctgtc
tatttcgttc atccatagtt gcctgactcc 3660 ccgtcgtgta gataactacg
atacgggagg gcttaccatc tggccccagt gctgcaatga 3720 taccgcgaga
cccacgctca ccggctccag atttatcagc aataaaccag ccagccggaa 3780
gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct attaattgtt
3840 gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt
gttgccattg 3900 ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc
ttcattcagc tccggttccc 3960 aacgatcaag gcgagttaca tgatccccca
tgttgtgcaa aaaagcggtt agctccttcg 4020 gtcctccgat cgttgtcaga
agtaagttgg ccgcagtgtt atcactcatg gttatggcag 4080 cactgcataa
ttctcttact gtcatgccat ccgtaagatg cttttctgtg actggtgagt 4140
actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct tgcccggcgt
4200 caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc
attggaaaac 4260 gttcttcggg gcgaaaactc tcaaggatct taccgctgtt
gagatccagt tcgatgtaac 4320 ccactcgtgc acccaactga tcttcagcat
cttttacttt caccagcgtt tctgggtgag 4380 caaaaacagg aaggcaaaat
gccgcaaaaa agggaataag ggcgacacgg aaatgttgaa 4440 tactcatact
cttccttttt caatattatt gaagcattta tcagggttat tgtctcatga 4500
gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg cgcacatttc
4560 cccgaaaagt gccacctgac gcgccctgta gcggcgcatt aagcgcggcg
ggtgtggtgg 4620 ttacgcgcag cgtgaccgct acacttgcca gcgccctagc
gcccgctcct ttcgctttct 4680 tcccttcctt tctcgccacg ttcgccggct
ttccccgtca agctctaaat cgggggctcc 4740 ctttagggtt ccgatttagt
gctttacggc acctcgaccc caaaaaactt gattagggtg 4800 atggttcacg
tagtgggcca tcgccctgat agacggtttt tcgccctttg acgttggagt 4860
ccacgttctt taatagtgga ctcttgttcc aaactggaac aacactcaac cctatctcgg
4920 tctattcttt tgatttataa gggattttgc cgatttcggc ctattggtta
aaaaatgagc 4980 tgatttaaca aaaatttaac gcgaatttta acaaaatatt
aacgtttaca atttcccatt 5040 cgccattcag gctgcgcaac tgttgggaag
ggcgatcggt gcgggcctct tcgctattac 5100 gccagcccaa gctaccatga
taagtaagta atattaaggt acgggaggta cttggagcgg 5160 ccgcaataaa
atatctttat tttcattaca tctgtgtgtt ggttttttgt gtgaatcgat 5220
agtactaaca tacgctctcc atcaaaacaa aacgaaacaa aacaaactag caaaataggc
5280 tgtccccagt gcaagtgcag gtgccagaac atttctctat cgata 5325 51 5108
DNA Artificial Sequence Description of Artificial Sequence
pGHRH1-29WTCMV construct 51 gctgtgcctt ctagttgcca gccatctgtt
gtttgcccct cccccgtgcc ttccttgacc 60 ctggaaggtg ccactcccac
tgtcctttcc taataaaatg aggaaattgc atcgcattgt 120 ctgagtaggt
gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat 180
tgggaagaca atagcaggca tgctggggat gcggtgggct ctatgggtac ccaggtgctg
240 aagaattgac ccggttcctc ctgggccaga aagaagcagg cacatcccct
tctctgtgac 300 acaccctgtc cacgcccctg gttcttagtt ccagccccac
tcataggaca ctcatagctc 360 aggagggctc cgccttcaat cccacccgct
aaagtacttg gagcggtctc tccctccctc 420 atcagcccac caaaccaaac
ctagcctcca agagtgggaa gaaattaaag caagataggc 480 tattaagtgc
agagggagag aaaatgcctc caacatgtga ggaagtaatg agagaaatca 540
tagaatttct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
600 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
gggataacgc 660 aggaaagaac atgtgagcaa aaggccagca aaaggccagg
aaccgtaaaa aggccgcgtt 720 gctggcgttt ttccataggc tccgcccccc
tgacgagcat cacaaaaatc gacgctcaag 780 tcagaggtgg cgaaacccga
caggactata aagataccag gcgtttcccc ctggaagctc 840 cctcgtgcgc
tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc 900
ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
960 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
gctgcgcctt 1020 atccggtaac tatcgtcttg agtccaaccc ggtaagacac
gacttatcgc cactggcagc 1080 agccactggt aacaggatta gcagagcgag
gtatgtaggc ggtgctacag agttcttgaa 1140 gtggtggcct aactacggct
acactagaag aacagtattt ggtatctgcg ctctgctgaa 1200 gccagttacc
ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg 1260
tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
1320 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
cacgttaagg 1380 gattttggtc atgagattat caaaaaggat cttcacctag
atccttttaa attaaaaatg 1440 aagttttaaa tcaatctaaa gtatatatga
gtaaacttgg tctgacagtt accaatgctt 1500 aatcagtgag gcacctatct
cagcgatctg tctatttcgt tcatccatag ttgcctgact 1560 cggggggggg
gggcgctgag gtctgcctcg tgaagaaggt gttgctgact cataccaggc 1620
ctgaatcgcc ccatcatcca gccagaaagt gagggagcca cggttgatga gagctttgtt
1680 gtaggtggac cagttggtga ttttgaactt ttgctttgcc acggaacggt
ctgcgttgtc 1740 gggaagatgc gtgatctgat ccttcaactc agcaaaagtt
cgatttattc aacaaagccg 1800 ccgtcccgtc aagtcagcgt aatgctctgc
cagtgttaca accaattaac caattctgat 1860 tagaaaaact catcgagcat
caaatgaaac tgcaatttat tcatatcagg attatcaata 1920 ccatattttt
gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat 1980
aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct
2040 attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg
agtgacgact 2100 gaatccggtg agaatggcaa aagcttatgc atttctttcc
agacttgttc aacaggccag 2160 ccattacgct cgtcatcaaa atcactcgca
tcaaccaaac cgttattcat tcgtgattgc 2220 gcctgagcga gacgaaatac
gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa 2280 tgcaaccggc
gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat 2340
tcttctaata cctggaatgc tgttttcccg gggatcgcag tggtgagtaa ccatgcatca
2400 tcaggagtac ggataaaatg cttgatggtc ggaagaggca taaattccgt
cagccagttt 2460 agtctgacca tctcatctgt aacatcattg gcaacgctac
ctttgccatg tttcagaaac 2520 aactctggcg catcgggctt cccatacaat
cgatagattg tcgcacctga ttgcccgaca 2580 ttatcgcgag cccatttata
cccatataaa tcagcatcca tgttggaatt taatcgcggc 2640 ctcgagcaag
acgtttcccg ttgaatatgg ctcataacac cccttgtatt actgtttatg 2700
taagcagaca gttttattgt tcatgatgat atatttttat cttgtgcaat gtaacatcag
2760 agattttgag acacaacgtg gctttccccc cccccccatt attgaagcat
ttatcagggt 2820 tattgtctca tgagcggata catatttgaa tgtatttaga
aaaataaaca aataggggtt 2880 ccgcgcacat ttccccgaaa agtgccacct
gacgtctaag aaaccattat tatcatgaca 2940 ttaacctata aaaataggcg
tatcacgagg ccctttcgtc ctcgcgcgtt tcggtgatga 3000 cggtgaaaac
ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga 3060
tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg
3120 gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgc
ggtgtgaaat 3180 accgcacaga tgcgtaagga gaaaataccg catcagattg
gctattggcc attgcatacg 3240 ttgtatccat atcataatat gtacatttat
attggctcat gtccaacatt accgccatgt 3300 tgacattgat tattgactag
ttattaatag taatcaatta cggggtcatt agttcatagc 3360 ccatatatgg
agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc 3420
aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg
3480 actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt
ggcagtacat 3540 caagtgtatc atatgccaag tacgccccct attgacgtca
atgacggtaa atggcccgcc 3600 tggcattatg cccagtacat gaccttatgg
gactttccta cttggcagta catctacgta 3660 ttagtcatcg ctattaccat
ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag 3720 cggtttgact
cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt 3780
tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa
3840 atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctcgttt
agtgaaccgt 3900 cagatcgcct ggagacgcca tccacgctgt tttgacctcc
atagaagaca ccgggaccga 3960 tccagcctcc gcggccggga acggtgcatt
ggaacgcgga ttccccgtgc caagagtgac 4020 gtaagtaccg cctatagact
ctataggcac acccctttgg ctcttatgca tgctatactg 4080 tttttggctt
ggggcctata cacccccgct tccttatgct ataggtgatg gtatagctta 4140
gcctataggt gtgggttatt gaccattatt gaccactccc ctattggtga cgatactttc
4200 cattactaat ccataacatg gctctttgcc acaactatct ctattggcta
tatgccaata 4260 ctctgtcctt cagagactga cacggactct gtatttttac
aggatggggt cccatttatt 4320 atttacaaat tcacatatac aacaacgccg
tcccccgtgc ccgcagtttt tattaaacat 4380 agcgtgggat ctccacgcga
atctcgggta cgtgttccgg acatgggctc ttctccggta 4440 gcggcggagc
ttccacatcc gagccctggt cccatgcctc cagcggctca tggtcgctcg 4500
gcagctcctt gctcctaaca gtggaggcca gacttaggca cagcacaatg cccaccacca
4560 ccagtgtgcc gcacaaggcc gtggcggtag ggtatgtgtc tgaaaatgag
cgtggagatt 4620 gggctcgcac ggctgacgca gatggaagac ttaaggcagc
ggcagaagaa gatgcaggca 4680 gctgagttgt tgtattctga taagagtcag
aggtaactcc cgttgcggtg ctgttaacgg 4740 tggagggcag tgtagtctga
gcagtactcg ttgctgccgc gcgcgccacc agacataata 4800 gctgacagac
taacagactg ttcctttcca tgggtctttt ctgcagtcac cgtcgtcgac 4860
acgtgtgatc agatatcgcg gccgctctag accaggcgcc tggatccgcc accatgccac
4920 tctgggtgtt cttctttgtg atcctcaccc tcagcaacag ctcccactgc
tccccacctc 4980 cccctttgac cctcaggatg cggcggtatg cagatgccat
cttcaccaac agctaccgga 5040 aggtgctggg ccagctgtcc gcccgcaagc
tgctccagga catcatgagc aggtagagat 5100 ccagatct 5108 52 5108 DNA
Artificial Sequence Description of Artificial Sequence
pGHRH1-29YWTCMV construct 52 gctgtgcctt ctagttgcca gccatctgtt
gtttgcccct cccccgtgcc ttccttgacc 60 ctggaaggtg ccactcccac
tgtcctttcc taataaaatg aggaaattgc atcgcattgt 120 ctgagtaggt
gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat 180
tgggaagaca atagcaggca tgctggggat gcggtgggct ctatgggtac ccaggtgctg
240 aagaattgac ccggttcctc ctgggccaga aagaagcagg cacatcccct
tctctgtgac 300 acaccctgtc cacgcccctg gttcttagtt ccagccccac
tcataggaca ctcatagctc 360 aggagggctc cgccttcaat cccacccgct
aaagtacttg gagcggtctc tccctccctc 420 atcagcccac caaaccaaac
ctagcctcca agagtgggaa gaaattaaag caagataggc 480 tattaagtgc
agagggagag aaaatgcctc caacatgtga ggaagtaatg agagaaatca 540
tagaatttct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
600 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
gggataacgc 660 aggaaagaac atgtgagcaa aaggccagca aaaggccagg
aaccgtaaaa aggccgcgtt 720 gctggcgttt ttccataggc tccgcccccc
tgacgagcat cacaaaaatc gacgctcaag 780 tcagaggtgg cgaaacccga
caggactata aagataccag gcgtttcccc ctggaagctc 840 cctcgtgcgc
tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc 900
ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
960 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
gctgcgcctt 1020 atccggtaac tatcgtcttg agtccaaccc ggtaagacac
gacttatcgc cactggcagc 1080 agccactggt aacaggatta gcagagcgag
gtatgtaggc ggtgctacag agttcttgaa 1140 gtggtggcct aactacggct
acactagaag aacagtattt ggtatctgcg ctctgctgaa 1200 gccagttacc
ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg 1260
tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
1320 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
cacgttaagg 1380 gattttggtc atgagattat caaaaaggat cttcacctag
atccttttaa attaaaaatg 1440 aagttttaaa tcaatctaaa gtatatatga
gtaaacttgg tctgacagtt accaatgctt 1500 aatcagtgag gcacctatct
cagcgatctg tctatttcgt tcatccatag ttgcctgact 1560 cggggggggg
gggcgctgag gtctgcctcg tgaagaaggt gttgctgact cataccaggc 1620
ctgaatcgcc ccatcatcca gccagaaagt gagggagcca cggttgatga gagctttgtt
1680 gtaggtggac cagttggtga ttttgaactt ttgctttgcc acggaacggt
ctgcgttgtc 1740 gggaagatgc gtgatctgat ccttcaactc agcaaaagtt
cgatttattc aacaaagccg 1800 ccgtcccgtc aagtcagcgt aatgctctgc
cagtgttaca accaattaac caattctgat 1860 tagaaaaact catcgagcat
caaatgaaac tgcaatttat tcatatcagg attatcaata 1920 ccatattttt
gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat 1980
aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct
2040 attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg
agtgacgact 2100 gaatccggtg agaatggcaa aagcttatgc atttctttcc
agacttgttc aacaggccag 2160 ccattacgct cgtcatcaaa atcactcgca
tcaaccaaac cgttattcat tcgtgattgc 2220 gcctgagcga gacgaaatac
gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa 2280 tgcaaccggc
gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat 2340
tcttctaata cctggaatgc tgttttcccg gggatcgcag tggtgagtaa ccatgcatca
2400 tcaggagtac ggataaaatg cttgatggtc ggaagaggca taaattccgt
cagccagttt 2460 agtctgacca tctcatctgt aacatcattg gcaacgctac
ctttgccatg tttcagaaac 2520 aactctggcg catcgggctt cccatacaat
cgatagattg tcgcacctga ttgcccgaca 2580 ttatcgcgag cccatttata
cccatataaa tcagcatcca tgttggaatt taatcgcggc 2640 ctcgagcaag
acgtttcccg ttgaatatgg ctcataacac cccttgtatt actgtttatg 2700
taagcagaca gttttattgt tcatgatgat atatttttat cttgtgcaat gtaacatcag
2760 agattttgag acacaacgtg gctttccccc cccccccatt attgaagcat
ttatcagggt 2820 tattgtctca tgagcggata catatttgaa tgtatttaga
aaaataaaca aataggggtt 2880 ccgcgcacat ttccccgaaa agtgccacct
gacgtctaag aaaccattat tatcatgaca 2940 ttaacctata aaaataggcg
tatcacgagg ccctttcgtc ctcgcgcgtt tcggtgatga 3000 cggtgaaaac
ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga 3060
tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg
3120 gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgc
ggtgtgaaat 3180 accgcacaga tgcgtaagga gaaaataccg catcagattg
gctattggcc attgcatacg 3240 ttgtatccat atcataatat gtacatttat
attggctcat gtccaacatt accgccatgt 3300 tgacattgat tattgactag
ttattaatag taatcaatta cggggtcatt agttcatagc 3360 ccatatatgg
agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc 3420
aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg
3480 actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt
ggcagtacat 3540 caagtgtatc atatgccaag tacgccccct attgacgtca
atgacggtaa atggcccgcc 3600 tggcattatg cccagtacat gaccttatgg
gactttccta cttggcagta catctacgta 3660 ttagtcatcg ctattaccat
ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag 3720 cggtttgact
cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt 3780
tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa
3840 atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctcgttt
agtgaaccgt 3900 cagatcgcct ggagacgcca tccacgctgt tttgacctcc
atagaagaca ccgggaccga 3960 tccagcctcc gcggccggga acggtgcatt
ggaacgcgga ttccccgtgc caagagtgac 4020 gtaagtaccg cctatagact
ctataggcac acccctttgg ctcttatgca tgctatactg 4080 tttttggctt
ggggcctata cacccccgct tccttatgct ataggtgatg gtatagctta 4140
gcctataggt gtgggttatt gaccattatt gaccactccc ctattggtga cgatactttc
4200 cattactaat ccataacatg gctctttgcc acaactatct ctattggcta
tatgccaata 4260 ctctgtcctt cagagactga cacggactct gtatttttac
aggatggggt cccatttatt 4320 atttacaaat tcacatatac aacaacgccg
tcccccgtgc ccgcagtttt tattaaacat 4380 agcgtgggat ctccacgcga
atctcgggta cgtgttccgg acatgggctc ttctccggta 4440 gcggcggagc
ttccacatcc gagccctggt cccatgcctc cagcggctca tggtcgctcg 4500
gcagctcctt gctcctaaca gtggaggcca gacttaggca cagcacaatg cccaccacca
4560 ccagtgtgcc gcacaaggcc gtggcggtag ggtatgtgtc tgaaaatgag
cgtggagatt 4620 gggctcgcac ggctgacgca gatggaagac ttaaggcagc
ggcagaagaa gatgcaggca 4680 gctgagttgt tgtattctga taagagtcag
aggtaactcc cgttgcggtg ctgttaacgg 4740 tggagggcag tgtagtctga
gcagtactcg ttgctgccgc gcgcgccacc agacataata 4800 gctgacagac
taacagactg ttcctttcca tgggtctttt ctgcagtcac cgtcgtcgac 4860
acgtgtgatc agatatcgcg gccgctctag accaggcgcc tggatccgcc accatgccac
4920 tctgggtgtt cttctttgtg atcctcaccc tcagcaacag ctcccactgc
tccccacctc 4980 cccctttgac cctcaggatg cggcggtatg cagatgccat
cttcaccaac agctaccgga 5040 aggtgctggg ccagctgtcc gcccgcaagc
tcctccagga catcatgagc aggtagagat 5100 ccagatct 5108 53 3954 DNA
Artificial Sequence Description of Artificial Sequence
pGHRH1-29YWTSK685 construct 53 ggtaccatcg ctggggagct gggggagggg
tcgccttcct gccctaccca ggactccggg 60 tgcgaccgct cctctatctc
tccagcccac caccactcca ccacttggac acgtctccct 120 cctccctgga
gtcgctctag agggtttggg ggtctgagta aagaacccga agtagggata 180
cagtgtggcg gcaccttcca gaggccccgg gcgcagggta gaccggggcg gggcggcccg
240 cggacaggtg cagccccagg cgcaggcgca ctcgcgcctc ccggcgcagg
cggtgaacct 300 cgccccaccc cagcccctcc ggggggcagc tgggccgggt
cgggaggggc ccaccagccc 360 gggagacact ccatatacgg ccaggcccgc
tttacctggg ctccggccag gccgctcctt 420 ctttggtcag cacaggggac
ccgggcgggg gcccaggccg ctaacccgcc gggggagggg 480 gctccagtgc
ccaacaccca aatatggctc gagaagggga gcgacattcc agtgaggcgg 540
ctcgggggga gaacccgcgg gctatataaa acctgagcgt ggggaccagc ggccaccgca
600 gcggacagcg ccgagagaag cctcgcttcc ctcccgcggc gaccagggcc
ccagccggag 660 agcagcaggt gtagccacca agcttgccac catgccactc
tgggtgttct tctttgtgat 720 cctcaccctc agcaacagct cccactgctc
cccacctccc cctttgaccc tcaggatgcg 780 gcggtattat gcagatgcca
tcttcaccaa cagctaccgg aaggtgctgg gccagctgtc 840 cgcccgcaag
ctcctccagg acatcatgag caggtagtct agagtcgggg cggccggccg 900
cttcgagcag acatgataag atacattgat gagtttggac aaaccacaac tagaatgcag
960 tgaaaaaaat gctttatttg tgaaatttgt gatgctattg ctttatttgt
aaccattata 1020 agctgcaata aacaagttaa caacaacaat tgcattcatt
ttatgtttca ggttcagggg 1080 gaggtgtggg aggtttttta aagcaagtaa
aacctctaca aatgtggtaa aatcgataag 1140 gatccgtcga ccgatgccct
tgagagcctt caacccagtc agctccttcc ggtgggcgcg 1200 gggcatgact
atcgtcgccg cacttatgac tgtcttcttt atcatgcaac tcgtaggaca 1260
ggtgccggca gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc
1320 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg gttatccaca
gaatcagggg 1380 ataacgcagg aaagaacatg tgagcaaaag gccagcaaaa
ggccaggaac cgtaaaaagg 1440 ccgcgttgct ggcgtttttc cataggctcc
gcccccctga cgagcatcac aaaaatcgac 1500 gctcaagtca gaggtggcga
aacccgacag gactataaag ataccaggcg tttccccctg 1560 gaagctccct
cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct 1620
ttctcccttc gggaagcgtg gcgctttctc aatgctcacg ctgtaggtat ctcagttcgg
1680 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc ccccgttcag
cccgaccgct 1740 gcgccttatc cggtaactat cgtcttgagt ccaacccggt
aagacacgac ttatcgccac 1800 tggcagcagc cactggtaac aggattagca
gagcgaggta tgtaggcggt gctacagagt 1860 tcttgaagtg gtggcctaac
tacggctaca ctagaaggac agtatttggt atctgcgctc 1920 tgctgaagcc
agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca 1980
ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga aaaaaaggat
2040 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc tcagtggaac
gaaaactcac 2100 gttaagggat tttggtcatg agattatcaa aaaggatctt
cacctagatc cttttaaatt 2160 aaaaatgaag ttttaaatca atctaaagta
tatatgagta aacttggtct gacagttacc 2220 aatgcttaat cagtgaggca
cctatctcag cgatctgtct atttcgttca tccatagttg 2280 cctgactccc
cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg 2340
ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca ataaaccagc
2400 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt atccgcctcc
atccagtcta 2460 ttaattgttg ccgggaagct agagtaagta gttcgccagt
taatagtttg cgcaacgttg 2520 ttgccattgc tacaggcatc gtggtgtcac
gctcgtcgtt tggtatggct tcattcagct 2580 ccggttccca acgatcaagg
cgagttacat gatcccccat gttgtgcaaa aaagcggtta 2640 gctccttcgg
tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg 2700
ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc ttttctgtga
2760 ctggtgagta ctcaaccaag tcattctgag aatagtgtat gcggcgaccg
agttgctctt 2820 gcccggcgtc aatacgggat aataccgcgc cacatagcag
aactttaaaa gtgctcatca 2880 ttggaaaacg ttcttcgggg cgaaaactct
caaggatctt accgctgttg agatccagtt 2940 cgatgtaacc cactcgtgca
cccaactgat cttcagcatc ttttactttc accagcgttt 3000 ctgggtgagc
aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga 3060
aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat cagggttatt
3120 gtctcatgag cggatacata tttgaatgta tttagaaaaa taaacaaata
ggggttccgc 3180 gcacatttcc ccgaaaagtg ccacctgacg cgccctgtag
cggcgcatta agcgcggcgg 3240 gtgtggtggt tacgcgcagc gtgaccgcta
cacttgccag cgccctagcg cccgctcctt 3300 tcgctttctt cccttccttt
ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc 3360 gggggctccc
tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg 3420
attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt cgccctttga
3480 cgttggagtc cacgttcttt aatagtggac tcttgttcca aactggaaca
acactcaacc 3540 ctatctcggt ctattctttt gatttataag ggattttgcc
gatttcggcc tattggttaa 3600 aaaatgagct gatttaacaa aaatttaacg
cgaattttaa caaaatatta acgtttacaa 3660 tttcccattc gccattcagg
ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt 3720 cgctattacg
ccagcccaag ctaccatgat aagtaagtaa tattaaggta cgggaggtac 3780
ttggagcggc cgcaataaaa tatctttatt ttcattacat ctgtgtgttg gttttttgtg
3840 tgaatcgata gtactaacat acgctctcca tcaaaacaaa acgaaacaaa
acaaactagc 3900 aaaataggct gtccccagtg caagtgcagg tgccagaaca
tttctctatc gata 3954 54 5163 DNA Artificial Sequence Description of
Artificial Sequence pGHRH1-29YWTSK2014 construct 54 ggtaccgcta
taggagagaa aagagctgca ctgagcaccc tccttcccct ttaaatgtca 60
acagattagg agtcagtgaa tgacagcaca cctcttgcta ccttagagac caaaatttaa
120 gctactcccc ttaagctata gctagagtgc acctgccagt gtctttagtc
cccactgatg 180 gaacaggacc caaggtattg aagatggaac atagttattc
attcatcctc taatttaaaa 240 agctggatat gctgtacagc agaaattgac
ggaacaatgt aaatcaacta taacagaaga 300 aataaaaacc tggggggaaa
gaagctgact atgaaacccc aggagctttc tacatgggcc 360 tggactcacc
aaactcttta ttttgtaatg gacttctgac atttttagga agggctgtcc 420
tgatgtgggc tatagaagag ggtttcacat gcttcttcaa gaggacccac actgtcccag
480 ttgctgagtc ccaccaccag atgctagtgg cagctatttg gggaacactt
aggcactaca 540 aaaaaatgag tgattccatt ctggctcaca ccatatccct
gatgtacccc ttaaagcatg 600 tcactgagtt catcacagaa aattgtttcc
cctgtgcctt ccacaacaag gttagagctg 660 tccttggggc caggggaagg
gggcagggag tgagaagcac caactggata acctcctctg 720 acccccactc
caccttacca taagtagatc caaatccttc tagaaaatta ggaaggcata 780
tccccatata tcagcgatat aaatagaact gcttcagcgc tctggtagac ggtgactctc
840 caaggtggac tgggaggcag cctggccttg gctgggcatc gtcctctaaa
tagaaagatg 900 aacttgttca gcctttccag aaggaaaact gctgcccagc
ctacagtgca acgtccttgt 960 cttccatctg gaggaagcac gggtgacata
tcatctagta agggcacctc tctgtttcca 1020 cctccaggtc gaggggtgtg
acccttactt ctcagcctca agggagggac actcaacccc 1080 ccaaaaagac
atgagggcgc tcagctcggc ccaccgcacc ccggaccgga gccgtcaccc 1140
cccgaaattc actcccttca caagccccca agcgcgttct ctggtgcgga ctgctccggg
1200 gccctggctt tgtgcccagc gttgtcagag ccaccgccct gagcctgtcc
ccgggagccc 1260 cgcgcctcct cccaccgctc cgctctcgcg ccccgcggcc
agttgtctgc cccgagacag 1320 ctgcgcgccc tcccgctgcc ggtggccctc
tccggtgggg gtggggaccg acagggtcag 1380 ccctccggat ccggggcgct
ccgggtagcg gggagaagtg atcgctgggg agctggggga 1440 ggggtcgcct
tcctgcccta cccaggactc cgggtgcgac cgctcctcta tctctccagc 1500
ccgggcgcag ggtagaccgg ggcggggcgg cccgcggaca ggtgcagccc caggcgcagg
1560 cgcactcgcg cctcccggcg caggcggtga acctcgcccc accccagccc
ctccgggggg 1620 cagctgggcc gggtcgggag gggcccacca gcccgggaga
cactccatat acggccaggc 1680 ccgctttacc tgggctccgg ccaggccgct
ccttctttgg tcagcacagg ggacccgggc 1740 gggggcccag gccgctaacc
cgccggggga gggggctcca gtgcccaaca cccaaatatg 1800 gctcgagaag
gggagcgaca ttccagtgag gcggctcggg gggagaaccc gcgggctata 1860
taaaacctga gcgtggggac cagcggccaa gcttgccacc atgccactct gggtgttctt
1920 ctttgtgatc ctcaccctca gcaacagctc ccactgctcc ccacctcccc
ctttgaccct 1980 caggatgcgg cggtattatg cagatgccat cttcaccaac
agctaccgga aggtgctggg 2040 ccagctgtcc gcccgcaagc tcctccagga
catcatgagc aggtagtcta gagtcggggc 2100 ggccggccgc ttcgagcaga
catgataaga tacattgatg agtttggaca aaccacaact 2160 agaatgcagt
gaaaaaaatg ctttatttgt gaaatttgtg atgctattgc tttatttgta 2220
accattataa gctgcaataa acaagttaac aacaacaatt gcattcattt tatgtttcag
2280 gttcaggggg aggtgtggga ggttttttaa agcaagtaaa acctctacaa
atgtggtaaa 2340 atcgataagg atccgtcgac cgatgccctt gagagccttc
aacccagtca gctccttccg 2400 gtgggcgcgg ggcatgacta tcgtcgccgc
acttatgact gtcttcttta tcatgcaact 2460 cgtaggacag gtgccggcag
cgctcttccg cttcctcgct cactgactcg ctgcgctcgg 2520 tcgttcggct
gcggcgagcg gtatcagctc actcaaaggc ggtaatacgg ttatccacag 2580
aatcagggga taacgcagga aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc
2640 gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg cccccctgac
gagcatcaca 2700 aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg
actataaaga taccaggcgt 2760 ttccccctgg aagctccctc gtgcgctctc
ctgttccgac cctgccgctt accggatacc 2820 tgtccgcctt tctcccttcg
ggaagcgtgg cgctttctca atgctcacgc tgtaggtatc 2880 tcagttcggt
gtaggtcgtt cgctccaagc tgggctgtgt gcacgaaccc cccgttcagc 2940
ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc caacccggta agacacgact
3000
tatcgccact ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg
3060 ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca
gtatttggta 3120 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt
tggtagctct tgatccggca 3180 aacaaaccac cgctggtagc ggtggttttt
ttgtttgcaa gcagcagatt acgcgcagaa 3240 aaaaaggatc tcaagaagat
cctttgatct tttctacggg gtctgacgct cagtggaacg 3300 aaaactcacg
ttaagggatt ttggtcatga gattatcaaa aaggatcttc acctagatcc 3360
ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg
3420 acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta
tttcgttcat 3480 ccatagttgc ctgactcccc gtcgtgtaga taactacgat
acgggagggc ttaccatctg 3540 gccccagtgc tgcaatgata ccgcgagacc
cacgctcacc ggctccagat ttatcagcaa 3600 taaaccagcc agccggaagg
gccgagcgca gaagtggtcc tgcaacttta tccgcctcca 3660 tccagtctat
taattgttgc cgggaagcta gagtaagtag ttcgccagtt aatagtttgc 3720
gcaacgttgt tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt
3780 cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg
ttgtgcaaaa 3840 aagcggttag ctccttcggt cctccgatcg ttgtcagaag
taagttggcc gcagtgttat 3900 cactcatggt tatggcagca ctgcataatt
ctcttactgt catgccatcc gtaagatgct 3960 tttctgtgac tggtgagtac
tcaaccaagt cattctgaga atagtgtatg cggcgaccga 4020 gttgctcttg
cccggcgtca atacgggata ataccgcgcc acatagcaga actttaaaag 4080
tgctcatcat tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga
4140 gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct
tttactttca 4200 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc
cgcaaaaaag ggaataaggg 4260 cgacacggaa atgttgaata ctcatactct
tcctttttca atattattga agcatttatc 4320 agggttattg tctcatgagc
ggatacatat ttgaatgtat ttagaaaaat aaacaaatag 4380 gggttccgcg
cacatttccc cgaaaagtgc cacctgacgc gccctgtagc ggcgcattaa 4440
gcgcggcggg tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc
4500 ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt
ccccgtcaag 4560 ctctaaatcg ggggctccct ttagggttcc gatttagtgc
tttacggcac ctcgacccca 4620 aaaaacttga ttagggtgat ggttcacgta
gtgggccatc gccctgatag acggtttttc 4680 gccctttgac gttggagtcc
acgttcttta atagtggact cttgttccaa actggaacaa 4740 cactcaaccc
tatctcggtc tattcttttg atttataagg gattttgccg atttcggcct 4800
attggttaaa aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa
4860 cgtttacaat ttcccattcg ccattcaggc tgcgcaactg ttgggaaggg
cgatcggtgc 4920 gggcctcttc gctattacgc cagcccaagc taccatgata
agtaagtaat attaaggtac 4980 gggaggtact tggagcggcc gcaataaaat
atctttattt tcattacatc tgtgtgttgg 5040 ttttttgtgt gaatcgatag
tactaacata cgctctccat caaaacaaaa cgaaacaaaa 5100 caaactagca
aaataggctg tccccagtgc aagtgcaggt gccagaacat ttctctatcg 5160 ata
5163 55 5111 DNA Artificial Sequence Description of Artificial
Sequence pGHRH1-29Yala1522CMV construct 55 gctgtgcctt ctagttgcca
gccatctgtt gtttgcccct cccccgtgcc ttccttgacc 60 ctggaaggtg
ccactcccac tgtcctttcc taataaaatg aggaaattgc atcgcattgt 120
ctgagtaggt gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat
180 tgggaagaca atagcaggca tgctggggat gcggtgggct ctatgggtac
ccaggtgctg 240 aagaattgac ccggttcctc ctgggccaga aagaagcagg
cacatcccct tctctgtgac 300 acaccctgtc cacgcccctg gttcttagtt
ccagccccac tcataggaca ctcatagctc 360 aggagggctc cgccttcaat
cccacccgct aaagtacttg gagcggtctc tccctccctc 420 atcagcccac
caaaccaaac ctagcctcca agagtgggaa gaaattaaag caagataggc 480
tattaagtgc agagggagag aaaatgcctc caacatgtga ggaagtaatg agagaaatca
540 tagaatttct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc
ggctgcggcg 600 agcggtatca gctcactcaa aggcggtaat acggttatcc
acagaatcag gggataacgc 660 aggaaagaac atgtgagcaa aaggccagca
aaaggccagg aaccgtaaaa aggccgcgtt 720 gctggcgttt ttccataggc
tccgcccccc tgacgagcat cacaaaaatc gacgctcaag 780 tcagaggtgg
cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc 840
cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc
900 ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt
cggtgtaggt 960 cgttcgctcc aagctgggct gtgtgcacga accccccgtt
cagcccgacc gctgcgcctt 1020 atccggtaac tatcgtcttg agtccaaccc
ggtaagacac gacttatcgc cactggcagc 1080 agccactggt aacaggatta
gcagagcgag gtatgtaggc ggtgctacag agttcttgaa 1140 gtggtggcct
aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa 1200
gccagttacc ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg
1260 tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag
gatctcaaga 1320 agatcctttg atcttttcta cggggtctga cgctcagtgg
aacgaaaact cacgttaagg 1380 gattttggtc atgagattat caaaaaggat
cttcacctag atccttttaa attaaaaatg 1440 aagttttaaa tcaatctaaa
gtatatatga gtaaacttgg tctgacagtt accaatgctt 1500 aatcagtgag
gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact 1560
cggggggggg gggcgctgag gtctgcctcg tgaagaaggt gttgctgact cataccaggc
1620 ctgaatcgcc ccatcatcca gccagaaagt gagggagcca cggttgatga
gagctttgtt 1680 gtaggtggac cagttggtga ttttgaactt ttgctttgcc
acggaacggt ctgcgttgtc 1740 gggaagatgc gtgatctgat ccttcaactc
agcaaaagtt cgatttattc aacaaagccg 1800 ccgtcccgtc aagtcagcgt
aatgctctgc cagtgttaca accaattaac caattctgat 1860 tagaaaaact
catcgagcat caaatgaaac tgcaatttat tcatatcagg attatcaata 1920
ccatattttt gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat
1980 aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc
aatacaacct 2040 attaatttcc cctcgtcaaa aataaggtta tcaagtgaga
aatcaccatg agtgacgact 2100 gaatccggtg agaatggcaa aagcttatgc
atttctttcc agacttgttc aacaggccag 2160 ccattacgct cgtcatcaaa
atcactcgca tcaaccaaac cgttattcat tcgtgattgc 2220 gcctgagcga
gacgaaatac gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa 2280
tgcaaccggc gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat
2340 tcttctaata cctggaatgc tgttttcccg gggatcgcag tggtgagtaa
ccatgcatca 2400 tcaggagtac ggataaaatg cttgatggtc ggaagaggca
taaattccgt cagccagttt 2460 agtctgacca tctcatctgt aacatcattg
gcaacgctac ctttgccatg tttcagaaac 2520 aactctggcg catcgggctt
cccatacaat cgatagattg tcgcacctga ttgcccgaca 2580 ttatcgcgag
cccatttata cccatataaa tcagcatcca tgttggaatt taatcgcggc 2640
ctcgagcaag acgtttcccg ttgaatatgg ctcataacac cccttgtatt actgtttatg
2700 taagcagaca gttttattgt tcatgatgat atatttttat cttgtgcaat
gtaacatcag 2760 agattttgag acacaacgtg gctttccccc cccccccatt
attgaagcat ttatcagggt 2820 tattgtctca tgagcggata catatttgaa
tgtatttaga aaaataaaca aataggggtt 2880 ccgcgcacat ttccccgaaa
agtgccacct gacgtctaag aaaccattat tatcatgaca 2940 ttaacctata
aaaataggcg tatcacgagg ccctttcgtc ctcgcgcgtt tcggtgatga 3000
cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga
3060 tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt
gtcggggctg 3120 gcttaactat gcggcatcag agcagattgt actgagagtg
caccatatgc ggtgtgaaat 3180 accgcacaga tgcgtaagga gaaaataccg
catcagattg gctattggcc attgcatacg 3240 ttgtatccat atcataatat
gtacatttat attggctcat gtccaacatt accgccatgt 3300 tgacattgat
tattgactag ttattaatag taatcaatta cggggtcatt agttcatagc 3360
ccatatatgg agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc
3420 aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac
gccaataggg 3480 actttccatt gacgtcaatg ggtggagtat ttacggtaaa
ctgcccactt ggcagtacat 3540 caagtgtatc atatgccaag tacgccccct
attgacgtca atgacggtaa atggcccgcc 3600 tggcattatg cccagtacat
gaccttatgg gactttccta cttggcagta catctacgta 3660 ttagtcatcg
ctattaccat ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag 3720
cggtttgact cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt
3780 tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc
attgacgcaa 3840 atgggcggta ggcgtgtacg gtgggaggtc tatataagca
gagctcgttt agtgaaccgt 3900 cagatcgcct ggagacgcca tccacgctgt
tttgacctcc atagaagaca ccgggaccga 3960 tccagcctcc gcggccggga
acggtgcatt ggaacgcgga ttccccgtgc caagagtgac 4020 gtaagtaccg
cctatagact ctataggcac acccctttgg ctcttatgca tgctatactg 4080
tttttggctt ggggcctata cacccccgct tccttatgct ataggtgatg gtatagctta
4140 gcctataggt gtgggttatt gaccattatt gaccactccc ctattggtga
cgatactttc 4200 cattactaat ccataacatg gctctttgcc acaactatct
ctattggcta tatgccaata 4260 ctctgtcctt cagagactga cacggactct
gtatttttac aggatggggt cccatttatt 4320 atttacaaat tcacatatac
aacaacgccg tcccccgtgc ccgcagtttt tattaaacat 4380 agcgtgggat
ctccacgcga atctcgggta cgtgttccgg acatgggctc ttctccggta 4440
gcggcggagc ttccacatcc gagccctggt cccatgcctc cagcggctca tggtcgctcg
4500 gcagctcctt gctcctaaca gtggaggcca gacttaggca cagcacaatg
cccaccacca 4560 ccagtgtgcc gcacaaggcc gtggcggtag ggtatgtgtc
tgaaaatgag cgtggagatt 4620 gggctcgcac ggctgacgca gatggaagac
ttaaggcagc ggcagaagaa gatgcaggca 4680 gctgagttgt tgtattctga
taagagtcag aggtaactcc cgttgcggtg ctgttaacgg 4740 tggagggcag
tgtagtctga gcagtactcg ttgctgccgc gcgcgccacc agacataata 4800
gctgacagac taacagactg ttcctttcca tgggtctttt ctgcagtcac cgtcgtcgac
4860 acgtgtgatc agatatcgcg gccgctctag accaggcgcc tggatccgcc
accatgccac 4920 tctgggtgtt cttctttgtg atcctcaccc tcagcaacag
ctcccactgc tccccacctc 4980 cccctttgac cctcaggatg cggcggtatt
atgcagatgc catcttcacc aacagctacc 5040 ggaaggtgct ggcccagctg
tccgcccgca aggccctcca ggacatcatg agcaggtaga 5100 gatccagatc t 5111
56 3327 DNA Artificial Sequence Description of Artificial Sequence
pGHRH1-29Yala1522SK construct 56 ggtaccgagc tcttacgcgt gctagcccgg
gctcgagatc tgcgatctaa gtaagcttgc 60 caccatgcca ctctgggtgt
tcttctttgt gatcctcacc ctcagcaaca gctcccactg 120 ctccccacct
ccccctttga ccctcaggat gcggcggtat tatgcagatg ccatcttcac 180
caacagctac cggaaggtgc tggcccagct gtccgcccgc aaggccctcc aggacatcat
240 gagcaggtag tctagagtcg gggcggccgg ccgcttcgag cagacatgat
aagatacatt 300 gatgagtttg gacaaaccac aactagaatg cagtgaaaaa
aatgctttat ttgtgaaatt 360 tgtgatgcta ttgctttatt tgtaaccatt
ataagctgca ataaacaagt taacaacaac 420 aattgcattc attttatgtt
tcaggttcag ggggaggtgt gggaggtttt ttaaagcaag 480 taaaacctct
acaaatgtgg taaaatcgat aaggatccgt cgaccgatgc ccttgagagc 540
cttcaaccca gtcagctcct tccggtgggc gcggggcatg actatcgtcg ccgcacttat
600 gactgtcttc tttatcatgc aactcgtagg acaggtgccg gcagcgctct
tccgcttcct 660 cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
agcggtatca gctcactcaa 720 aggcggtaat acggttatcc acagaatcag
gggataacgc aggaaagaac atgtgagcaa 780 aaggccagca aaaggccagg
aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc 840 tccgcccccc
tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga 900
caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc
960 cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc
gtggcgcttt 1020 ctcaatgctc acgctgtagg tatctcagtt cggtgtaggt
cgttcgctcc aagctgggct 1080 gtgtgcacga accccccgtt cagcccgacc
gctgcgcctt atccggtaac tatcgtcttg 1140 agtccaaccc ggtaagacac
gacttatcgc cactggcagc agccactggt aacaggatta 1200 gcagagcgag
gtatgtaggc ggtgctacag agttcttgaa gtggtggcct aactacggct 1260
acactagaag gacagtattt ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa
1320 gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt
ttttttgttt 1380 gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
agatcctttg atcttttcta 1440 cggggtctga cgctcagtgg aacgaaaact
cacgttaagg gattttggtc atgagattat 1500 caaaaaggat cttcacctag
atccttttaa attaaaaatg aagttttaaa tcaatctaaa 1560 gtatatatga
gtaaacttgg tctgacagtt accaatgctt aatcagtgag gcacctatct 1620
cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg tagataacta
1680 cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga
gacccacgct 1740 caccggctcc agatttatca gcaataaacc agccagccgg
aagggccgag cgcagaagtg 1800 gtcctgcaac tttatccgcc tccatccagt
ctattaattg ttgccgggaa gctagagtaa 1860 gtagttcgcc agttaatagt
ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt 1920 cacgctcgtc
gtttggtatg gcttcattca gctccggttc ccaacgatca aggcgagtta 1980
catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca
2040 gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat
aattctctta 2100 ctgtcatgcc atccgtaaga tgcttttctg tgactggtga
gtactcaacc aagtcattct 2160 gagaatagtg tatgcggcga ccgagttgct
cttgcccggc gtcaatacgg gataataccg 2220 cgccacatag cagaacttta
aaagtgctca tcattggaaa acgttcttcg gggcgaaaac 2280 tctcaaggat
cttaccgctg ttgagatcca gttcgatgta acccactcgt gcacccaact 2340
gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa
2400 atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata
ctcttccttt 2460 ttcaatatta ttgaagcatt tatcagggtt attgtctcat
gagcggatac atatttgaat 2520 gtatttagaa aaataaacaa ataggggttc
cgcgcacatt tccccgaaaa gtgccacctg 2580 acgcgccctg tagcggcgca
ttaagcgcgg cgggtgtggt ggttacgcgc agcgtgaccg 2640 ctacacttgc
cagcgcccta gcgcccgctc ctttcgcttt cttcccttcc tttctcgcca 2700
cgttcgccgg ctttccccgt caagctctaa atcgggggct ccctttaggg ttccgattta
2760 gtgctttacg gcacctcgac cccaaaaaac ttgattaggg tgatggttca
cgtagtgggc 2820 catcgccctg atagacggtt tttcgccctt tgacgttgga
gtccacgttc tttaatagtg 2880 gactcttgtt ccaaactgga acaacactca
accctatctc ggtctattct tttgatttat 2940 aagggatttt gccgatttcg
gcctattggt taaaaaatga gctgatttaa caaaaattta 3000 acgcgaattt
taacaaaata ttaacgttta caatttccca ttcgccattc aggctgcgca 3060
actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagccc aagctaccat
3120 gataagtaag taatattaag gtacgggagg tacttggagc ggccgcaata
aaatatcttt 3180 attttcatta catctgtgtg ttggtttttt gtgtgaatcg
atagtactaa catacgctct 3240 ccatcaaaac aaaacgaaac aaaacaaact
agcaaaatag gctgtcccca gtgcaagtgc 3300 aggtgccaga acatttctct atcgata
3327 57 3954 DNA Artificial Sequence Description of Artificial
Sequence pGHRH1-29Yala1522SK construct 57 ggtaccatcg ctggggagct
gggggagggg tcgccttcct gccctaccca ggactccggg 60 tgcgaccgct
cctctatctc tccagcccac caccactcca ccacttggac acgtctccct 120
cctccctgga gtcgctctag agggtttggg ggtctgagta aagaacccga agtagggata
180 cagtgtggcg gcaccttcca gaggccccgg gcgcagggta gaccggggcg
gggcggcccg 240 cggacaggtg cagccccagg cgcaggcgca ctcgcgcctc
ccggcgcagg cggtgaacct 300 cgccccaccc cagcccctcc ggggggcagc
tgggccgggt cgggaggggc ccaccagccc 360 gggagacact ccatatacgg
ccaggcccgc tttacctggg ctccggccag gccgctcctt 420 ctttggtcag
cacaggggac ccgggcgggg gcccaggccg ctaacccgcc gggggagggg 480
gctccagtgc ccaacaccca aatatggctc gagaagggga gcgacattcc agtgaggcgg
540 ctcgggggga gaacccgcgg gctatataaa acctgagcgt ggggaccagc
ggccaccgca 600 gcggacagcg ccgagagaag cctcgcttcc ctcccgcggc
gaccagggcc ccagccggag 660 agcagcaggt gtagccacca agcttgccac
catgccactc tgggtgttct tctttgtgat 720 cctcaccctc agcaacagct
cccactgctc cccacctccc cctttgaccc tcaggatgcg 780 gcggtattat
gcagatgcca tcttcaccaa cagctaccgg aaggtgctgg cccagctgtc 840
cgcccgcaag gccctccagg acatcatgag caggtagtct agagtcgggg cggccggccg
900 cttcgagcag acatgataag atacattgat gagtttggac aaaccacaac
tagaatgcag 960 tgaaaaaaat gctttatttg tgaaatttgt gatgctattg
ctttatttgt aaccattata 1020 agctgcaata aacaagttaa caacaacaat
tgcattcatt ttatgtttca ggttcagggg 1080 gaggtgtggg aggtttttta
aagcaagtaa aacctctaca aatgtggtaa aatcgataag 1140 gatccgtcga
ccgatgccct tgagagcctt caacccagtc agctccttcc ggtgggcgcg 1200
gggcatgact atcgtcgccg cacttatgac tgtcttcttt atcatgcaac tcgtaggaca
1260 ggtgccggca gcgctcttcc gcttcctcgc tcactgactc gctgcgctcg
gtcgttcggc 1320 tgcggcgagc ggtatcagct cactcaaagg cggtaatacg
gttatccaca gaatcagggg 1380 ataacgcagg aaagaacatg tgagcaaaag
gccagcaaaa ggccaggaac cgtaaaaagg 1440 ccgcgttgct ggcgtttttc
cataggctcc gcccccctga cgagcatcac aaaaatcgac 1500 gctcaagtca
gaggtggcga aacccgacag gactataaag ataccaggcg tttccccctg 1560
gaagctccct cgtgcgctct cctgttccga ccctgccgct taccggatac ctgtccgcct
1620 ttctcccttc gggaagcgtg gcgctttctc aatgctcacg ctgtaggtat
ctcagttcgg 1680 tgtaggtcgt tcgctccaag ctgggctgtg tgcacgaacc
ccccgttcag cccgaccgct 1740 gcgccttatc cggtaactat cgtcttgagt
ccaacccggt aagacacgac ttatcgccac 1800 tggcagcagc cactggtaac
aggattagca gagcgaggta tgtaggcggt gctacagagt 1860 tcttgaagtg
gtggcctaac tacggctaca ctagaaggac agtatttggt atctgcgctc 1920
tgctgaagcc agttaccttc ggaaaaagag ttggtagctc ttgatccggc aaacaaacca
1980 ccgctggtag cggtggtttt tttgtttgca agcagcagat tacgcgcaga
aaaaaaggat 2040 ctcaagaaga tcctttgatc ttttctacgg ggtctgacgc
tcagtggaac gaaaactcac 2100 gttaagggat tttggtcatg agattatcaa
aaaggatctt cacctagatc cttttaaatt 2160 aaaaatgaag ttttaaatca
atctaaagta tatatgagta aacttggtct gacagttacc 2220 aatgcttaat
cagtgaggca cctatctcag cgatctgtct atttcgttca tccatagttg 2280
cctgactccc cgtcgtgtag ataactacga tacgggaggg cttaccatct ggccccagtg
2340 ctgcaatgat accgcgagac ccacgctcac cggctccaga tttatcagca
ataaaccagc 2400 cagccggaag ggccgagcgc agaagtggtc ctgcaacttt
atccgcctcc atccagtcta 2460 ttaattgttg ccgggaagct agagtaagta
gttcgccagt taatagtttg cgcaacgttg 2520 ttgccattgc tacaggcatc
gtggtgtcac gctcgtcgtt tggtatggct tcattcagct 2580 ccggttccca
acgatcaagg cgagttacat gatcccccat gttgtgcaaa aaagcggtta 2640
gctccttcgg tcctccgatc gttgtcagaa gtaagttggc cgcagtgtta tcactcatgg
2700 ttatggcagc actgcataat tctcttactg tcatgccatc cgtaagatgc
ttttctgtga 2760 ctggtgagta ctcaaccaag tcattctgag aatagtgtat
gcggcgaccg agttgctctt 2820 gcccggcgtc aatacgggat aataccgcgc
cacatagcag aactttaaaa gtgctcatca 2880 ttggaaaacg ttcttcgggg
cgaaaactct caaggatctt accgctgttg agatccagtt 2940 cgatgtaacc
cactcgtgca cccaactgat cttcagcatc ttttactttc accagcgttt 3000
ctgggtgagc aaaaacagga aggcaaaatg ccgcaaaaaa gggaataagg gcgacacgga
3060 aatgttgaat actcatactc ttcctttttc aatattattg aagcatttat
cagggttatt 3120 gtctcatgag cggatacata tttgaatgta tttagaaaaa
taaacaaata ggggttccgc 3180 gcacatttcc ccgaaaagtg ccacctgacg
cgccctgtag cggcgcatta agcgcggcgg 3240 gtgtggtggt tacgcgcagc
gtgaccgcta cacttgccag cgccctagcg cccgctcctt 3300 tcgctttctt
cccttccttt ctcgccacgt tcgccggctt tccccgtcaa gctctaaatc 3360
gggggctccc tttagggttc cgatttagtg ctttacggca cctcgacccc aaaaaacttg
3420 attagggtga tggttcacgt agtgggccat cgccctgata gacggttttt
cgccctttga 3480 cgttggagtc cacgttcttt aatagtggac tcttgttcca
aactggaaca acactcaacc 3540 ctatctcggt ctattctttt gatttataag
ggattttgcc gatttcggcc tattggttaa 3600 aaaatgagct gatttaacaa
aaatttaacg cgaattttaa caaaatatta acgtttacaa 3660 tttcccattc
gccattcagg ctgcgcaact gttgggaagg gcgatcggtg cgggcctctt 3720
cgctattacg ccagcccaag ctaccatgat aagtaagtaa tattaaggta cgggaggtac
3780 ttggagcggc cgcaataaaa tatctttatt ttcattacat ctgtgtgttg
gttttttgtg 3840 tgaatcgata gtactaacat acgctctcca tcaaaacaaa
acgaaacaaa acaaactagc 3900 aaaataggct gtccccagtg caagtgcagg
tgccagaaca tttctctatc gata 3954 58 5283 DNA Artificial Sequence
Description of Artificial Sequence pGHRH1-29Yala1522SK2014
construct 58 ggtaccgcta taggagagaa aagagctgca ctgagcaccc tccttcccct
ttaaatgtca 60 acagattagg agtcagtgaa tgacagcaca cctcttgcta
ccttagagac caaaatttaa 120 gctactcccc ttaagctata gctagagtgc
acctgccagt gtctttagtc cccactgatg 180 gaacaggacc caaggtattg
aagatggaac atagttattc attcatcctc taatttaaaa 240 agctggatat
gctgtacagc agaaattgac ggaacaatgt aaatcaacta taacagaaga 300
aataaaaacc tggggggaaa gaagctgact atgaaacccc aggagctttc tacatgggcc
360 tggactcacc aaactcttta ttttgtaatg gacttctgac atttttagga
agggctgtcc 420 tgatgtgggc tatagaagag ggtttcacat gcttcttcaa
gaggacccac actgtcccag 480 ttgctgagtc ccaccaccag atgctagtgg
cagctatttg gggaacactt aggcactaca 540 aaaaaatgag tgattccatt
ctggctcaca ccatatccct gatgtacccc ttaaagcatg 600 tcactgagtt
catcacagaa aattgtttcc cctgtgcctt ccacaacaag gttagagctg 660
tccttggggc caggggaagg gggcagggag tgagaagcac caactggata acctcctctg
720 acccccactc caccttacca taagtagatc caaatccttc tagaaaatta
ggaaggcata 780 tccccatata tcagcgatat aaatagaact gcttcagcgc
tctggtagac ggtgactctc 840 caaggtggac tgggaggcag cctggccttg
gctgggcatc gtcctctaaa tagaaagatg 900 aacttgttca gcctttccag
aaggaaaact gctgcccagc ctacagtgca acgtccttgt 960 cttccatctg
gaggaagcac gggtgacata tcatctagta agggcacctc tctgtttcca 1020
cctccaggtc gaggggtgtg acccttactt ctcagcctca agggagggac actcaacccc
1080 ccaaaaagac atgagggcgc tcagctcggc ccaccgcacc ccggaccgga
gccgtcaccc 1140 cccgaaattc actcccttca caagccccca agcgcgttct
ctggtgcgga ctgctccggg 1200 gccctggctt tgtgcccagc gttgtcagag
ccaccgccct gagcctgtcc ccgggagccc 1260 cgcgcctcct cccaccgctc
cgctctcgcg ccccgcggcc agttgtctgc cccgagacag 1320 ctgcgcgccc
tcccgctgcc ggtggccctc tccggtgggg gtggggaccg acagggtcag 1380
ccctccggat ccggggcgct ccgggtagcg gggagaagtg atcgctgggg agctggggga
1440 ggggtcgcct tcctgcccta cccaggactc cgggtgcgac cgctcctcta
tctctccagc 1500 ccaccaccac tccaccactt ggacacgtct ccctcctccc
tggagtcgct ctagagggtt 1560 tgggggtctg agtaaagaac ccgaagtagg
gatacagtgt ggcggcacct tccagaggcc 1620 ccgggcgcag ggtagaccgg
ggcggggcgg cccgcggaca ggtgcagccc caggcgcagg 1680 cgcactcgcg
cctcccggcg caggcggtga acctcgcccc accccagccc ctccgggggg 1740
cagctgggcc gggtcgggag gggcccacca gcccgggaga cactccatat acggccaggc
1800 ccgctttacc tgggctccgg ccaggccgct ccttctttgg tcagcacagg
ggacccgggc 1860 gggggcccag gccgctaacc cgccggggga gggggctcca
gtgcccaaca cccaaatatg 1920 gctcgagaag gggagcgaca ttccagtgag
gcggctcggg gggagaaccc gcgggctata 1980 taaaacctga gcgtggggac
cagcggccaa gcttgccacc atgccactct gggtgttctt 2040 ctttgtgatc
ctcaccctca gcaacagctc ccactgctcc ccacctcccc ctttgaccct 2100
caggatgcgg cggtattatg cagatgccat cttcaccaac agctaccgga aggtgctggc
2160 ccagctgtcc gcccgcaagg ccctccagga catcatgagc aggtagtcta
gagtcggggc 2220 ggccggccgc ttcgagcaga catgataaga tacattgatg
agtttggaca aaccacaact 2280 agaatgcagt gaaaaaaatg ctttatttgt
gaaatttgtg atgctattgc tttatttgta 2340 accattataa gctgcaataa
acaagttaac aacaacaatt gcattcattt tatgtttcag 2400 gttcaggggg
aggtgtggga ggttttttaa agcaagtaaa acctctacaa atgtggtaaa 2460
atcgataagg atccgtcgac cgatgccctt gagagccttc aacccagtca gctccttccg
2520 gtgggcgcgg ggcatgacta tcgtcgccgc acttatgact gtcttcttta
tcatgcaact 2580 cgtaggacag gtgccggcag cgctcttccg cttcctcgct
cactgactcg ctgcgctcgg 2640 tcgttcggct gcggcgagcg gtatcagctc
actcaaaggc ggtaatacgg ttatccacag 2700 aatcagggga taacgcagga
aagaacatgt gagcaaaagg ccagcaaaag gccaggaacc 2760 gtaaaaaggc
cgcgttgctg gcgtttttcc ataggctccg cccccctgac gagcatcaca 2820
aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg actataaaga taccaggcgt
2880 ttccccctgg aagctccctc gtgcgctctc ctgttccgac cctgccgctt
accggatacc 2940 tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca
atgctcacgc tgtaggtatc 3000 tcagttcggt gtaggtcgtt cgctccaagc
tgggctgtgt gcacgaaccc cccgttcagc 3060 ccgaccgctg cgccttatcc
ggtaactatc gtcttgagtc caacccggta agacacgact 3120 tatcgccact
ggcagcagcc actggtaaca ggattagcag agcgaggtat gtaggcggtg 3180
ctacagagtt cttgaagtgg tggcctaact acggctacac tagaaggaca gtatttggta
3240 tctgcgctct gctgaagcca gttaccttcg gaaaaagagt tggtagctct
tgatccggca 3300 aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa
gcagcagatt acgcgcagaa 3360 aaaaaggatc tcaagaagat cctttgatct
tttctacggg gtctgacgct cagtggaacg 3420 aaaactcacg ttaagggatt
ttggtcatga gattatcaaa aaggatcttc acctagatcc 3480 ttttaaatta
aaaatgaagt tttaaatcaa tctaaagtat atatgagtaa acttggtctg 3540
acagttacca atgcttaatc agtgaggcac ctatctcagc gatctgtcta tttcgttcat
3600 ccatagttgc ctgactcccc gtcgtgtaga taactacgat acgggagggc
ttaccatctg 3660 gccccagtgc tgcaatgata ccgcgagacc cacgctcacc
ggctccagat ttatcagcaa 3720 taaaccagcc agccggaagg gccgagcgca
gaagtggtcc tgcaacttta tccgcctcca 3780 tccagtctat taattgttgc
cgggaagcta gagtaagtag ttcgccagtt aatagtttgc 3840 gcaacgttgt
tgccattgct acaggcatcg tggtgtcacg ctcgtcgttt ggtatggctt 3900
cattcagctc cggttcccaa cgatcaaggc gagttacatg atcccccatg ttgtgcaaaa
3960 aagcggttag ctccttcggt cctccgatcg ttgtcagaag taagttggcc
gcagtgttat 4020 cactcatggt tatggcagca ctgcataatt ctcttactgt
catgccatcc gtaagatgct 4080 tttctgtgac tggtgagtac tcaaccaagt
cattctgaga atagtgtatg cggcgaccga 4140 gttgctcttg cccggcgtca
atacgggata ataccgcgcc acatagcaga actttaaaag 4200 tgctcatcat
tggaaaacgt tcttcggggc gaaaactctc aaggatctta ccgctgttga 4260
gatccagttc gatgtaaccc actcgtgcac ccaactgatc ttcagcatct tttactttca
4320 ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc cgcaaaaaag
ggaataaggg 4380 cgacacggaa atgttgaata ctcatactct tcctttttca
atattattga agcatttatc 4440 agggttattg tctcatgagc ggatacatat
ttgaatgtat ttagaaaaat aaacaaatag 4500 gggttccgcg cacatttccc
cgaaaagtgc cacctgacgc gccctgtagc ggcgcattaa 4560 gcgcggcggg
tgtggtggtt acgcgcagcg tgaccgctac acttgccagc gccctagcgc 4620
ccgctccttt cgctttcttc ccttcctttc tcgccacgtt cgccggcttt ccccgtcaag
4680 ctctaaatcg ggggctccct ttagggttcc gatttagtgc tttacggcac
ctcgacccca 4740 aaaaacttga ttagggtgat ggttcacgta gtgggccatc
gccctgatag acggtttttc 4800 gccctttgac gttggagtcc acgttcttta
atagtggact cttgttccaa actggaacaa 4860 cactcaaccc tatctcggtc
tattcttttg atttataagg gattttgccg atttcggcct 4920 attggttaaa
aaatgagctg atttaacaaa aatttaacgc gaattttaac aaaatattaa 4980
cgtttacaat ttcccattcg ccattcaggc tgcgcaactg ttgggaaggg cgatcggtgc
5040 gggcctcttc gctattacgc cagcccaagc taccatgata agtaagtaat
attaaggtac 5100 gggaggtact tggagcggcc gcaataaaat atctttattt
tcattacatc tgtgtgttgg 5160 ttttttgtgt gaatcgatag tactaacata
cgctctccat caaaacaaaa cgaaacaaaa 5220 caaactagca aaataggctg
tccccagtgc aagtgcaggt gccagaacat ttctctatcg 5280 ata 5283 59 5188
DNA Artificial Sequence Description of Artificial Sequence
pGHRH1-44YWTCMV construct 59 gctgtgcctt ctagttgcca gccatctgtt
gtttgcccct cccccgtgcc ttccttgacc 60 ctggaaggtg ccactcccac
tgtcctttcc taataaaatg aggaaattgc atcgcattgt 120 ctgagtaggt
gtcattctat tctggggggt ggggtggggc aggacagcaa gggggaggat 180
tgggaagaca atagcaggca tgctggggat gcggtgggct ctatgggtac ccaggtgctg
240 aagaattgac ccggttcctc ctgggccaga aagaagcagg cacatcccct
tctctgtgac 300 acaccctgtc cacgcccctg gttcttagtt ccagccccac
tcataggaca ctcatagctc 360 aggagggctc cgccttcaat cccacccgct
aaagtacttg gagcggtctc tccctccctc 420 atcagcccac caaaccaaac
ctagcctcca agagtgggaa gaaattaaag caagataggc 480 tattaagtgc
agagggagag aaaatgcctc caacatgtga ggaagtaatg agagaaatca 540
tagaatttct tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg
600 agcggtatca gctcactcaa aggcggtaat acggttatcc acagaatcag
gggataacgc 660 aggaaagaac atgtgagcaa aaggccagca aaaggccagg
aaccgtaaaa aggccgcgtt 720 gctggcgttt ttccataggc tccgcccccc
tgacgagcat cacaaaaatc gacgctcaag 780 tcagaggtgg cgaaacccga
caggactata aagataccag gcgtttcccc ctggaagctc 840 cctcgtgcgc
tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc 900
ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt
960 cgttcgctcc aagctgggct gtgtgcacga accccccgtt cagcccgacc
gctgcgcctt 1020 atccggtaac tatcgtcttg agtccaaccc ggtaagacac
gacttatcgc cactggcagc 1080 agccactggt aacaggatta gcagagcgag
gtatgtaggc ggtgctacag agttcttgaa 1140 gtggtggcct aactacggct
acactagaag aacagtattt ggtatctgcg ctctgctgaa 1200 gccagttacc
ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg 1260
tagcggtggt ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga
1320 agatcctttg atcttttcta cggggtctga cgctcagtgg aacgaaaact
cacgttaagg 1380 gattttggtc atgagattat caaaaaggat cttcacctag
atccttttaa attaaaaatg 1440 aagttttaaa tcaatctaaa gtatatatga
gtaaacttgg tctgacagtt accaatgctt 1500 aatcagtgag gcacctatct
cagcgatctg tctatttcgt tcatccatag ttgcctgact 1560 cggggggggg
gggcgctgag gtctgcctcg tgaagaaggt gttgctgact cataccaggc 1620
ctgaatcgcc ccatcatcca gccagaaagt gagggagcca cggttgatga gagctttgtt
1680 gtaggtggac cagttggtga ttttgaactt ttgctttgcc acggaacggt
ctgcgttgtc 1740 gggaagatgc gtgatctgat ccttcaactc agcaaaagtt
cgatttattc aacaaagccg 1800 ccgtcccgtc aagtcagcgt aatgctctgc
cagtgttaca accaattaac caattctgat 1860 tagaaaaact catcgagcat
caaatgaaac tgcaatttat tcatatcagg attatcaata 1920 ccatattttt
gaaaaagccg tttctgtaat gaaggagaaa actcaccgag gcagttccat 1980
aggatggcaa gatcctggta tcggtctgcg attccgactc gtccaacatc aatacaacct
2040 attaatttcc cctcgtcaaa aataaggtta tcaagtgaga aatcaccatg
agtgacgact 2100 gaatccggtg agaatggcaa aagcttatgc atttctttcc
agacttgttc aacaggccag 2160 ccattacgct cgtcatcaaa atcactcgca
tcaaccaaac cgttattcat tcgtgattgc 2220 gcctgagcga gacgaaatac
gcgatcgctg ttaaaaggac aattacaaac aggaatcgaa 2280 tgcaaccggc
gcaggaacac tgccagcgca tcaacaatat tttcacctga atcaggatat 2340
tcttctaata cctggaatgc tgttttcccg gggatcgcag tggtgagtaa ccatgcatca
2400 tcaggagtac ggataaaatg cttgatggtc ggaagaggca taaattccgt
cagccagttt 2460 agtctgacca tctcatctgt aacatcattg gcaacgctac
ctttgccatg tttcagaaac 2520 aactctggcg catcgggctt cccatacaat
cgatagattg tcgcacctga ttgcccgaca 2580 ttatcgcgag cccatttata
cccatataaa tcagcatcca tgttggaatt taatcgcggc 2640 ctcgagcaag
acgtttcccg ttgaatatgg ctcataacac cccttgtatt actgtttatg 2700
taagcagaca gttttattgt tcatgatgat atatttttat cttgtgcaat gtaacatcag
2760 agattttgag acacaacgtg gctttccccc cccccccatt attgaagcat
ttatcagggt 2820 tattgtctca tgagcggata catatttgaa tgtatttaga
aaaataaaca aataggggtt 2880 ccgcgcacat ttccccgaaa agtgccacct
gacgtctaag aaaccattat tatcatgaca 2940 ttaacctata aaaataggcg
tatcacgagg ccctttcgtc ctcgcgcgtt tcggtgatga 3000 cggtgaaaac
ctctgacaca tgcagctccc ggagacggtc acagcttgtc tgtaagcgga 3060
tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt gtcggggctg
3120 gcttaactat gcggcatcag agcagattgt actgagagtg caccatatgc
ggtgtgaaat 3180 accgcacaga tgcgtaagga gaaaataccg catcagattg
gctattggcc attgcatacg 3240 ttgtatccat atcataatat gtacatttat
attggctcat gtccaacatt accgccatgt 3300 tgacattgat tattgactag
ttattaatag taatcaatta cggggtcatt agttcatagc 3360 ccatatatgg
agttccgcgt tacataactt acggtaaatg gcccgcctgg ctgaccgccc 3420
aacgaccccc gcccattgac gtcaataatg acgtatgttc ccatagtaac gccaataggg
3480 actttccatt gacgtcaatg ggtggagtat ttacggtaaa ctgcccactt
ggcagtacat 3540 caagtgtatc atatgccaag tacgccccct attgacgtca
atgacggtaa atggcccgcc 3600 tggcattatg cccagtacat gaccttatgg
gactttccta cttggcagta catctacgta 3660 ttagtcatcg ctattaccat
ggtgatgcgg ttttggcagt acatcaatgg gcgtggatag 3720 cggtttgact
cacggggatt tccaagtctc caccccattg acgtcaatgg gagtttgttt 3780
tggcaccaaa atcaacggga ctttccaaaa tgtcgtaaca actccgcccc attgacgcaa
3840 atgggcggta ggcgtgtacg gtgggaggtc tatataagca gagctcgttt
agtgaaccgt 3900 cagatcgcct ggagacgcca tccacgctgt tttgacctcc
atagaagaca ccgggaccga 3960 tccagcctcc gcggccggga acggtgcatt
ggaacgcgga ttccccgtgc caagagtgac 4020 gtaagtaccg cctatagact
ctataggcac acccctttgg ctcttatgca tgctatactg 4080 tttttggctt
ggggcctata cacccccgct tccttatgct ataggtgatg gtatagctta 4140
gcctataggt gtgggttatt gaccattatt gaccactccc ctattggtga cgatactttc
4200 cattactaat ccataacatg gctctttgcc acaactatct ctattggcta
tatgccaata 4260 ctctgtcctt cagagactga cacggactct gtatttttac
aggatggggt cccatttatt 4320 atttacaaat tcacatatac aacaacgccg
tcccccgtgc ccgcagtttt tattaaacat 4380 agcgtgggat ctccacgcga
atctcgggta cgtgttccgg acatgggctc ttctccggta 4440 gcggcggagc
ttccacatcc gagccctggt cccatgcctc cagcggctca tggtcgctcg 4500
gcagctcctt gctcctaaca gtggaggcca gacttaggca cagcacaatg cccaccacca
4560 ccagtgtgcc gcacaaggcc gtggcggtag ggtatgtgtc tgaaaatgag
cgtggagatt 4620 gggctcgcac ggctgacgca gatggaagac ttaaggcagc
ggcagaagaa gatgcaggca 4680 gctgagttgt tgtattctga taagagtcag
aggtaactcc cgttgcggtg ctgttaacgg 4740 tggagggcag tgtagtctga
gcagtactcg ttgctgccgc gcgcgccacc agacataata 4800 gctgacagac
taacagactg ttcctttcca tgggtctttt ctgcagtcac cgtcgtcgac 4860
acgtgtgatc agatatcgcg gccgctctag accaggcgcc tggatccgcc accatgccac
4920 tctgggtgtt cttctttgtg atcctcaccc tcagcaacag ctcccactgc
tccccacctc 4980 cccctttgac cctcaggatg cggcggtatt atgcagatgc
catcttcacc aacagctacc 5040 ggaaggtgct gggccagctg tccgcccgca
agctgctcca ggacatcatg agcaggcagc 5100 agggagagag aaaccaagag
caaggagcaa gggtgcggct ttgaagatct tagtagtagt 5160 aggcggccgc
tctagaggat ccagatct 5188 60 5254 DNA Artificial Sequence
Description of Artificial Sequence pGHRH1-44WTGHpep construct 60
gctgtgcctt ctagttgcca gccatctgtt gtttgcccct cccccgtgcc ttccttgacc
60 ctggaaggtg ccactcccac tgtcctttcc taataaaatg aggaaattgc
atcgcattgt 120 ctgagtaggt gtcattctat tctggggggt ggggtggggc
aggacagcaa gggggaggat 180 tgggaagaca atagcaggca tgctggggat
gcggtgggct ctatgggtac ccaggtgctg 240 aagaattgac ccggttcctc
ctgggccaga aagaagcagg cacatcccct tctctgtgac 300 acaccctgtc
cacgcccctg gttcttagtt ccagccccac tcataggaca ctcatagctc 360
aggagggctc cgccttcaat cccacccgct aaagtacttg gagcggtctc tccctccctc
420 atcagcccac caaaccaaac ctagcctcca agagtgggaa gaaattaaag
caagataggc 480 tattaagtgc agagggagag aaaatgcctc caacatgtga
ggaagtaatg agagaaatca 540 tagaatttct tccgcttcct cgctcactga
ctcgctgcgc tcggtcgttc ggctgcggcg 600 agcggtatca gctcactcaa
aggcggtaat acggttatcc acagaatcag gggataacgc 660 aggaaagaac
atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt 720
gctggcgttt ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag
780 tcagaggtgg cgaaacccga caggactata aagataccag gcgtttcccc
ctggaagctc 840 cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga
tacctgtccg cctttctccc 900 ttcgggaagc gtggcgcttt ctcatagctc
acgctgtagg tatctcagtt cggtgtaggt 960 cgttcgctcc aagctgggct
gtgtgcacga accccccgtt cagcccgacc gctgcgcctt 1020 atccggtaac
tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc 1080
agccactggt aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa
1140 gtggtggcct aactacggct acactagaag aacagtattt ggtatctgcg
ctctgctgaa 1200 gccagttacc ttcggaaaaa gagttggtag ctcttgatcc
ggcaaacaaa ccaccgctgg 1260 tagcggtggt ttttttgttt gcaagcagca
gattacgcgc agaaaaaaag gatctcaaga 1320 agatcctttg atcttttcta
cggggtctga cgctcagtgg aacgaaaact cacgttaagg 1380 gattttggtc
atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg 1440
aagttttaaa tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt
1500 aatcagtgag gcacctatct cagcgatctg tctatttcgt tcatccatag
ttgcctgact 1560 cggggggggg gggcgctgag gtctgcctcg tgaagaaggt
gttgctgact cataccaggc 1620 ctgaatcgcc ccatcatcca gccagaaagt
gagggagcca cggttgatga gagctttgtt 1680 gtaggtggac cagttggtga
ttttgaactt ttgctttgcc acggaacggt ctgcgttgtc 1740 gggaagatgc
gtgatctgat ccttcaactc agcaaaagtt cgatttattc aacaaagccg 1800
ccgtcccgtc aagtcagcgt aatgctctgc cagtgttaca accaattaac caattctgat
1860 tagaaaaact catcgagcat caaatgaaac tgcaatttat tcatatcagg
attatcaata 1920 ccatattttt gaaaaagccg tttctgtaat gaaggagaaa
actcaccgag gcagttccat 1980 aggatggcaa gatcctggta tcggtctgcg
attccgactc gtccaacatc aatacaacct 2040 attaatttcc cctcgtcaaa
aataaggtta tcaagtgaga aatcaccatg agtgacgact 2100 gaatccggtg
agaatggcaa aagcttatgc atttctttcc agacttgttc aacaggccag 2160
ccattacgct cgtcatcaaa atcactcgca tcaaccaaac cgttattcat tcgtgattgc
2220 gcctgagcga gacgaaatac gcgatcgctg ttaaaaggac aattacaaac
aggaatcgaa 2280 tgcaaccggc gcaggaacac tgccagcgca tcaacaatat
tttcacctga atcaggatat 2340 tcttctaata cctggaatgc tgttttcccg
gggatcgcag tggtgagtaa ccatgcatca 2400 tcaggagtac ggataaaatg
cttgatggtc ggaagaggca taaattccgt cagccagttt 2460 agtctgacca
tctcatctgt aacatcattg gcaacgctac ctttgccatg tttcagaaac 2520
aactctggcg catcgggctt cccatacaat cgatagattg tcgcacctga ttgcccgaca
2580 ttatcgcgag cccatttata cccatataaa tcagcatcca tgttggaatt
taatcgcggc 2640 ctcgagcaag acgtttcccg ttgaatatgg ctcataacac
cccttgtatt actgtttatg 2700 taagcagaca gttttattgt tcatgatgat
atatttttat cttgtgcaat gtaacatcag 2760 agattttgag acacaacgtg
gctttccccc cccccccatt attgaagcat ttatcagggt 2820 tattgtctca
tgagcggata catatttgaa tgtatttaga aaaataaaca aataggggtt 2880
ccgcgcacat ttccccgaaa agtgccacct gacgtctaag aaaccattat tatcatgaca
2940 ttaacctata aaaataggcg tatcacgagg ccctttcgtc ctcgcgcgtt
tcggtgatga 3000 cggtgaaaac ctctgacaca tgcagctccc ggagacggtc
acagcttgtc tgtaagcgga 3060 tgccgggagc agacaagccc gtcagggcgc
gtcagcgggt gttggcgggt gtcggggctg 3120 gcttaactat gcggcatcag
agcagattgt actgagagtg caccatatgc ggtgtgaaat 3180 accgcacaga
tgcgtaagga gaaaataccg catcagattg gctattggcc attgcatacg 3240
ttgtatccat atcataatat gtacatttat attggctcat gtccaacatt accgccatgt
3300 tgacattgat tattgactag ttattaatag taatcaatta cggggtcatt
agttcatagc 3360 ccatatatgg agttccgcgt tacataactt acggtaaatg
gcccgcctgg ctgaccgccc 3420 aacgaccccc gcccattgac gtcaataatg
acgtatgttc ccatagtaac gccaataggg 3480 actttccatt gacgtcaatg
ggtggagtat ttacggtaaa ctgcccactt ggcagtacat 3540 caagtgtatc
atatgccaag tacgccccct attgacgtca atgacggtaa atggcccgcc 3600
tggcattatg cccagtacat gaccttatgg gactttccta cttggcagta catctacgta
3660 ttagtcatcg ctattaccat ggtgatgcgg ttttggcagt acatcaatgg
gcgtggatag 3720 cggtttgact cacggggatt tccaagtctc caccccattg
acgtcaatgg gagtttgttt 3780 tggcaccaaa atcaacggga ctttccaaaa
tgtcgtaaca actccgcccc attgacgcaa 3840 atgggcggta ggcgtgtacg
gtgggaggtc tatataagca gagctcgttt agtgaaccgt 3900 cagatcgcct
ggagacgcca tccacgctgt tttgacctcc atagaagaca ccgggaccga 3960
tccagcctcc gcggccggga acggtgcatt ggaacgcgga ttccccgtgc caagagtgac
4020 gtaagtaccg cctatagact ctataggcac acccctttgg ctcttatgca
tgctatactg 4080 tttttggctt ggggcctata cacccccgct tccttatgct
ataggtgatg gtatagctta 4140 gcctataggt gtgggttatt gaccattatt
gaccactccc ctattggtga cgatactttc 4200 cattactaat ccataacatg
gctctttgcc acaactatct ctattggcta tatgccaata 4260 ctctgtcctt
cagagactga cacggactct
gtatttttac aggatggggt cccatttatt 4320 atttacaaat tcacatatac
aacaacgccg tcccccgtgc ccgcagtttt tattaaacat 4380 agcgtgggat
ctccacgcga atctcgggta cgtgttccgg acatgggctc ttctccggta 4440
gcggcggagc ttccacatcc gagccctggt cccatgcctc cagcggctca tggtcgctcg
4500 gcagctcctt gctcctaaca gtggaggcca gacttaggca cagcacaatg
cccaccacca 4560 ccagtgtgcc gcacaaggcc gtggcggtag ggtatgtgtc
tgaaaatgag cgtggagatt 4620 gggctcgcac ggctgacgca gatggaagac
ttaaggcagc ggcagaagaa gatgcaggca 4680 gctgagttgt tgtattctga
taagagtcag aggtaactcc cgttgcggtg ctgttaacgg 4740 tggagggcag
tgtagtctga gcagtactcg ttgctgccgc gcgcgccacc agacataata 4800
gctgacagac taacagactg ttcctttcca tgggtctttt ctgcagtcac cgtcgtcgac
4860 acgtgtgatc agatatcgcg gccgctctag accaggcgcc tggatccgcc
accatgccac 4920 tctgggtgtt cttctttgtg atcctcaccc tcagcaacag
ctcccactgc tccccacctc 4980 cccctttgac cctcaggatg cggcggtatt
atgcagatgc catcttcacc aacagctacc 5040 ggaaggtgct gggccagctg
tccgcccgca agctgctcca ggacatcatg agcaggcagc 5100 agggagagag
aaaccaagag caaggagcaa gggtgcggct tgggcggaaa gtagaaacgt 5160
ttctgcgtat tgtacagtgt cgtagcgtag aagggagctg tgggttttga agatcttagt
5220 agtagtaggc ggccgctcta gaggatccag atct 5254 61 39 DNA
Artificial Sequence Description of Artificial Sequence Primer 61
agatctgcca ccatgccact ctgggtgttc ttctttgtg 39 62 36 DNA Artificial
Sequence Description of Artificial Sequence Primer 62 ggatccaagc
cgcacccttg ctccttgctc ttggtt 36 63 492 DNA Artificial Sequence
Description of Artificial Sequence Primer 63 ggttttttgt ggatccaagg
ccgagacgta cctgcgggtc atgaagtgtc gccgcttcgt 60 ggaaagcagc
tgtgccttca cctacaaaga gtttgagcgg gcgtacatcc ccgagggaca 120
gaggtactcc atccagaacg cgcaggccgc cttctgcttc tcggagacca tcccggcccc
180 cacgggcaag gacgaggccc agcagcgatc cgacgtggag ctgctccgct
tctccctgct 240 gctcatccag tcgtggctcg ggcccgtgca gtttctcagc
agggtcttca ccaacagcct 300 ggtgttcggc acctcagacc gagtctacga
gaagctcaag gacctggagg aaggcatcca 360 agccctgatg cgggagctgg
aagatggcag tccccgggcc gggcagatcc tgaagcagac 420 ctacgacaag
tttgacacga acctgcgcag tgacgatgcg ctgcttaaga actacgggct 480
gctctcctgc tt 492 64 69 DNA Artificial Sequence Description of
Artificial Sequence Primer 64 ggatccgaag gcacagctgc tttccacgaa
gcggcgacac ttcatgaccc gcaggtacgt 60 ctcggcctt 69 65 102 DNA
Artificial Sequence Description of Artificial Sequence Primer 65
agatcttcaa agccgcaccc ttgctccttg ctcttggttt ctctctccct gctgcctgct
60 catgatgtcc tggagcagct tgcgggcgga cagctggccc ag 102 66 21 DNA
Artificial Sequence Description of Artificial Sequence Primer 66
ccgcggcatc ctgagggtca a 21 67 21 DNA Artificial Sequence
Description of Artificial Sequence Primer 67 tatgcagatg ccatcttcaa
c 21
* * * * *